METHODS IN MOLECULAR BIOLOGY™

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 Neurotrophic Factors
Methods and Protocols

Edited by

Stephen D. Skaper
Department of Pharmacology and Anesthesiology, University of Padova, Padova, Italy
Editor
Stephen D. Skaper, Ph.D.
Department of Pharmacology and Anesthesiology
University of Padova
Padova, Italy
[email protected]

ISSN 1064-3745 e-ISSN 1940-6029

ISBN 978-1-61779-535-0 e-ISBN 978-1-61779-536-7
DOI 10.1007/978-1-61779-536-7
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Preface

Neuroscience has been described by Nobel laureate and neurobiologist Eric Kandel as the
“last frontier” of science. Nervous system development evolves from the well-orchestrated
processes of neural induction, cell proliferation, differentiation, cell migration, survival, and
synapse formation. Among these environmental cues, neurotrophic factors are secreted
proteins that promote neurite outgrowth, neuronal cell differentiation and survival both
in vivo and in vitro. Nerve growth factor (NGF) is the founding and best-characterized
member of the neurotrophin family of neurotrophic polypeptides and was discovered more
than half a century ago. Since their initial discovery, neurotrophic factors have raised expec-
tations that their clinical application to neurodegenerative diseases might provide an effec-
tive therapy for what are now untreatable conditions.
Exploring nervous system function and dysfunction is oftentimes impractical in humans,
and the availability of ex vivo and in vivo models which mimic, as closely as possible, how
neural cells act and interact among themselves is of critical importance in neurobiological
research. This volume of Methods in Molecular Biology aims to provide the reader, special-
ist and novice alike, with a selection of protocols and procedures which make use of cellular,
tissue, and whole animal models which can be applied to the investigation of neurotrophic
factors and other agents impacting on these systems. The book begins with a number of
chapters dealing with the culture of neurons and glia from the central and peripheral ner-
vous systems, neuron–glia coculture models, and cell-based assays for the evaluation of
neuroprotective molecules, as well as assays which can be applied to the study of agents
with neuroregenerative potential. Protocols describing viral- and nanoparticle-based deliv-
ery methods to neural cells are also presented, followed by chapters dealing with organo-
typic slice culture protocols. Lastly, several chapters are dedicated to in vivo lesion models
of relevance to nervous system pathology, which can be applied to the investigation of neu-
rotrophic factors and peptides.
I would like to gratefully acknowledge the contributors for their excellent cooperation
and patience during the course of this project. While extensive, this volume is by no means
intended to be all-inclusive, given the field’s vastness and publication space limitations.
Even so, I sincerely hope that this book will be useful to a broad spectrum of readers as they
explore nervous system physiology and pathology.

Padova, Italy Stephen D. Skaper

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

1 The Neurotrophin Family of Neurotrophic Factors: An Overview . . . . . . . . . . 1

Stephen D. Skaper
2 Neuronal Growth-Promoting and Inhibitory Cues
in Neuroprotection and Neuroregeneration . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Stephen D. Skaper
3 Culture of Rat Cerebellar Granule Neurons and Application
to Identify Neuroprotective Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Laura Facci and Stephen D. Skaper
4 Isolation and Culture of Neural Progenitor Cells from Rat
Postnatal Cerebellum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Morena Zusso and Patrizia Debetto
5 Culture of Rodent Cortical and Hippocampal Neurons . . . . . . . . . . . . . . . . . . 49
Laura Facci and Stephen D. Skaper
6 Amyloid b-Peptide Neurotoxicity Assay Using Cultured
Rat Cortical Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Laura Facci and Stephen D. Skaper
7 Culture of Neonatal Rodent Microglia, Astrocytes,
and Oligodendrocytes from Cortex and Spinal Cord . . . . . . . . . . . . . . . . . . . . 67
Stephen D. Skaper, Carla Argentini, and Massimo Barbierato
8 Central Nervous System Neuron-Glia Co-culture Models . . . . . . . . . . . . . . . . 79
Stephen D. Skaper and Laura Facci
9 Culture and Characterization of Rat Mesencephalic
Dopaminergic Neurons. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Stephen D. Skaper, Giulia Mercanti, and Laura Facci
10 Preparation of Adult Spinal Cord Motor Neuron Cultures
Under Serum-Free Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Jose V. Montoya-Gacharna, Jhon Jairo Sutachan,
Wai Si Chan, Alexandra Sideris, Thomas J.J. Blanck,
and Esperanza Recio-Pinto
11 Rodent Retinal Ganglion Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Stephen D. Skaper
12 Culture of Purified Glial Cell Populations from Optic Nerve . . . . . . . . . . . . . . 131
Stephen D. Skaper
13 Isolation and Culture of Rat Cone Photoreceptor Cells . . . . . . . . . . . . . . . . . . 147
Stephen D. Skaper

vii
viii Contents

14 Culture of Rat Retina Pigmented Epithelial Cells . . . . . . . . . . . . . . . . . . . . . . 159

Stephen D. Skaper
15 Mammalian Growth Cone Turning Assays Identify Distinct
Cell Signalling Mechanisms That Underlie Axon Growth,
Guidance and Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Andrew J. Murray, Andrew G. Peace, Steven J. Tucker,
and Derryck A. Shewan
16 Culture of Dissociated Sensory Neurons from Dorsal Root
Ganglia of Postnatal and Adult Rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Davina E. Owen and Julie Egerton
17 Culture and Proliferation of Highly Purified Adult Schwann Cells
from Rat, Dog, and Man. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Kirsten Haastert-Talini
18 Use of PC12 Cells and Rat Superior Cervical Ganglion
Sympathetic Neurons as Models for Neuroprotective Assays
Relevant to Parkinson’s Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Cristina Malagelada Grau and Lloyd A. Greene
19 Compartmented Chambers for Studying Neurotrophic Factor Action . . . . . . . 213
Stephen D. Skaper
20 Preparation and Culture of Adrenal Chromaffin Cells . . . . . . . . . . . . . . . . . . . 223
Natalia Domínguez, Miriam Rodríguez, J. David Machado,
and Ricardo Borges
21 Indirect Immunofluorescence Staining of Cultured Neural Cells . . . . . . . . . . . 235
Massimo Barbierato, Carla Argentini, and Stephen D. Skaper
22 Neurite Outgrowth Assessment Using High Content
Analysis Methodology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Nicholas M. Radio
23 Dissociated Cell Culture for Testing Effects of Carbon Nanotubes
on Neuronal Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
William Lee and Vladimir Parpura
24 High-Resolution Imaging and Evaluation of Spines in Organotypic
Hippocampal Slice Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Frederik Sündermann, Nataliya Golovyashkina, Christian Tackenberg,
Roland Brandt, and Lidia Bakota
25 Imaging Amyloid Precursor Protein In Vivo:
An Axonal Transport Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Tomás L. Falzone and Gorazd B. Stokin
26 The Use of Specific AAV Serotypes to Stably Transduce Primary
CNS Neuron Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
Saafan Z. Malik, Margaret A. Maronski, Marc A. Dichter,
and Deborah J. Watson
27 Preparation and Characterization of Biocompatible Chitosan
Nanoparticles for Targeted Brain Delivery of Peptides . . . . . . . . . . . . . . . . . . . 321
Secil Caban, Yılmaz Capan, Patrick Couvreur,
and Turgay Dalkara
 Contents ix

28 [3H]Serotonin Release Assay Using Antigen-Stimulated

Rat Peritoneal Mast Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Stephen D. Skaper and Laura Facci
29 Rat Hippocampal Slice Culture Models for the Evaluation
of Neuroprotective Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Elisabetta Gerace, Elisa Landucci, Tania Scartabelli,
Flavio Moroni, and Domenico E. Pellegrini-Giampietro
30 A 6-Hydroxydopamine In Vivo Model of Parkinson’s Disease . . . . . . . . . . . . . 355
Giulia Mercanti, Gianfranco Bazzu, and Pietro Giusti
31 Brain Microdialysis in Freely Moving Animals . . . . . . . . . . . . . . . . . . . . . . . . . 365
Gianfranco Bazzu, Alice Biosa, Donatella Farina,
Ylenia Spissu, Giammario Calia, Sonia Dedola,
Gaia Rocchitta, Rossana Migheli, Pier Andrea Serra,
and Maria Speranza Desole
32 Evaluating Motor Neuron Death in Neonatal Rats Subjected
to Sciatic Nerve Lesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Andre Schwambach Vieira, Alexandre Cesar Santos de Rezende,
and Fabio Rogerio
33 Rodent Spinal Cord Injury Model and Application of Neurotrophic
Factors for Neuroprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
Hari Shanker Sharma and Aruna Sharma

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 417
Contributors

CARLA ARGENTINI • Department of Pharmacology and Anesthesiology,

University of Padova, Padova, Italy
LIDIA BAKOTA • Department of Neurobiology, University of Osnabrück,
Osnabrück, Germany
MASSIMO BARBIERATO • Department of Pharmacology and Anesthesiology,
University of Padova, Padova, Italy
GIANFRANCO BAZZU • Department of Neuroscience, Medical School,
University of Sassari, Sassari, Italy
THOMAS J.J. BLANCK • Department of Anesthesiology, Langone Medical Center,
New York University, New York, NY, USA
ALICE BIOSA • Department of Neuroscience, Medical School, University of Sassari,
Sassari, Italy
RICARDO BORGES • Unidad de Farmacología, Facultad de Medicina,
Universidad de La Laguna, La Laguna, Tenerife, Spain
ROLAND BRANDT • Department of Neurobiology, University of Osnabrück,
Osnabrück, Germany
SECIL CABAN • Department of Pharmaceutical Technology, Hacettepe University,
Ankara, Turkey
GIAMMARIO CALIA • Department of Neuroscience, Medical School, University of Sassari,
Sassari, Italy
YILMAZ CAPAN • Department of Pharmaceutical Technology, Hacettepe University,
Ankara, Turkey
WAI SI CHAN • Department of Anesthesiology, Langone Medical Center,
New York University, New York, NY, USA
PATRICK COUVREUR • Physico-Chimie, Pharmacotechnie, Biopharmacie,
Facult e de Pharmacie, Universit e Paris Sud, UMR Centre National
de la Recherche Scientifique 8612, Chatenay-Malabry, France
TURGAY DALKARA • Department of Neurology, Hacettepe University, Ankara, Turkey
PATRIZIA DEBETTO • Department of Pharmacology and Anesthesiology,
University of Padova, Padova, Italy
SONIA DEDOLA • Department of Neuroscience, Medical School, University of Sassari,
Sassari, Italy
MARIA SPERANZA DESOLE • Department of Neuroscience, Medical School,
University of Sassari, Sassari, Italy
MARC A. DICHTER • Department of Neurology, University of Pennsylvania
School of Medicine, Philadelphia, PA, USA
NATALIA DOMÍNGUEZ • Unidad de Farmacología, Facultad de Medicina,
Universidad de La Laguna, La Laguna, Tenerife, Spain

xi
xii Contributors

JULIE EGERTON • GlaxoSmithKline R&D Ltd, Gunnels Wood Road, Stevenage, UK

LAURA FACCI • Department of Pharmacology and Anesthesiology, University of Padova,
Padova, Italy
TOMÁS L. FALZONE • Instituto de Biología Celular y Neurociencias, CONICET,
Facultad de Medicina, UBA, Buenos Aires, Argentina
DONATELLA FARINA • Department of Neuroscience, Medical School,
University of Sassari, Sassari, Italy
ELISABETTA GERACE • Dipartimento di Farmacologia Preclinica e Clinica
“Mario Aiazzi Mancini”, Università di Firenze, Florence, Italy
PIETRO GIUSTI • Department of Pharmacology and Anesthesiology,
University of Padova, Padova, Italy
NATALIYA GOLOVYASHKINA • Department of Neurobiology, University of Osnabrück,
Osnabrück, Germany
LLOYD A. GREENE • Department of Pathology and Cell Biology, Columbia University,
New York, NY, USA
KIRSTEN HAASTERT-TALINI • Hannover Medical School, Institute of Neuroanatomy,
Hannover, Germany
ELISA LANDUCCI • Dipartimento di Farmacologia Preclinica e Clinica
“Mario Aiazzi Mancini”, Università di Firenze, Florence, Italy
WILLIAM LEE • Department of Neurobiology, Center for Glial Biology in Medicine,
Atomic Force Microscopy and Nanotechnology Laboratories, Civitan International
Research Center, Evelyn F. McKnight Brain Institute, University of Alabama,
Birmingham, AL, USA
J. DAVID MACHADO • Unidad de Farmacología, Facultad de Medicina,
Universidad de La Laguna, La Laguna, Tenerife, Spain
CRISTINA MALAGELADA GRAU • Department of Pharmacology, University of Barcelona,
Barcelona, Spain
SAAFAN Z. MALIK • Department of Neurosurgery, University of Pennsylvania
School of Medicine, Philadelphia, PA, USA
MARGARET A. MARONSKI • Department of Neurology, University of Pennsylvania
School of Medicine, Philadelphia, PA, USA
GIULIA MERCANTI • Department of Pharmacology and Anesthesiology,
University of Padova, Padova, Italy
ROSSANA MIGHELI • Department of Neuroscience, Medical School, University of Sassari,
Sassari, Italy
JOSE V. MONTOYA-GACHARNA • Department of Anesthesiology, Langone Medical Center,
New York University, New York, NY, USA
FLAVIO MORONI • Dipartimento di Farmacologia Preclinica e Clinica
“Mario Aiazzi Mancini”, Università di Firenze, Florence, Italy
ANDREW J. MURRAY • Department of Biochemistry and Molecular Biophysics,
Columbia University, New York, NY, USA
DAVINA E. OWEN • Convergence Pharmaceuticals Ltd, Babraham Research Campus,
Cambridge, UK
 Contributors xiii

VLADIMIR PARPURA • Department of Neurobiology, Center for Glial Biology

in Medicine, Atomic Force Microscopy and Nanotechnology Laboratories,
Civitan International Research Center, Evelyn F. McKnight Brain Institute,
University of Alabama, Birmingham, AL, USA
ANDREW G. PEACE • School of Medical Sciences, College of Life Sciences and Medicine,
University of Aberdeen, Foresterhill, Aberdeen, UK
DOMENICO E. PELLEGRINI-GIAMPIETRO • Dipartimento di Farmacologia Preclinica
e Clinica “Mario Aiazzi Mancini”, Università di Firenze, Florence, Italy
NICHOLAS M. RADIO • Thermo Fisher Scientific, Pittsburgh, PA, USA
ESPERANZA RECIO-PINTO • Department of Anesthesiology, Langone Medical Center,
New York University, New York, NY, USA
ALEXANDRE CESAR SANTOS DE REZENDE • Department of Anatomy, Cellular Biology,
Physiology and Biophysics, State University of Campinas, Campinas, SP, Brazil
GAIA ROCCHITTA • Department of Neuroscience, Medical School, University of Sassari,
Sassari, Italy
MIRIAM RODRÍGUEZ • Unidad de Farmacología, Facultad de Medicina,
Universidad de La Laguna, La Laguna, Tenerife, Spain
FABIO ROGERIO • Department of Pathology, State University of Campinas,
Campinas, SP, Brazil
TANIA SCARTABELLI • Dipartimento di Farmacologia Preclinica e Clinica
“Mario Aiazzi Mancini”, Università di Firenze, Florence, Italy
PIER ANDREA SERRA • Department of Neuroscience, Medical School,
University of Sassari, Sassari, Italy
HARI SHANKER SHARMA • Department of Surgical Sciences, Anesthesiology
and Intensive Care Medicine, Laboratory of Cerebrovascular and Pain Research,
University Hospital, Uppsala University, Uppsala, Sweden
ARUNA SHARMA • Department of Surgical Sciences, Anesthesiology and Intensive Care
Medicine, Laboratory of Cerebrovascular and Pain Research, University Hospital,
Uppsala University, Uppsala, Sweden
DERRYCK A. SHEWAN • School of Medical Sciences, College of Life Sciences and Medicine,
University of Aberdeen, Foresterhill, Aberdeen, UK
ALEXANDRA SIDERIS • Department of Anesthesiology, Langone Medical Center,
New York University, New York, NY, USA
STEPHEN D. SKAPER • Department of Pharmacology and Anesthesiology,
University of Padova, Padova, Italy
YLENIA SPISSU • Department of Neuroscience, Medical School, University of Sassari,
Sassari, Italy
GORAZD B. STOKIN • Studenec 48 and Division of Neurology, University
Psychiatric Hospital, University Medical Center, Ljubljana, Slovenia
FREDERIK SÜNDERMANN • Department of Neurobiology, University of Osnabrück,
Osnabrück, Germany
JHON JAIRO SUTACHAN • Department of Anesthesiology, Langone Medical Center,
New York University, New York, NY, USA
xiv Contributors

CHRISTIAN TACKENBERG • Department of Neurobiology, University of Osnabrück,

Osnabrück, Germany
STEVEN J. TUCKER • School of Medical Sciences, College of Life Sciences and Medicine,
University of Aberdeen, Foresterhill, Aberdeen, UK
ANDRE SCHWAMBACH VIEIRA • Department of Anatomy, Cellular Biology,
Physiology and Biophysics, State University of Campinas, Campinas,
SP, Brazil
DEBORAH J. WATSON • Department of Neurosurgery, University of Pennsylvania
School of Medicine, Philadelphia, PA, USA
MORENA ZUSSO • Department of Pharmacology and Anesthesiology,
University of Padova, Padova, Italy
 Chapter 1

The Neurotrophin Family of Neurotrophic

Factors: An Overview
Stephen D. Skaper

Abstract
The neurotrophins are a family of closely related proteins that were first identified as survival factors for
sympathetic and sensory neurons and have since been shown to control a number of aspects of survival,
development, and function of neurons in both the central and peripheral nervous systems. Limiting quan-
tities of neurotrophins during development control the numbers of surviving neurons to ensure a match
between neurons and the requirement for a suitable density of target innervation. Biological effects of each
of the four mammalian neurotrophins are mediated through activation of one or more of the three mem-
bers of the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases (TrkA, TrkB, and TrkC).
In addition, all neurotrophins activate the p75 neurotrophin receptor, a member of the tumor necrosis
factor receptor superfamily. Neurotrophin engagement of Trk receptors leads to activation of Ras, phos-
phatidylinositol 3-kinase, phospholipase C-γ1, and signaling pathways controlled through these proteins,
including the mitogen-activated protein kinases. Neurotrophin availability is required into adulthood,
where they control synaptic function and plasticity and sustain neuronal cell survival, morphology, and
differentiation. This chapter will provide an overview of neurotrophin biology, their receptors, and signal-
ing pathways.

Key words: Neurotrophic factors, Nerve growth factor, Brain-derived neurotrophic factor,
Neurotrophin-3, Neurotrophin-4/5, Glial cell line–derived neurotrophic factor, Tropomyosin-related
kinase, Receptor tyrosine kinases, Neurodegeneration, Neuroregeneration

1. Introduction

Neurotrophic factors are secreted proteins that promote neurite

outgrowth, neuronal cell differentiation, and survival both in vivo
and in vitro. Nerve growth factor (NGF) is the founding and best
characterized member of the neurotrophin family (1) of neu-
rotrophic polypeptides and was discovered more than half a cen-
tury ago (2) during a search for survival factors that could explain
the deleterious effects of target tissue ablation on the subsequent
survival of motor and sensory neurons (1). NGF is also present

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_1, © Springer Science+Business Media, LLC 2012

1
2 S.D. Skaper

in the central nervous system (CNS) where it serves a trophic

function in the development and maintenance of basal forebrain
cholinergic neurons (3).
Neurotrophins are synthesized at a considerable distance from
the cell body by peripheral tissues or neurons (“targets”) that are
contacted by axons of the neurotrophin-sensitive neurons. In the
periphery, the tissue sources of neurotrophins are typically non-
neuronal cells, whereas in the CNS, they are synthesized predomi-
nantly by neurons under physiological conditions (4). During
development, a retrograde flow of a neurotrophin is established,
transporting the protein from the target into the nerve terminal
and up the axon to the cell body (5). Those neurons that establish
this flow survive the period of neuronal cell death, while those that
do not, degenerate. Once the retrograde flow of neurotrophin is
established, it must continue for the lifetime of the neuron to
maintain the functional differentiated state of the neuron (6).
Recent studies on the expression and actions of the neurotrophin
family indicate that, in addition to target-derived factor acquisi-
tion, autocrine and non-target-derived paracrine modes of factor
presentation are likely to be important (7) (see Fig. 1).
Since their initial discovery, neurotrophic factors have raised
expectations that their clinical application to neurodegenerative
diseases might provide an effective therapy for what are now
untreatable conditions. Indeed, there is an impressive volume of
evidence for neuroprotective effects of neurotrophic factors in ani-
mal models of neurodegenerative diseases (see, e.g., ref. (8)). This
chapter is intended to introduce the reader to the basic concepts of
neurotrophic factor biology which operate to affect nervous system
function.

Fig. 1. Target-derived, autocrine, and paracrine modes of neurotrophin presentation. During the period of target innervation,
neurotrophins support the survival of a restricted number of neurons expressing the appropriate tropomyosin-related
kinase (Trk) receptors. Limiting amounts of secreted neurotrophins do not allow for the survival of all neurons, which can,
however, be rescued by the administration of exogenous neurotrophins.
 1 The Neurotrophin Family of Neurotrophic Factors: An Overview 3

2. Neurotrophin
Biology
The generality of the phenomenon of programmed cell death after
target deprivation (axotomy) has suggested that most neurons
respond to and are regulated by neurotrophic factors (9). This
hypothesis was validated by the subsequent isolation from pig brain
of a second neurotrophic factor, designated brain-derived neu-
rotrophic factor (BDNF) (10). Molecular cloning of the BDNF
gene (11) revealed its structural similarity to NGF, leading to the
concept of the neurotrophin family. Two additional members,
neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5), were
later identified (12). The term neurotrophin-4/5 resulted from
uncertainties about whether the human neurotrophin-5 (13) was a
species homolog of the NT-4 found in Xenopus (14). The nomen-
clature NT-4/5 was subsequently adopted by a number of investi-
gators to denote the mammalian form of Xenopus NT-4 when it
was shown that the differences between the two factors were
only due to phylogenetic variation (15). Two novel neurotrophins
from the platyfish and carp have been cloned and designated
neurotrophin-6 (16) and neurotrophin-7 (17), respectively. These
do not have orthologs in mammals or birds and appear to interact
with the same receptors as the mammalian proteins. The neu-
rotrophins exhibit actions on distinct, as well as partially overlap-
ping, subsets of peripheral and central neurons (12, 18). Individual
neurons may also be responsive to more than one neurotrophin at
a given time or at subsequent times during development.
The mature neurotrophin proteins are noncovalently associ-
ated homodimers. The neurotrophins share a highly homologous
structure and are members of a large superfamily of growth factors
that contain a tertiary fold and cysteine “knot.” These features are
present in transforming growth factor-β, platelet-derived growth
factor, human chorionic gonadotropin, vascular endothelial growth
factor, and others. The cysteine knot consists of three disulfide
bonds that form a true knot of the polypeptide chain. Two cysteines
that make up the knot are missing from human neurotrophin-6.
Neurotrophin residues are generally divided into two categories,
conserved or variable, based on sequence alignments (12). Amino
acid residues implicated in neurotrophin binding that are conserved
are likely to represent a common interface to the tropomyosin-related
kinase (Trk) receptors, while the unique ones may represent elements
of specificity (19). The dimer interface is composed of β-strands
that maintain the conformation; these hydrophobic core residues
are highly conserved (20–22). In contrast to the β-strands, the
β-loops are highly variable. Detailed discussions of neurotrophin
structure and molecular evolution have been published (23–26).
4 S.D. Skaper

In addition to the neurotrophins, a number of polypeptide

factors have been shown to possess neurotrophic activities. These
include ciliary neurotrophic factor (CNTF) (27), glial cell line–
derived neurotrophic factor (GDNF) (23), insulin-like growth
factor (28), and basic fibroblast growth factor (29). Transforming
growth factor-β (30) and Sonic hedgehog (31) are other proteins
capable of promoting survival of specific CNS neuron populations
and protecting these cells from toxic insults.

3. Neurotrophin
Receptors
The neurotrophins interact with two entirely distinct classes of
receptors, Trks and p75NTR, the first discovered member of the
tumor necrosis factor receptor superfamily. The former was initially
identified as a low-affinity receptor for NGF (32) but was subse-
quently shown to bind each of the neurotrophins with approxi-
mately equal nanomolar affinity (14, 33, 34). While p75
neurotrophin receptor (p75NTR) does not contain a catalytic motif,
it interacts with several proteins that relay signals important for
regulating neuronal cell survival, differentiation, and synaptic plas-
ticity. Each of the four cysteine-rich repeats of p75NTR participates
in binding to NGF (35). p75NTR binds NGF along the interface
between two NGF monomers, and binding results in a conforma-
tional change in NGF that alters the monomeric interface on the
opposite side of the NGF dimer, eliminating the potential for bind-
ing of one NGF dimer to two p75NTR monomers.
In mammals, the Trk subfamily of receptor tyrosine kinases
constitutes the second major class of neurotrophin receptors. The
extracellular domain of each of the Trks consists of a cysteine-rich
cluster followed by three leucine-rich repeats, another cysteine-rich
cluster, and two Ig-like domains (see Fig. 2). Each receptor has a
single transmembrane region that terminates in a cytoplasmic,
tyrosine kinase-containing domain surrounded by several tyrosine
residues that serve as phosphorylation-dependent docking sites for
cytoplasmic adaptors and enzymes. The neurotrophins dimerize
their cognate Trk receptor, resulting in activation via transphos-
phorylation of the cytoplasmic domain kinases. Specificity of neu-
rotrophin action is believed to be achieved in part by the selective
interaction between members of the Trk family of receptors and
the different neurotrophins. Thus, NGF binds to TrkA (36, 37),
TrkB binds BDNF and NT-4/5 with high affinity (38, 39), and
TrkC binds NT-3 (40). NT-3 can also interact, albeit with less effi-
ciency, with TrkA and TrkB (see Fig. 2) (15, 39).
Trk receptor function is modulated by p75NTR on several levels—
by promoting ligand binding, by promoting accessibility to neurotro-
phins through promotion of axonal growth and target innervation,
 1 The Neurotrophin Family of Neurotrophic Factors: An Overview 5

Fig. 2. Neurotrophins and their receptors. The neurotrophins display specific interactions with the three Trk receptors: NGF
binds TrkA, BDNF and NT-4 bind TrkB, and NT-3 binds TrkC. In some cellular contexts, NT-3 can also activate TrkA and TrkB,
albeit with less efficiency. All neurotrophins bind to and activate p75 neurotrophin receptor (p75NTR). CR1–CR4 cysteine-rich
motifs, C1/C2 cysteine-rich clusters, LRR1–3 leucine-rich repeats, Ig1/Ig2 immunoglobulin-like domains.

and by promoting endocytosis and retrograde transport to

membrane compartments where internal engagement of neurotro-
phins with Trk receptors may promote signaling. For example,
p75NTR inhibits activation of Trk receptors by nonpreferred
neurotrophins both in vivo and in vitro (41, 42).While p75NTR
potentiates activation of TrkA by suboptimal concentrations of NGF,
it does not potentiate the activation of other Trk receptors similarly
by their ligands (43). In addition, p75NTR collaborates with TrkA to
form high-affinity (10−11 M) binding sites for NGF (44). Besides
facilitating the binding of NGF to TrkA, p75NTR can promote retro-
grade transport of several neurotrophins (45). p75NTR may reduce
ligand-induced Trk receptor ubiquitination, thereby delaying Trk
internalization and degradation (46), or promote Trk receptor
endocytosis through polyubiquitination and subsequent internaliza-
tion to endosomal compartments, resulting in enhanced signaling
(47). These findings each suggest a mechanism by which p75NTR
may promote axon growth and target innervation in vivo and in vitro
(48, 49). Sensory and sympathetic deficits are seen in mice lacking
p75NTR (50–52).
The major site at which neurotrophins interact with the Trk
receptors is in the membrane-proximal Ig-like domain. The three-
dimensional structures of the domain in each of the Trk receptors
have been solved (53). In addition, the structure of NGF bound to
the TrkA Ig domain has also been determined (19). These results
have made it possible to identify residues in the neurotrophins and
6 S.D. Skaper

the Trk receptors that account for the specificity observed in their
interactions (54). Such information may prove useful in the design
of small molecule mimetic ligands for neurotrophin receptors.

4. Neurotrophin
Signaling
Many extracellular signals transduce their cellular responses by reg-
ulating tyrosine phosphorylation of their target proteins. Ligand-
induced oligomerization of receptor protein tyrosine kinases and
autophosphorylation have been established as a general mechanism
for the activation of growth factor receptors, as well as many other
families of cell surface receptors (55). The Trk receptors are typical
receptor tyrosine kinases whose activation is stimulated by neu-
rotrophin-mediated dimerization and transphosphorylation of
activation loop kinases (56). Trk receptors are activated specifically
by the mature and not the pro-forms of the neurotrophin gene
products (57). Thus, the proteases that control processing of
proneurotrophins control Trk receptor responsiveness. The cyto-
plasmic domains of the Trk receptors contain several additional
tyrosines that are also substrates for phosphorylation by each recep-
tor’s tyrosine kinase. When phosphorylated, these residues form
the cores of binding sites that serve as a scaffolding for the recruit-
ment of a variety of adaptor proteins and enzymes that ultimately
propagate the neurotrophin signal (58). Within the activated Trk
molecule, the phosphotyrosines and their surrounding amino acid
residues create binding sites for proteins containing phosphoty-
rosine-binding or Src homology 2 domains. The major pathways
activated by the Trk receptors are Ras, Rac, phosphatidylinositol
3-kinase, phospholipase C-γ1, and their downstream effectors
(56, 58). In addition, endocytosis and transfer of Trk receptors to
different membrane compartments control the efficiency and dura-
tion of Trk-mediated signaling in part because many adaptor
proteins are localized to specific membrane compartments (59).
Activation of p75NTR results in activation of the nuclear factor-κB
and Jun kinase, as well as other signaling pathways (see Fig. 3).
Transactivation of receptor tyrosine kinases by G protein–cou-
pled receptors (GPCRs) is now recognized as an important signal-
ing mechanism allowing the cell to respond to a vast array of
extracellular stimuli (60, 61). Activation of TrkA and TrkB recep-
tors can also occur via a GPCR mechanism, in the absence of NGF
or BDNF (62, 63). Two GPCR ligands, adenosine and pituitary
adenylate cyclase–activating peptide, can activate Trk receptor
activity to increase the survival of neuronal cells through stimula-
tion of protein kinase B (Akt) activity. The effects of adenosine and
pituitary adenylate cyclase–activating peptide can be blocked by
K252a, an inhibitor of Trk tyrosine kinases. In contrast to
 1 The Neurotrophin Family of Neurotrophic Factors: An Overview 7

Fig. 3. Neurotrophin signaling. Depicted are interactions of NGF (exemplar neurotrophin) with Trk and major intracellular
signaling pathways activated. Neurotrophin binding to Trk receptor leads to dimerization and autophosphorylation. The
linker Shc binds to phospho-Y490 on Trk and to a Grb2-SOS complex. SOS is a nucleotide exchange factor that activates
Ras by replacing GDP with GTP. Activated Ras interacts directly with the serine–threonine kinase Raf. The activated Raf
leads to the sequential activation of MAPK kinase (MEK), the mitogen-activated protein kinase-ERK kinase (MAPK). MAPK
translocates to the nucleus, where it phosphorylates transcription factors, promoting neuronal cell differentiation. Activation
of phosphatidylinositol 3-kinase through Ras or Gab1 promotes survival and growth of neurons. Activation of phospholi-
pase C-γ1 (PLC-γ1) results in activation of Ca2+- and protein kinase C-regulated pathways that promote synaptic
plasticity.

neurotrophin treatment, Trk receptor activation by adenosine

analogs and pituitary adenylate cyclase–activating peptide is sensi-
tive to transcriptional and translational inhibitors, with the major-
ity of the transactivated Trk receptors found in intracellular
membranes, suggesting that receptor signaling may occur and per-
sist inside of neuronal cells (64). More recent studies indicate that
the tyrosine kinase Fyn is activated by GPCR stimulation and is
responsible for transactivation of Trk receptors on intracellular
membranes (65).
Several excellent reviews describe recent progress in under-
standing the signaling pathways stimulated by the neurotrophins
that affect the survival, differentiation, and function of cell within
the nervous system (66–69).

5. Why Study
Neurotrophins?
The power of neurotrophic factors to regulate neuronal cell sur-
vival in the developing nervous system and to promote also survival
after injury or protect neurons in toxin-mediated disease models in
8 S.D. Skaper

animals has encouraged the idea that such proteins could be

harnessed for the treatment of neurodegenerative disease. For
example, the traditional perspective of applying neurotrophins in
the context of Alzheimer’s disease is based on the premise that
neurotrophins are capable of upregulating cholinergic function
and of rendering neurons less vulnerable to certain processes caus-
ing degeneration (70, 71). Another example is that of GDNF,
which acts on dopaminergic neurons. Administered intracerebrally,
GDNF has neuroprotective and neurorestorative effects in toxin-
induced rodent and nonhuman primate models of Parkinson’s
disease (72, 73). Neurotrophic factor treatment of CNS diseases
presents an especially complex problem since these polypeptides
have poor pharmacokinetics and bioavailability and are not able to
cross the blood–brain barrier. Neurotrophic proteins can be modi-
fied to increase blood–brain barrier transport (74), and intrave-
nous administration of BDNF, conjugated to an anti-transferrin
antibody, is able to traverse the blood–brain barrier in rats and
provide neuroprotection in focal transient brain ischemia (75, 76).
Viral vector or cell-based gene therapy approaches may also prove
advantageous for the targeted delivery of lower doses of neu-
rotrophic factors, and dopaminergic neuroprotection by viral-
mediated delivery of GDNF has been demonstrated in animals
(72, 77). In the case of spinal cord injury, the transplantation of
genetically modified cells (e.g., viral expression of neurotrophins
either in vivo or ex vivo in stem cells) to lesion sites has been
investigated as a means to augment axonal regeneration (78, 79).
A cell-based approach for NGF delivery has shown promise in a
phase I trial in Alzheimer’s disease (80). In the case of CNTF, a
fusion protein consisting of mature human CNTF and an 11-amino
acid protein transduction domain rescued the learning and memory
impairments induced by amyloid β-peptide in mice (81), as did
infusion of recombinant cells secreting CNTF encapsulated in
alginate polymers (82).
Peripheral nervous system disorders should also be amenable
to a neurotrophic protein therapeutic. NGF, which is selectively
trophic for small fiber sensory and sympathetic neurons, was
selected as a potential therapy for diabetic polyneuropathy because
of the serious consequences associated with degeneration of those
neuronal populations in this condition. In addition, a reduced
NGF availability may contribute to the pathogenesis of diabetic
neuropathy (83), and animal models of neuropathy respond to
the exogenous administration of NGF (84). Two sets of phase II
clinical trials suggested that recombinant human NGF administra-
tion was effective in ameliorating the symptoms associated with
both diabetic polyneuropathy (85) and human immunodeficiency
virus–related neuropathy (86). Clinical trials are continuing in this
direction.
 1 The Neurotrophin Family of Neurotrophic Factors: An Overview 9

Clearly, methodologies which are directed to understanding

neurotrophic protein–receptor interactions, biological activities,
and neuroprotective/neurorestorative effects are of great impor-
tance in advancing this critical area of neuroscience. The protocols
detailed in this volume have been written with this goal in mind.

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 Chapter 2

Neuronal Growth-Promoting and Inhibitory Cues

in Neuroprotection and Neuroregeneration
Stephen D. Skaper

Abstract
During development of the nervous system, neurons extend axons over considerable distances in a highly
stereospecific fashion in order to innervate their targets in an appropriate manner. This involves the recog-
nition, by the axonal growth cone, of guidance cues that determine the pathway taken by the axons. These
guidance cues can act to promote and/or repel growth cone advance. The directed growth of axons is
partly governed by cell adhesion molecules (CAMs) on the neuronal growth cone that bind to CAMs on
the surface of other axons or nonneuronal cells. In vitro assays have established the importance of the
CAMs ((neural cell adhesion molecule NCAM), N-cadherin, and L1) in promoting axonal growth over
cells. Compelling evidence implicates the fibroblast growth factor receptor tyrosine kinase as the primary
signal transduction molecule in the CAM pathway. CAMs are important constituents of synapses, and they
appear to play important and diverse roles in regulating synaptic plasticity associated with learning and
memory. Synthetic NCAM peptide mimetics corresponding to the binding site of NCAM for the fibro-
blast growth factor receptor promote synaptogenesis, enhance presynaptic function, and facilitate memory
consolidation. Dimeric versions of functional binding motifs of N-cadherin behave as N-cadherin agonists,
promoting both neuritogenesis and neuronal cell survival. Negative extracellular signals that physically
direct neurite growth have also been described. The latter include the myelin inhibitory proteins, Nogo,
myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein. Potentiation of outgrowth-
promoting signals, together with antagonism of myelin proteins or their convergent receptor, NgR, and
its second messenger pathways, may provide new opportunities in the rational design of treatments for
acute brain injury and neurodegenerative disorders.

Key words: Cell adhesion molecules, Fibroblast growth factor receptor, Neuroprotection,
Neuroregeneration, Myelin, Nogo, Rho kinase

1. Introduction

Nervous system development evolves from the well-orchestrated

processes of neural induction, cell proliferation, differentiation,
cell migration, survival, and synapse formation (1, 2). The directed
growth of axons is fundamental to synapse formation. Receptors
for guidance molecules are present on growth cones and their

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_2, © Springer Science+Business Media, LLC 2012

13
14 S.D. Skaper

filopodia, and interactions with cognate ligands could result in

promotion, inhibition, attraction, or repulsion of the growth cone.
A broad spectrum of putative growth-promoting and/or guidance
molecules has been identified to date. These include neurotrophic
factors (3, 4), chemoattractants such as netrin 1 and 2 (5), chemore-
pellents from the collapsin/semaphorin family (6–8), and cell
adhesion molecules (CAMs) (2, 9). The importance of the last
class of molecules is evidenced by the severe neurological deficits
found in humans with mutations in the L1 gene, which can include
a complete absence of corticospinal tracts (10).
Growth inhibitory molecules present in the central nervous
system (CNS) myelin contribute, at least in part, to the inability of
mammalian CNS axons to regenerate upon injury (11). In recent
years, some molecular determinants of axonal regeneration and
plasticity in the adult brain have been defined (12). Three proteins,
Nogo-A (13–15), myelin-associated glycoprotein (MAG) (16, 17),
and oligodendrocyte-myelin glycoprotein (OMgp) (18), appear to
be responsible for this inhibition of axonal growth. This myelin
inhibitory biology leads to the hypothesis that modulation of the
interactions of these myelin proteins with their axonal receptor(s)
would overcome the inhibitory effects of CNS myelin and pro-
mote axon regeneration, leading to improved functional recovery
after CNS injury (see Fig. 1). Much recent work has focused upon
the identification and characterization of signal transduction path-
ways for both CAMs and myelin-associated inhibitors.

2. CAMs, Neuronal
Plasticity, and
Neuroprotection
Cell–cell interactions mediated by CAMs are fundamental to
numerous developmental processes. In the nervous system, the
ability of neurons to extend axons and innervate their targets in an
appropriate manner is governed to a large extent by the binding of
CAMs on the surface of other axons or nonneuronal cells (19, 20).
The neuronal receptors important for general cell contact–depen-
dent axonal growth are the β1-integrins, which recognize extracel-
lular matrix molecules; and in mammals, three CAMs, namely, the
neural cell adhesion molecule (NCAM), N-cadherin, and L1, pro-
mote axonal growth during development (2).
N-Cadherin is a member of the classical cadherin family of
transmembrane glycoproteins that mediate cellular recognition
via homophilic (binding with other N-cadherin molecules on
neighboring cells) interaction (21). In the nervous system, N-cadherin
function has been implicated in cell migration (22), axonal growth
and guidance (23), and synapse formation and synaptic plasticity
(24, 25). In addition to homophilic binding, cadherins have been
shown to interact with many adaptor or signaling molecules,
 2 Neuronal Growth-Promoting and Inhibitory Cues in Neuroprotection… 15

Fig. 1. Strategies for central nervous system regeneration. Mechanical injury or traumatic injury, or chronic neurodegen-
erative disease, can result in neurons being “disconnected” from their innervation fields, leading to a loss of critical tissue-
derived trophic support. Regeneration may be promoted by replacement of neurotrophic factors, by stimulation of axonal
outgrowth and/or cell survival with adhesion molecules, and by agents capable of overriding the outgrowth inhibitory
environment. MAG myelin-associated glycoprotein, OMgp oligodendrocyte-myelin glycoprotein.

including the fibroblast growth factor receptor (FGFR). Neurite

outgrowth stimulated by N-cadherin is inhibited by a variety of
agents that block FGFR function in neurons (26–28). Soluble
forms of some adhesion molecules (29), including N-cadherin
(30), are effective also in promoting axonal growth. The homophilic
binding site resides in extracellular domain 1 (ECD1) (31), and
peptide mimetics of two linear sequences from ECD1 (HAVDI
and INPISGQ) function as specific N-cadherin antagonists (32, 33).
This information was then used to design cyclic peptides con-
taining a tandem repeat of the individual motifs, which function
as N-cadherin agonists and stimulate neurite outgrowth (34).
A recent study has shown that a dimeric version (SW4 peptide) of
a short N-cadherin binding motif (HAVDI) is capable of promot-
ing the survival of several populations of CNS neurons, under
distinct paradigms of injury, in an FGFR-dependent manner (35).
An example of this is illustrated in Fig. 2 for hippocampal neurons
subjected to glutamate-induced excitotoxicity. The neuroprotective
16 S.D. Skaper

90

Neuronal cell survival (%)

75

60

45

30

15

0
0 1 3 10 30 50
SW4 (ug/ml)

Fig. 2. The dimeric HAVDI peptide SW4 attenuates glutamate-induced toxicity in culture
hippocampal neurons. Cultures (days in vitro, 7–9) were pretreated with the indicated
concentrations of SW4 peptide for 48 h and then exposed to 50 μM glutamate, and neu-
ronal cell survival was quantified 24 h later by a colorimetric reaction (MTT). Data are
means ± SD (three experiments) expressed relative to no glutamate (100%). **P < 0.05 or
**
P < 0.01 vs. glutamate only. Reproduced with permission from Elsevier (75).

effects of SW4 displayed a concentration-dependence similar to

those inducing neuritogenesis (34). The responses to the dimeric
agonist peptide were inhibited by a monomeric version of the
same motif (itself a highly specific N-cadherin antagonist) (32) and
by a specific FGFR antagonist. These data suggest that the dimeric
agonist peptide functions by binding to and clustering N-cadherin
in neurons, and thereby activating an N-cadherin/FGFR signal-
ing cascade (35). The NCAMs, L1 and CHL1 (36), and the syn-
thetic NCAM peptide ligand, C3d (37), have been reported to
display survival-promoting effects (albeit modest) on cultured
CNS neurons. The neurotropic and neurotrophic effects of CAMs
strengthen the emerging role for adhesion molecules in synaptic
plasticity (38, 39).
CAMs are important constituents of synapses, with well-
recognized roles in building and maintaining synaptic structure
during brain development. Growing evidence indicates that
CAMs play important and diverse roles in regulating synaptic
plasticity associated with learning and memory (40, 41). The role
of NCAM in cognitive processes is demonstrated by studies in
which interference with NCAM function through the administra-
tion of antibodies or gene inactivation has resulted in impaired
long-term potentiation (42, 43) and learning and memory deficits
(44, 45). As with N-cadherin, synthetic NCAM peptide mimetics
have now become available (46). After homophilic binding, NCAM
promotes neurite outgrowth through mechanisms involving inter-
action with the FGFR (47, 48). The recently identified FG loop
 2 Neuronal Growth-Promoting and Inhibitory Cues in Neuroprotection… 17

(FGL) peptide, a 15-amino acid sequence corresponding to the

binding site of NCAM for the FGFR1, has been shown to bind to
and activate FGFR1 and to stimulate neurite outgrowth (48).
Intracerebroventricular administration of the NCAM mimetic FGL
peptide increases memory strength in rats and enhances presynap-
tic function in primary hippocampal neurons (49). These results
provide the first evidence for a memory-facilitating effect resulting
from a treatment that mimics NCAM function.

3. Growth
Inhibitory
Molecules
In contrast to fish, amphibia, and the mammalian peripheral nerves
and developing central nerves, adult central mammalian neurons
do not regrow functional axons after damage. It is not the absence
of growth-promoting molecules in the CNS, but rather the pres-
ence of axonal outgrowth inhibitors in CNS astroglial “scars” (50)
and CNS myelin (51, 52) that suppresses the regrowth of damaged
axons. In particular, two components present in CNS myelin have
been characterized as potent inhibitors of axonal growth: MAG
(16, 17) and Nogo-A, the largest transcript of the recently identi-
fied nogo gene (formerly called NI-220) (13–15). Other inhibitors
include chondroitin sulfate proteoglycans (53) and OMgp (18).
Nogo is a member of the Reticulon family and occurs in three
forms, Nogo-A, -B, and -C, which are generated from alternate
splicing (13–15). Nogo-A, -B, and -C all contain a 66-amino acid
ECD (Nogo-66) that alone can inhibit neurite outgrowth and
induce growth cone collapse (13–15). MAG is a type I membrane
protein composed of five extracellular immunoglobulin (Ig)–like
domains (16, 17), whereas OMgp is a glycosylphosphatidylinosi-
tol-anchored protein (18). Remarkably, a single neuronal protein,
the Nogo-66 receptor (NgR), binds Nogo, MAG, and OMgp
(54–57). The p75 low-affinity neurotrophin receptor protein has
been implicated in transducing a myelin/NgR signal to the axonal
interior (58, 59) (see Fig. 3).
The presence of myelin-derived inhibitors suggests that block-
ing their action might allow the intrinsic growth potential of CNS
axons to be unmasked (60). Indeed, neutralizing Nogo-A with
IN-1 antibody induced CNS axon regeneration and improved
recovery after various lesions (61, 62). Targeting the axonal NgR
with a competitive antagonist compound has the potential to block
the action of the three known myelin inhibitors. Both intrathecal
(63) and delayed systemic application (64) of NEP1–40 (Nogo
extracellular peptide, residues 1–40) produced significant axonal
regrowth after spinal cord hemitransection injury, as well as
enhanced locomotor recovery (64). However, these reagents only
target a single myelin protein, Nogo, which may not be sufficient to
18 S.D. Skaper

Oligodendrocyte C

NogoA MAG
?
? OMgp

Nogo-66
N

NgR
NgR3
NgR2
? p75 Neurite
outgrowth
inhibition
GPI GPI Axon PI

Rho A

ROCK Cytoskeleton

Fig. 3. The Nogo receptor and inhibition of axonal regeneration. The leucine-rich repeat
domains of the Nogo receptor (NgR) are necessary for interaction with Nogo-66, MAG, and
OMgp. NgR does not transduce signals directly but utilizes coreceptor molecules, for
example, p75 or others. Coreceptor activation, in turn, activates the Rho and ROCK path-
way to modulate the cytoskeleton and neurite growth. While structurally similar to NgR1,
NgR2 and NgR3 display essentially no binding to known myelin-derived NgR ligands. GPI
glycosylphosphatidylinositol, ROCK Rho-associated kinase.

facilitate maximal CNS axonal regeneration because other inhibitors

such as MAG and OMgp are present in the CNS myelin environ-
ment. In rats with middle cerebral artery occlusion, both the
recovery of motor skills and corticofugal axonal plasticity are
promoted by intracerebroventricular administration of a function-
blocking NgR fragment, sNgR310-Fc (an Ig-fusion protein con-
taining a soluble fragment of NgR encompassing the ligand binding
domain (sNgR310)) (65). Stroke lesion size was not significantly
reduced in the sNgR310-Fc protein–treated group (65), suggesting
that reduction of NgR function after stroke allows increased
anatomical plasticity and improved motor performance that are
not attributable to neuroprotection. Comparable results were
 2 Neuronal Growth-Promoting and Inhibitory Cues in Neuroprotection… 19

obtained in ngr−/− mice with photothrombotic cortical lesion (65).

A newly described neutralizing anti-Nogo-66 receptor monoclonal
antibody 7E11 was reported to be a more potent inhibitor than
sNgR310-Fc in in vitro competition binding assays and in a neurite
outgrowth assay against CNS myelin (66). Further experiments in
relevant animal models may prove the relevance of anti-NgR anti-
bodies in promoting CNS axonal regeneration (67).
The signaling mechanisms responsible for the transduction of
the inhibitory properties of Nogo-A and MAG domains are not
well understood. Recent evidence supports the notion that cytoskel-
etal components required for proper axonal pathfinding and the
formation of axons and dendrites are differentially regulated by
members of the Rho family, including RhoA, Rac1, and Cdc42
(68). Rho proteins serve as a molecular switch by cycling between
an inactive GDP-bound state and an active GTP-bound state. The
most important effector of RhoA in the growth cone is probably
the serine–threonine kinase Rho-kinase ROCK (69). Data indicate
that MAG activates RhoA by increasing the proportion of the pro-
tein bound to GTP (70); Nogo-66 and myelin utilize this same
signaling pathway (see Fig. 3) (71, 72). Pharmacological studies
in vitro and in vivo indicate that ROCK plays a prime role in medi-
ating myelin-induced inhibition via NgR (72, 73) and that Rho
pathway inactivation can promote spinal cord repair (73, 74) and
enhance axonal regeneration after corticospinal tract lesions in the
adult rat (71).

4. Nerve
Regeneration
and Neurotrophic
Factors Information is encoded in the CNS through networks of neurons
that are functionally connected by synapses. Upon injury, factors
in CNS myelin inhibit neurite outgrowth. Understanding of how
signals from extracellular factors associated with myelin and the
injury site are integrated with outgrowth-promoting signals and
neurotrophic factors to regulate axonal elongation should further
facilitate the development of interventions to improve the outcome
of both acute CNS injury and chronic neurodegenerative
disorders. Methodologies which can be applied to understanding
how neurite stimulatory signals and neurotrophic factors interact
will be of considerable importance in advancing nerve regenera-
tion efforts. This volume describes as well protocols which have
this goal in mind.
20 S.D. Skaper

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 Chapter 3

Culture of Rat Cerebellar Granule Neurons

and Application to Identify Neuroprotective Agents
Laura Facci and Stephen D. Skaper

Abstract
In primary culture of the early postnatal cerebellum, glutamatergic granule cells are highly enriched and
recapitulate many properties characteristic of developing granule neurons in vivo. For example, withdrawal
of K+ from differentiated rat primary cerebellar granule neurons results in the apoptotic death of the majority
of cells after 48 h. Removal of cerebellar granule neurons from depolarizing culture conditions with high
K+ is thought to reflect the regulation of trophic action of neuronal activity and has found widespread
application as a model for studying the mechanisms of survival factor withdrawal-induced neuronal cell
apoptosis and the neuroprotective action of trophic agents. This chapter presents a protocol for the culture
of postnatal rat cerebellar granule neurons and results in a preparation containing 95% glutamatergic granule
cells and its application to the evaluation of corticotropin receptor agonists as neuroprotective agents.

Key words: Cerebellum, Granule neurons, Cell culture, Rat, Depolarization, Development,
Phosphatidylinositol 3-kinase, Glycogen synthase kinase-3, Apoptosis, Neuroprotection,
Neurotrophic factors

1. Introduction

Like many other cell types, neurons require survival factors to

suppress apoptotic machinery and prevent death (1). Apoptosis is
observed not only in neurodegenerative disorders but also during
nervous system development. In the developing cerebellum, granule
cells proliferate at the outer part of the external granular layer and
become postmitotically differentiated at the inner part of this layer
(2). In primary culture of the early postnatal cerebellum, granule
cells are highly enriched and recapitulate many properties charac-
teristic of developing granule neurons in vivo (3, 4). For example,
withdrawal of K+ from differentiated rat primary cerebellar granule
neurons (CGNs) results in the apoptotic death of the majority of

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_3, © Springer Science+Business Media, LLC 2012

23
24 L. Facci and S.D. Skaper

cells after 48 h (3, 5, 6). Removal of CGN from depolarizing

culture conditions with high K+ is thought to reflect the regulation
of trophic action of neuronal activity and has found widespread
application as a model for studying the mechanisms of survival factor
withdrawal-induced neuronal cell apoptosis (7–11). Moreover, the
in vivo correlate of CGN apoptosis has been described (12, 13).
Apoptosis is a morphologically distinct form of cell death char-
acterized by cellular fragmentation, chromatin condensation and
margination, and internucleosomal cleavage of genomic DNA
(14). The apoptosis of mature CGNs deprived of depolarizing
levels of extracellular K+ is prevented by insulin-like growth factor
I, brain-derived neurotrophic factor, basic fibroblast growth factor,
or cyclic AMP (11, 15–17). Neuronal survival in this model is
abolished by the phosphatidylinositol 3-kinase (PI 3-kinase) inhib-
itors LY294002 or wortmannin (7, 8), indicating that PI 3-kinase
is a central component of the survival machinery of these neurons.
Recombinant gene overexpression studies have indicated that
protein kinase B (PKB; also known as Akt), a downstream effector
of PI 3-kinase, can play a critical role in regulating the survival of
CGNs (18). Among several known targets of PKB, glycogen synthase
kinase-3 is inhibited by PKB-mediated phosphorylation (19).
Recombinant overexpression studies using active or dominant-neg-
ative proteins (10, 20) or small molecule inhibitors (8) have led to
the proposal that glycogen synthase kinase-3 may be a relevant
effector of PI 3-kinase-mediated cell survival (see Fig. 1).

Cerebellar granule neuron model of apoptotic death

in vitro culture requires depolarizing concentrations KCl (25mM) RTK

K+ withdrawal induces apoptotic death. PI-3K

LY294002
Death mimicked by PI-3K inhibitor LY294002.
PDK1
Compound added concommitant with death inducer.
Ser473 P
MTT assay cell viability 48 hours post treatment. Akt /PKB

Ser9/21 P
GSK-3 GSK-3
Less active

Fig. 1. Schematic representation of the phosphatidylinositol 3-kinase (PI3K)/glycogen synthase kinase (GSK-3) signaling
pathway involved in cerebellar granule neuron survival promoted by a high-potassium environment. Phosphorylation of
protein kinase B (PKB or Akt) on Ser473 leads to its activation, with subsequent phosphorylation (and inactivation) of GSK-3
on Ser9 and Ser21. The active state of GSK-3 promotes apoptosis, while GSK-3 inactivation is antiapoptotic. RTK receptor
tyrosine kinase; PDK1 phosphoinositide-dependent kinase-1.
 3 Culture of Rat Cerebellar Granule Neurons and Application… 25

This chapter describes a protocol for culturing CGNs from

postnatal rat brain, followed by a procedure for inducing their
apoptotic death based on pharmacological inhibition of PI 3-kinase,
combined with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay for cell viability. An example of neu-
roprotection is also presented, using corticotropin-releasing factor.

2. Materials

2.1. Equipment 1. Stereo dissecting microscope (backlighting of stage is preferred)

and Labware with fiber optic light source
2. Laminar flow cabinet for dissections
3. Laminar flow biological safety cabinet (CL2)
4. Humidified, water-jacketed culture incubator at 37°C and 5%
CO2/95% air
5. Water bath set at 37°C
6. Dissecting tools (Fine Science Tools)
7. Bench centrifuge to accommodate 15- and 50-mL tubes
8. 15- and 50-mL polypropylene plastic centrifuge tubes (sterile)
9. 0.5-mL microfuge tubes
10. Vacuum-driven filter units (0.45 μm pore size, 250 and 500 mL
capacity) from Millipore (Stericup®)
11. 10 and 6 cm ∅ sterile Petri plastic dishes
12. 10 cm ∅ sterile tissue culture dishes
13. Microtest 96 (96-well plate), tissue culture plastic
14. 0.22 and 0.45 μm filters (Millipore)
15. Neubauer hemocytometer (Fisher Scientific)
16. 96-well poly-D-lysine-treated tissue culture plates (BD
Biosciences)

2.2. Reagents 1. Water, sterile, tissue culture grade (Invitrogen)

2. Phosphate-buffered saline, pH 7.4 (Invitrogen)
3. Basal medium Eagle (BME) (Invitrogen)
4. Neurobasal-A medium (Invitrogen)
5. Neurobasal-A medium, phenol red-free (Invitrogen)
6. B27 supplement, 50×, with antioxidants (Invitrogen)
7. Fetal calf serum (FCS) (Invitrogen) (see Note 1)
8. L-Glutamine (200 mM stock), sterile, for cell culture
(Invitrogen)
26 L. Facci and S.D. Skaper

9. Sodium pyruvate (100 mM stock), sterile, for cell culture

(Sigma)
10. Penicillin/streptomycin, 10,000 U/mL penicillin + 10,000 μg/mL
streptomycin (100× stock), sterile, for cell culture (Invitrogen)
11. Gentamicin, 50 mg/mL, sterile, for cell culture (Sigma)
12. HEPES, 1 M, sterile (Sigma)
13. Cytosine β-D-arabinofuranoside (Ara-C) (Sigma)
14. Papain digestion kit (Worthington Biochemicals)
15. Trypsin, hog pancreas, for cell culture (Sigma)
16. Trypsin inhibitor, soybean, for cell culture (Sigma)
17. DNase from bovine pancreas, type II (Sigma)
18. Bovine serum albumin (BSA) (Sigma)
19. Poly-L-lysine, sterile, for cell culture (MW 30,000–70,000)
(Sigma)
20. Phenol red (0.5% stock) (Sigma)
21. Trypan blue stain 0.4% (Invitrogen)
22. Erythrosin B (Sigma)
23. LY294002 (Merck4Biosciences)
24. MTT formazan (Sigma)

2.3. Culture Media 1. Krebs Buffer (10×). Add the following components and
and Other Solutions amounts: NaCl (17.67 g), KCl (0.900 g), KH2PO4 (0.415 g),
D-glucose (6.425 g), NaHCO3 (5.350 g), 0.5% phenol red
(5 mL), dissolve in water and bring to 250 mL, and filter-
sterilize. The solution can be stored at 4°C for 3 months.
2. Solution K. Dilute the 10× Krebs buffer stock 1:10 in double-
distilled water, add MgCl2 to 0.5 mM and 0.3% (w/v) BSA.
Filter-sterilize. Can be stored for 1 month at 4°C.
3. 0.15 M Borate buffer, pH 8.4. Dissolve 28.6 g of sodium borate
(Na2B4O7 ⋅ 10 H2O) in 500 mL water (pH will be ~9.2). Adjust
pH to 8.4 with 5 N HCl. Filter-sterilize and store at 4°C (up
to 6 months).
4. Ara-C stock solution (10 mM). Prepare Ara-C as 10-mM stock
solution in PBS, filter-sterilize, and store as aliquots at −20°C
for up to 6 months. Once thawed, the aliquot tube may be
kept at 4°C for 4 weeks (see Note 2).
5. Serum-free culture medium. This consists of Neurobasal-A
medium (see Note 3) with the addition of B27 supplements
(1:50 of a 50× stock solution), 1 mM sodium pyruvate (from
a 100-mM stock solution), 2 mM L-glutamine (from a 200-
mM stock solution), 20 mM KCl (1:50 from a 1-M stock
solution), 100 U/mL penicillin-100 μg/mL streptomycin
 3 Culture of Rat Cerebellar Granule Neurons and Application… 27

(1:100 from a stock solution containing 10,000 U/mL penicillin

and 10,000 μg/mL streptomycin). Medium should be stored
at 4°C and used within 2 months.
6. Serum-containing culture medium. Culture medium consists
of BME (with Earle’s salts) and glutamine, and supplemented
by adding 20 mM KCl (1:50 from a 1-M stock solution),
NaHCO3 (2.2 g/L), gentamicin (1 mL/L of 50 mg/mL
stock), and 10% (v/v) heat-inactivated FCS (see Note 1).
Medium should be stored at 4°C and used within 2 months.
7. MTT stock solution. Prepare a 3-mg/mL stock solution of MTT
in PBS and filter through a 0.22-μm filter attached to a syringe
(syringe size depends on volume being filtered). Aliquot and
store at −20°C.

2.4. Poly-L-Lysine 1. Dissolve a 5-mg bottle of poly-L-lysine in 5 mL of 0.15 M

Substratum borate buffer, pH 8.4 (see Note 4). Dilute this solution to
50 μg/mL with sterile, tissue culture-grade water (see Note 5).
Coat desired dishes or multiwall plates with 150–200 μL/cm2
of surface area. Place the dishes/plates in a 37°C incubator
overnight (see Note 6). Aspirate poly-L-lysine solution and
leave plates or dishes uncovered in a vertical laminar flow
hood to dry. Coated and dried plates/dishes can be stored at
4°C for a month.
2. For serum-free cultures, the day prior to use, incubate poly-L-
lysine-coated dishes/plates with culture medium (choice of
medium is not important) containing 10% FCS. Keep in 37°C
incubator until used (aspirate FCS-containing medium imme-
diately before seeding with cells—do not allow to dry).

2.5. Papain 1. The kit consists of the following components: vial (1)—100 mL
Dissociation System: EBSS (Earle’s balanced salt solution); vial (2)—papain con-
Solutions taining L-cysteine and EDTA; vial (3)—DNase; vial (4)—ovo-
mucoid protease inhibitor with BSA.
2. Add 2 mL of 1 M HEPES buffer to the entire contents of vial
(1)—this will still be referred to as “EBSS.”
3. Solution A: Add 5 mL of EBSS to vial (2) (20 U of papain/mL
in 1 mM L-cysteine with 0.5 mM EDTA).
4. Solution B: Add to vial (3) 0.5 mL of EBSS (2,000 U/mL
DNase).
5. Solution C: Add 32 mL of EBSS to vial (4) (10 mg ovomucoid
inhibitor and 10 mg BSA/mL). This solution is stable when
stored at 2–8°C.
6. Solution D: Add 250 μL of solution B to 5 mL of solution A.
28 L. Facci and S.D. Skaper

2.6. Stock Solutions 1. Solution B: Dissolve 20 mg of hog pancreas trypsin in 25 mL

for Trypsin solution K and filter-sterilize.
Dissociation (Prepare 2. Solution C: Add 2.4 mg DNase, 15.6 mg trypsin inhibitor,
on Day of Use) and 0.3 mL of 0.15 M MgSO4⋅7 H2O to solution K, and bring
volume to 30 mL with solution K. Filter-sterilize.
3. Solution D: Combine 4 mL of solution C and 21 mL of
solution K.
4. Solution E: To 25 mL of solution K, add 0.2 mL of 0.15
MgSO4⋅7 H2O and 0.2 mL of 0.012 M CaCl2 2 H2O.

3. Methods

3.1. Tissue Dissection 1. Dissect out the cerebellum from 7- to 8-day-old rat pups of
and Dissociation both sexes (CD strain, Sprague Dawley) under aseptic condi-
(Papain Dissociation tions following decapitation. Pups are sacrificed in accordance
Procedure) with appropriate institutional and national guidelines for the
care and use of laboratory animals. Standard techniques and
anatomical landmarks are followed.
2. Make an incision under the skin along the midline of the dorsal
surface and peel back the skin. Make a second incision close to
the midline along the exposed dorsal surface (the cut should
be along the complete anteroposterior axis of the lateral ven-
tricles). Splay open the hemispheres along the cut surface and
continue cut along the dorsal surface of the hemisphere.
3. Remove the cerebellum to a 10-cm ∅ dish containing solution
K. Free the tissue of meninges with the use of a stereo dissect-
ing microscope.
4. Remove solution K and mince the tissue with a flamed razor
blade. Collect the tissue pieces with solution K and transfer to
a 15-mL centrifuge tube.
5. Allow the tissue pieces to settle by gravity, remove the medium,
and add 5 mL of solution D. Incubate for 15 min at 37°C with
occasional agitation.
6. Vigorously titrate with a cotton-plugged, sterile Pasteur pipette
(attached to an automatic pipettor) until devoid of clumps (see
Notes 7 and 8).
7. Centrifuge at 200 × g for 5 min.
8. During step 5, prepare two tubes with 2.7 mL EBSS + 0.3 mL
solution C + 0.15 mL solution B (3.15 mL total = EBSS Mix);
5 mL solution C.
9. After centrifugation, discard the supernatant and resuspend
the pellet in 3 mL EBSS Mix. Using a Pasteur pipette, carefully
layer the cell suspension on top of the 5 mL ovomucoid.
10. Centrifuge at 150 × g for 5 min.
 3 Culture of Rat Cerebellar Granule Neurons and Application… 29

11. Remove and discard the supernatant and resuspend the pellet
in culture medium (either serum-containing or serum-free).
12. It will be necessary to dilute the cell suspension before counting.
A density of 1.5–2 million cells/mL for hemacytometer counting
is convenient. See Subheading 3.3 for details on counting with
the hemacytometer. Typical yields are 20–23 million cells/
cerebellum for 7–8-day-old animals.
13. Dilute the cell suspension with complete culture medium to
give the desired plated density. Generally, it is advisable to plate
2.5–3.0 × 105 cells/cm2 of culture surface area. Survival of
CGN is density-dependent, and plating the cells below 1 × 105
cells/cm2 will result in reduced cell viability (see Notes 9–11).
14. Add Ara-C 18–22 h after cell plating to a final concentration of
10 μM (1:1,000 from a 10-mM stock solution in PBS). Ara-C is
an antimitotic and is added to inhibit the growth of nonneurons.

3.2. Preparation 1. Collect cerebellar tissue from 7- to 8-day-old rat pups into
of CGNs Using Trypsin solution K, as described in Subheading 3.1. Mince tissue with
Dissociation a razor blade and collect in a 15-mL tube, then add solution B
(1 mL/cerebellum). If more than 10 cerebella are processed,
it is advisable to perform this step using a 50-mL tube.
2. Incubate for 15 min (37°C) with gentle swirling.
3. Add solution D (1 mL/cerebellum) to the tube and centrifuge
at 300 × g for 3 min.
4. Remove as much as possible the supernatant; the pellet may
not be very compact. It is possible to transfer the pellet to a
new tube, thereby eliminating most of the supernatant.
5. Add 2 mL of solution C. Pipette C down 5 times with a 5-mL
graduated pipette to break up the largest tissue pieces. Dissociate
the tissue using a long (9-in.) glass Pasteur pipette (cotton
plugged, sterile) with the tip slightly flame-constricted—25
up-and-down strokes should be sufficient (see Note 12).
6. Add 3 mL of solution C per tube and apply a further 15 strokes
with the Pasteur pipette.
7. Add 6 mL of solution E per tube.
8. Centrifuge at 200 × g for 7 min.
9. Resuspend the cell pellet in 2–3 mL complete serum-containing
culture medium, using a Pasteur pipette as above. The remaining
steps are the same as steps 10–12 in Subheading 3.1.

3.3. Cell Counting 1. Hemocytometers are modified glass slides engraved with grids
with a Hemocytometer of fixed area at each side of a central trough, which are covered
with a coverslip prior to use. The sample of cells to be counted
is harvested and applied to the slide surface with a Pasteur
pipette or pipetman, and allowed to move under the coverslip
by capillary action.
30 L. Facci and S.D. Skaper

2. The most commonly used counting chamber is a neubauer

hemocytometer. When the coverslip is in the correct position,
the area within the grid corresponds to 1 mm2. If all the cells
on the central 5 × 5 grid are counted, the number of cells in
1 cm3 is equal to the cell count × 104. Since there will always be
cells located on the lines of the square, the convention is to
count all cells on the top and left-hand lines, and ignore all
those on the bottom and right-hand lines. A minimum of 200
cells should be counted where possible to minimize inaccuracies
of the technique.
3. Cell viability is easily checked by dye exclusion, which relies on
the premise that living cells prevent certain agents crossing the
membrane, while dead cells are permeable to a selection of
stains. Erythrosin B is the dye of choice here, although the
most commonly used is trypan blue due to ease (although it
may give a high background in serum-containing media) (see
Notes 13 and 14). We have not noticed this when dealing with
primary neurons, and routinely utilize trypan blue. A 0.4%
solution of erythrosin B is prepared by dissolving 0.4 g erythrosin
B in 95 mL PBS (heated to boiling point). When dissolved,
cool and bring volume to 100 mL. Filter-sterilize and aliquot.
4. Thoroughly clean surface of the hemocytometer with 70%
ethanol. Secure a clean coverslip onto the top of the slide
centrally by moistening the edges of the coverslip and pressing
down firmly (see Note 15).
5. Using a 0.4-mL Eppendorf tube, mix 10 μL of a 0.4% (v/v)
trypan blue solution and 90 μL of cell suspension using a 200-
μL pipetman.
6. Carefully pipette a small volume (10 μL seems to work fine)
onto the surface of the slide to draw the cells into the counting
chamber. Repeat on the opposite edge of the coverslip.
7. Use a light microscope at low magnification to focus on the
counting chamber (see Note 16).
8. Count both the number of cells in the 5 × 5 grid that exclude
trypan blue (viable) and the number of cells that do not
exclude trypan blue (they are blue and not viable). Count
both grids.
9. Calculate viability as a percentage by dividing the number of
live cells counted by the number of dead cells, and multiplying
the answer by 100.
10. Calculate the cell count by multiplying the live cells counted
by 104 to give a cell number/mL and multiply by 1.1 to allow
for the dilution factor of adding the dye.
11. Clean the hemocytometer immediately, by gently wiping off
with a Kimwipe moistened with 70% ethanol.
 3 Culture of Rat Cerebellar Granule Neurons and Application… 31

3.4. Setting Up 1. Prepare one or more 96-well tissue culture plastic plates coated
the CGN Cultures with poly-L-lysine.
for Neuroprotection 2. Dilute the CGN cell dissociate to 400,000 cells/mL, using
Assay BME supplemented with 10% heat-inactivated FCS, 2 mM
L-glutamine, 50 μg/mL gentamicin, and 25 mM KCl (see
Note 17).
3. Add to each culture well 0.1 mL of the above cell suspension.
This will give a density of 40,000 CGN/well.
4. Incubate cultures at 37°C in a humidified atmosphere with
5% CO2.
5. Eighteen to twenty-four hours after cell plating, add cytosine
β-D-arabinofuranoside (Ara-C) to a final concentration of
10 μM. To do this, remove 10 μL of medium from each well
and add 10 μL of fresh medium containing 100 μM Ara-C
(diluted 1:100 from a 10-mM stock solution).

3.5. Induction 1. At 8 days in vitro, completely exchange the CGN culture

of Apoptosis medium for fresh medium lacking Ara-C (100 μL/well) and
containing 5 mM KCl or 25 mM KCl.
2. Alternatively, exchange the medium for fresh serum-free
medium containing 25 mM KCl ± 75 μM LY294002 (added
from a 37.5-mM stock solution in dimethyl sulfoxide). Under
these conditions, maximal CGN cell death is achieved with
50–75 μM LY294002 (7) (see Note 18).
3. Include the test compound with the medium change at this
time (see Note 19).
4. Continue incubation for 48 h.
5. Assess cell viability by the MTT assay.

3.6. MTT Analysis Ideally, a colorimetric assay for living cells should utilize a colorless
of Cell Viability substrate that is modified to a colored product by any living cell,
of LY294002-Treated but not by dead cells, their lytic debris, or tissue culture medium.
CGN: Protection Tetrazolium salts are attractive candidates for this purpose since
by Corticotropin- they measure the activity of various dehydrogenase enzymes (21).
Releasing Factor The tetrazolium ring is cleaved in active mitochondria, and so the
reaction occurs only in living cells. A rapid colorimetric assay is
based on the tetrazolium salt MTT that measures only living cells
and can be read on a scanning multiwall spectrophotometer (ELISA
reader) (22, 23).
1. At 8 days in culture, exchange the culture medium for serum-
free BME (or Neurobasal-A) containing 25 mM KCl ± 75 μM
LY294002 ± 30 nM corticotropin-releasing factor. After 6, 24,
and 48 h, cellular vitality is evaluated by MTT assay.
2. Remove the culture medium (aspirate under vacuum, using a
Pasteur pipette).
32 L. Facci and S.D. Skaper

3. Add to each well 100 μL of serum-free BME or Neurobasal-A

(with 25 mM KCl) containing 0.15 mg/mL MTT (which
represents a 1:20 dilution of the 3 mg/mL stock) (see Note 20).
4. Incubate the MTT-containing cultures for 1 h at 37°C (see
Note 21).
5. Examination of the cultures under a light microscope will show
that viable cells contain blue crystals. This is the formazan reac-
tion product.
6. Remove the culture medium by aspiration and add 100 μL of
dimethyl sulfoxide/well. This is to dissolve the reaction product.
Gently tap the microwell plate from underneath to mix the blue
reaction product uniformly with the solvent (see Note 22).
7. Read the plate on a microplate (ELISA) reader. We use a
SpectraMax M2 microplate reader from Molecular Devices,
although there are a number of quality plate readers on the
market. For optimal results, the plate should be read at a wave-
length of 570 nm (test wavelength), followed by 630 nm (ref-
erence wavelength). The difference (A570−A630) is used as the
final absorbance value generated by the sample. Correcting for
optical imperfections in the microplates by subtracting A630 is
recommended, but is not an essential procedure.
8. Normalize the absolute MTT values by scaling to the mean
of cultures in serum-free medium with 25 mM KCl (defined
as 100%).
9. An example of the outcome of this illustrative experiment is
shown in Table 1. Note the time-dependent loss of cell viability
in the LY294002-treated cultures.
10. Cell vitality should be routinely confirmed by morphological
observation using a light microscope with phase contrast optics.

3.7. Cell Culture 1. A horizontal laminar flow hood is recommended for dissection
Safety Issues of tissues. Once dissection is complete, all work should be
moved to a Class 2 vertical laminar flow safety cabinet.
2. The antibiotics and enzymes used should be considered as
noxious agents. Suitable precautions should be taken in the use
and disposal of these products.
3. All liquids containing live cells should be aspirated into a waste
vessel containing MicroSol +3 (a broad spectrum disinfectant)
(Anachem), and pipettes placed in containers with MicroSol.
Plasticware is then disposed of via autoclaving.
4. Accumulated culture liquid waste should then be transferred
to a 20-L carboy containing ~100 g of Virkon powder (VK734,
Medisave, UK). Virkon is a multipurpose disinfectant and con-
tains oxone (potassium peroxymonosulfate), sodium dodecyl-
benzenesulfonate, sulfamic acid, and inorganic buffers. Virkon
 3 Culture of Rat Cerebellar Granule Neurons and Application… 33

Table 1
Corticotropin-releasing factor protects cerebellar granule
neurons from LY294002-induced death

Treatment MTT
6h
LY 77 ± 6
LY + CRF 92 ± 3∗
24 h
LY 64 ± 5†
LY + CRF 87 ± 6∗∗
48 h
LY 28 ± 1
LY + CRF 84 ± 5∗∗∗
Cerebellar granule neurons were cultured for 8 days, then shifted to serum-free
BME containing 25 mM KCl and 75 μ M LY294002 (“LY”) ± 30 nM
corticotropin-releasing factor (CRF). After the times indicated, cellular vitality
was evaluated by MTT assay. Data are means ± S.D. (n = 3)
∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <0.0001 (LY + CRF vs. LY); p < 0.05 or p < 0.001
vs. LY at 24 and 48 h, respectively; †p < 0.001 vs. LY at 48 h
Adapted (with substantial modification) from Neuropharmacology 45(5), L.
Facci, D.A. Stevens, M. Pangallo, D. Franceschini, S.D. Skaper, P.J.L.M.
Strijbos, Corticotropin-releasing factor (CRF) and related peptides confer
neuroprotection via type 1 CRF receptors, 623–636 (Table 3), Copyright©
(2003), with permission from Elsevier

has a wide spectrum of activity against viruses, some fungi, and

bacteria. The pink color is useful in that it helps gage the
concentration when preparing the Virkon, and importantly, as
the Virkon ages, it discolors, making it obvious when it needs
to be replaced. The solution is generally stable for 5–7 days.
Virkon is relatively safe in terms of skin contact but can cause
eye damage and should not be used as a hand-washing liquid.
Wear appropriate personal protective clothing.

4. Notes

1. Heat inactivation of fetal calf serum is recommended to destroy

heat-labile complement. Thaw the bottle of serum in advance,
using a 37°C water bath. Next, immerse the serum bottle in
the water bath after reequilibrating to 56°C and leave for
30 min. Swirl the bottle occasionally to ensure proper mixing.
Allow the serum to cool to room temperature, aliquot into
50-mL tubes, and store at −20°C.
34 L. Facci and S.D. Skaper

2. Cytosine arabinoside is an antimitotic and potential carcinogen.

Usage should be restricted to fume cupboards and biological
safety cabinets. Avoid weighing out powder, or if not possible,
then weigh out in a suitable closed environment. Culture
media can be disposed of by normal routes. Stock/concen-
trated solutions should be enclosed within a leakproof con-
tainer and put into a burn bin for disposal.
3. There are two Neurobasal medium formulations—without
and with the suffix “A.” The principal difference is in their
osmolarity: Neurobasal “A” is intended for postnatal and adult
neuron cultures, which the other formulation is preferable for
culturing embryonic neurons.
4. Poly-L-lysine (and poly-L-ornithine) is very hygroscopic and
difficult to weigh. It is advisable to purchase the material as a
5–10-mg quantity, and then dissolve the entire amount in
borate buffer. The solutions can be stored at 4°C for up to
6 months.
5. Working solutions of poly amino acids should be prepared in
sterile tissue culture plastic polypropylene tubes. TAKE CARE:
The poly amino acid will adhere to glass surfaces, and also to
polystyrene plastic.
6. In a pinch, the coating time with poly-L-lysine can be limited
to 2 h—the poly amino acid sticks rapidly to tissue culture
plastic-treated surfaces.
7. Avoid pulling air through the Pasteur pipette and creating
bubbles/foam—this can cause cell damage.
8. If necessary, and to clear the suspension of any remaining small
clumps, this may be passed through a cell strainer (70 μm, BD
Falcon #352350) placed over the mouth of a 50-mL centri-
fuge tube.
9. To maintain long-term CGN cultures, it is necessary to add
glucose after the first 10 days and weekly thereafter. Glucose is
prepared as a 0.5-M stock in water, filter-sterilized, and added
at 1:100 to the cultures. Alternatively, a 0.5-M glucose solu-
tion for cell culture can be purchased from Sigma.
10. It may be a good idea to check periodically the volume of the
cultures. Should evaporation be noted, the lost volume can be
replaced by adding sterile water. This is more likely to occur if
the cultures are carried for more than 2 weeks. Never change
the culture medium on CGN, especially when culturing in
serum-containing medium—the CGNs condition their medium,
and serum contains glutamate which is toxic to mature CGN.
11. On the subject of evaporation, when culturing in 96-well
plates, the empty wells should be filled with water—this will
help to reduce volume loss on the edge wells.
3 Culture of Rat Cerebellar Granule Neurons and Application… 35

12. Depending on the dissociation volume, it may be advisable to

divide the suspension into 2–3 parts prior to dissociation. The
dissociation efficiency of the Pasteur pipette is reduced when
the volume exceeds 4 mL.
13. Erythrosin B is cytotoxic, and cells should not be left in the dye
for prolonged times prior to counting.
14. Be sure to add the trypan blue to the cells just before count-
ing—after several minutes, it is toxic to the cells.
15. As an alternative, you can lightly moisten the edges of the
hemocytometer before placing the coverslip—this will prevent
the cover from sliding off prior to loading with cells.
16. The hemocytometer chamber will accept about 10 μL—never
overfill, as this can cause erroneous cell counts.
17. Alternatively, you can use the Neurobasal-A-based serum-free
culture medium. In this case, remember to pretreat overnight
the polylysine-coated plates with medium containing 10% FCS,
prior to cell seeding.
18. If at all possible, we recommend not exceeding a dimethyl sul-
foxide concentration of 0.4%. When treating cells with
LY294002 and test compound, each is added from a 500×
stock solution. When only LY294002 or test compound is
used, the dimethyl sulfoxide concentration should be brought
to 0.4% final by addition of solvent.
19. If the compound being tested is made up in nonaqueous
medium (e.g., in dimethyl sulfoxide or ethanol), then control
cultures should be included (medium with 5 mM and 25 mM
KCl) which contain the same amounts of organic solvent.
20. The stock solution (3 mg/mL) of MTT, once thawed, can be
refrozen and used at least three additional times without com-
promising its efficacy.
21. The incubation time for obtaining optimal MTT color yield
will vary as a function of cell type and plating density, as well as
MTT concentration and time of incubation (14). We have
established the 1-h time used here for CGN under the culture
conditions specified. The experimenter is advised to conduct
trials with the cell type being used to establish optimal condi-
tions for color yield. Note that excessive incubation times and/
or concentrations of MTT can be cytotoxic.
22. The initial MTT procedure for cultured primary neurons used
a solution of isopropanol-0.08 N HCl to dissolve the MTT
reaction product. We find that this procedure is very slow to
dissolve the blue crystals and requires rather vigorous agitation
of the microwell plate. In contrast, the action of dimethyl sul-
foxide is quite rapid, taking only a matter of seconds.
36 L. Facci and S.D. Skaper

Acknowledgments

L. Facci was supported by Fondazione CARIPARO Progetto

Dottorati di Ricerca Anno 2009.

References

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2. Hatten ME, and Heintz N (1995) Mechanisms of cell death by insulin-like growth factor I
of neural patterning and specification in the and cAMP. Proc Natl Acad Sci USA 90,
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18, 385–408 12. Wood KA, Dipasquale B, and Youle RJ (1993)
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Brain Res 41, 482–488 3366–3374
5. Thangnipon W, Kingsbury A, Webb M, and 14. Bredesen DE (1995) Neuronal apoptosis. Ann
Balázs R (1983) Observations on rat cerebellar Neurol 38, 839–851
granule cells in vitro: influence of substratum, 15. Fernandez-Sanchez MT, and Novelli A (1993)
potassium concentration and relationship Basic fibroblast growth factor protects cerebellar
between neurones and astrocytes. Brain Res neurons in primary culture from NMDA and
313, 177–189 non-NMDA receptor mediated neurotoxicity.
6. Ginham R, Harrison DC, Facci L, Skaper SD, FEBS Lett 335, 124–131
and Philpott K (2001) Upregulation of death 16. Kubo T, Nonomura T, Enokido Y, and
pathways in rat cerebellar granule neurons Hatanaka H (1995) Brain-derived neurotrophic
undergoing apoptosis. Neurosci Lett 302, factor (BDNF) can prevent apoptosis of rat
113–116 cerebellar granule neurons in culture. Dev
7. Miller TM, Tansey MG, Johnson EM Jr, and Brain Res 16, 249–258
Creedon DR (1997) Inhibition of phosphati- 17. Shimoke K, Yamagishi S, Yamada M, Ikeuchi
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tion- and insulin-like growth factor 1-mediated phatidylinositol 3-kinase activity elevates c-Jun
survival of cerebellar granule cells. J Biol Chem N-terminal kinase activity in apoptosis of
272, 9847–9853 cultured cerebellar granule neurons. Dev Brain
8. Cross DAE, Culbert AA, Chalmers KA, Facci Res 112, 245–253
L, Skaper SD, and Reith AD (2001) Selective 18. Crowder RJ, and Freeman RS (2000) Glycogen
small-molecule inhibitors of glycogen synthase synthase kinase-3b activity is critical for neu-
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 Chapter 4

Isolation and Culture of Neural Progenitor Cells

from Rat Postnatal Cerebellum
Morena Zusso and Patrizia Debetto

Abstract
The mammalian brain contains undifferentiated, mitotically active, and multipotent neural stem/progenitor
cells that in vivo contribute new neurons and glia to specific areas of the mature brain. When isolated under
the appropriate conditions, these cells maintain in vitro the ability to proliferate and differentiate into cells
that express neuronal and glial markers. Neural stem/progenitor cells have been identified and isolated
from many regions of the embryonic, postnatal, and adult central nervous system, including cerebellum.
This chapter details techniques to isolate and culture neural progenitor cells from rat postnatal cerebellum,
which can be used as an in vitro model to study the molecular mechanisms underlying proliferation and
differentiation into mature neural cells induced by various stimuli including pharmacological agents.

Key words: Neurogenesis, Neural stem/progenitor cells, Cerebellum, Postnatal, Rat

1. Introduction

During development of the mammalian central nervous system

(CNS), neural cells are generated by the proliferation of multipo-
tent stem or progenitor cells that migrate, find their site of final
destination, and ultimately differentiate. Neural stem/progenitor
cells are most abundant at early developmental stages; however,
they persist throughout development, and it is now known that the
in vivo genesis of new neurons from endogenous pools of mitoti-
cally active neural stem/progenitors (i.e., neurogenesis) continues
in the adult CNS of all mammalian organisms, including humans
(1). Adult neurogenesis mainly occurs in two constitutive neuro-
genic brain regions: the subventricular zone (SVZ) of the lateral
ventricles, which generates neurons destined for the olfactory bulb,
and the subgranular zone of the dentate gyrus of the hippocampus,
which generates neurons that functionally integrate into the existing
circuitry (2–5). However, neural stem/progenitor cells have been

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_4, © Springer Science+Business Media, LLC 2012

39
40 M. Zusso and P. Debetto

identified also in several mammalian embryonic and adult brain

regions that are normally nonneurogenic, including cerebral cor-
tex, substantia nigra, spinal cord, and cerebellum (6–9). Neural
stem/progenitor cells can be isolated, propagated, and differenti-
ated in vitro both from constitutive and latent areas. In particular,
the mammalian cerebellum is one of the brain regions exhibiting
protracted postnatal neurogenesis. Cerebellar neurons are gener-
ated from different sources and at different developmental stages.
Granule cells, the most abundant interneurons, are generated
almost entirely during the first 2 postnatal weeks from proliferating
progenitors located in the external granule layer (for review, see ref. (10)).
This germinal layer is active during early postnatal periods whose
duration is strictly species dependent (11–14). Recently, it has been
shown that stem cells can be isolated from both postnatal (15, 16)
and adult (17) cerebellum, suggesting that latent neurogenic
potential may be present in this brain structure.
In this chapter, we describe a method that we developed for
the isolation, culturing, and differentiation of neural progenitor
cells from rat postnatal cerebellum. This method typically yields
1 × 106 viable progenitor cells from a single postnatal cerebellum.
Once isolated and cultured, cerebellar progenitors provide a
suitable in vitro model to identify the critical molecules that reg-
ulate cell proliferation and subsequent differentiation induced
by pharmacological stimuli (e.g., serotonergic antidepressant
drugs) (16).

2. Materials

2.1. Dissection 1. Animals: Sprague Dawley rat pups; age of pups = postnatal day
and Cell Cultures 7. We define postnatal day 0 as the day of birth.
2. HBSS/BSA: Add 3 mg/mL bovine serum albumin (BSA) to
calcium- and magnesium-free Hank’s balanced salt solution
(HBSS; Gibco, Life Technologies, Rockville, MD) and filter
sterilize through 0.22 mm filter. Store at 4°C for up to
1 month.
3. Trypsin solution: Add 0.8 mg/mL trypsin to HBSS/BSA and
filter sterilize (see Note 1).
4. Trypsin inhibitor solution: Add 0.5 mg/mL trypsin inhibitor
and 0.1 mg/mL deoxyribonuclease I (DNase) to HBSS/BSA.
Filter sterilize (see Note 1).
5. Growth factor stock solutions: Reconstitute epidermal growth
factor (EGF) (human recombinant) and basic fibroblast growth
factor (bFGF) (Sigma-Aldrich) in order to have a 500 mg/mL
 4 Isolation and Culture of Neural Progenitor Cells from Rat Postnatal Cerebellum 41

stock of each. Aliquot into sterile tubes and store at −20°C.

The shelf life of the stock solution is 1 year at −20°C.
6. Culture medium: Serum-free growth medium (500 mL). To
480 mL of Neurobasal medium (Gibco), add 10 mL 50× B-27
supplement (Gibco), 5 mL 200-mM L-glutamine, 5 mL 100×
Pen-Strep (10,000 IU/mL penicillin and 10,000 mg/mL
streptomycin) (Gibco), and 20 mL EGF and bFGF stock solu-
tions (final concentrations 20 ng/mL). Store at 4°C for
<1 month; prewarm to 37°C before use.

2.2. Differentiation 1. 100× Poly-D-lysine stock solution: Dissolve 20 mg/mL poly-

of Cerebellar Neural D-lysine hydrobromide (MW > 40,000; Sigma-Aldrich) in ster-
Progenitor Cells ile tissue culture water. Sterilize by 0.22-mm filter and store at
4°C for up to 1 month.
2. Poly-D-lysine-coated chamber slides: Dilute the poly-D-lysine
stock solution to a final concentration of 200 mg/mL in sterile
tissue culture water. Add enough diluted solution to entirely
cover the surface of chamber slides (Permanox slide, Lab Tek,
Nalge Nunc International) and incubate at room temperature
for 1 h. Remove the poly-D-lysine solution and allow to dry
under the laminar flow hood.
3. Culture medium: Differentiation medium (500 mL). To 480-
mL Basal Medium Eagle (Sigma-Aldrich), add 10 mL 10%
heat-inactivated fetal bovine serum (see Note 2), 5 mL 200-
mM L-glutamine, 5 mL 2.5-M KCl, and 5 mL 100× Pen-Strep
(10,000 IU/mL penicillin, 10,000 mg/mL streptomycin).
Store at 4°C for <1 month; prewarm to 37°C before use.

2.3. Immunostaining 1. Phosphate-buffered saline (PBS): To 800-mL double distilled

of Cerebellar Neural water, add 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g
Progenitor Cells KH2PO4. Dissolve, adjust the pH to 7.4 with HCl, and bring
to 1,000 mL with water. Store at room temperature.
2. 4% Paraformaldehyde solution: Heat 50 mL distilled water to
70°C (do not boil), add 4 g paraformaldehyde, and then add
1-N NaOH drop by drop, until the solution clears. When solu-
tion is cool, adjust the pH to 7.4 with HCl and bring to
100 mL with PBS (see Note 3).
3. Antibodies for characterization of cell phenotype: Primary anti-
bodies: mouse antineurofilament 200 (Sigma-Aldrich), mouse
antimicrotubule-associated protein-2 (Millipore Corporation),
mouse anti-b-tubulin isotype III (Millipore Corporation), rabbit
antiglial fibrillary acidic protein (Millipore Corporation), and
mouse anti-O4 (Millipore Corporation). Fluorescent secondary
antibodies: Alexa Fluor 555–conjugated goat anti-mouse and
anti-rabbit and Alexa Fluor 488–conjugated goat anti-mouse
and anti-rabbit (Molecular Probes) (see Note 4).
42 M. Zusso and P. Debetto

4. 4¢,6-Diamidino-2-phenylindole (DAPI) stock solution: Dissolve

1 mg/mL DAPI (Sigma-Aldrich) in ultrapure water. Aliquot
and store at −20°C. The stock solution is stable for several
months and can be used repeatedly if stored protected from light.

3. Methods

3.1. Dissection 1. Rapidly decapitate 7-day-old rat pups (see Note 5) by cutting
of Cerebellum the neck with sterile scissors large enough to ensure the head is
from Postnatal Rats removed in one cut. Discard the body into a plastic bag for
and Establishment disposal. Transfer the heads to a sterile 100-mm ∅ Petri dish
of Primary Cultures containing cold HBSS/BSA and cut the skin on top of the
head (see Note 6).
2. Under the dissection hood, cut the skull along the midline,
starting from the back of the head to the eyes. Make a superfi-
cial cut and be careful not to damage the underlying brain tis-
sue. Using forceps, lift up the skull to expose the brain.
3. Using a pair of curved forceps, remove the cerebellum and
immediately transfer it to a sterile Petri dish containing cold
HBSS/BSA (see Note 7). Repeat step 2 until all cerebella have
been collected.
4. Under the dissecting microscope, carefully remove the menin-
ges from each cerebellum using two forceps. The meninges
will appear as a delicate membrane containing blood vessels.
Place the cleaned cerebella into a new dish containing cold
HBSS/BSA and keep on ice.
5. Once all the cerebella have been processed, under the tissue
culture hood, remove the HBSS/BSA from the dish and mince
the tissue into small pieces (0.5–1 mm3) with a flamed razor
blade (see Note 8). Transfer dissected tissues into a 50-mL
conical tube containing 1-mL HBSS/BSA/cerebellum and
centrifuge at 1,000 × g for 3 min.
6. Carefully aspirate the HBSS/BSA (see Note 9), resuspend the
tissue pieces in 1-mL trypsin solution/cerebellum, and incu-
bate for 15 min in a 37°C water bath under gentle shaking to
keep the tissue pieces suspended.
7. Stop the enzymatic reaction by adding the trypsin inhibitor
solution (1 mL/cerebellum). Pellet the tissue by centrifuga-
tion at 1,000 × g for 7 min and remove the supernatant by
gentle aspiration (see Note 9).
8. Add 5 mL HBSS/BSA and dissociate the tissue by triturating
with a sterile, fire-polished, cotton-plugged glass Pasteur pipette
 4 Isolation and Culture of Neural Progenitor Cells from Rat Postnatal Cerebellum 43

until the suspension looks cloudy and only small pieces of tissue
are left (see Note 10).
9. Pellet the cells by centrifugation at 1,000 × g for 7 min. Discard
the supernatant and resuspend the cells in 10 mL of pre-
warmed (37°C) culture medium. Make an appropriate dilu-
tion of an aliquot of the cell suspension in trypan blue solution
and count the number of viable cells using a hematocytometer
(see Note 11).
10. Plate the cells at a density of 2 × 105 viable cells/cm2 in uncoated
tissue culture plates, in culture medium containing 20 ng/mL
of EGF and bFGF.
11. Incubate at 37°C, 5% CO2, in a humidified incubator.
12. After 5–6 days, replace half of the medium with fresh medium.
Add a double amount of EGF and bFGF to give a final concen-
tration of 20 ng/mL.

3.2. Culture Cerebellar progenitors proliferate to form floating aggregates,

Propagation which are usually ready for subculturing 10–12 days after plating:
1. Dislodge aggregates by taping sides of plates; transfer content
of the plates to 15-mL sterile, conical tubes, using a sterile
plastic pipette. Use fresh medium to rinse plates and add rinse
to the tube.
2. Pellet cell suspension by centrifugation at 1,000 × g for 7 min.
Remove the supernatant without disturbing the cell pellet.
3. Resuspend cells in 1 mL culture medium. Using a P200
(200 mL) Pipetman, gently dissociate the pellet (30–40×) to
produce a single cell suspension (see Note 12).
4. Count viable cells by trypan blue exclusion and plate cells at
the density of 5 × 103 cells/cm2 onto uncoated tissue culture
plates, in culture medium containing appropriate growth fac-
tors (EGF and bFGF, 20 ng/mL) (see Note 13).
5. Subculture when the aggregates start to lift off and float in
suspension. This will require 10–12 days (see Note 14).

3.3. Differentiation When maintained in growth medium, cerebellar neural progenitors

of Cerebellar Neural display a round shape (Fig. 1a) and an undifferentiated state, as
Progenitors evidenced by their immunoreactivity for several immature neuronal
cell markers (Fig. 1b). Nevertheless, plating cerebellar progenitors
onto an adhesive substrate and growing in serum-containing
medium without growth factors (differentiation medium) is suffi-
cient to promote a differentiation process into the three CNS cell
types (Fig. 1c, d):
1. Dislodge aggregates by taping sides of plates and transfer con-
tent of the plates to 15-mL sterile, plastic, conical tubes, using
44 M. Zusso and P. Debetto

Fig. 1. Morphology and characterization of cerebellar neural progenitors. (a) Representative phase contrast photomicrograph
of a cerebellar neural progenitor cell culture maintained in growth medium for 10 days. (b) Fluorescent photomicro-
graph of the specific immunoreactivity for nestin. Cells were replated onto poly-D-lysine-coated chamber slides in differentiation
medium. After 3 days, cells were fixed with 4% paraformaldehyde and stained with specific antibodies. (c, d) Fluorescent
photomicrographs of neurofilament- (NF), glial fibrillary acidic protein- (GFAP), and O4-positive cells. DAPI staining shows
the nuclei. Scale bars = 50 mm (adopted in part from ref. (16), with modifications).

a sterile plastic pipette. Rinse plates with fresh medium, add

rinse to the tube, and centrifuge cell suspension at 1,000 × g
for 7 min.
2. To wash cells from growth factors, remove supernatant and
resuspend cells in 10 mL of differentiation medium. Centrifuge
at 1,000 × g for 7 min.
3. Remove the supernatant and resuspend cells in 1 mL of dif-
ferentiation medium. Using a P200 Pipetman, gently triturate
the pellet (30–40×) (see Note 15).
4. Count viable cells by trypan blue exclusion. Resuspend cells in
the appropriate volume of culture medium (containing 10%
fetal bovine serum) and plate them at the density of 1 × 104
cells/cm2 onto poly-D-lysine-coated chamber slides.
5. Allow the cells to differentiate for 3–5 days and analyze the
phenotype by immunocytochemical determination of cell-type-
specific markers for neurons, astrocytes, and oligodendrocytes.
 4 Isolation and Culture of Neural Progenitor Cells from Rat Postnatal Cerebellum 45

3.4. Immuno- All staining procedures are carried out at room temperature except
cytochemical Staining where indicated. Washing steps are done for 5 min each:
for Characterization
1. Remove culture medium and carefully wash cultures twice
of Differentiation
with PBS.
2. Fix cells for 10 min in 4% paraformaldehyde at room tempera-
ture and wash three times with PBS (see Note 16).
3. Block nonspecific staining by incubation for 1 h in PBS con-
taining 5% normal donkey serum (Jackson ImmunoResearch
Laboratories) and 0.1% Triton X-100 (blocking solution) at
room temperature (see Note 17).
4. Incubate with primary antibodies diluted in blocking solution for
2 h at room temperature or overnight at 4°C (see Note 18).
5. Wash cells three times with PBS and incubate for 1 h at room
temperature in the dark with species-specific secondary anti-
bodies (conjugated to the desired fluorophores) diluted in
blocking solution (see Note 18).
6. Wash cells three times in PBS, incubate in PBS containing
0.1 mg/mL DAPI for 2 min (for nuclear counterstain), and
then coverslip in Fluoromount mounting medium (Sigma-
Aldrich) (see Note 19).
7. Analyze cell phenotype by confocal or fluorescence microscopy.

4. Notes

1. Dissecting solutions (trypsin and trypsin inhibitor solutions)

must be prepared on the day of dissection.
2. Heat-inactivated fetal bovine serum: Heat inactivation of
serum is important to destroy heat-labile complement.
Degradation of these proteins by raising the temperature of
the serum to 56°C for 30 min will ensure that antibody bind-
ing will not lyse the cells. Heat inactivation may change the
appearance of the fetal bovine serum and result in precipita-
tion. This is normal and does not indicate that the serum has
been compromised:
– Thaw the bottle of fetal bovine serum overnight at room
temperature.
– Immerse the serum bottle in the 56°C water bath for
30 min. Swirl the bottle every 5 min to ensure proper
mixing.
– Allow serum to cool for 1 h at room temperature, aliquot
into 50-mL sterile tubes, and store at −20°C.
3. This fixative solution should be made up fresh. Paraformaldehyde
is toxic and may be carcinogenic; therefore, prepare the solution
46 M. Zusso and P. Debetto

under a fume hood and wear gloves and mask. Disposal should
follow chemical safety rules.
4. All the antibodies should be freshly diluted just before use. Do
not store them as diluted solutions.
5. For a good cell yield, use at least one large litter of rats (12 or
more). Rats are generally sacrificed by cervical dislocation prior
to decapitation or by decapitation alone (this depends on the
animal care protocols of your institution).
6. In Subheading 3.1, step 1 can be performed at the lab bench.
To prevent contamination, use sterile instruments (autoclaved
using autoclave bags and opened only when needed) and solu-
tions and take precautions to maintain sterility, including the
wearing of gloves. Speed is also important.
7. The Petri dish should be maintained on ice for the duration of
dissection.
8. Chop the tissues with a rapid chopping motion, moving the
dish in a circular fashion so that all tissue pieces are evenly cut.
9. Do not use the vacuum since there is a risk of sucking out the
tissue.
10. Normally, 20–30 strokes are sufficient, but perform a second
trituration of 15 strokes in case undissociated tissue is still vis-
ible. During triturating, always avoid foaming and bubbles.
11. Initially try a 1:5 dilution. The number of viable cells yielded
by this protocol, using a single postnatal day 7 rat cerebellum,
is 10 × 106 cells. Approximately 5–10% of plated cells form pro-
genitor cell aggregates.
12. Always avoid foaming and bubbles.
13. Feed cells with fresh medium (replace half of the medium)
every 5–6 days.
14. Isolated cerebellar neural progenitors can be subcultured for
three passages.
15. Trituration into a single cell suspension is critical in the differ-
entiation protocol. A drop of cell suspension should be added
to a Petri dish to check that the suspension is free from large
aggregates before plating. Trituration in trypsin/EDTA should
be avoided as it damages the cell membrane and can cause
adhesion defects.
16. Fixed cells can be stored in PBS at 4°C for about a week or can
be processed immediately for immunocytochemistry.
17. Make blocking solution with serum from the species in which
the secondary antibodies were raised. Since blocking solution
contains serum, it can be contaminated. Blocking solution is
stable for 1 week at 4°C; if the solution becomes cloudy, it has
become contaminated and should not be used.
 4 Isolation and Culture of Neural Progenitor Cells from Rat Postnatal Cerebellum 47

18. Primary and secondary antibody concentrations should always

be titrated for the particular experiment, to obtain the best
possible staining.
19. Fluoromount stabilizes the fluorescence well for long periods.
Refrigerated, Fluoromuont-mounted slides kept in the dark
will retain their fluorescence for months.

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9. Klein C, Butt SJ, Machold RP, Johnson JE, and 17. Sottile V, Li M, and Scotting PJ (2006) Stem
Fishell G (2005) Cerebellum- and forebrain- cell marker expression in the Bergmann glia
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character. Development 132, 4497–4508 1099, 8–17
 Chapter 5

Culture of Rodent Cortical and Hippocampal Neurons

Laura Facci and Stephen D. Skaper

Abstract
Neurons cultured from rodent central nervous system tissues represent an important tool in the study of
neurodegenerative disease mechanisms and neuroregenerative processes, including the survival- and axon
growth–promoting properties of neurotrophic factors. This chapter presents a detailed protocol for the
preparation of rat and mouse cortical and hippocampal neuron cell cultures using either embryonic or
postnatal tissue with enzymatic digestion.

Key words: Cortex, Hippocampus, Neurons, Cell culture, Rat, Mouse, Embryonic, Newborn

1. Introduction

The study of central nervous system diseases has been greatly facili-
tated by the use of ex vivo models which are based on the culture
of neurons from discrete brain areas, for example, cortex, hip-
pocampus, and striatum. Procedures have been developed to allow
for the survival and development of cultured central neurons largely
free of unwanted glial cell elements, in particular the now widely
used B27/Neurobasal formulation (1). Such cultures allow one to
investigate in detail the molecular events involved in amyloid
β-peptide toxicity, excitatory amino acid toxicity, oxidative stress,
the survival-promoting effects of growth factors and cell adhesion
molecule mimetics, kinase signaling pathways, and electrophysio-
logical behaviors (2–13). The following chapter describes a proto-
col for the culture of cortical and hippocampal neurons from
embryonic and newborn rodent (rat, mouse) tissues, based on the
B27/Neurobasal serum-free medium.

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_5, © Springer Science+Business Media, LLC 2012

49
50 L. Facci and S.D. Skaper

2. Materials

2.1. Equipment 1. Stereo dissecting microscope (backlighting of stage is pre-

and Labware ferred) with fiber optic light source
2. Laminar flow cabinet for dissections
3. Laminar flow biological safety cabinet (CL2)
4. Humidified, water-jacketed culture incubator at 37°C and 5%
CO2/95% air
5. Water bath set at 37°C
6. Dissecting tools (Fine Science Tools)
7. Bench centrifuge to accommodate 15- and 50-mL tubes
8. 15- and 50-mL Polypropylene plastic centrifuge tubes (sterile)
9. 10-cm ∅ Sterile tissue culture dishes
10. 0.22-μm Filters (Millipore)
11. Neubauer hemocytometer (Fisher Scientific)
12. 96-Well poly-D-lysine-treated tissue culture plates (BD Biosciences)

2.2. Reagents 1. Hank’s balanced salt solution, without CaCl2 and MgCl2,
sterile (HBSS) (Invitrogen)
2. Neurobasal medium (Invitrogen)
3. Neurobasal-A medium (Invitrogen)
4. B27 supplement, 50×, with antioxidants (Invitrogen)
5. B27 supplement, 50×, without antioxidants (Invitrogen)
6. Fetal calf serum (FCS) (Invitrogen) (see Note 1)
7. L-Glutamine (200 mM stock), sterile, for cell culture (Invitrogen)
8. Sodium pyruvate (100 mM stock), sterile, for cell culture (Sigma)
9. Penicillin/streptomycin, 10,000 U/mL penicillin + 10,000 μg/
mL streptomycin (100× stock), sterile, for cell culture
(Invitrogen)
10. MgCl2, 1 M, sterile (Sigma)
11. KCl, 3 M, sterile (Sigma)
12. HEPES, 1 M, sterile (Sigma)
13. Cytosine β-D-arabinofuranoside
14. Papain digestion kit (Worthington Biochemicals)

2.3. Culture Media 1. HBSS for dissection. To 490 mL of calcium- and magnesium-
and Other Solutions free HBSS, add 2.25 g of D-glucose, 5 mL of 100-mM sodium
pyruvate solution, and 5 mL of 1-M HEPES solution. Filter-
sterilize.
 5 Culture of Rodent Cortical and Hippocampal Neurons 51

2. Embryonic neurons. Neurobasal medium containing B27 supplement

(1×), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL
streptomycin: To 500 mL Neurobasal medium, add 10 mL 50×
B27 supplement, 5 mL 200-mM glutamine, and 5 mL 10,000-U/
mL penicillin–10,000 μg/mL streptomycin. Complete medium
can be stored at +4°C for up to 2 months.
3. Neonatal neurons. Neurobasal-A medium containing B27
supplement (1×), 1 mM sodium pyruvate, 2 mM L-glutamine,
25 mM KCl, 5 mM MgCl2, penicillin (100 U/mL), and
streptomycin (100 μg/mL): To 500 mL Neurobasal-A medium,
add 10 mL 50× B27 supplement, 5 mL 100-mM sodium
pyruvate, 5 mL 200-mM glutamine, 2 mL 1-M MgCl2, 3.3 mL
3-M KCl, and 5 mL 10,000-U/mL penicillin–10,000 μg/mL
streptomycin. Complete medium can be stored at +4°C for up
to 2 months.
4. Cytosine b-D-arabinofuranoside (araC) stock solution (10 mM).
Prepare araC as a 10-mM stock solution in phosphate buffered
saline, filter-sterilize, and store aliquots at −20°C (good for up
to 6 months). Once thawed, the aliquot tube can be kept at
4°C for 1 month (see Note 2).

3. Methods

3.1. Dissection of Rat 1. Rat cultures are routinely performed using animals at 18 days
Embryonic Cortex of gestation. Euthanize the pregnant rat (CD strain, Sprague
and Hippocampus Dawley) following appropriate national and/or institutional
guidelines for animal sacrifice (see Note 3).
2. Place the animal dorsal side down and wipe thoroughly with a
tissue soaked in 70% ethanol. Make an incision through the
skin along the midventral line from the sternum to the pubis.
3. Make an incision through the belly wall muscles. While push-
ing away the muscle, pick up one of the uterine horns with
forceps and cut the attachment of the uterine horns to the
dorsal body wall.
4. Place the uterine horns in a 10-cm ∅ Petri dish containing cold
HBSS for dissection.
5. Remove the second uterine horn in a similar manner.
6. Remove the embryos from the uterine segments and place in a
new Petri dish containing ice-cold HBSS for dissection.
7. Decapitate embryos and store heads in HBSS (for dissection)
on ice until/during dissection.
8. Remove brain from head by cutting the scalp along the mid-
brain line with fine scissors.
52 L. Facci and S.D. Skaper

9. Scoop the brain out using a small spatula.

10. Place the brain in a 10-cm ∅ Petri dish containing fresh, ice-
cold HBSS for dissection.
11. Remove and discard the cerebellum, brainstem, and optic
nerve stumps.
12. Separate the two cerebral hemispheres and remove and discard
the meninges from the cortex (see Note 4).
13. Using angled forceps, scoop the white matter from the under-
side of the cortex and the striatum which lies below this, in
direct contact with the cortex.
14. The hippocampus, which appears as a banana-shaped area, is
removed and transferred to a 15-mL centrifuge tube contain-
ing HBSS for dissection; place tube on ice (see Note 5).
15. Transfer the cortices to a 10-cm ∅ Petri dish containing fresh,
ice-cold HBSS for dissection. Using a fine forceps, gently pince
the tissue in thirds. Use a Pasteur pipette to transfer the pieces
to a conical 15-mL sterile polypropylene tube.
16. Store on ice until all the brains have been processed.

3.2. Papain Digestion 1. The papain dissociation kit contains all needed reagents men-
of Dissected Cortical tioned below except HEPES.
and Hippocampal 2. Add 2 mL of 1-M HEPES buffer to 100 mL of EBSS (vial 1 in
Tissue and kit). This will still be referred to as EBSS.
Preparation of a Single 3. Solution C: Add 32 mL of EBSS to the albumin–ovomucoid
Cell Suspension inhibitor mixture (vial 4 in kit, containing 10 mg ovomucoid
and 10 mg albumin/mL) and allow to dissolve.
4. Solution A: Add 5 mL of EBSS to the papain vial (vial 2 in kit,
containing 20 U of papain/mL in 1 mM L-cysteine with
0.5 mM EDTA).
5. Solution B: Add 500 μL of EBSS to the DNase vial (vial 3 in
kit, giving 2,000 U DNAse/mL) and mix gently.
6. Solution D: To solution A, add 250 μL of solution B.
7. Remove excess HBSS from the cortical tissue and add 5 mL of
solution D.
8. Incubate at 37°C for 15 min with occasional gentle agitation.
9. Vigorously triturate tissue with a long (9-in.) Pasteur pipette
until the cell suspension is devoid of visible clumps.
10. Although not usually necessary, small clumps of tissue remain-
ing can be removed by passing the suspension over a 70-μm
mesh filter (Falcon-BD Biosciences), having first inserted the
strainer into the mouth of a 50-mL conical centrifuge tube.
11. Centrifuge the cell suspension (or filtrate) at 200 × g for 5 min.
 5 Culture of Rodent Cortical and Hippocampal Neurons 53

12. While the cells are centrifuging, in a 15-mL tube, mix 2.7 mL
EBSS with 0.3 mL solution C and add 0.15 mL solution B
(“EBSS mix”).
13. Place 5 mL solution C in a second 15-mL conical centrifuge
tube.
14. Following centrifugation, discard the supernatant and resus-
pend the cell pellet in the above “EBSS mix.”
15. Using a 5-mL pipette, carefully layer the cell suspension on top
of the 5 mL ovomucoid solution.
16. Centrifuge the ovomucoid gradient at 150 × g for 5 min.
17. Discard the supernatant and resuspend the cell pellet as required
in Neurobasal medium for embryonic or neonatal cells.
18. Optimal cell attachment and survival can be obtained by pre-
coating the poly-D-lysine culture surface with culture medium
containing 10% (v/v) FCS. It is sufficient to expose the surface
to FCS-containing medium for 1 h at 37°C. The FCS-
containing medium should be removed just prior to cell
plating.
19. Count cells (see Chap. 3) and dilute as required. For a 96-well
plate, recommended density is 35,000 cells/well in 0.1 mL
medium.
20. Average yields are 5–6 × 106 cells and 6–8 × 105 cells/rat embry-
onic day 18 cortex and hippocampus, respectively (see Note 6).
21. In our experience, such cultures can be maintained for up to
1 month (see Note 7).

3.3. Preparation 1. Use newborn pups (both sexes) within 24 h of birth. It is our
of Newborn (Rat, experience that neuron cell survival diminishes markedly if the
Mouse) Cortical tissue is older than 1 day postnatal.
Neuron Cell Cultures 2. Euthanize the pups strictly following appropriate institution-
ally approved guidelines.
3. The dissection differs in some respects from that used for
embryos. Cerebral hemispheres are dissected out following
standard techniques and anatomical landmarks.
4. Following sacrifice and decapitation, place heads on a disinfec-
tant wipe and spray with 70% ethanol.
5. Make an incision under the skin along the midline of the dorsal
surface of the head and peel back the skin.
6. Make a second incision close to the midline along the exposed
dorsal surface (the cut should be along the complete antero-
posterior axis of the lateral ventricles).
7. Splay open the hemisphere along the cut surface and continue
cut along the dorsal surface of the hemisphere.
54 L. Facci and S.D. Skaper

8. Remove the cerebral hemispheres (minus cerebellum) and peel

off meninges.
9. Place the hemispheres into a separate dish of cold HBSS for dis-
section and repeat for the other hemisphere and remaining pups.
10. The hippocampus and striatum are dissected away but can be
saved if needed.
11. Papain dissociation of the tissue, cell counting, and plating are
carried out as described earlier for embryonic tissue, except that
Neurobasal-A medium is used.
12. The following day, add an equal volume of plating medium
containing 20-μM araC (10 μM final; diluted 1:500 from the
10-mM stock). araC is added to retard growth of nonneuronal
cell elements, especially astrocytes. The period of gliogenesis
occurs around this time developmentally, which is why glial
contamination is less of an issue when culturing cells from
embryonic tissue (and hence araC is not used).
13. These cultures are generally used within 7–8 days and do not
survive as long as those derived from embryonic tissues.

4. Notes

1. Heat inactivation of FCS is recommended to destroy heat-

labile complement. Thaw the bottle of serum in advance using
a 37°C water bath. Next, immerse the serum bottle in the
water bath after reequilibrating to 56°C and leave for 30 min.
Swirl the bottle occasionally to ensure proper mixing. Allow
the serum to cool to room temperature, aliquot into 50-mL
tubes, and store at −20°C.
2. Cytosine arabinoside is an antimitotic and potential carcino-
gen. Usage should be restricted to fume cupboards and bio-
logical safety cabinets. Avoid weighing out powder, or if not
possible, then weigh out in a suitable closed environment.
Culture media can be disposed of by normal routes. Stock/
concentrated solutions should be enclosed within a leak-proof
container and put into a burn bin for disposal.
3. It is preferable to flame-sterilize dissecting instruments by
dipping in a solution of 70% ethanol and passing through a
flame. However, if using a horizontal laminar flow cabinet for
sterile work, it is possible with care to carry out the dissection
with instruments swiped using a disinfectant wipe (e.g., Clinet
101 from Interscience, although this type of product can be
obtained from any supplier of lab products).
 5 Culture of Rodent Cortical and Hippocampal Neurons 55

4. Removal of meninges is a critical step, as their inclusion can

lead to cultures contaminated with nonneuronal cell elements.
We find that immobilizing the hemisphere with one pair of fine
forceps while using a second pair of forceps to peel away the
meninges works well. In particular, it is suggested to start at
one end rather than in the middle of the tissue.
5. The hippocampal area is best visualized using a stereo dissecting
microscope with illumination from below. This permits the
hippocampal area to be easily distinguished from the cortical
area due to differences in tissue contrast, with the hippocampus
appearing darker.
6. An analogous procedure can be used to prepare cortical and
hippocampal cell cultures from mouse embryonic tissue.
However, optimal cell survival is achieved with tissue derived
from embryonic day 15 fetuses. This is because mouse central
nervous system development is precocious with respect to rat.
7. When attempting to maintain cultures for more than 10 days,
the following is recommended: Evaporation can be an issue.
If using 96-well plates, it is best to avoid plating cells in the
outermost wells, that is, leave a “frame” in which sterile water
is added. This will minimize volume loss. If larger wells are
being used, for example, 24-, 12-, or 6-well plates, periodically
check medium volume in the wells and top up with sterile tis-
sue culture grade water if needed. Lastly, medium should be
replenished weekly, by removing one half the volume and
adding back a like volume of fresh plating medium.

Acknowledgment

L. Facci was supported by Fondazione CARIPARO “Progetto

Dottorati di Ricerca” Anno 2009.

References
1. Brewer GJ (1995) Serum-free B27/neurobasal mimetics activate TrkB signaling and prevent
medium supports differentiated growth of neuronal degeneration in rodents. J Clin Invest
neurons from the striatum, substantia nigra, 120, 1774–1785. doi: 10.1172/JCI41356
septum, cerebral cortex, cerebellum, and den- 4. Edwards D, Das M, Molnar P, and Hickman JJ
tate gyrus. J Neurosci Res 42, 674–683 (2010) Addition of glutamate to serum-free
2. Hussain I, Harrison DC, Hawkins J, Chapman culture promotes recovery of electrical activity
T, Marshall I., Facci L, et al (2011) TASTPM in adult hippocampal neurons in vitro. J
mice expressing APP and PS-1 mutant trans- Neurosci Methods 190, 155–163
genes are sensitive to γ-secretase modulation 5. Baptista MS, Melo CV, Armelão M, Herrmann
and Aβ42 lowering by GSM-10h. Neurodege- D, Pimentel DO, Leal G, et al (2010) Role of
nerative Dis 8, 15–24 the proteasome in excitotoxicity-induced cleav-
3. Massa SM, Yang T, Xie Y, Shi J, Bilgen M, age of glutamic acid decarboxylase in cultured
Joyce JN, et al (2010) Small molecule BDNF hippocampal neurons. PLoS One 5, e10139
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6. Franceschini D, Guisti P and Skaper SD (2006) neurons: a possible role for mast cells. J Neurochem
MEK inhibition exacerbates ischemic calcium 76, 47–55
imbalance and neuronal cell death in rat corti- 10. Chapman GA, Moores K, Harrison D,
cal cultures. Eur J Pharmacol 553, 18–27 Campbell CA, Stewart BR, and Strijbos, PJ
7. Skaper SD, Facci L, Williams G, Williams E-J, (2000) Fractalkine cleavage from neuronal
Walsh FS, and Doherty P (2004) A dimeric membranes represents an acute event in the
version of the short N-cadherin binding motif inflammatory response to excitotoxic brain
HAVDI promotes neuronal cell survival by damage. J Neurosci 20, RC87
activating an N-cadherin/fibroblast growth 11. Skaper SD, Ancona B, Facci L, Franceschini D,
factor receptor signalling cascade. Mol Cell and Giusti P (1998) Melatonin prevents the
Neurosci 26, 17–23 delayed death of hippocampal neurons induced
8. Hughes JP, Staton PC, Wilkinson MG, by enhanced excitatory neurotransmission and
Strijbos PJLM, Skaper SD, Arthur JSC, et al the nitridergic pathway. FASEB J 12, 725–731
(2003) Mitogen and stress response kinase-1 12. Skaper SD, Leon A, and Facci L (1991) Death of
(MSK1) mediates excitotoxic induced death cultured hippocampal pyramidal neurons induced
of hippocampal neurones. J Neurochem 86, by pathological activation of N-methyl-D-
25–32 aspartate receptors is reduced by monosialogan-
9. Skaper SD, Facci L, Kee WJ, and Strijbos PJLM gliosides. J Pharmacol Exp Ther 259, 452–457
(2001) Potentiation by histamine of synaptically- 13. Choi DW (1987) Ionic dependence of gluta-
mediated excitotoxicity in cultured hippocampal mate toxicity. J Neurosci 7, 369–379
 Chapter 6

Amyloid b-Peptide Neurotoxicity Assay

Using Cultured Rat Cortical Neurons
Laura Facci and Stephen D. Skaper

Abstract
Neurons cultured from rodent central nervous system tissues represent an important tool in the study of
neurodegenerative disease mechanisms and neuroregenerative processes, including the survival- and axon
growth-promoting properties of neurotrophic factors. This chapter presents a detailed protocol for the
preparation of rat and mouse cortical and hippocampal neuron cell cultures, using either embryonic or
postnatal tissue.

Key words: Amyloid, Cortical neurons, Neurotoxicity, Lactate dehydrogenase assay, Luminescence
assay, Cell culture, Rat, Embryonic

1. Introduction

A major pathological hallmark of Alzheimer’s disease is the presence

of extracellular senile plaques and intracellular neurofibrillary
triangles in the brain (1, 2). The main protein components of
senile plaques are amyloid-β (Aβ) peptides predominantly of 40 or
42 amino acids in length (Aβ40, Aβ42) which are generated following
sequential cleavage of the amyloid precursor protein by β-site
amyloid precursor protein cleaving enzyme 1 (β-secretase) and
γ-secretase (3). A number of findings indicate the longer Aβ42
peptide to be the key pathogenic species in Alzheimer’s disease.
Aβ42 has a greater tendency to aggregate than Aβ40 and acts as the
seed for Aβ deposition in the brain (4, 5). Aβ42 oligomers are
neurotoxic to cell lines and neurons and can inhibit long-term
potentiation in hippocampal slices (6, 7). In addition, a number of
mutations in presenilin-1 that cause early onset familial Alzheimer’s
disease have been shown to specifically elevate production of
Aβ42 (8).

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_6, © Springer Science+Business Media, LLC 2012

57
58 L. Facci and S.D. Skaper

Ex vivo models utilizing rodent-derived central nervous system

neurons, in particular rat and mouse cortical and hippocampal
neurons, have found widespread application in identifying small-
molecule nonpeptide compounds with neuroprotective activity
against Aβ42-induced death (9–13), as well as peptidomimetics
of neurotrophic factors (14) and neurotrophins themselves.
Compounds with this protective action would be potentially disease-
modifying and thus highly desirable as therapeutics for Alzheimer’s
disease. This chapter describes a protocol for quantifying Aβ42-
induced death (15, 16) of cultured rat embryonic cortical neurons,
based on the measurement of either lactate dehydrogenase (LDH)
release into the culture medium (colorimetric assay) or the intrac-
ellular content of ATP (luminescence assay).

2. Materials

2.1. Equipment 1. Laminar flow biological safety cabinet (CL2).

and Labware 2. Humidified, water-jacketed culture incubator at 37°C and 5%
CO2/95% air.
3. Water bath set at 37°C.
4. 96-well poly-D-lysine treated tissue culture plates (BD
Biosciences).
5. Microplate reader capable of reading absorbance at 490 nm.
6. Wallac 1450 MICROBETA PLUS Liquid Scintillation Counter
(TopCount) or similar instrument for luminescence readout.
7. 3-mL glass weighing vials with cap.
8. Bench centrifuge to accommodate 15- and 50-mL tubes.
9. 15- and 50-mL polypropylene plastic centrifuge tubes, sterile.

2.2. Reagents 1. Hank’s balanced salt solution, with CaCl2 and MgCl2, sterile
(HBSS) (Invitrogen).
2. Neurobasal medium (Invitrogen).
3. B27 supplement, 50×, with antioxidants (Invitrogen).
4. B27 supplement, 50×, without antioxidants (Invitrogen).
5. Fetal calf serum (FCS) (Invitrogen) (see Note 1).
6. L-Glutamine (200 mM stock), sterile, for cell culture
(Invitrogen).
7. Sodium pyruvate (100 mM stock), sterile, for cell culture
(Sigma).
8. Penicillin/streptomycin, 10,000 U/mL penicillin + 10,000 μg/
mL streptomycin (100× stock), sterile, for cell culture
(Invitrogen).
 6 Amyloid b-Peptide Neurotoxicity Assay Using Cultured Rat Cortical Neurons 59

9. HEPES, 1 M, sterile (Sigma).

10. Human β-amyloid peptide 1–42 (Aβ42) (California Peptide
Research).
11. Hexafluoroisopropanol (HFIP) (Sigma).
12. Dimethylsulfoxide (DMSO) for cell culture (Sigma).
13. CytoTox 96® nonradioactive cytotoxicity assay kit (Promega).
14. CellTiter-Glo® luminescent cell viability assay kit (Promega).
15. ATP standard solution, supplied as 100 mM stock
(BioVision).

2.3. Culture Medium 1. Neurobasal medium containing B27 supplement (1×), 2 mM

L-glutamine, 100 U/mL penicillin, and 100 μg/mL strepto-
mycin: To 500 mL Neurobasal medium, add 10 mL 50× B27
supplement, 5 mL 200 mM glutamine, and 5 mL 5,000 U/
mL penicillin to 5,000 μg/mL streptomycin. Complete
medium can be stored at +4°C for up to 2 months.

2.4. Ab42 Solution 1. Weigh the desired amount of Aβ42 into a 3-mL glass weighing
vial (see Notes 2 and 3).
2. In a CL2 cabinet, add the required volume of HFIP to the
Aβ42 (use 400 μL HFIP per mg of Aβ42) (see Note 4).
3. Briefly mix and leave at room temperature for 10 min to allow
peptide to fully dissolve (the solution should be clear at this
stage).
4. Evaporate the HFIP under a gentle stream of air, for example,
by attaching a Pasteur pipette to the tube delivering the air
stream. Perform this operation under a fume cupboard.
5. At this point, a film of peptide should be visible around the
base of the vial.
6. Resuspend the dried Aβ42 peptide in tissue culture grade
DMSO 1 mg peptide in 44 μL DMSO, ensuring that the
DMSO is pipetted around the base of the eppendorf tip where
the peptide film is visible.
7. This produces a solution of 5-mM peptide, to which is now
added 0.1% trifluoroacetic acid (73 μL/mg peptide).
8. The Aβ42 peptide solution is now diluted directly into pre-
warmed (37°C) culture medium, to give a concentration of
80 μM (taking into account the volume contributed by DMSO
and trifluoroacetic acid).
9. The cortical neuron cell cultures receive one-fourth volume of
the Aβ42 solution (80 μM) in medium, to yield a final peptide
concentration of 20 μM in the cultures.
60 L. Facci and S.D. Skaper

3. Methods

3.1. Treatment of 1. Cortical (or hippocampal) neurons prepared from embryonic

Neuronal Cell Cultures rat (or mouse) are plated in 96-well poly-D-lysine-coated wells,
with Test Agent(s) 30,000 cells per well in 0.1 mL of Neurobasal/B27 plating
medium (see Chapter 5 for cell preparation protocol).
2. Day 2 in culture: Add to each well 0.1 mL of Neurobasal
medium supplemented with antioxidant-free B27. The medium
formulation is identical to that described in Subheading 2.3,
except that B27 with antioxidants is substituted for by the anti-
oxidant-free version.
3. Day 6 in culture: Remove medium from the wells and add
50 μL Neurobasal/B27 medium (antioxidant-free).
4. Next, add to the well 25 μL of the same medium containing
test compound at 4× the desired final concentration (0.1%
DMSO contributed by compound).
5. Incubate cells with compound for 30 min (or longer, as appro-
priate to target) at 37°C.
6. Next, add to each well (except controls) 25 μL of Aβ42
solution (80 μM) to give a final concentration of 20 μM (0.4%
DMSO contribution). Total final concentration of DMSO in
all wells from test compound + amyloid is 0.5% (v/v).
7. Controls receive the same solutions but without Aβ42 (DMSO
is maintained at 0.5%).
8. Return the plate(s) to the 5% CO2 incubator at 37°C for 3 days.

3.2. Lactate The assay is composed of two component part numbers—G1781

Dehydrogenase and G1782—and is sufficient to process 5- × 96-well plates.
Release Assay Contents
Protocol (CytoTox 96® G1781
Nonradioactive ● G179A: substrate mix (lyophilized diaphorase, lactate, and
Cytotoxicity Assay) NAD+), five vials.
● G180A: assay buffer (Tris-buffered tetrazolium dye (INT-
chloride) and Triton X-100), 60 mL.
G1782
● G181A: LDH-positive control (slurry in ammonium sul-
fate, pH 6.0), 25 μL.
● G182A: lysis solution (10×), 3 mL.
● G183A: stop solution, 65 mL.
The assay procedure is as follows:
1. Remove 12 mL of assay buffer and promptly store the unused
portion at −20°C.
6 Amyloid b-Peptide Neurotoxicity Assay Using Cultured Rat Cortical Neurons 61

2. Add the 12 mL of assay buffer (room temperature) to a bottle

of substrate mix. Once resuspended, protect the solution from
direct light and use immediately.
3. Dilute the LDH-positive control (bovine heart LDH, 800 U/mL)
according to the scheme shown in the table below:
# LDH (mU/mL) mL Medium (mL)

B 160 2 μL (*) 10,000

C 112 2,000 (B) 857
D 80 2,000 (C) 800
E 64 2,000 (D) 500
F 48 2,000 (E) 667
G 32 2,000 (F) 1,000
H 16 2,000 (G) 2,000
I 8 2,000 (H) 2,000
0 0 0 1,000

Add 50 μL of LDH standard-curve sample according to the

template illustrated below. The stock solution should be
prepared fresh each time.
1 2 3 4 5 6 7 8 9 10 11 12

A 0 0 0 I I I H H H G G G
B
C
D
E
F
G
H F F F E E E D D D C C C

4. Collect from each well the full amount (100 μL) of culture
medium and set aside.
5. Transfer 50 μL of sample medium to a flat bottom clear micro-
titer clear 96-well plate.
6. Lyse cells by adding to each well 100 μL culture medium
containing 1% lysis solution (provided in the kit). Leave in the
37°C incubator for at least 10 min (see Note 5).
7. Transfer 10 μL of cell lysate to the flat-bottom 96-well plate.
Add 40 μL culture medium to each of these wells.
8. Add 50 μL reconstituted substrate to all wells.
62 L. Facci and S.D. Skaper

0.4

0.3

O.D. (490 nm)

0.2

0.1

0.0

-0.1
0 25 50 75 100 125
LDH (mU/ml)

Fig. 1. Sample LDH standard curve.

9. Cover the plate with aluminum foil to protect from light.

10. Incubate at room temperature (20–23°C) for 30 min.
11. Add 50 μL of stop solution to each well (final volume
−150 μL).
12. Read samples in a plate reader at A490 within 1 h of stopping
the assay (see Note 6).
13. Calculate LDH values from the standard curve (see Fig. 1 for
a “representative” example) (see Note 7).

3.3. ATP Cell Content 1. Allow CellTiter-Glo® substrate and buffer to come to room
Assay Protocol temperature (see Notes 8 and 9).
(CellTiter-Glo® 2. Starting with the 100 mM ATP stock solution, prepare a work-
Luminescent Cell ing stock solution of 1 mM ATP using Neurobasal medium
Viability Assay) with B27 supplement (no antioxidants).
3. Prepare serial dilutions of ATP in the above culture medium,
covering the range 0.003–2 μM. A 96-well template may be
used, analogous to that described above for LDH assay but
modified to accommodate the concentration range of ATP
standard dilutions.
4. Add 100 μL per well of ATP standards (using empty wells but
in the same 96-well plate with cells).
5. Add to each well (cells or ATP standards) 100 μL of CellTiter-
Glo solution.
6. Allow at least 10 min for cell to lyse and come to equilibrium.
7. Read plate in a Wallac 1450 MICROBETA PLUS liquid
scintillation counter (TopCount) or similar instrument for
luminescence.
8. Calculate ATP values from the standard curve (see Fig. 2 for a
“representative” example).
 6 Amyloid b-Peptide Neurotoxicity Assay Using Cultured Rat Cortical Neurons 63

200000

Luminescence
100000

-100000
0 500 1000 1500 2000 2500
ATP (nM)

Fig. 2. Sample ATP standard curve.

4. Notes

1. Heat inactivation of fetal calf serum is recommended to destroy

heat-labile complement. Thaw the bottle of serum in advance
using a 37°C water bath. Next, immerse the serum bottle in
the water bath after reequilibrating to 56°C and leave for
30 min. Swirl the bottle occasionally to ensure proper mixing.
Allow the serum to cool to room temperature, aliquot into
50-mL tubes, and store at −20°C.
2. We have tested Aβ42 from different suppliers and find that
California Peptide Research gives the most consistent results
from batch to batch, and is easiest to dissolve in HFIP com-
pared to Aβ42 from other sources (e.g., Sigma, Calbiochem,
Bachem). If one intends to perform these assays on a routine
basis (e.g., structure-activity screening of compounds rather
than a “one off”) we suggest requesting a 1 mg sample from the
company to test for neurotoxicity. If satisfactory, a large amount
of that peptide batch should be purchased and set aside.
3. Aβ42 (at least that from California Peptide) is supplied lyo-
philized and is rather hygroscopic. It should be stored at −20°C,
desiccated, and under vacuum. The parent bottle should be
allowed to warm to room temperature before opening.
4. We recommend purchasing HFIP in small quantities, for
example, as 25-mL bottles from Sigma-Aldrich. Once opened,
the bottle should be used for no more than 2 weeks and then
discarded if not consumed. The HFIP solvent absorbs mois-
ture from the air, and this will interfere with the proper solubi-
lization of Aβ42.
64 L. Facci and S.D. Skaper

5. If sufficient lysis solution is not available, you can use 0.5%

Triton X-100.
6. The presence of bubbles, especially large ones, may cause spu-
rious absorbance readings. These can be eliminated by using a
hypodermic needle to pierce and break the bubble. For smaller
bubbles, one method which works quite well is to first heat the
needle tip, then touch the hot tip to the bubble(s).
7. A test compound which is cytotoxic per se can cause a false-
positive result (indicating an apparent neuroprotective effect).
This is especially true when the test compound causes a rapid
release of LDH, which is then degraded over the 3-day incuba-
tion period. This leads to a false low value of LDH released and
an apparent “neuroprotective” effect. To minimize the risk of
such false positives, we strongly recommend measuring the
LDH content of both the culture medium and in the remain-
ing cells, and calculating LDH release as a percent of total
(extra- and intracellular) LDH in the culture well.
8. For long-term storage, it is best to keep CellTiter-Glo ®
substrate and buffer at −20°C.
9. Reconstituted CellTiter-Glo® reagent can be stored at +4°C
with approximately 5% reduction in luminescence intensity or
4 days at +4°C with approximately a 20% decrease in intensity.
The reconstituted reagent may be frozen at −20°C for storage.
Ten freeze-thaw cycles decrease the luminescence intensity by
only 5–10%. The frozen reagent should be thawed in a 37°C
water bath prior to assay.

Acknowledgments

L. Facci was supported by Fondazione CARIPARO Progetto

Dottorati di Ricerca’ Anno 2009.

References

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120, 885–890 The carboxy terminus of the beta amyloid protein
2. Goedert M, Wischik CM, Crowther RA, Walker is critical for the seeding of amyloid formation:
JE, and Klug A (1988) Cloning and sequenc- implications for the pathogenesis of Alzheimer’s
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disease: identification as the microtubule- Eriksen J, Yu C, et al (2005) Aβ42 is essential
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85, 4051–4055 tion in mice. Neuron 47, 191–199
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6. Dahlgren KN, Manelli AM, Stine Jr WB, Baker non-peptide p75NTR ligands inhibit Aβ-induced
LK, Krafft GA, and LaDu MJ (2002) neurodegeneration and synaptic impairment.
Oligomeric and fibrillar species of amyloid-β- PLoS One 3, e3604
peptides differentially affect neuronal viability. 12. Liu F, Gong X, Zhang G, Marquis K, Reinhart
J Biol Chem 277, 32046–32053 P, and Andree TH (2005) The inhibition of
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Anwyl R, Wolfe MS, et al (2002) Naturally glutamate receptor 5 mediated pathway confers
secreted oligomers of amyloid β protein neuroprotection to Aβ peptides. J Neurochem
potently inhibit hippocampal long-term poten- 95, 1363–1372
tiation in vivo. Nature 416, 535–539 13. Chen J, Zhou Y, Mueller-Steiner S, Chen LF,
8. Scheuner D, Eckman C, Jensen M, Song X, Kwon H, Yi S, Mucke L, et al (2005) SIRT1
Citron M, Suzuki N, et al (1996) Secreted protects against microglia-dependent amyloid-
amyloid beta-protein similar to that in the β toxicity through inhibiting NF-κB signaling.
senile plaques of Alzheimer’s disease is increased J Biol Chem 280, 40364–40374
in vivo by the presenilin 1 and 2 and APP muta- 14. Massa SM, Yang T, Xie Y, Shi J, Bilgen M,
tions linked to familial Alzheimer’s disease. Nat Joyce JN, et al (2010) Small molecule BDNF
Med 2, 864–870 mimetics activate TrkB signaling and prevent
9. Bozyczko-Coyne D, O’Kane TM, Wu ZL, neuronal degeneration in rodents. J Clin
Dobrzanski P, Murthy S, Vaught JL, et al Invest 120, 1774-1785. doi: 10.1172/
(2001) CEP-1347/KT-7515, an inhibitor of JCI41356
SAPK/JNK pathway activation, promotes sur- 15. Pym LJ, Buckingham SD, Tsetlin V, Boyd CA,
vival and blocks multiple events associated with and Sattelle DB (2007) The Aβ1-42M35C
Abeta-induced cortical neuron apoptosis. J mutated amyloid peptide Aβ1-42 and the 25-35
Neurochem 3, 849–863 fragment fail to mimic the subtype-specificity
10. Hussain I, Harrison DC, Hawkins J, Chapman of actions on recombinant human nicotinic
T, Marshall I, Facci L, et al (2011) TASTPM acetylcholine receptors (α7, α4β2, α3β4).
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11. Yang T, Knowles JK, Lu Q, Zhang H, Arancio ponent of amyloid-mediated neuronal cell
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 Chapter 7

Culture of Neonatal Rodent Microglia, Astrocytes,

and Oligodendrocytes from Cortex and Spinal Cord
Stephen D. Skaper, Carla Argentini, and Massimo Barbierato

Abstract
The protocol described in this chapter covers the preparation and culture of enriched populations of
microglia, astrocytes, and oligodendrocytes from the cortex and spinal cord of neonatal rat and mouse.
The procedure is based on the enzymatic digestion of tissue, followed by the culture of a mixed glial cell
population which is then utilized as the starting point for the isolation, via differential attachment, of the
different cell types.

Key words: Microglia, Astrocytes, Oligodendrocytes, Cortex, Spinal cord, Rat, Cell culture

1. Introduction

Glia, which outnumber neurons by as much as 10 to 1 in some

regions of the human brain, were at one time thought to function
solely by providing physical support and housekeeping for the neu-
rons. We now know that during brain development, glia guide
migrating neurons to their destinations and instruct them to form
synapses. In the adult brain, glia talk back to neurons, releasing
neurotransmitters and other signals that regulate synaptic strength.
The emerging realization is that glia may have key roles in central
nervous system disorders from neuropathic pain and epilepsy to
neurodegenerative disorders such as Alzheimer’s—and may even
contribute to schizophrenia, depression, and other psychiatric dis-
orders. The brain’s glia can be divided into three principle types:
microglia (the brain’s resident macrophages), astrocytes, and oli-
godendrocytes. Each glial cell type displays distinct properties and
behaviors. For example, oligodendrocytes, the glia that form a fatty
myelin sheath around the axons of neurons in the brain and spinal
cord (1), are sensitive to injury from glutamate-induced oxidative

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_7, © Springer Science+Business Media, LLC 2012

67
68 S.D. Skaper et al.

stress (2, 3) and excitotoxicity (4, 5) and are also capable of

producing mature amyloid β-peptide (6). Microglia, depending on
the context in which they occur, can provide both neurotrophic
support (7) and neurotoxins under pathological conditions (8–10).
Astrocytes, which are the most abundant glial cell type in the brain,
are known to synthesize and release low (11) and high (12) molec-
ular weight neurotrophic agents, as well as reactive nitrogen oxide
species (13), glutamate, and pro-inflammatory cytokines (14).
Cultured astrocytes have also proven useful for the study of cellular
differentiation signals (15) and gene transcription (16). Clearly,
enriched populations of these glial cell types are of considerable use
in the study of nervous system development, injury, and therapeu-
tic targets. The following chapter presents a protocol for the isola-
tion and culture of rodent central nervous system glia.

2. Materials

2.1. Equipment 1. Stereo dissecting microscope (backlighting of stage is pre-

and Labware ferred) with fiber-optic light source.
2. Horizontal flow cabinet for dissections.
3. Laminar flow biological safety cabinet (CL2).
4. Humidified, water-jacketed culture incubator at 37°C and 5%
CO2/95% air.
5. Water bath set at 37°C.
6. Dissecting tools (Fine Science Tools (InterFocus Ltd) are very
high quality for the price).
7. Bench centrifuge to accommodate 15- and 50-mL tubes.
8. Orbital shaker with enclosed, temperature controlled chamber.
9. 10-cm ∅ sterile tissue culture dishes (any supplier).
10. 15- and 50-mL polypropylene plastic centrifuge tubes (sterile).
11. Sterilin 10-cm ∅ sterile plastic petri dishes (Bibby-Sarstedt).
12. Tissue culture flasks (T-75; 75 cm2) with canted neck and 0.2 μm
vented cap (Corning) or without vented cap (BD Falcon).
13. Cell scrapers, 25-cm handle, 1.8-cm blade (BD Falcon).
14. 0.22-μm filters (Millipore Corporation).

2.2. Reagents 1. Phosphate-buffered saline (pH 7.4, sterile) (Invitrogen).

2. Dulbecco’s modified Eagle’s medium (DMEM) (+4.5 g/L
glucose, L-glutamine, pyruvate) (Invitrogen).
3. N2 supplement (Invitrogen).
4. Trypsin inhibitor (Sigma).
 7 Culture of Neonatal Rodent Microglia, Astrocytes, and Oligodendrocytes… 69

5. DNAse (Type I) (Sigma).

6. L-15 medium (+L-glutamine, L-amino acids) (Invitrogen).
7. Trypsin inhibitor, type I from soybean (Sigma).
8. Papain (Worthington (Lorne)).
9. Bovine serum albumin, Cohen fraction V (Sigma).
10. T3 (3,3¢,5-triiodo-L-thyronine, sodium salt), cell-culture-
tested (Sigma).
11. T4 (L-thyroxine sodium salt pentahydrate), γ-irradiated, cell-
culture-tested (Sigma).
12. Penicillin/streptomycin (100× stock), sterile, for cell culture
(Invitrogen).
13. Gentamicin (50 mg/mL stock), sterile, for cell culture (Sigma).
14. Fetal calf serum (Invitrogen).
15. Poly-L-lysine (MW 70,000–150,000), sterile, for cell culture
(Sigma P-6282).
16. L-Glutamine (200 mM stock), sterile, for cell culture
(Invitrogen).
17. Sodium pyruvate (100 mM stock), sterile, for cell culture
(Sigma).
18. HEPES (1 M), sterile for cell culture (Sigma).
19. L-Leucine methyl ester HCl (Sigma).

2.3. Culture Media 1. T3: Dissolve 4 mg of T3 in 10 mL of 1 N NaOH (1,000× stock,

and Other Solutions 400 μg/mL), filter-sterilize, aliquot, and store at −20°C.
2. T4: Dissolve 4 mg of T4 in 10 mL of tissue culture grade water
(1,000× stock, 400 μg/mL), filter-sterilize, aliquot, and store
at −20°C.
3. DNase: Dissolve 40 mg of DNase in 10 mL of L-15 medium
(100× stock, 4 mg/mL), filter-sterilize, aliquot, and store at
−20°C.
4. L-Cysteine: Dissolve 24 mg of L-cysteine in 1 mL of L-15
medium (100× stock). Prepare fresh each time.
5. Trypsin inhibitor: Dissolve 100 mg of trypsin inhibitor in 1 mL
(100× stock) of L-15 medium. Filter-sterilize, aliquot, and
store at −20°C.
6. Bovine serum albumin: Dissolve 5 mg of bovine serum albu-
min in 1 mL (100× stock) of L-15 medium. Filter-sterilize,
aliquot, and store at −20°C.
7. 0.15 M borate buffer pH 8.4: Dissolve 28.6 g of sodium borate
(Na2B4O7, 10 H2O) in 500 mL water (pH will be ~9.2). Adjust
pH to 8.4 with 5 N HCl. Filter-sterilize and store at 4°C (up
to 6 months).
70 S.D. Skaper et al.

8. Plating medium/growth (maintenance) medium: To a 500-mL

bottle of DMEM, add 50 mL fetal calf serum (10% final),
5 mL stock penicillin/streptomycin (100 U/mL penicil-
lin + 100 μg/mL streptomycin final), and 0.5 mL of gentamicin
stock (50 μg/mL final).
9. Sato medium (for oligodendrocytes): To 500 mL DMEM, add
5 mL N2 stock (1:100), 0.5 mL T3 stock (1:1,000), 0.5 mL
T4 stock (1:1,000), 5 mL penicillin/streptomycin stock
(1:100), and 2.5 mL fetal calf serum (0.5% final concentra-
tion). Store at +4°C for up to 2 months. Note: the defined
medium supplement contains components which have been
isolated from human sources (e.g., transferrin), and their haz-
ard is not known.

2.4. Preparation 1. Dissolve a 5-mL bottle of poly-L-lysine (MW 70,000–150,000)

of Substratum in 5 mL 0.15 M borate buffer (pH 8.4). This 1-mg/mL stock
solution can be stored at +4°C for up to 2 months.
2. Dilute the 1-mg/mL stock solution of poly-L-lysine 1:100
(1 mL in 99 mL sterile water) and add 10 mL to each of the
T-75 culture flasks. Rock the flask to cover the entire culture
surface and place flask in the 5% CO2 incubator (37°C)
overnight.
3. The following day aspirate the poly-L-lysine coating solution
and leave the flask(s) under the CL2 cabinet uncapped to dry.
This will usually take several hours. Once dried, the poly-L-
lysine-coated flasks can be stored at +4°C for 1 month.

3. Methods

3.1. Dissection of Rat 1. Rat cultures are routinely performed using animals at postnatal
Cortical Tissue day 1–2. Following appropriate national and institutional
guidelines for animal sacrifice, pregnant rats (CD strain,
Sprague Dawley) are euthanized (see Note 1).
2. Dissect out cerebral hemispheres following standard tech-
niques and anatomical landmarks. The procedure is applicable
to either rat or mouse pups.
3. Make an incision under the skin along the midline of the dorsal
surface and peel the skin back.
4. Make a second incision close to the midline along the exposed
dorsal surface (the cut should be along the complete antero-
posterior axis of the lateral ventricles).
5. Splay open the hemisphere along the cut surface and continue
the cut along the dorsal surface of the hemisphere.
 7 Culture of Neonatal Rodent Microglia, Astrocytes, and Oligodendrocytes… 71

6. Remove the cerebral hemispheres (minus cerebellum and brain

stem) and peel off meninges. The latter step is best accom-
plished by using two fine-tipped #5 Dumont forceps, using
one pair to anchor the hemisphere while peeling off the menin-
ges with the second pair. (see Note 2).
7. Place the dissected hemisphere into a separate 10-cm ∅ dish of
cold L-15 medium.
8. Repeat this procedure for the other hemisphere and remaining
pups.
9. The hippocampal area can be dissected away and retained or
discarded, depending on need.

3.2. Enzymatic 1. Grip a straight-edge razor blade with a hemostat, dip in 70%
Digestion of Cortical ethanol, and pass through a flame to sterilize.
Tissue and 2. Mince the collected cortical tissue with the razor blade.
Preparation of Cell
3. Collect the minced tissue with L-15 and transfer to a 15-mL
Suspension for Plating centrifuge tube.
4. Centrifuge collected cortical tissue for 30 s (200 × g) to com-
pact the minced tissue.
5. While the tissue is centrifuging, prepare the following: 3 mL of
L-15 medium containing 140 μL stock papain solution (as pur-
chased), 30 μL of 100× stock L-cysteine solution, and 30 μL of
100× DNase stock solution. Filter-sterilize before use.
6. Remove L-15 medium from the tube with cortical tissue pel-
let, and add the L-15 solution containing papain, DNase, and
L-cysteine.

7. Incubate tissue for 60 min in a 37°C water bath with occa-

sional swirling.
8. During this time, prepare the ovomucoid solution: to 3 mL of
L-15 medium add 30 μL of 100× stock DNase solution, 30 μL
of 100× stock bovine serum albumin solution, and 30 μL of
100× stock trypsin inhibitor solution.
9. Upon completion of the enzyme incubation step, remove the
supernatant and replace with 1.5 mL ovomucoid solution (see
Note 3).
10. Incubate for 2 min in a 37°C water bath.
11. Centrifuge at 250 × g for 3 min.
12. Remove the supernatant and add 1.5 mL fresh ovomucoid
solution.
13. Triturate 20–25 times with a long (9-in.) cotton-plugged
Pasteur pipette (see Note 4).
14. Add 4 mL plating medium and centrifuge at 200 × g for
5 min.
72 S.D. Skaper et al.

15. Using a 5-mL tissue culture pipette, resuspend the cell pellet in
3 mL plating medium. Add an additional amount of plating
medium such that the final volume is equivalent to 2 mL × num-
ber of T-75 flasks to be used (e.g., 20 mL final for 10 flasks).
16. Add 18 mL plating medium to each flask, followed by 2 mL of
cell suspension. Normally, we seed cultures at a ratio of 1.5
brains/flask (see Note 5).
17. The following day, replace the culture medium with 15 mL
fresh plating (growth) medium.
18. Twice per week, replace one-half the volume of culture medium
with an equal volume of fresh growth medium.
19. The mixed glial cell cultures are incubated for 10–14 days,
after which time, confluence will have been reached and they
can be taken for harvesting different cell populations (astro-
cytes, microglia, oligodendrocytes) (see Note 6).

3.3. Isolation 1. Remove 3 mL medium from each T-75 flask; this should leave
of Oligodendrocyte about 12 mL of medium.
Precursors and 2. As the culture medium is bicarbonate-buffered, to prevent pH
Microglia excursions, close the flask cap (or cover with parafilm if the cap
is of the filter type).
3. Place the flasks on an orbital shaker fitted with a temperature-
controlled chamber. Flasks can be fastened to the shaker plat-
form using double-sided adhesive tape, or taped directly to the
platform using high-strength industrial ducting tape.
4. Shake the flasks for 2 h at 200 cycles/min (37°C).
5. Remove the culture medium (containing mainly microglia)
from the flasks and transfer to 10-cm ∅ Sterilin plastic petri
dishes. A volume of 20–25 mL/dish is best (see Notes 7
and 8).
6. Add 13 mL maintenance medium to the flasks and place on
the orbital shaker overnight at 200 cycles/min.
7. Place the Sterilin dishes in the 5% CO2 incubator (37°C) and
leave for 45–60 min.
8. Aspirate the medium on the Sterilin plates and add 6 mL fresh
maintenance medium.
9. Use a cell scraper to remove the attached microglia from the
Sterilin dishes. Transfer the medium with cells to a 50-mL cen-
trifuge tube. Rinse the dishes with 5 mL maintenance medium
and transfer to the collection tube.
10. Resuspend the microglia cell pellet in maintenance medium and
plate into poly-L-lysine-coated multiwall plates as dictated by the
experimental design. Typically, we plate 105 cells in a 96-well
plate (equivalent to 3.5 × 105 cells/cm2). For cell counting
 7 Culture of Neonatal Rodent Microglia, Astrocytes, and Oligodendrocytes… 73

procedure, see Chap. 3. Microglia obtained are ³97% pure.

Cultures will maintain their viability for approximately 10 days.
11. Collect culture medium from the overnight shaking of flasks
and transfer to Sterilin dishes as in step 5 above.
12. Place the Sterilin dishes in the 5% CO2 incubator (37°C) and
leave for 45–60 min.
13. Collect the medium from this last step (containing mainly non-
adherent oligodendrocytes) and centrifuge at 200 × g for 5 min.
The Sterilin dishes contain a limited number of microglia and
can be discarded.
14. Resuspend the oligodendrocyte cell pellet in Sato medium,
count, and dilute in Sato medium to the desired cell density for
plating. Cultures prepared in this manner contain approxi-
mately 93% oligodendrocytes and 3% cells positive for the
astrocytic marker glial fibrillary acidic protein. The remaining
cells do not stain by any of these antibodies, including markers
specific for microglia (7). The oligodendrocytes can be cul-
tured for up to 10 days before loss of viability begins.
15. Oligodendrocytes are normally plated on a poly-D-lysine-
coated tissue culture plastic surface: this substratum is prepared
as described for coating with poly-L-lysine, except that the
1 mg/mL poly-D-lysine stock solution in borate buffer is
diluted 1:20 for coating.

3.4. Isolation of an 1. The T-75 flasks remaining after shaking to recover oligoden-
Enriched Population drocytes and microglia are used as a source of highly enriched
of Astrocytes astrocytes.
2. Aspirate the medium from the flask and rinse the cell mono-
layer with 10-mL sterile phosphate-buffered saline.
3. Add to the flask 3 mL of 0.25% trypsin/EDTA solution, rock
the flask to spread the trypsin solution over the entire mono-
layer, and then remove by aspiration. This will leave a thin film
of trypsin solution over the cells.
4. Incubate the flask(s) at 37°C for 10 min.
5. Tap the flask against the palm of the hand to dislodge cells and
add 5 mL maintenance medium per flask; the fetal calf serum
in the medium inactivates the trypsin.
6. Rinse the flask with the medium added, collect cells, and trans-
fer to a 15- or 50-mL centrifuge tube, as needed.
7. Pellet cells by centrifugation at 200 × g for 5 min, re-suspend
pellet in maintenance medium and count. The typical cell yield
is 4–5 × 106 cells/T-75 flask. On this basis, dilute the cell sus-
pension to 1–1.5 × 106 cells/mL for ease of counting. Astrocytes
thus obtained are ³95% pure.
74 S.D. Skaper et al.

8. If needed, dilute further the cell suspension for plating into

poly-L-lysine-coated culture vessels are dictated by the experi-
mental design. Typically, we plate 5 × 104 cells in a 96-well plate
(equivalent to 1.75 × 105 cells/cm2) (see Note 9).

3.5. Preparation 1. The procedure is essentially as that described for cortical tissue,
of Spinal Cord Glia except for the dissection.
2. Sacrifice pups by decapitation.
3. Place the body face down on a paper towel. Spray 70% ethanol
over the back.
4. Remove the back skin with surgical scissors to expose the ver-
tebral column.
5. Entering from the anterior side of the body, make a cut through
the midline as far as the tail.
6. Using a fine pair of surgical scissors trim back, at a 45° angle,
the tissue on either side of the spinal canal (being careful not
to disrupt the spinal cord itself).
7. Carefully strip off the spinal cord with a fine pair of forceps and
transfer to a 10-cm ∅ dish of cold L-15 medium.
8. Repeat this procedure for the remaining pups.
9. Peel off the meninges using fine tipped forceps, and transfer
the tissue pieces to a 15-mL centrifuge tube. It is not necessary
to mince the tissue as for cortex; the spinal cord is much smaller
and will already have been fragmented to some extent in the
course of removing the meninges.
10. Centrifuge the collected tissue for 30 s (200 × g) to compact.
11. Remove L-15 medium from the tube and add the L-15 solu-
tion containing papain, DNase, and L-cysteine.
12. Incubate tissue for 60 min in a 37°C water bath with occa-
sional swirling.
13. Proceed with tissue dissociation as for cortex, except the final
5-min centrifugation (Subheading 3.2, step 14). Doing so
reduces the risk of losing material, which is limited in compari-
son to cortex.
14. Following dissociation, plate the spinal cord cell suspension in
medium and distribute in poly-L-lysine-coated T-75 flasks, five
spinal cords per flask.
15. For harvesting microglia, we find that spinal cord mixed glia
cultures mature more slowly than do the cortical cultures. As
such, optimal recovery of microglia is achieved after 10–11
days in vitro. Isolation of separate populations of microglia,
astrocytes, and oligodendrocytes is carried out as described for
cortical cultures.
 7 Culture of Neonatal Rodent Microglia, Astrocytes, and Oligodendrocytes… 75

4. Notes

1. When preparing cortical glia cultures from mouse, we recom-

mend using pups at 6–7 days of age. The yield of microglia and
oligodendrocytes is superior to that obtained with younger
animals.
2. We find that removal of meninges is facilitated by using a dis-
secting microscope with backlighting (i.e., under the stage, so
that the tissue is illuminated from beneath). In this way, menin-
ges have a different contrast from the underlying cortex, which
makes it easier to delineate. This principle is valid also for delin-
eating the hippocampal region from the cortex.
3. If there is no clear boundary between the incubation solution
and tissue, you can centrifuge for 1 min at 200 × g to achieve a
better separation.
4. The number of up-and-down strokes to achieve complete dis-
persal of the tissue can vary; the value given here is normally
sufficient but can be increased if tissue pieces are still evident.
If necessary, and to clear the suspension of any remaining small
clumps, this may be passed through a cell strainer (70 μm, BD
Falcon) placed over the mouth of a 50-mL centrifuge tube.
5. The choice of flask type, i.e., with or without vented cap, is
largely immaterial. The advantage of the vented cap is that it
eliminates the risk of forgetting to leave the cap loose, to allow
for CO2 exchange in the incubator. The disadvantage is that
one needs to remember to cover the cap with parafilm before
placing on the orbital shaker (and remove when returning to
the incubator).
6. The time to reach maturity can vary between labs, which may
be a reflection of differences in reagent source, batch number
(e.g., serum, enzymes), and animal (sub)strain. In our former
lab, microglia could be harvested after 10–14 days following
setting up of the initial mixed glial culture. In our present lab,
this timing appears to be accelerated, with optimal recovery of
microglia (numbers, vitality) after 7–8 days. We would suggest
that investigators determine the best time in their hands.
7. Sterilin plastic petri dishes appear to work best for collecting
microglia by differential attachment. We have used other types
(sources) of bacterial plastic petri dishes, but observed that the
microglia recovered from these had a lower vitality than when
using Sterilin.
8. The efficiency of the differential plating step over Sterilin
plastic petri dishes depends on the volume in the dish and time
of incubation. We find that a volume of 20–25 mL/dish is
optimal, when combined with an incubation time of 45–60 min.
76 S.D. Skaper et al.

Larger volumes tend to decrease efficiency, as do shorter

(<30 min) incubation times. Incubation times longer than 1 h
can increase the percent of nonmicroglial cellular elements
(hence inpurities).
9. Residual microglia can be removed by treating the astrocyte
monolayers with medium containing 50 mM L-leucine methyl
ester for 60 min (17). This medium is then removed and
replaced with fresh maintenance medium and the cultures
incubated for at least one day prior to use. L-Leucine methyl
ester, a lysosomotropic agent originally used to selectively
destroy macrophages (18), has also been employed to deplete
microglia from neural cell cultures including astrocytes (19)
and oligodendrocytes (20).

References

1. Schwab ME, and Schnell L (1991) Channeling 9. Culbert AA, Skaper SD, Howlett DR, Evans
of developing rat corticospinal tract axons by NA, Facci L, Soden PE, et al (2006) MAPKAP
myelin-associated neurite growth inhibitors. kinase 2 deficiency in microglia inhibits pro-
J Neurosci 11, 709–721 inflammatory mediator release and resultant
2. Rosin C, Colombo S, Calver AA, Bates TE, neurotoxicity: Relevance to neuroinflammation
and Skaper SD (2005) Dopamine D2 and D3 in a transgenic mouse model of Alzheimer’s
receptor agonists limit oligodendrocyte injury disease. J Biol Chem 281, 23658–23667
caused by glutamate oxidative stress and oxygen/ 10. Skaper SD, Facci L, Culbert A, Evans NA,
glucose deprivation. Glia 52, 336–343 Chessell I, Davis JB, et al (2006) P2X7 recep-
3. Rosin C, Bates TE, and Skaper SD (2004) tors on microglial cells mediate injury to corti-
Excitatory amino acid induced oligodendro- cal neurons in vitro. Glia 54, 234–242
cyte cell death in vitro: receptor-dependent 11. Varon S, Skaper SD, Barbin G, Selak I, and
and-independent mechanisms. J Neurochem Manthorpe M (1984) Low molecular weight
90, 1173–1185 agents support survival of cultured neurons
4. Matute C, Alberdi E, Domercq M, Perez-Cerda from the central nervous system. J Neurosci 4,
F, Perez-Samartin A, and Sanchez-Gomez MV 654–658
(2001) The link between excitotoxic oligoden- 12. Shen LH, Li Y, and Chopp M (2010) Astrocytic
droglial death and demyelinating diseases. endogenous glial cell derived neurotrophic fac-
Trends Neurosci 24, 224–230 tor production is enhanced by bone marrow
5. Gallo V, and Ghiani CA (2000) Glutamate stromal cell transplantation in the ischemic
receptors in glia: new cells, new inputs and boundary zone after stroke in adult rats. Glia
new functions. Trends Pharmacol Sci 21, 58, 1074–1081
252–258 13. Skaper SD, Facci L, and Leon A (1995)
6. Skaper SD, Evans NA, Soden PE, Rosin C, Inflammatory mediator stimulation of astro-
Facci L, and Richardson JC (2009) Oligoden- cytes and meningeal fibroblasts induces neu-
drocytes are a novel source of amyloid peptide ronal degeneration via the nitridergic pathway.
generation. Neurochem Res 34, 2243–2250 J Neurochem 64, 266–276
(doi: 10.1007/s11064-009-0022-9) 14. Miller G (2005) The dark side of glia. Science
7. Trang T, Beggs S, Wan X, and Salter MW 308, 778–781
(2009) P2X4-receptor-mediated synthesis and 15. Facci L, Skaper SD, Favaron M, and Leon A
release of brain-derived neurotrophic factor (1988) A role for gangliosides in astroglial cell
in microglia is dependent on calcium and differentiation in vitro. J Cell Biol 106, 821–828
p38-mitogen-activated protein kinase activation. 16. Gabellini N, Facci L, Milani D, Negro A,
J Neurosci 29, 3518–3528 Callegaro L, Skaper S.D, et al (1991)
8. Zhang P, Hatter A, and Liu B (2007) Differences in induction of c-fos transcription
Manganese chloride stimulates rat microglia to by cholera toxin-derived cyclic AMP and Ca2+
release hydrogen peroxide. Toxicol Lett 173, signals in astrocytes and 3 T3 fibroblasts. Exp
88–100 Cell Res 194, 210–217
 7 Culture of Neonatal Rodent Microglia, Astrocytes, and Oligodendrocytes… 77

17. Hamby ME, Uliasz TF, Hewett SJ, and Hewett to the lysosomotropic agent, L-leucine methyl
JA (2006) Characterization of an improved ester. J Immunol 131, 2282–2290
procedure for the removal of microglia from 19. Giulian D, Vaca K, and Corpuz M (1993)
confluent monolayers of primary astrocytes. Brain glia release factors with opposing actions
J Neurosci Methods 150, 128–137 upon neuronal survival. J Neurosci 13, 29–37
18. Thiele DL, Kurosaka M, and Lipsky PE (1983) 20. Hewett JA, Hewett SJ, Winkler S, and Pfeiffer SE
Phenotype of the accessory cell necessary for (1999) Inducible nitric oxide synthase expression
mitogen-activated T and B cell responses in human in cultures enriched for mature oligodendrocytes
peripheral blood: delineation by its sensitivity is due to microglia. J Neurosci Res 56, 189–198
 Chapter 8

Central Nervous System Neuron-Glia Co-culture Models

Stephen D. Skaper and Laura Facci

Abstract
Glial cell activation plays an important role in the pathogenesis of various neurodegenerative disorders.
This article presents a protocol for the preparation of cultures consisting of rat embryonic cortical neurons
grown in the presence of cortical microglia, in which the glia are present in physical contact with the neu-
rons or separated by a semi-permeable membrane barrier.

Key words: Neurons, Microglia, Astrocytes, Cortex, Co-culture, Rat, Cell culture

1. Introduction

Our appreciation of inflammation’s role in neurodegenerative

disease pathogenesis has increased rapidly in recent years. Neuroin-
flammation in disorders such as Alzheimer’s disease has previously
been viewed as an epiphenomenon, with inflammation occurring
when damaged neurons provoke an activation response from glia.
Accumulating evidence, however, is now challenging this earlier
perspective and points to a more active role of neuroinflammation
in pathophysiology onset and progression. In the central nervous
system, glial cells (microglia, astroglia, and oligodendroglia) serve
supportive and nutritive roles for neurons; in the healthy brain,
they often respond to stress and insults by transiently upregulating
inflammatory processes. These processes are kept in check by other
endogenous anti-inflammatory and neuroprotective responses
that return the brain to homeostasis. Otherwise “normal” glial
functions can sometimes result in a more severe and chronic neu-
roinflammatory cycle that actually promote or propagate neurode-
generative disease (1, 2). The delicate balance in this homeostasis
can be disturbed, resulting in disease or exacerbation of initiating
factors that result in disease (i.e., the neuroinflammation hypothesis)

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_8, © Springer Science+Business Media, LLC 2012

79
80 S.D. Skaper and L. Facci

(3). A corollary of the neuroinflammation hypothesis is that sup-

pression of neurotoxic products by glial activation will result in
neuroprotection (4, 5). Indeed, activated microglia can cause neu-
ronal cell death in vitro (6–12), and inhibiting the former can limit
neuron injury (cf. (5, 10–12)). Therefore, controlling glial activa-
tion and, hence, inflammation is of great therapeutic interest in
mitigating neuronal cell death in different neurological disorders.
This chapter describes a protocol for preparing cultures of rat
embryonic cortical neurons grown in the presence of cortical
microglia, either as co-cultures in which the two cell populations
are in physical contact or with the use of a transwell insert system
whereby glia are cultured on a semi-permeable membrane in close
proximity to the underlying monolayer of neurons.

2. Materials

2.1. Equipment 1. Laminar flow biological safety cabinet (CL2).

and Labware 2. Humidified, water-jacketed culture incubator at 37°C and 5%
CO2/95% air.
3. Water bath set at 37°C.
4. Dissecting tools (Fine Science Tools are very high quality for
the price).
5. Bench centrifuge to accommodate 15- and 50-mL tubes.
6. Orbital shaker with enclosed, temperature-controlled chamber.
7. 10-cm ∅ sterile tissue culture dishes.
8. 15-mL and 50-mL polypropylene plastic centrifuge tubes
(sterile).
9. Sterilin 10-cm ∅ sterile petri plastic dishes (Bibby-Sarstedt).
10. Tissue culture flasks (T-75; 75 cm2) with canted neck and 0.2-μm
vented cap (Corning) or without vented cap (BD Falcon).
11. Cell scrapers, 25-cm handle, 1.8-cm blade (BD Falcon).
12. 0.22-μm filters (Millipore Corporation).
13. Cell culture inserts for 24-well plate, PET (polyethylene
terephthalate) transparent membrane, 0.4-μm pore size, BD
Falcon™ (BD Biosciences).
14. Tissue culture plate, 24-well plate, BD Falcon™. Tissue-
culture-treated polystyrene, flat bottom, with low-evaporation
lid (BD Biosciences).

2.2. Reagents 1. Hank’s balanced salt solution (HBSS), with CaCl2 and MgCl2
(Invitrogen).
2. Neurobasal medium (Invitrogen).
 8 Central Nervous System Neuron-Glia Co-culture Models 81

3. Phenol red-free Neurobasal medium (Invitrogen).

4. B27 supplement, 50×, with antioxidants (Invitrogen).
5. B27 supplement, 50×, without antioxidants (Invitrogen).
6. Dulbecco’s modified Eagle’s medium (DMEM), containing
4.5 g/L glucose, L-glutamine, and pyruvate (Invitrogen).
7. Fetal calf serum (FCS) (Invitrogen) (see Note 1).
8. L-Glutamine (200 mM stock), sterile, for cell culture
(Invitrogen).
9. Sodium pyruvate (100 mM stock), sterile, for cell culture
(Sigma).
10. Penicillin/streptomycin (100× stock), sterile, for cell culture
(Invitrogen).
11. Gentamicin (50 mg/mL stock), sterile, for cell culture
(Sigma).
12. Poly-D-lysine hydrobromide, γ-irradiated, cell culture–tested,
mol. wt. 70,000–150,000 (Sigma).
13. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide (MTT) (Sigma).
14. CytoTox® non-radioactive cytotoxicity assay kit for lactate
dehydrogenase (LDH) (Promega).

2.3. Culture Media 1. Culture medium for neurons : Neurobasal medium containing
and Solutions B27 supplement (1×), 2 mM L-glutamine, 100 U/mL penicillin,
and 100 μg/mL streptomycin: To 500 mL Neurobasal
medium, add 10 mL 50× B27 supplement, 5 mL 200 mM
glutamine, and 5 mL 10,000 U/ml penicillin to 10,000 μg/mL
streptomycin. Complete medium can be stored at +4°C for up
to 2 months.
2. Culture medium for microglia: To a 500-mL bottle of DMEM,
add 50 mL FCS (10% final), 5 mL stock penicillin/streptomy-
cin (100 U/mL penicillin + 100 μg/mL streptomycin final),
and 0.5 mL of gentamicin stock (50 μg/ml final).
3. 0.15 M borate buffer pH 8.4: Dissolve 28.6 g of sodium borate
(Na2B4O7·10H2O) in 500 mL water (pH will be ~9.2). Adjust
pH to 8.4 with 5 N HCl. Filter-sterilize and store at 4°C (up
to 6 months).

2.4. Preparation 1. Dissolve a 5 mL bottle of poly-D-lysine (MW 70,000–150,000)

of Poly-D-Lysine in 5 mL 0.15 M borate buffer (pH 8.4). This 1 mg/mL stock
Substratum solution can be stored at +4°C for up to 2 months (see Note 2).
2. Dilute the 1-mg/mL stock solution of poly-D-lysine 1:10 (1 in
9 mL sterile water) and add 0.5 mL to each well of a 48-well
plate or 1 mL to each well of a 24-well plate. Place the plate(s)
in the 5% CO2 incubator (37°C) overnight.
82 S.D. Skaper and L. Facci

3. The following day aspirate the poly-D-lysine coating solution

and leave the open plate(s) under the CL2 cabinet to dry.
The poly-D-lysine-coated plate(s) can be stored at +4°C for
1 month.

3. Methods

3.1. Neuron-Glia 1. Prepare primary cerebral cortical neuron cell cultures from
Co-cultures: Physical gestational day 18 fetuses of time-mated CD rats (Charles
Contact Model River) (see Chap. 5).
2. Re-suspend the final cell pellet in Neurobasal medium contain-
ing 2% B27 supplements, 1 mM sodium pyruvate, 2 mM
L-glutamine, and penicillin (100 units/mL)/streptomycin
(100 μg/mL).
3. Plate cells in poly-D-lysine-coated 48-well plates, 1 × 105 cells/
well in 0.35 mL of the same medium. Place cultures in a
humidified incubator at 37°C in a 5% CO2/95% air
atmosphere.
4. Expose the poly-D-lysine-coated culture wells overnight to
medium containing 10% FCS prior to cell seeding.
5. After 2 days, add to each culture well 0.35 mL of plating
medium, but containing B27 supplement without antioxidants.
Neuron cell cultures cells are normally used on day 6 for
experiments.
6. Culture microglia from the cortex of newborn CD (Charles
River) rat pups, as described in Chap. 7.
7. Collect microglia from the 10-cm ∅ Sterilin plates by mechan-
ically scraping into culture medium for glia (see Chap. 7).
8. Pellet microglia by centrifugation (200 × g for 5 min) and
re-suspend the cell pellet in Neurobasal medium containing
2% B27 supplements (without antioxidants), 1 mM sodium
pyruvate, 2 mM L-glutamine, and penicillin/streptomycin.
Use 1 mL medium for each Sterilin plate of microglia collected,
and re-suspend pellet using a long (9-in.) Pasteur pipette
attached to an automatic pipettor. Eight or nine up-and-down
strokes are usually sufficient to fully disperse the cell pellet.
Determine the cell density in the suspension by counting with a
hemocytometer (Sec Chap. 3).
9. To prepare the co-cultures, dilute the microglia cell suspension
to 285,000 cells/mL, using the same medium for suspension of
the starting cell pellet.
10. Take the 48-well plate of neurons set up 6 days ago and remove
0.35 mL of medium. Now add to each well 0.35 mL of the
 8 Central Nervous System Neuron-Glia Co-culture Models 83

microglia cell suspension, which gives a neuron to microglia

ratio of 1:1. Return the plate to the 5% CO2 incubator.
11. After incubation for 30 min, wash the plate with 1 mL of
Neurobasal/B27 (antioxidant-free) medium (point 8 above)
to remove non-adherent microglia and debris, then add to
each well 0.7 mL of the same medium. Return the plate to the
5% CO2 incubator; wait several hours before commencing
treatments.
12. Co-cultures are usually utilized the same day. Of course, the
treatment regimen will be determined by the experimental
plan one wishes to carry out. One example may be to treat
with an agent (lipopolysaccharide) which is known to stimulate
microglia to release neurotoxic factors, and then add potential
anti-inflammatory drugs to test for neuroprotection. However,
description of such material is beyond the scope of this chapter
(see Note 3).

3.2. Assessment 1. LDH release into the culture medium was used as a measure of
of Neurotoxicity cell death and has been utilized previously to quantify neuronal
in Physical Contact cell injury in microglia-neuron co-cultures (8, 9).
Neuron-Microglia 2. LDH activity in the cell culture supernatants is determined
Co-cultures after 72 h, using the CytoTox® non-radioactive cytotoxicity
assay kit.
3. Collect 100 μL of medium from each well of the co-cultures
for assay. Use 50 μL of this sample for assay, and proceed with
the assay as described in Chap. 6 (see Note 4).
4. Express values of LDH release as milliunits/mL (see Notes 5
and 6).
5. Establish separate cultures of microglia without neurons in
poly-D-lysine-coated 48-well plates (100,000 cells/well) in
0.7 mL Neurobasal/B27 (antioxidant-free) at the time micro-
glia are added to the neuron cultures. Incubate these microglia
cultures for 72 h in the presence of stimuli, and then measure
LDH release from the microglia alone ± stimuli.
6. Subtract the microglia only LDH values obtained from the LDH
values obtained for the neuron-microglia co-cultures receiving
the same treatments (see Note 7). The purpose of this step is to
correct for the contribution of microglia LDH release.

3.3. Neuron-Glia 1. This model allows one to culture glia on a porous membrane,
Co-cultures: Transwell which is then placed in a well containing an established mono-
Insert Model layer of neurons. Application of a stimulus to the glia layer
allows for glia-derived agents to diffuse across the membrane
and act on the underlying neurons. The insert with glia can
then be removed, permitting morphological or biochemical
analysis of the neurons. Figure 1 shows a typical commercially
84 S.D. Skaper and L. Facci

Fig. 1. A typical cell culture insert chamber.

Transwell insert co-culture model

Transwell Insert

Microglial Cells

Neurons

Fig. 2. Schematic representation of the transwell insert neuron-glia co-culture model.

available insert chamber, and Fig. 2 illustrates schematically

the principal of this co-culture model.
2. Prepare primary cerebral cortical neuron cell cultures from
gestational day 18 fetuses of time-mated CD rats (Charles
River) (see Chap. 5).
3. Re-suspend the final cell pellet in Neurobasal medium containing
2% B27 supplements, 1 mM sodium pyruvate, 2 mM L-glutamine,
and penicillin (100 units/mL)/streptomycin (100 μg/mL).
 8 Central Nervous System Neuron-Glia Co-culture Models 85

4. Plate cells in poly-D-lysine-coated 24-well plates, 2 × 105 cells/

well in 0.5 mL of the same medium. Place cultures in a humid-
ified incubator at 37°C in a 5% CO2/95% air atmosphere.
5. Expose the poly-D-lysine-coated culture wells overnight to
medium containing 10% FCS prior to cell seeding.
6. After 2 days, add to each culture well 0.5 mL of plating
medium, but containing B27 supplement without antioxidants.
Neuron cell cultures cells are normally used on day 6 for
experiments.
7. Culture microglia from the cortex of newborn CD (Charles
River) rat pups (see Chap. 7).
8. Collect microglia from the Sterilin plates by mechanically
scraping into culture medium for microglia. This should be
done on the day prior to intended transfer of inserts to wells
containing neurons (the neuron cultures will be 5 days old at
this point) (see Chap. 5).
9. Pellet microglia by centrifugation (200 × g, 5 min) and re-suspend
cell pellet in culture medium for microglia. Use 1 mL medium
for each Sterilin plate of microglia collected, and re-suspend
pellet using a 9-in. Pasteur pipette. Determine cell density of
the suspension (see Chap. 3).
10. To prepare the co-cultures, dilute the microglia cell suspension
to 187,500 cells/mL, using the same medium for suspension
of the starting cell pellet.
11. To an empty 24-well plate, add 0.8 mL/well of culture medium
for microglia.
12. Using flame-sterilized forceps, transfer an empty cell culture
insert to each well of the above 24-well plate.
13. Add to each insert 0.4 mL of microglia suspension containing
75,000 cells.
14. The following day, wash the inserts containing microglia once
with 0.5 mL of Neurobasal medium containing 2% B27 supple-
ments (without antioxidants), 1 mM sodium pyruvate, 2 mM
L-glutamine, and penicillin/streptomycin.

15. Transfer the inserts to the 24-well plate of cortical neurons (set
up 6 days ago), without changing the medium in the well.
The porous membrane allows free diffusion of molecules.
The distance between the neuron monolayer and microglia
on the insert membrane is 1 mm. Return the plate to the 5%
CO2 incubator.
16. The insert co-cultures are now ready for use. The treatment
regimen will, of course, be determined by the experimental
plan one wishes to carry out. For example, one can treat the
upper (microglia-containing) chamber with amyloid β-peptide,
using a concentration sufficient to stimulate microglia without
causing injury to the underlying neurons (10), in the presence
86 S.D. Skaper and L. Facci

or absence of potential anti-inflammatory drugs to test for

neuroprotection. Generally, we terminate the experiment 3 days
after addition of the microglia stimulus (see Note 8).

3.4. Assessment 1. At the end of the incubation period, remove the microglia-
of Neurotoxicity containing inserts and evaluate neuronal cell viability by the
in Transwell Insert colorimetric method based upon conversion of 3-(4,5-dimeth-
Co-cultures ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by
mitochondrial dehydrogenases to a blue formazan product.
2. Add to each well 400 μL of phenol red-free Neurobasal
medium containing 2% B27 supplements (without antioxidants),
1 mM sodium pyruvate, 2 mM L-glutamine, and penicillin/
streptomycin, plus 0.15 mg/mL MTT (which represents a 1:20
dilution of the 3 mg/mL stock). We find that this MTT concen-
tration gives optimal color yield (under these conditions).
3. Incubate the MTT-containing cultures for 1 h at 37°C. This
incubation time gives optimal color yield (under these
conditions).
4. Examination of the cultures under a light microscope will show
that viable cells contain blue crystals. This is the formazan
reaction product.
5. Remove the culture medium by aspiration, and add 400 μL of
dimethyl sulfoxide per well. This is to dissolve the reaction
product. Gently rock the plate to mix the blue reaction product
uniformly with the solvent. This should take only 1–2 min.
6. Transfer 100 μL of the colored dimethyl sulfoxide solution
from each well to the well of a clear polystyrene plastic 96-well
microplate (any brand will do). Read the plate on a microplate
(ELISA) reader. We use a SpectraMax M2 microplate reader
from Molecular Devices, although there are a number of qual-
ity plate readers on the market. For optimal results, the plate
should be read at a wavelength of 570 nm (test wavelength),
followed by 630 nm (reference wavelength). The difference
(A570−A630) is used as the final absorbance value generated by
the sample. Correcting for optical imperfections in the
microplates by subtracting A630 is recommended but is not an
essential procedure.
7. Normalize the absolute MTT values by scaling to the mean of
cultures which have not been treated with test compound
(defined as 100%).
8. Cell vitality should be routinely confirmed by morphological
observation using a light microscope with phase contrast optics
(see Note 9).
 8 Central Nervous System Neuron-Glia Co-culture Models 87

4. Notes

1. Heat inactivation of fetal calf serum is recommended to destroy

heat-labile complement. Thaw the bottle of serum in advance,
using a 37°C water bath. Next, immerse the serum bottle in
the water bath after re-equilibrating to 56°C and leave for
30 min. Swirl the bottle occasionally to ensure proper mixing.
Allow the serum to cool to room temperature, aliquot into
50-mL tubes and store at −20°C.
2. The molecular weight of the poly-D-lysine used is critical.
Optimal culture of neurons on poly-D-lysine is a function of
cell type and should be determined by the experimenter. We
find that the poly-D-lysine used here (Sigma P-6407) works
best for cortical, hippocampal, and striatal neuron cell cultures.
A molecular weight lower or higher than this will result in cell
clumping, which is most undesirable.
3. Neuron-microglia co-cultures (“physical contact”) as described
are not limited to the use of rat cortical neurons but can also
be carried out using hippocampal neurons, or cortical, or
hippocampal neurons prepared from mouse embryonic tissues.
In addition, one can make use of microglia derived from mouse,
rather than rat cortex. These variations can be of particular utility
if one wishes to examine the influence of gene target deletion.
We find that this system performs equally well even when using
“heterogeneous” cell populations, for example, co-culturing rat
cortical neurons and mouse microglia, or vice versa.
4. As a starting point, we recommend using 50 μL of culture
medium for the LDH assay. If the value obtained is outside the
standard curve, dilute the culture medium sample twofold in
culture medium and repeat the assay.
5. Values of absolute LDH release will most likely vary from
experiment to experiment, even for control (untreated) cul-
tures. This is not unexpected, given that microglia in culture
are never in a truly “resting” state. Their tonic level of activa-
tion will influence the absolute LDH values obtained. We find
that data can be normalized to achieve a reasonable consis-
tently across experiments by expressing values as a percent of
the control value for each individual trial.
6. Our historical experience in using these neuron-microglia
co-cultures is that in a minority of preparations, (about 1 in
10) the microglia are in a “highly” activated state per se, and
the neurons will degenerate in the presence of the microglia
without addition of any stimulus. We recommend observing
the co-cultures microscopically for appearance of the neurons
prior to commencing treatments; neuronal cell injury (if present)
will be evident morphologically by disintegration of the neurite
network and cell bodies.
88 S.D. Skaper and L. Facci

7. The purpose of this step is to correct for the contribution of

microglia LDH release. In our experience, most experimental
treatments (e.g., lipopolysaccharide, anti-inflammatory drugs)
have little or no influence on this LDH release. However, it is
recommended to do in every case, as the data thus generated
will represent a more rigorous analysis. This procedure will
double the number of microglia cells needed for an experi-
ment. One can also perform the same using neuron-only cul-
tures. In our experience, this is not necessary as LDH release
from neurons in the absence of microglia is virtually below the
limit of detection.
8. Determination of the optimal time of exposure of neurons to
the injury-inducing effects of activated microglia is empirical.
Using, for example, lipopolysaccharide to stimulate the microglia,
degeneration of the underlying neurons is largely complete by
3 days. With incubation times of less than 2 days, neuronal cell
injury in our hands was not evident.
9. A limitation of the insert system is the following: The “foot-
print” of the culture insert is smaller than the area of the under-
lying well (approximately 50%). When microscopically
examining the neuron monolayer after removing the micro-
glia-containing insert, one will notice that damage to neurons
(degeneration of neurite network and cell bodies) tends to be
limited to the area immediately under the insert. Possibly, the
activated microglia release neurotoxic agents with a limited
diffusion or lifetime, although we have not explored this. One
thus needs to keep in mind that the measure of neuronal cell
viability loss obtained is an underestimate, as only those cells in
the vicinity of the insert will “register” with the MTT assay.

Acknowledgments

L. Facci was supported by Fondazione CARIPARO Progetto

Dottorati di Ricerca’ Anno 2009.

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13309–13317 12676–12689
 Chapter 9

Culture and Characterization of Rat Mesencephalic

Dopaminergic Neurons
Stephen D. Skaper, Giulia Mercanti, and Laura Facci

Abstract
Dopaminergic neuronal cell degeneration is the principal characteristic feature of the neuropathology of
Parkinson’s disease. Cultures of mesencephalic neurons are widely used as a source of dopaminergic neu-
rons for the study of mechanisms implicated in dopaminergic cell death and for the evaluation of potential
dopaminergic neuroprotective agents, including neurotrophic factors. This chapter presents a detailed
protocol for the preparation of rat mesencephalic cell cultures and their application to evaluating the neu-
roprotective action of brain-derived neurotrophic factor.

Key words: Mesencephalic, Ventral tegmentum, Rat, Brain-derived neurotrophic factor, Dopamine
uptake, Tyrosine hydroxylase, Parkinson’s disease

1. Introduction

Parkinson’s disease is the most common neurodegenerative move-

ment disorder, whose neuropathological hallmarks are character-
ized by progressive and profound loss of neuromelanin-containing
dopaminergic neurons in the substantia nigra pars compacta with
the presence of eosinophilic intracytoplasmic, proteinaceous inclu-
sions termed Lewy bodies and dystrophic Lewy neurites in surviv-
ing neurons (1, 2). Brain-derived neurotrophic factor (BDNF)
enhances the survival of and differentiation of dopaminergic neu-
rons in vitro (3, 4), and the intranigral transplantation of epigeneti-
cally induced BDNF-secreting human mesenchymal stem cells in a
dopamine neurotoxin model in rats rescued some neurological defi-
cits (5). In addition, BDNF is neuroprotective against a number of
insults (6–8). Mesencephalic dopaminergic neurons in vitro respond
also to basic fibroblast growth factor (9) and their striatal target
cells (10). This chapter describes in detail a protocol for culturing

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_9, © Springer Science+Business Media, LLC 2012

91
92 S.D. Skaper et al.

rat embryonic mesencephalic cells and identifying the dopaminergic

neuron component. The response of the dopamine cells to BDNF
will also be presented, to demonstrate an application of these
cultures.

2. Materials

2.1. Equipment 1. Stereo dissecting microscope (backlighting of stage is pre-

and Labware ferred) with fiber optic light source.
2. Laminar flow cabinet for dissections.
3. Laminar flow biological safety cabinet (CL2).
4. Humidified, water-jacketed culture incubator at 37°C and 5%
CO2/95% air.
5. Water bath set at 37°C.
6. Dissecting tools (Fine Science Tools (InterFocus Ltd.) are very
high quality for the price).
7. Bench centrifuge to accommodate 15- and 50-mL tubes.
8. 10-cm ∅ sterile tissue culture dishes (any supplier).
9. Glass coverslips (12 mm ∅ #1, Menzel-Gläser, Menzel GmbH,
Braunschweig, Germany) (see Note 1).
10. 15- and 50-mL polypropylene plastic centrifuge tubes (sterile).
11. 0.22-μm filters (Millipore).

2.2. Culture Media, 1. Hank’s balanced salt solution (HBSS, with CaCl2 and MgCl2)
Supplements, (Invitrogen).
and Additives 2. Dulbecco’s modified Eagle’s medium, high glucose with
GlutaMAX I, and sodium pyruvate (Invitrogen).
3. Neurobasal medium (Invitrogen).
4. B27 supplement (with antioxidants, 50×) (Invitrogen).
5. B27 supplement (without antioxidants, 50×) (Invitrogen).
6. N2 supplement (100×) (Invitrogen).
7. Fetal calf serum (Invitrogen).
8. L-Glutamine (200 mM, sterile, for cell culture) (Invitrogen).
9. Non-essential amino acids (100×, sterile) (Invitrogen).
10. Penicillin/streptomycin (100×, sterile) (Invitrogen).
11. Poly-D-lysine (MW 30,000–70,000, sterile, for cell culture)
(Sigma).
12. [3H]Dopamine (DA) (Amersham Biosciences).
13. [γ-14C]Aminobutyric (GABA) (Amersham Biosciences).
 9 Culture and Characterization of Rat Mesencephalic Dopaminergic Neurons 93

14. Mazindol (Sigma).

15. L-2,4-Diaminobutyric acid·2HCl (Sigma).
16. Anti–tyrosine hydroxylase (TH) rabbit polyclonal antibody
(Abcam).
17. Donkey polyclonal to rabbit IgG-H&L (Rhodamine) (Abcam).
18. 2,4,5-Trihydroxyphenylalanine (TOPA) (Sigma).
19. BDNF (human, recombinant) (Peprotech).

2.3. Culture Media 1. Medium 1: To 500 mL of Neurobasal medium, add B27

and Other Solutions (10 mL of a 50×, containing antioxidants), 25 μM L-glutamate
final (0.5 mL of a 1,000× stock in H2O), 0.5 mM L-glutamine
final (1.25 mL of a 200 mM stock), 1 mM sodium pyruvate final
(5 mL of a 100 mM stock), and 100 U/mL penicillin/100 μg/mL
streptomycin final (5 mL of a 100× stock). Store at 4°C for
1 month.
2. Medium 2: To 500 mL of Neurobasal medium, add B27
(10 mL of a 50×, without antioxidants), 0.5 mM L-glutamine
final (1.25 mL of a 200 mM stock), 1 mM sodium pyruvate final
(5 mL of a 100 mM stock), and 100 U/mL penicillin/100 μg/mL
streptomycin final (5 mL of a 100× stock). Store at 4°C for
1 month (see Note 2).
3. Medium 3: To 500 mL of Dulbecco’s modified medium high
glucose with GlutaMAX I and sodium pyruvate, add 5% (v/v)
fetal calf serum final, 1× non-essential amino acids (5 mL of a
100× stock), and 50 U/mL penicillin/50 μg/mL streptomy-
cin final (2.5 mL of a 100× stock). Store at 4°C for 1 month
(see Note 3).
4. Medium 4: To 500 mL of Hank’s balanced salt solution (with
CaCl2 and MgCl2), add 1 mM sodium pyruvate final (5 mL of
a 100 mM stock), 10 mM HEPES final (5 mL of a 1 M stock),
0.035% NaHCO3 final (2.3 mL of a 7.5% stock), and 100 U/mL
penicillin/100 μg/mL streptomycin final (5 mL of a 100× stock).
Can be stored for 3 months at 4°C.
5. Krebs-Ringer buffer (pH 7.4): 16 mM NaH2PO4⋅H2O (2.20 g/L),
16 mM Na2HPO4, (2.27 g/L), 119 mM NaCl (6.95 g/L),
0.35 g 4.7 mM KCl (0.35 g/L), 0.26 g 1.8 mM CaCl2⋅2H2O
(0.26 g/L), 1.2 mM MgSO4 (0.14 g/L), mM EDTA·Na2⋅2H2O
(0.48 g/L), and 5.6 mM D-glucose (1.00 g/L).
6. Borate buffer (pH 8.4): Dissolve 28.6 g of sodium borate
(Na2B4O7⋅10H2O) in 500 mL water (pH will be ~9.2). Adjust
pH to 8.4 with 5 N HCl. This gives a concentration of 0.15 M.
Filter-sterilize and store at 4°C (up to 6 months).
94 S.D. Skaper et al.

3. Methods

3.1. Isolation 1. Euthanize pregnant rats (CD strain, Sprague Dawley) following
of Mesencephalic appropriate national and institutional guidelines. Under asep-
Cells tic conditions, remove the whole brain from the rat embryos
(gestational age 14–15 days) and collect in a petri dish (10 cm
∅) containing ice-cold Medium 4. Remove the blood vessels
and meninges.
2. After having collected all brains, dissect out the ventral two-
thirds of the mesencephalon. The dissected regions include
dopaminergic neurons in the substantia nigra and the ventral
tegmental area but not the noradrenergic neurons in the locus
ceruleus.
3. When all cortices have been dissected, mechanically dissociate
the tissue in 2 mL of Neurobasal medium containing 10%
(v/v) fetal calf serum, using a series of three flame-polished
Pasteur pipettes of decreasing bore size (see Notes 4 and 5).
4. Count the cell suspension (see Chapter 3). A typical yield is
106 viable cells per embryo.
5. Use a poly-L-lysine substratum for culture. The method of
preparation follows that described in Chapter 3 (see Note 6).
6. Centrifuge the cell suspension (200 × g, 5 min), remove the
supernatant, and resuspend the cell pellet in prewarmed (37°C)
Medium 1. Plate at 1,100–2,500 cells/mm2.
7. After 5 days in culture, replace 1/2 of the plating medium with
Medium 2 (this may not be necessary—check pH).
8. Replace 1/2 of the culture medium twice a week with Medium
2, until the cultures are used.

3.2. Mesencephalic 1. Follow the procedure detailed in Subheading 3.1 up to and

Neuron-Glia Cultures including step 5. Resuspend the cell pellet in Medium 3, count
for cell number, and adjust cell density 1 × 106 cells/mL (see
Note 7).
2. Plate cells on poly-L-lysine-coated tissue culture plastic surfaces
at a density of 2.5 × 105 cells/cm2.
3. Change culture medium every second day.
4. During the course of treatment, the culture medium is not
supplemented with amino acids to avoid the cytotoxicity of
glutamate.
5. The composition for major cell types in the culture can be esti-
mated by visual counting of immunostained cells, using indi-
rect immunofluorescence (see Chapter 20 for protocol).
Seven-day-old cultures will “typically” contain the following cel-
lular composition: ~45% astrocytes (glial fibrillary acidic protein+,
 9 Culture and Characterization of Rat Mesencephalic Dopaminergic Neurons 95

1:400, Sigma), ~40% neurons (βIII-tubulin+, 1:200, Upstate),

~1% dopaminergic neurons (tyrosine hydroxylase+, 1:200,
Chemicon), and the remainder being microglia (MAC-1+,
1:200, Serotec). Information in parentheses refers to primary
antibody, suggested dilution, and source. This culture compo-
sition is consistent with that described by others (11, 12).

3.3. High-Affinity 1. Initially seed mesencephalic cells (either as neuron-enriched

[3H]DA Uptake cultures or neuron-glia cultures) in poly-L-lysine-coated
24-well plates, 5 × 105 cells per well.
2. At the desired culture age (days in vitro; will depend on the
experiment being performed), wash the cells twice with 1 mL
of Krebs-Ringer buffer.
3. Incubate each culture well with 1 μCi (~50 nM) [3H]DA in
Krebs-Ringer buffer (0.4 mL/well) for 15 min at 37°C.
4. Determine nonspecific uptake of DA in parallel wells receiving
both (3H)DA and 10 μM mazindol (1:1,000, from a 10 mM
stock in H2O), an inhibitor of neuron DA uptake (see Notes 8
and 9).
5. Wash the cells 3 times with ice-cold Krebs-Ringer buffer
(1 mL/well).
6. Obtain blank values by incubating cells under the same condi-
tions at 0°C.
7. Lyse the cells with 1 N NaOH (0.5 mL/well).
8. Mix the lysate with 3 mL of scintillation fluid (there are a number
of brands on the market which accommodate aqueous samples).
9. Determine radioactivity with a liquid scintillation counter.
10. Calculate the specific DA uptake by subtracting the amount of
radioactivity observed in the presence of mazindol from that
observed in the absence of mazindol.

3.4. High-Affinity [14C] 1. Incubate mesencephalic cells at 37°C for 15 min with 0.1 μM
GABA Uptake [14C]GABA in Krebs-Ringer buffer in the presence of 2 mM
β-alanine (1:250 from a 0.5 M stock solution in H2O), an
inhibitor of glial high-affinity GABA uptake.
2. Verify high-affinity neuron-specific (14C)GABA uptake using
the inhibitor L-2,4-diaminobutyric acid (1 mM final, 1:500
from a 0.5 M stock in H2O).
3. Washing and extraction procedures are as for (3H)DA uptake.

3.5. Catecholamine 1. Dissociated mesencephalic neurons can be analyzed by cate-

Cytofluorescence cholamine fluorescence using a glyoxylic acid technique.
2. Grow cells on 12-mm ∅ glass coverslips previously coated with
poly-L-lysine (or poly-L-ornithine), the coverslips having been
96 S.D. Skaper et al.

placed within the individual wells of a 24-well plate. Plating

density is the same as for (3H)DA uptake measurement.
3. At the desired culture age, rinse the cells once with ice-cold
Krebs-Ringer buffer (1 mL/well).
4. Incubate cells with ice-cold 2% glyoxylic acid solution in Krebs-
Ringer buffer (pH 7.4) for 5 min (see Note 10).
5. As an index of dopaminergic specificity, parallel cultures can be
preincubated for 15 min with 10 μM mazindol, prior to fixa-
tion with glyoxylic acid.
6. Wash twice with Krebs-Ringer buffer to remove excess glyoxy-
lic acid.
7. Dry the coverslips for 10 min under warm air.
8. Heat coverslips for 10 min at 95°C.
9. Mount coverslips on glass slides with liquid paraffin.
10. View under a fluorescence microscope equipped for cate-
cholamine epifluorescence and phase-contrast optics.
11. Using prefixed coordinates, count the number of glyoxylic
acid–positive neurons corresponding to at least 3% of the total
surface area.

3.6. BDNF In the case of Parkinson’s disease, it is intriguing that a substance

as a Neuroprotectant closely related to the therapeutic agent 3,4-dihydroxydopamine
for Mesencephalic (L-DOPA) acts as an excitatory agent. The 6-hydroxylated deriva-
Dopaminergic Neurons tive of L-DOPA (2,4,5-trihydroxyphenylalanine or TOPA) in
solution elicits both electrophysiological responses and neurotox-
icity when applied to central nervous system neurons (13, 14),
including mesencephalic-derived dopaminergic neurons (15).
This paradigm can be utilized to show the neuroprotective action
of BDNF:
1. Seed mesencephalic cell dissociates into poly-L-lysine-coated
24-well plates, without (for (3H)DA and (14C)GABA uptake)
or with 13-mm ∅ glass coverslips previously coated with poly-
L-lysine (for TH immunostaining). The plating density is
5 × 105 cells/well in all cases. Use culture medium as described
in Subheading 3.1.
2. The day after cell plating, add BDNF to the cultures (1–50
ng/mL final) (see Note 11).
3. At the seventh day in culture, add TOPA to a final concentra-
tion of 10 μM (1:200 from a 2 mM stock—see Note 12).
TOPA has LD50 values of 3.3 ± 0.5 and 3.8 ± 0.6 μM, respec-
tively, for general cell viability (MTT oxidation) and dopamin-
ergic cell survival (TH immunostaining) (15).
9 Culture and Characterization of Rat Mesencephalic Dopaminergic Neurons 97

Fig. 1. Rat mesencephalic dopaminergic neurons at 7 days in culture, immunostained for

tyrosine hydroxylase.

4. On the eighth day in culture (i.e., 24 h after TOPA addition),

process the cultures for dopaminergic neuron survival, using
tyrosine hydroxylase (TH) immunostaining and neurotrans-
mitter uptake.
5. For TH immunostaining, drain cells of medium and fix by add-
ing 1 mL cold (−20°C) methanol per well for 6 min.
6. Incubate with anti-TH antibody (60 min, 37°C) diluted
1:1,000 in PBS (pH 7.4) containing 0.5% BSA.
7. Wash twice with PBS (1 mL/well).
8. Stain with rhodamine-conjugated donkey anti-rabbit second-
ary antibodies (1:200 in PBS) for 30 min at 37°C.
9. Mount cover glasses as described in Chapter 20.
10. For observation, a photomicroscope equipped with rhodamine
epifluorescence filters and phase-contrast optics will be required.
Using prefixed coordinates, the number of TH+ neurons,
corresponding to at least 10% of the total surface area of the
cover glass, should be counted. Only immunoreactive cells having
round, smooth bodies and intact, non-fragmented neurites
are scored as positive (see Fig. 1).
11. Figure 2 and Table 1 illustrate the results of a typical experi-
ment, using this model and the described assay procedures.
98 S.D. Skaper et al.

6000 **

TH+ Cell Number (Cells per dish)

5000 **

4000

3000

*
2000

1000

0
Ctrl BDNF TOPA TOPA+ TOPA+ TOPA+ TOPA+
BDNF BDNF BDNF BDNF
(1) (3) (10) (50)

Fig. 2. BDNF protects cultured mesencephalic dopaminergic neurons from the neurotoxic
effects of TOPA. After 24 h in vitro, cultures received BDNF (1–50 ng/mL). Cultures were
kept until seven DIV, before being exposed to 10 μM TOPA for 24 h. At the end of the
experiment (8 days in total), all cover glasses were processed for TH immunoreactivity,
and the number of TH+ cells was determined in each group (mean ± SD, n = 6, 3 experi-
ments). Values in parentheses indicate BDNF concentration (ng/mL). *P < 0.05 or **P < 0.01
vs. TOPA. The “TOPA + BDNF (10)” and “TOPA + BDNF (50)” groups were not significantly
different from each other or from the no-treatment group (CTRL). Reprinted with permis-
sion from Wiley-Blackwell (15).

Table 1
Effect of BDNF on [3H]dopamine and [14C]GABA uptake
in control and TOPA-treated mesencephalic cells

Treatment Dopamine (pmol/well) GABA (nmol/well)

5 None 50 ± 9 76 ± 11
BDNF 63 ± 14 86 ± 18
TOPA 6.9 ± 2.6* 9.3 ± 3.4*
BDNF/TOPA 43 ± 7** 15 ± 4
Mesencephalic cells were plated (5 × 105) in 25 mm diameter cluster wells (24-well
plate) and treated with 50 ng/mL BDNF on day 1. At 7 days, TOPA, where indicated,
was added to a final concentration of 10 μM. Twenty-four hours later, specific uptake
of [3H]dopamine and [14C]GABA was determined
*P < 0.01 vs. control (“none”) or BDNF-treated cultures; **P < 0.01 vs. TOPA-treated
cultures (n = 6). Reprinted with permission from Wiley-Blackwell (15)
 9 Culture and Characterization of Rat Mesencephalic Dopaminergic Neurons 99

4. Notes

1. The source of glass is very important for the coverslips: German

suppliers are highly recommended, in particular the one listed
here.
2. Neurobasal medium/B27 medium yields cultures that contain
relatively few glial cells. Should a mixed neuron/glial cell cul-
ture be desired (approximately 30% glia), replace B27 supple-
ment with N2 supplement of 10% fetal calf serum, and increase
L-glutamine to 3 mM.

3. Heat inactivation of fetal calf serum is recommended to destroy

heat-labile complement. Thaw the bottle of serum in advance,
using a 37°C water bath. Next, immerse the serum bottle in
the water bath after re-equilibrating to 56°C and leave for
30 min. Swirl the bottle occasionally to ensure proper mixing.
Allow the serum to cool to room temperature, aliquot into
50-mL tubes, and store at −20°C.
4. When mechanically dissociating the mesencephalic tissue, it is
important to use medium with protein (e.g., fetal calf serum).
Alternatively, one can use Neurobasal medium with 1% (w/v)
bovine serum albumin. The presence of protein protects cells
from damage caused by trituration.
5. It is preferable to use a long (9 in.) Pasteur pipette for dissocia-
tion, rather than a shorter (e.g., 6 in.) pipette. The dissociation
of tissue occurs mainly as a result of the shear force within the
narrow bore (lower) part of the pipette, and the longer pipette
facilitates the process.
6. Poly-L-ornithine (30,000–70,000 MW) can be substituted for
poly-L-lysine.
7. In some cases, one may find that fetal calf serum is cytotoxic
for neurons. It is advisable to test several batches of serum
from different suppliers before initiating a series of experiments
making use of neuron-glia cultures.
8. Benztropine (0.5 μM) can also be used to specifically block
(3H)DA uptake.
9. As additional controls, (3H)DA uptake can be assessed in the
presence of either desmethylimipramine (0.5 μM) or fluox-
etine (1 μM), drugs which block the transport of catecholamines
in noradrenergic terminals and amines in serotoninergic nerve
terminals, respectively.
10. Alternatively, cultures can be fixed in 2% glyoxylic acid/0.5%
paraformaldehyde in Krebs-Ringer bicarbonate buffer (pH 7)
for 15 min.
100 S.D. Skaper et al.

11. BDNF is supplied as a solid. When reconstituting, it is impor-

tant to use a solution with carrier protein, for example, PBS
with 0.1% BSA. Neurotrophins (NGF, BDNF, NT-3, NT-4)
have a very high isoelectric point (which means they are quite
basic) and will stick to glass and plastic (especially polystyrene)
surfaces. We recommend reconstitution in the container pro-
vided by the supplier to a concentration between 25 and
100 μg/mL. Aliquot this stock into small Eppendorf tubes
(10–20 μL/tube) and store at −20°C for up to 6 months. Once
thawed, an aliquot can be safely kept in the refrigerator for up
to 4 weeks. Do not subject BDNF to repeated freeze-thaw cycles.
12. When preparing solutions of TOPA, the following are recom-
mended: (1) in 1 N HCl, up to 50 mg/mL. Solutions should
be freshly prepared and protected from exposure to light.
(2) In water, up to 3 mg/mL. Dissolve in oxygen-free boiled
water containing 0.1% sodium metabisulfite or other antioxi-
dant. Solutions should be freshly prepared and protected from
exposure to light.

Acknowledgments

L. Facci was supported by “Fondazione CARIPARO Progetto

Dottorati di Ricerca” Anno 2009.

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 Chapter 10

Preparation of Adult Spinal Cord Motor Neuron Cultures

Under Serum-Free Conditions
Jose V. Montoya-Gacharna, Jhon Jairo Sutachan, Wai Si Chan,
Alexandra Sideris, Thomas J.J. Blanck, and Esperanza Recio-Pinto

Abstract
Spinal cord motor neuron cultures are an important tool for the study of mechanisms involved in motor
neuron survival, degeneration and regeneration, volatile anesthetic-induced immobility, motor neuron
disorders such as amyotrophic lateral sclerosis or spinal muscular atrophy as well as in spinal cord injury.
Embryonic spinal cord motor neurons derived from rats have been successfully cultured; unfortunately,
the culture of adult motor neurons has been problematic due to their short-term survival. Recently, by
using a cocktail of target-derived factors, neurotrophins (brain-derived neurotrophic factor and glial cell
line-derived neurotrophic factor) and a permeable cyclic adenosine monophosphate analog, we have established
a reproducible protocol for long-term cultures of healthy and functional adult motor neurons (Exp Neurol
220:303–315, 2009). Here, we now describe in detail the steps that we used for the optimization of the
process of isolation and maintenance of adult rat ventral horn motor neurons in vitro.

Key words: Motor neurons, Spinal cord, Brain-derived neurotrophic factor, Glial cell line-derived
neurotrophic factor, Cyclic AMP, Conditioned medium, Adult rat

1. Introduction

Embryonic spinal cord motor neuron cultures have been a very

important research tool that has provided insights on the mechanisms
involved in their physiology (1–6) and in several motor neuron
diseases such as amyotrophic lateral sclerosis (6–8). However, the
biology of embryonic motor neurons differs from their adult
counterpart. Several physiological changes occur in motor neurons
after birth (9–13) making an in vitro model of adult spinal cord
motor neurons desirable. For a long time, it has been known that
embryonic spinal cord motor neurons (SCMNs) require target-
derived factors to survive in vitro (14–25). Interestingly, we recently
found that adult rat spinal cord motor neurons survive and become

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_10, © Springer Science+Business Media, LLC 2012

103
104 J.V. Montoya-Gacharna et al.

electrically functional in the presence of soluble muscle-derived

factors but also require the presence of two neurotrophins, brain-
derived neurotrophic factor (BDNF) and glial cell line-derived
neurotrophic factor (GDNF), and a cell-permeable cyclic adenos-
ine monophosphate analog (cAMP) (26). Adult mouse motor
neurons can also survive for 24 h in the presence of soluble muscle-
derived factors (25) and for longer times with a mixture of
muscle-conditioned medium, BDNF, GDNF, ciliary neurotrophic
factor, and cAMP (data not published). This supports the view that
mature SCMNs are strongly dependent on their interactions with
neurotrophins, cAMP, and soluble factors from their target tissue.
A similar trophic dependence of mature SCMNs has also been
described by Das et al. (27, 28). Adult rat SCMNs survive in the
presence of a complex cocktail of neurotrophins (27) and they reach
electrical functionality in the presence of neurotransmitters (28).
Here, we describe a practical and detailed protocol for the isola-
tion and maintenance of adult SCMNs derived from rats (Figs. 1
and 2). This protocol requires rapid isolation and dissection of the
spinal cord at low temperature, separation of myelin and purification
of the neuronal cell fraction with a density gradient according to
Brewer and Torricelli (29) (Fig. 2), cell attachment on poly-D-
lysine (PDL) substrate, and maintenance with a serum-free
neuronal culture medium (Neurobasal-A with B-27 supplement)
containing myocyte-conditioned medium generated from cultur-
ing C2C12 cells (mouse myoblast cell line), BDNF, GDNF, and
cAMP. The adult SCMNs obtained using this protocol (Fig. 3)
express proteins and transcription factors selective to SCMNs such
as the non-phosphorylated neurofilament H, the transcription
factors HB9 and Islet-1, and the enzyme choline acetyltransferase
responsible for synthesis of the neurotransmitter acetylcholine
(26). These SCMNs also express the major synaptic protein
synaptophysin (Fig. 4) (data not published) that has been involved
in regulating activity-dependent synapse formation (30). Cultured
SCMNs are also electrically active: They express outward K+ currents
as early as 1 h after plating, and, fast and slow inward currents become
detectable after 1 week in culture (26). Action potentials can also
be evoked (26). Calcium transients have been detected after 1 week
of culture (Fig. 5) (data not published) using calcium imaging
techniques. This in vitro system provides rapid and efficient
serum-free cultures of adult SCMNs and hence represents a
valuable tool for future studies on motor neuron survival, synapse
formation, anesthetic-induced immobility, motor neuron diseases,
and spinal cord injury.
 10 Preparation of Adult Spinal Cord Motor Neuron Cultures… 105

Anesthesia and perfusion (30 min)

Spinal cord extrusion (Fig. 2A) (2 min)

Ventral horn dissection (Fig. 2B) (15 min)

Mechanical and enzymatic dissociation (40 min)

Myelin (Fig. 2C) and neuron purification

(Fig. 2D) using a density gradient (30 min)

Plating of cells on glass cover slips and incubation

at 5% CO2 and 37 ºC (Fig. 3A) (5 min)

Feeding of cells with a cocktail of NB-B27, BDNF, GDNF, cAMP

and C2C12 conditioned medium (every other day)

Adult motor neurons ready for experiment

(1 week) (Fig. 3B)

Fig. 1. Flowchart protocol for the isolation and maintenance of adult rat spinal cord motor
neurons in vitro. Correlate the procedure with Fig. 2.

Fig. 2. Isolation and purification of spinal cord neurons. (a) Spinal cord obtained by hydraulic
spinal cord extrusion (modified from Meikle and Martin (34)). (b) Ventral horn segments in
neuronal isolation medium. (c) Density gradient for myelin removal (modified from Brewer (33)).
(d) Density gradient for neuron purification (modified from Brewer and Torricelli (29)).
106 J.V. Montoya-Gacharna et al.

Fig. 3. Adult rat spinal cord cultures 1 h (a) and 7 days (b) after plating. Cells were maintained with a cocktail of GDNF, BDNF,
cAMP, and C2C12 myocyte-conditioned medium (26). The arrows indicate large phase bright cells which are considered
morphologically as motor neurons. These neurons display extensive neurites in culture. Bar = 20 mm.

Fig. 4. Spinal cord neurons express a major synaptic protein, synaptophysin in vitro (7 days post plating). Cell cultures were
co-immunostained with microtubule-associated protein 2 (a) and synaptophysin (b). Synaptophysin puncta (arrows) are
found in the microtubule-associated protein 2-positive neurites. Inset : Neurites at higher magnification. Bar = 20 mm.

Fig. 5. KCl-evoked calcium cytoplasmic transients in dissociated adult rat spinal neurons (14 days in vitro). (a) The SCMNs
cultures were loaded with the calcium indicator Fura 2-AM (5 mM) (Molecular Probes) and stimulated with 100 mM KCl.
The excitation wavelengths for Fura (338 and 380 nm) were alternately (every 0.55 s) applied and the emitted fluorescence
was filtered with the fluorescence barrier filter BA 475 nm and collected with an intensified CCD camera (ATTO Instruments,
Rockville, MD, USA). (b) Phase bright light image of the spinal cord culture after KCl depolarization (40×).
 10 Preparation of Adult Spinal Cord Motor Neuron Cultures… 107

2. Materials

Prepare all solutions using 0.22-mm filtered double distilled water.

Every solution and medium in contact with cell cultures should be
equilibrated at least 1 h in the cell incubator at 5% CO2 and 37°C
unless specified otherwise. Follow all waste disposal regulations.

2.1. C2C12 Myocyte- 1. C2C12 mouse myoblast cell line (CRL-1772, ATCC, VA).
Conditioned Medium C2C12 is a sub-clone (31) of the mouse myoblast cell line
established by Yaffe and Saxel (32).
2. C2C12 cell proliferation medium: Dulbecco’s modified Eagle’s
medium (DMEM), 10% fetal calf serum and 1% penicillin/
streptomycin. Store at 4°C.
3. C2C12 differentiation medium: DMEM, 1% penicillin/strep-
tomycin supplemented with 10% horse serum. Store at 4°C.
4. Cryoprotectant medium: DMEM, 10% fetal calf serum supple-
mented with 5% (v/v) dimethylsulfoxide.
5. 0.25% Trypsin-0.53 mM EDTA. Prepare 5 mL aliquots in
15-mL conical tubes. Store at −20°C.

2.2. Intracardiac 1. Adult Sprague–Dawley rats (70–100 days old) (see Note 1).
Animal Perfusion 2. A dose of 80 mg/kg ketamine (Ketaset® 100 mg/mL; Fort
Dodge Animal Health, IA) and 12 mg/kg xylazine (AnaSed®
20 mg/mL; Shenandoah, IA) is used per animal. Store anes-
thetics at room temperature in a locked cabinet.
3. Artificial cerebrospinal fluid solution (ACSF): 0.4 mM dex-
tran, 15 mM glucose, 3 mM KCl, 125 mM sucrose, 2.1 mM
HEPES, 1.2 mM KH2PO4, 125 mM glycerol, 1.3 mM MgSO4,
and 26 mM NaHCO3. Store at 4°C. Use within a month. On
the day of the spinal cord isolation, bubble the ACSF solution
for 45 min with 95% O2/5% CO2 on ice. Adjust the pH to 7.4
on ice with 1 N NaOH (see Note 2).

2.3. Spinal Cord Cell 1. Neuronal isolation medium (NIM): Hibernate A Medium
Isolation (Brain Bits, IL), 2% B27 supplement (Gibco, CA, 50×),
0.5 mM Glutamax, 100 U/mL penicillin, and 100 mg/mL
streptomycin (Gibco, CA) (see Note 3). Store at 4°C.
2. 2% DNAse I (Worthington, NJ) stock solution in NIM: Use
within 1–2 weeks. For long-term storage in solution follow the
manufacturer’s instructions. Store at −20°C.
3. Enzyme digestion solution (EDS): Prepare the day of neuron
isolation. Pre-warm 10 mL of NIM at 37°C and dissolve 20 mg
of papain (36 U/mL) (Worthington, NJ) (see Note 4). Add
108 J.V. Montoya-Gacharna et al.

100 mL of 2% DNAse stock solution to 9,900 mL of papain-NIM

solution. Filter with a 0.22-mm membrane into a 15-mL conical
tube and place on ice.
4. Density gradient for myelin separation: Prepare OptiPrep™
1.32 (Axis-Shield, Oslo) stock solution: Mix homogeneously
4.95 mL OptiPrep with 5.05 mL Hibernate A with 2% B27 in
a 15-mL conical tube (modified from Brewer, (33)) (Figs. 1
and 2c). Store at 4°C. Prepare 6% OptiPrep 1.32 layer: Mix
homogeneously 2 mL Optiprep 1.32 stock solution with 8 mL
Hibernate A with 2% B27 in a 15-mL conical tube (modified
from Brewer (33)). Store at 4°C (see Note 5).
5. Density gradient for neuronal cell purification according to
Brewer and Torricelli (29) (see Note 6) (Figs. 1 and 2d).
6. Motor neuron basal medium: Neurobasal-A medium, 2% B27
supplement, 0.5 mM glutamine, 100 U/mL penicillin, and
100 mg/mL streptomycin. Prepare a total volume of 100 mL
per spinal cord isolation.
7. PDL solution: 50 mg/mL PDL (molecular weight 70,000–
150,000, Sigma, MO) dissolved in sterile water (see Note 7).
Store 1 mL PDL aliquots at −20°C.
8. Acid-washed glass cover slips: 24-h acid wash (1 N HCl) of
12 mm ∅ glass cover slips (Fisher HealthCare, TX). Rinse cover
slips with double distilled water at least five times. Air dry
cover slips for 3 h and sterilize them by autoclaving.
9. PDL-coated glass cover slips: Add 500 mL of PDL solution to
cover slips placed on 2 × 2Nunc® wells dishes (Invitrogen, CA)
for 3 h. Remove the solution and rinse with 1 mL of sterile
water three times. Air dry the cover slips for 2–3 h under a
sterile hood.

2.4. Motor Neuron 1. Concentrated stock solutions of growth factors (made under
Feeding Medium sterile conditions): 1.1 mg/mL BDNF (Invitrogen, CA) and
0.4 mg/mL GDNF (Invitrogen, CA) in 0.1% bovine serum
albumin (manufacturer’s recommendation) (Sigma, MO) (see
Note 8). Store at −20°C for up to 6 months.
2. Concentrated stock solution of the cell-permeable cAMP analog:
25 mg/mL 8-(4 chlorophenylthio) cyclic adenosine-3¢,
5¢-monophosphate sodium salt (cAMP) (Sigma, MO) in dou-
ble distilled water. Store at −20°C.
3. Motor neuron feeding medium (MFM): Motor neuron basal
medium, 30% of C2C12 cell-conditioned medium, 1 ng/mL
BDNF, 0.1 ng/mL GDNF, and 125 mM cAMP.
 10 Preparation of Adult Spinal Cord Motor Neuron Cultures… 109

3. Methods

Every medium or solution in contact with the cell culture should

be equilibrated in a controlled 5% CO2 incubator at 37°C for 1 h.

3.1. Preparation 1. Prepare C2C12 myocyte-conditioned medium at least 2 weeks

of C2C12 Conditioned before motor neuron isolation. Handle following the biosafety
Medium level I containment protocol.
2. Add 14 mL of cold C2C12 proliferation medium into a 15-mL
conical tube. Leave another empty 15-mL tube next to it.
3. Rapidly thaw C2C12 frozen cells by adding 1 mL of C2C12
proliferation medium to the aliquot of cells.
4. Pipette up and down and place that 1 mL of cell suspension
into the empty conical tube. Continue this process until all
C2C12 differentiation medium has been used.
5. Centrifuge the cell suspension at 1,315 × g for 5 min to obtain
a pellet.
6. Fill up two 12.5-cm2 flasks with 4 mL of DMEM containing
10% FCS.
7. After centrifugation carefully decant the supernatant and
resuspend the pellet in 2 mL of C2C12 proliferation medium.
8. Add 1 mL of this cell suspension to each 12.5-cm2 flask to
bring the final volume to 5 mL.
9. Place the cells in the incubator at 5% CO2 and 37°C.
10. Feed the cells every other day and let grow to confluence (1 week).
11. After approximately 1 week, fill up six 75-cm2 flasks with 15 mL
of C2C12 proliferation medium and place them in cell culture
incubator 5% CO2 at 37°C to equilibrate.
12. Remove and discard culture medium from the C2C12 culture.
13. Incubate the cells with a pre-warmed trypsin–EDTA solution
for 3 min at 37°C.
14. Add 4 mL of complete growth medium to stop the action of
trypsin. Aspirate cells by gently pipetting.
15. Prepare a 1:10 dilution with proliferation medium for a total
volume of 15 mL for each 75-cm2 flask.
16. Expand C2C12 cells in proliferation medium until they reach
30% confluence (see Note 9).
17. After 30% confluence is reached, rinse the cells three times with
motor neuron basal medium.
18. Add 15 mL of C2C12 differentiation medium (ATCC) to
differentiate the myoblasts into myocytes.
110 J.V. Montoya-Gacharna et al.

19. Within 3 days, replace the existing medium with 30 mL motor

neuron basal medium.
20. Collect C2C12 muscle-conditioned medium in 50-mL conical
tubes after 2 days of culture.
21. Filter the conditioned medium with a 0.22-mm membrane and
store at −80°C.

3.2. Intracardiac 1. Set up the perfusion pump system (Masterflex®, Cole Parmer,
Animal Perfusion IL). Attach the perfusion needle and the tubing to the pump.
Run double distilled water through the tubing to remove any
residue. Fill up the tubing with cold ACSF and adjust the pump
to a slow steady drip (20–40 mL/min). The volume of ACSF
perfused is usually 200–300 mL per animal (see Note 10).
2. Anesthetize the rats with an intraperitoneal injection containing
a mixture of ketamine and xylazine following standard proto-
cols for anesthesia of small animals.
3. Test the level of anesthesia with a firm toe pinch. All surgical
procedures should be done under deep anesthesia.
4. Place the animal on its back. Palpate the sternum and the inferior
border of the last rib. These landmarks define the inferior
aspect of the rib cage. Make a transverse incision with sharp
scissors in the upper abdomen below the rib cage. Identify the
diaphragm and make a transverse incision to access the thoracic
cavity. Rapidly cut the ribs toward the head at the point of the
anterior thoracic line. Open up the thoracic cavity. Hold the
beating heart with forceps and insert the needle into the apex
of the heart. Clamp the inserted needle near the entry point.
Incise the right atrium and perfuse cold oxygenated ACSF
through the heart. A volume of approximately 200–300 mL of
ACSF is used per animal. The perfusion is completed when the
liver changes color from a dark red to a gray color, and the
extremities become white.

3.3. Spinal Cord Cell Spinal cord cells are isolated following the protocol by Brewer and
Isolation Torricelli (29) with some modifications.
1. Place the rat on its abdomen and decapitate it using a guillotine.
Incise the dorsal skin longitudinally from head to tail. Make a
transverse incision in the thigh to localize the femur. Fracture
both femurs with scissors as well as the spinal column at the level
of the sacrum, approximately 4 cm from the base of the rat’s tail.
Pull up the sacral vertebra and identify the spinal canal.
2. Place a Petri dish filled with cold NIM at the cervical/decapitated
end of the rat. Then, insert a syringe filled with 10 mL of
cold ACSF in the sacral canal and apply hydraulic pressure to
extrude the spinal cord into the NIM (Figs. 1 and 2a). Quickly
compress the plunger of the syringe and the spinal cord should
 10 Preparation of Adult Spinal Cord Motor Neuron Cultures… 111

come out of the cervical end. Collect the spinal cord in cold
NIM (34) (see Note 11).
3. Immerse the spinal cord in the cold NIM (Fig. 2a) (see
Note 12).
4. The spinal cord should be immersed into a Petri dish containing
cold NIM at all times. Using a stereoscope, obtain transversal
sections of the spinal cord using a pair of iridectomy scissors
(Figs. 1 and 2b).
5. Tease away the meninges using small forceps.
6. Cut the spinal cord longitudinally through the posterior median
sulcus and the anterior median fissure to obtain two halves of
cord (each one containing a dorsal and a ventral column).
7. Identify a darker gray central area, which is butterfly shaped in
the cross-section of the spinal cord. The dorsal and ventral
horns are easily identified under a stereoscope. Separate the
dorsal and ventral columns by cutting longitudinally between
both ventral horns.
8. Cut the ventral horns in small pieces (2–3 mm) for better cell
dissociation.
9. Add 10 mL of EDS to a 15-mL conical tube. Transfer the ventral
horns to the conical tube and incubate for 5 min at room tem-
perature followed by 25 min at 30°C in a rotating incubator.
10. In the meantime, prepare 6 mL of 0.02% DNAse in EDS
medium by adding 60 mL of 2% DNAse to 6 mL of EDS
medium into a 15-mL conical tube.
11. Prepare three 2-mL fire-polished Pasteur pipettes (Fisher
brand) of decreasing diameter (large, medium, and small). The
Pasteur pipettes are prepared by flaming the pipette tips using
an alcohol burner.
12. Centrifuge the tissue at 1,228 × g for 5 min. All the following
steps (except for the centrifugation) should be done under a
sterile hood, and sterile techniques should be followed.
13. Remove the supernatant, and add 2 mL of NIM with 0.02%
DNAse to the pellet.
14. Triturate the tissue ten times with each of three Pasteur pipettes
starting with the pipette with the largest opening.
15. After each trituration, the homogenate is allowed to settle for
1 min and the supernatant is transferred into a separate 15-mL
conical tube.
16. Add an additional 2 mL of NIM to the precipitate and repeat
the process for a total of three times.
17. First gradient for myelin separation: Add 5 mL of 6% OptiPrep
1.32 to a 50-mL conical tube. Layer the final cell suspension
(6 mL) on top of the 6% OptiPrep 1.32 and centrifuge at
112 J.V. Montoya-Gacharna et al.

822 × g for 15 min at 4°C (Figs. 1 and 2c). Discard the

supernatant (6% OptiPrep) and the myelin layer (white band)
at the interface. Collect the cell-containing fractions (bottom
layer and the pellet). Resuspend and bring the volume to
20 mL with NIM.
18. Filter the cell suspension through a 70-mm nylon mesh.
19. Centrifuge the cell suspension at 480 × g for 5 min at 4°C.
20. Resuspend the pellet with 6 mL of NIM and place on ice.
21. Second gradient for neuron separation: Prepare an OptiPrep
1.32 density gradient according to Brewer and Torricelli (29).
Add the 6 mL cell suspension on top of this gradient (Figs. 1
and 2d).
22. Centrifuge the gradient at 1,027 × g (1,900 rpm Beckman
JS-4.2 rotor) for 15 min at 4°C.
23. Collect the neuronal cell fractions (Fig. 10.2d) (fractions 2 and
3 according to Brewer and Torricelli (29) which contain both
neurons and glia) in separate 15-mL conical tubes.
24. Pool the neuronal cell fractions and dilute them with 5 mL of
NIM and centrifuge at 244 × g for 2 min at 4°C to remove the
OptiPrep.
25. Resuspend each pellet separately with 150 mL of MFM. Pool
both cell suspensions together. Mix them by softly flicking the
bottom of the tube. Plate the cells with a 10-mL tip pipette.
Place a volume of 10 mL of cell suspension (6,000 cells) in the
middle of each 12-mm ∅ glass cover slip.
26. Allow the attachment of the cells for 1 h at 37°C and 5% CO2.
27. Place 500 mL of pre-warmed MFM into each culture plate by
resting the pipette tip on the side of the well.
28. Evaluate cell viability after dissociation by using the trypan
blue exclusion test (see Note 13).
29. Leave the spinal cord cultures in the incubator at 5% CO2 and
37°C (see Note 14).

3.4. Feeding of Motor The cells are fed the day after plating, and every other day until the
Neuron Cultures end of the experiment. Care must be taken to minimize the time
the cells are outside of the incubator and exposed to ambient CO2
levels, as it can cause cell death. The cells should not be out of the
incubator for more than 3 min at a time.
To feed the cells:
1. Under the sterile hood, remove approximately 350 mL of
MFM (see Note 14).
2. Carefully add 350 mL of MFM by resting the pipette tip on
the side of the well to avoid detachment of the cells.
 10 Preparation of Adult Spinal Cord Motor Neuron Cultures… 113

3. Place the cells in the incubator at 5% CO2 and 37°C.

Immunocytochemistry can be performed using several motor
neuron cell markers (26) at different time points. Electrically
functional SCMNs can be found after 1 week of culture (26)
and can be measured at different time points thereafter.

4. Notes

1. The age of the animal is a factor inversely related to the survival

of spinal cord MNs. Mostly we have evaluated the survival of
SCMNs derived from 70- to 100-day-old rats.
2. We usually prepare 1 L of ACSF solution. The dextran should
be the first chemical to be added to at least 500 mL of double
distilled water due to its low solubility. The solution should
also be filtered with 0.2-mm pore membrane and stored at 4°C.
On the day of the perfusion, the ACSF solution is bubbled for
at least 45 min with 95% O2/5% CO2 on ice and the pH
adjusted to 7.4 just prior to perfusing the animal.
3. B27 supplement is light sensitive and should be handled
according to the manufacturer’s instructions. It should be kept
frozen at −80°C indefinitely when not in use. Once thawed, it
should not be frozen again. Thawed B27 expires within a
month from the day it is opened and can be stored at 2–8°C in
the dark. We have observed cases where the survival of SCMNs
varied depending on the batch of B27 used. For a series of
extended experiments we recommend ordering enough B27
of the same lot in order to decrease the variability of the results.
Glutamax is stable in aqueous solution (Gibco). We recom-
mend storing 1 mL aliquots of Glutamax at −20°C. NIM solu-
tion should be prepared 1 day before isolation. A volume of
250 mL of NIM is prepared for each rat. Store the solution in
an aluminum foil covered bottle at 4°C.
4. The NIM should be warmed to 37°C before the papain is
added. Mix the solution well on a vortex for 5 min and filter it
with 0.22-mm membrane. As the papain powder is very light,
it is best to wear a mask to avoid inhaling the enzyme during
weighing. Store on ice until ready to use. This volume is suffi-
cient for one spinal cord.
5. Density stock solutions can be stored at 4°C and used within 1
month. Mix well by vortexing before preparing the density
gradient.
6. We recommend alternating between clear Hibernate A (with-
out phenol red) and red hibernate A (with phenol red) when
preparing the density gradients so as to facilitate the visualiza-
tion of the different fractions.
114 J.V. Montoya-Gacharna et al.

7. We have also evaluated several substrates like collagen,

poly-ornithine, laminin, and PDL. In our experience, PDL
gives the best results. Others have shown that the cell growth-
promoting organosilane substrate, N-1(3[trilnethoxysilyl]
propyl)-diethylenetriainine, coated on a glass surface is necessary
for the survival of these neurons (27, 28). PDL should not be
used after one freeze–thaw cycle. The PDL-coated glass cover
slips are prepared the day before cell isolation and can be stored
at 4°C. PDL-coated cover slips may be used up to 2 days later.
8. For growth factors storage follow manufacturer’s recommen-
dations. Do not store in glass, use polypropylene vials
(Invitrogen). Do not store diluted solutions and avoid adsorp-
tive loss using at least 0.1% bovine serum albumin (Invitrogen).
Avoid repeated freeze–thaw cycles of the stock solutions. In
our studies, we found that some of the essential factors for the
survival of dissociated SCMNs were cAMP and muscle-condi-
tioned medium. However, other molecules may be involved in
the survival of SCMNs. For instance, Das et al. (27, 28) showed
that a more complex mixture of factors (BDNF, GDNF, neurotro-
phin-3, heparin sulfate, cardiotrophin-1, vitronectin, and acidic
fibroblast growth factor) is necessary for the maintenance of
adult SCMNs under their conditions.
9. Do not let the C2C12 cells become confluent because the
myoblasts will be depleted (ATCC).
10. Make sure there are no bubbles in the tubing while perfusing
as they can destroy the rat tissues.
11. A 16G 1 1/2 needle is inserted into the spinal canal to form a
tight seal. The seal is important because otherwise the spinal
cord will not be extruded. Additionally, it is necessary to make
sure that approximately three quarters of the needle is inside
the spinal canal. When inserting the needle, there should be a
slight resistance. If it is too difficult to insert, stop, readjust the
needle and/or make another transverse cut of the sacral spine
if necessary. Never force the needle, because you will break the
bone, and extrusion may become impossible at this point as a
seal cannot be formed. On the other hand, be careful not to
cut the spinal column too high (lumbar region) because if the
needle slides inside the canal too easily, enough pressure can-
not be sustained, and extrusion will not be successful.
12. Keep the time between decapitation and immersion of the spinal
cord into the medium as short as possible (2 min) as the spinal
cord neurons are very sensitive to ischemia. Most of our exper-
iments were done with the cervical enlargement of the spinal
cord; however, any segment of the cord can be used.
13. The viability of the SCMNs cultures is more than 90% (26)
after plating.
 10 Preparation of Adult Spinal Cord Motor Neuron Cultures… 115

14. The SCMN cells are quite sensitive to temperature and pH,
and therefore it is essential to change the media quickly to
minimize the exposure to colder temperature and ambient pH
levels. If the cells remain outside for too long they are likely to
detach. We recommend the use of 2 × 2 Nunc® well plates
because they allow quick medium change. We also recommend
resting the well plate on a tube rack that is placed in the hood
(the night before, so it can be sterilized by the UV light)
because this reduces the contact the cells would have to the
relatively cold hood surface.

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Esquerda JE (1994) Skeletal muscle-derived Exp Neurol 209, 171–180
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 Chapter 11

Rodent Retinal Ganglion Cell Cultures

Stephen D. Skaper

Abstract
Central neurons lose the ability for axonal regrowth during development and typically do not regenerate
their axons following axotomy once they become mature unless given a growth-permissive environment,
for example, a peripheral nerve graft. Retinal ganglion cells (RGCs) of the optic nerve represent a highly
useful cell model for the study of neurotrophic factor responsiveness, although the presence of nonneu-
ronal cells in the retina makes it difficult to interpret the direct effects of tested factors on RGCs. Cultures
of purified RGCs thus represent an excellent tool for the study of optic nerve cell trophic responsiveness,
in terms of both survival and axonal regeneration.

Key words: Retina, Ganglion cells, Rat, Neonatal, Embryonic, Culture, Antibody panning, Neurotrophic
factors, Axonal regeneration

1. Introduction

Adult central nervous system (CNS) neurons typically do not

regenerate their axons following axotomy, with the consequence
that CNS injury results in permanent impairment. Studies have
concluded that it is the CNS environment that is inhibitory to
regenerative growth and that at least some of the adult CNS neu-
rons do have an intrinsic ability to regenerate if given a permissive
environment (1, 2). The optic nerve is a readily accessible CNS
tract that contains glial cells (oligodendrocytes, astrocytes, and
microglia) and only one type of axon, those arising from retinal
ganglion cells (RGCs). Neurotrophic factors applied intraocularly
or to the axotomized optic nerves enhance the survival of RGCs in
adult rodents during early stages after injury. Such factors include
brain-derived neurotrophic factor (BDNF) (3, 4), neutrophin-4/5
(5, 6), nerve growth factor (7, 8), ciliary neurotrophic factor
(CNTF) (9, 10), and fibroblast growth factor (11, 12). Although

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_11, © Springer Science+Business Media, LLC 2012

117
118 S.D. Skaper

RGCs can be studied as a flat-mounted preparation, as explants, or

as dissociated retinal cultures, the presence of nonneuronal cells in
the retina makes it difficult to interpret the direct effects of tested
factors on RGCs. Cultures of purified RGCs thus represent an
excellent tool for the study of optic nerve cell trophic responsive-
ness, in terms of both survival and axonal regeneration. Indeed, rat
RGCs display age-dependent responsiveness to isolated neu-
rotrophic factors, as well as Schwann cell–secreted factors (13).
The protocol described here outlines a procedure for the purifica-
tion of rat RGCs from a whole retina cell suspension (14). The
proteolytic enzyme papain is used to dissociate rat retinal tissue
(15–17). Purification is effected by the technique of antibody-
mediated plate adhesion, “panning,” developed by Wysocki and
Sato (18). This procedure yields a highly pure (>99%) population
of ganglion cells, with yields ranging from 25% to 50%.

2. Materials

2.1. Equipment 1. Stereo dissecting microscope with fiber optic light source.
and Labware 2. Laminar flow cabinet for dissections.
3. Laminar flow biological safety cabinet (CL2).
4. Humidified, water-jacketed culture incubator at 37°C and 5%
CO2/95% air.
5. Water bath set at 37°C.
6. Dissecting tools (Fine Science Tools).
7. Benchtop centrifuge to accommodate 15- and 50-mL tubes.
8. 15- and 50-mL polypropylene plastic centrifuge tubes (sterile).
9. 12-mL test tubes, sterile.
10. 6-mL flat bottom tubes (Bijou), sterile.
11. 1.5-mL Eppendorf tubes.
12. 10-cm ∅ sterile tissue culture dishes.
13. 6-, 10-, and 15-cm ∅ sterile Petri culture dishes.
14. #21- and #23-gauge hypodermic needles.
15. 5- and 10-mL syringes.
16. Nitex mesh, 20 μm (Tetko HC3-20).
17. 0.22- and 0.45-μm filters (Millipore).
18. Neubauer hemocytometer (Fisher Scientific).
19. 24- and 96-well tissue culture plates.
20. 24- and 96-well poly-D-lysine treated tissue culture plates (BD
Biosciences).
 11 Rodent Retinal Ganglion Cell Cultures 119

2.2. Reagents 1. Earle’s balanced salt solution (EBSS) (Invitrogen).

2. EBSS containing sodium bicarbonate and phenol red but lack-
ing Ca2+ and Mg2+ (CMF) (Invitrogen).
3. Dulbecco’s phosphate-buffered saline (D-PBS) (Invitrogen).
4. Minimal essential medium with Earle’s salts (MEM) (Invitrogen).
5. Dulbecco’s modified Eagle’s medium (DMEM), containing
4.5 g/L glucose, L-glutamine, and pyruvate (Invitrogen).
6. DMEM (American Type Culture Collection, ATCC).
7. Neurobasal medium (Invitrogen).
8. B27 supplement, 50×, with antioxidants (Invitrogen).
9. Fetal calf serum (FCS) (Invitrogen) (see Note 1).
10. Penicillin/streptomycin, 10,000 U/mL penicillin + 10,000
μg/mL streptomycin (100× stock), sterile, for cell culture
(Invitrogen).
11. L-Glutamine (200 mM stock), sterile, for cell culture
(Invitrogen).
12. Sodium pyruvate (100 mM stock), sterile, for cell culture
(Sigma).
13. Papain (Worthington (Lorne)).
14. DNase, type I (Worthington (Lorne)).
15. Trypsin, 2.5% solution, sterile, for cell culture (Sigma).
16. Trypsin (0.25%)-EDTA, sterile, for cell culture (Invitrogen).
17. Poly-D-lysine (MW 70,000–150,000), sterile, for cell culture
(Sigma).
18. Merosin, human (Invitrogen).
19. Bovine serum albumin (BSA), essentially fatty acid and globu-
lin-free (Sigma).
20. Ovomucoid (trypsin inhibitor from chicken egg white, type
III-O, free of ovoinhibitor) (Sigma).
21. L-Cysteine hydrochloride (Sigma).
22. Insulin, from bovine pancreas (Sigma).
23. Transferrin, human, partially iron saturated, tested for cell cul-
ture (Sigma).
24. Progesterone (Sigma).
25. Putrescine (Sigma).
26. Sodium selenite (Sigma).
27. T3 (3,3¢,5-triiodo-L-thyronine, sodium salt), cell culture tested
(Sigma).
28. Thyroxine (T4) (Sigma).
29. Affinity-purified goat anti-mouse IgG + IgM (H + L) (Jackson
ImmunoResearch).
120 S.D. Skaper

30. Affinity-purified goat anti-mouse IgM, mu-chain specific

(Jackson ImmunoResearch).
31. Anti-rat-macrophage antiserum (Accurate/Axell).
32. RAN-2 hybridoma TIB-119™ (ATCC).
33. Thy 1.1 IgM hybridoma TIB-103™ (name T11D7e2) (ATCC).
34. BDNF, human, recombinant (PeproTech).
35. CNTF, human, recombinant (PeproTech).
36. Glial cell line–derived neurotrophic factor, human, recombi-
nant (GDNF) (PeproTech).
37. Basic fibroblast growth factor (PeproTech).
38. Forskolin (Sigma).
39. Trypan blue stain 0.4%, for cell culture (Invitrogen).
40. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide (MTT) (Sigma).
41. Dimethyl sulfoxide, for cell culture (Sigma).

2.3. Solutions for 1. Papain solution. Immediately before the start of the dissection,
Tissue Dissociation prepare a papain solution by adding 165 units (for postnatal day
8 (P8) rats, for embryonic, much less is needed) of papain to
10 mL of EBSS (or P-PBS or MEM) in a 15-mL blue-top centri-
fuge tube. Next, add 100 μL of a 4 mg/mL solution of DNase.
Dissolve the latter by placing the mixture, which is in a 15-mL
centrifuge tube, in a test tube rack in a 37°C water bath.
About 10 min before use, mix the papain solution with 2.4 mg
of L-cysteine hydrochloride and pass through a 0.22-μM filter
into a sterile scintillation vial or universal (Bijou) tube.
2. Ovomucoid solution. Dissolve 13 mg of ovomucoid and 13 mg
of BSA in 13 mL of MEM, and then add 0.13 mL of a 4 mg/mL
solution of DNase; adjust to pH 7.4 and filter-sterilize.
3. Trypsin (0.125%). Add 200 μL of a 2.5% trypsin stock to 4 mL
of CMF. Be sure to prepare the trypsin aliquots immediately
upon trypsin arrival; store at −20°C until use.
4. Trypsin inactivating solution. Combine 3 mL of FCS plus
10 mL DMEM.
5. Sato 100× stock solution. To 20 mL of Neurobasal medium, add
the following (final concentrations in brackets): 200 mg transferrin
(100 μg/mL), 200 mg BSA (100 μg/mL), 5 μL of stock pro-
gesterone solution (stock is 2.5 mg/100 μL ethanol) (60 ng/mL
or 0.2 μM), 32 mg putrescine (16 μg/mL), 200 μL of stock
sodium selenite (stock is 40 mg in 1 mL of 0.1 N NaOH plus
10 mL Neurobasal-A) (40 ng/mL), 10 μL of stock T3 (stock
is 8 mg in 1 mL of 0.1 N NaOH, 30 ng/mL), and 10 μL of
stock T4 (stock is 8 mg in 1 mL of 0.1 N NaOH, 40 ng/mL).
 11 Rodent Retinal Ganglion Cell Cultures 121

Aliquot the above 100× stock solutions (200 μL) and store
at −20°C. The ethanol stocks for progesterone, sodium selenite,
T3, and T4 should be prepared fresh each time a new batch of
100× Sato medium is made.

2.4. Sato Serum-Free 1. To 20 mL of Neurobasal medium (19), add the following:

Culture Medium 200 μL Sato 100× stock solution, 400 μL 50× B27 supple-
ment, 200 μL sodium pyruvate 100× stock, 200 μL glu-
tamine 100× stock, 200 μL of 100× penicillin/streptomycin
stock (stored at −20°C), and 20 μL N-acetyl cysteine
1,000× stock.
2. Next, add 200 μL of 100× insulin stock (stored at 4°C; stock
must be prepared every 4–6 weeks—do not freeze). The 100×
insulin stock (0.5 mg/mL) is prepared by adding 2 mg of
insulin to 4 mL of tissue culture grade water containing 20 μL
of 1 N HCl. Filter the stock through a 0.22-μm filter and
store at 4°C.
3. Filter the above medium through a 0.22-μm filter. Before fil-
tering the medium, filter about 5 mL of Neurobasal medium
to rinse away traces of detergent in the filter. The quality of the
filter matters—Millex-GV from Millipore is best. Falcon plastic
is preferable, along with Monoject syringes.
4. Recommended trophic factors for mixed optic nerve cultures:
insulin (5 μg/mL) plus CNTF (10 ng/mL). Do not filter the
medium once the growth factors are added due to the risk of
their sticking to the filter. In addition, forskolin (5 μM) can be
included in the medium, as it promotes survival of mixed and
purified cultures.
5. For purified RGCs, add BDNF (50 ng/mL), CNTF (10 ng/
mL), and forskolin (5 μM final concentration; use a 1,000×
stock prepared in dimethyl sulfoxide). Optional: Add basic
fibroblast growth factor (10 ng/mL) for low-density culture.

3. Methods

3.1. Dissection 1. Normally, one to ten rats from a Sprague-Dawley or Long-

Evans litter (Charles River) are used, typically between P8 and
P10 (see Note 2).
2. Remove the retinas from the eye in situ. Sacrifice the animal by
decapitation (following approved institutional protocols), and
remove the skin overlying the eyes.
3. Pin back the head to a wax block and remove the anterior
tissues of the eye with a #11 scalpel blade while the cornea is
held with a pair of rat-tooth, microdissecting forceps. Remove
122 S.D. Skaper

the lens and vitreous humor with forceps. Then, gently lift the
retina away with a small spatula.
4. Store the retinas at room temperature in EBSS containing cal-
cium and magnesium, pH 7.4, until retinas are removed from
all animals.

3.2. Tissue 1. Upon completion of the dissection, remove the EBSS with a
Dissociation sterile Pasteur pipette and replace with 2 mL of the papain
solution (see Notes 3 and 4).
2. Transfer the retinas to the vial by pouring, and place the vial in
a test-tube rack in the water bath at 37°C. Every 10 min or so,
gently swirl the retinas. The incubation time with the papain
solution is 30 min (see Note 5).
3. Remove the vial from the water bath and transfer the retinas (by
pouring) to a 15-mL centrifuge tube. Allow the retinas to set-
tle, remove the papain solution with a pipette, and rinse the
tissue gently with 3 mL of ovomucoid solution. Allow the tis-
sue pieces to settle and remove the rinse solution (see Note 6).
4. Triturate the tissue with a 1-mL pipette in the ovomucoid
solution. The following steps are repeated ten times. Add
1 mL of ovomucoid solution, gently pull the tissue up into a
1-mL pipette and expel. Centrifuge the tissue dissociate for a
few seconds at 200 × g to remove pieces of tissue from the cell
suspension, and collect the supernatant. The retinal tissue is
further triturated by adding 1 mL of fresh ovomucoid solu-
tion, making one pass through the 1-mL plastic pipette, spin-
ning down the undissociated tissue, and collecting the
supernatant. After repeating this trituration procedure 6–10
times, the retinas should be completely broken up. A cell
count at this point should show a cell yield of about 20 million
cells/retina (see Note 7).
5. Centrifuge the resulting retinal cell suspension at 400 × g for
10 min to separate the retinal cells from the ovomucoid
solution.
6. Discard the supernatant and resuspend cells in 10 mL of MEM
containing 0.05% (w/v) BSA. The BSA serves to slow the cells’
settling time on the panning dishes. The cells are now ready for
the panning step.

3.3. RGC Purification 1. Incubate each of two 6-cm Petri dishes with 5 mL of 50 mM
by Panning Trizma buffer, pH 9.5, with affinity-purified goat anti-mouse
IgM, mu-chain specific (final concentration is 5–10 μg/mL)
3.3.1. Preparation
for 12 h at 4°C. To sterilize, pass the Tris solution through a
of the Panning Dish
filter into a sterile dish, and then add the azide-containing anti-
body directly to this; otherwise, much of the protein will be
lost to the filter (see Notes 8 and 9).
 11 Rodent Retinal Ganglion Cell Cultures 123

2. Similarly, incubate two 15-cm Petri dishes with 15 mL of

anti-rabbit IgG H + L (5–10 μg/mL in Tris buffer, pH 9.5). Wash
each dish three times with 8 mL of D-PBS (see Note 10).
3. Incubate the 10-cm dishes with 10 mL of Thy 1.1 IgM mono-
clonal supernatant for at least 1 h at room temperature. The
15-cm dishes are not incubated with any other antibodies.
4. Remove the supernatant and wash the plates three times with
D-PBS.
5. In order to prevent nonspecific binding of cells to the panning
dish, 5–15 mL of MEM with BSA (0.2%) is placed on each
plate for at least 20 min; this solution should remain on the
plate until the plate is needed.

3.3.2. Panning Procedure 1. Incubate the retinal suspension in anti-rat-macrophage antise-

rum (1:100 in MEM) for 20 min, centrifuge, resuspend in
MEM, and incubate on a 15-cm anti-rabbit IgG panning plate
at room temperature for 45 min total.
2. After 20 min, swirl the plate to ensure access of all cells to the
surface of the plate. If cells from more than eight retinas are
being panned, at the end of 45 min, transfer the nonadherent
cells to another 15-cm anti-rabbit IgG panning plate for a fur-
ther 30 min.
3. Remove the nonadherent cells (in suspension) and filter
through a 20-μm Nitex mesh to remove small clumps of cells.
Sterilize the mesh before use (UV exposure for a few minutes
is sufficient), prewet the mesh on the bottom side, then form a
cone with the filter on top of a test tube, and pass 1 mL of
MEM through it before using a Pasteur pipette to transfer the
cell suspension through the filter.
4. Clumps could occur either because they were not broken into
single cells during trituration or because they were clumped by
the antimacrophage antiserum. The latter type of clumping
represents the single greatest impediment to final RGC purity
as they can bring down unwanted contaminating cell types.
5. Transfer the cell suspension from the previous step to the
Thy1.1 panning dishes. The use of two 10-cm dishes improves
the final yield, although the use of more than this is not help-
ful. Leave the cell suspension on the dish(es) for 1 h while
swirling every 20 min.
6. Wash the dishes at least eight times with 6 mL of D-PBS (dis-
card after each wash) and swirl rather vigorously each wash to
dislodge nonadherent cells. Monitor the progress of nonad-
herent cell removal by observing the dishes under a light
microscope. Washing is considered complete when only adher-
ent cells remain on the dish.
124 S.D. Skaper

3.3.3. Removing Adherent 1. Incubate adherent cells on the panning dish with 0.125%
Cells from the Panning trypsin solution for 10 min in a 37°C CO2 incubator.
Dish
2. Remove the dish from the incubator and squirt off the cells
with a 1-mL pipetman while they are still in the trypsin solu-
tion. Mark one point on the outside of the dish with marker.
Squirt from this point, once around the dish, by squirting from
the peripheral edge toward the center, rotate the dish, squirt
again, etc., until the entire surface area has been covered once.
Keep to a minimum the amount of squirting necessary to
remove the cells (see Note 11).
3. Transfer the cell suspension from the dish to a 15-mL centri-
fuge tube. Add 1 mL of the trypsin inactivating solution to
these cells and add another 4 mL to the dish. Again squirt the
dish. Check quickly under the microscope to make sure most
of the cells have been released (if not, this probably indicates
that the trypsin has lost activity).
4. Collect this 4 mL and transfer to the centrifuge tube (total
volume: 9 mL). Gently invert the tube several times to mix the
cells and remove a 100 μL aliquot for counting. Pour the
remainder of the trypsin inactivating solution into the tube
with the cells to top up the tube. Centrifuge at 200 × g for
5 min to collect the cells.
5. Resuspend the cells in Sato medium with growth factors for
purified RGCs, mix a 20 μL aliquot with an equal volume of
0.4% trypan blue, count, and plate. Expected yield: 40,000
RGCs viable (trypan blue–excluding cells) per P8 rat pup (see
Notes 12–15).
6. Culture the cells on a poly-D-lysine/merosin substrate. Dissolve
a 5-mg bottle of poly-D-lysine in 5 mL of 0.15 M borate buf-
fer, pH 8.4. Add 200 μL of this poly-D-lysine stock (1 mg/
mL) to 20 mL of tissue culture grade sterile water. Use 0.2 mL
poly-D-lysine per cm2 surface area. Incubate with poly-D-lysine
at room temperature (20–22°C) for 60 min. Merosin coating
is done after rinsing off excess poly-D-lysine: Add a 2 μg/mL
solution in Neurobasal medium and incubate at 37°C for sev-
eral hours to overnight (if overnight, add penicillin/strepto-
mycin) in a 5% CO2 incubator. The cells can be plated at 5,000
cells per well of a 96-well plate (high density) or at low density
on 6-cm tissue culture dishes, as desired.

3.4. Modifications 1. Use CD1 mice or other Thy1.2-positive strains, between 8 and
for Purifying Mouse P8 10 days postnatal age.
RGCs Rather than Rat 2. Instead of using an IgM Thy1.1 antibody, use an IgM Thy 1.2
antibody for the final panning dish. For example, the F7D5
Thy1.2 antibody, which is a mouse monoclonal (Accurate
Chemical Co., Cat No. MAS 731).
 11 Rodent Retinal Ganglion Cell Cultures 125

3. Add 5 mL of 0.2% BSA in D-PBS containing F7D5 ascites

fluid at a dilution of 1:1,000 or 1:500.
4. All other steps remain the same.

3.5. Modifications 1. Decapitate embryos with a pair of #5 microdissecting forceps.

for Purifying 2. Remove skins overlying the eyes with the same forceps.
Embryonic Day 18
3. Make an insertion on the cornea with a pair of #55 microdis-
(E18) RGCs Rather
secting forceps.
than P8 RGCs
4. Peel off the cornea with another pair of #55 forceps to expose
3.5.1. Dissection the lens.
5. Remove the lens with the #55 forceps.
6. Lift away the retina with a small spatula.

3.5.2. Dissociation 1. Use 50 U of papain in 10 mL of D-PBS (for 30 min, same

length of time as for P8).
2. The yield of purified E18 RGCs will be substantially less than
for P8 because they have not all been generated yet and because
they express less Thy1.

3.5.3. Culture The same Neurobasal serum-free Sato medium, use of B27 at 1:50
is key, as well as BDNF, which will only save about a quarter of the
cells in any case. The E18 RGCs do not respond to the other
trophic factors.

3.6. MTT Survival 1. The MTT assay is frequently used to quantify neuronal cell
Assay survival. MTT reacts with mitochondrial dehydrogenases to
produce a blue formazan product in living cells, but not in
dying cells or their lytic debris (20, 21). Because of the low
density of RGC cultures, MTT-labeled cells are counted, rather
than dissolving the reaction product in dimethyl sulfoxide and
reading absorbance spectrophotometrically.
2. An MTT stock solution is prepared by dissolving MTT in
D-PBS at 5 mg/mL. Sterilize by passage through a 0.22-μm
filter. The stock solution can be stored at −20°C for up to
6 months.
3. Add 10 μL of the MTT stock to each 100-μL well of a 96-well
plate. Return the plate to the 37°C incubator for at least 1 h
(maximum 2 h). Viable cells with active mitochondria cleave
the tetrazolium ring into a visible dark blue/black formazan
reaction product (see Note 16).
4. Count the percentage of viable cells in each well by counting
at least 200 cells per well. Ideally, at least three wells per test
condition should be counted. Use an eyepiece reticle with a
counting grid. Cells must be counted immediately after the
MTT incubation to avoid the formation of large crystals of
MTT and toxicity of MTT to the cells.
126 S.D. Skaper

4. Notes

1. Heat inactivation of fetal calf serum is recommended to destroy

heat-labile complement. Thaw the bottle of serum in advance,
using a 37°C water bath. Next, immerse the serum bottle in
the water bath after reequilibrating to 56°C and leave for
30 min. Swirl the bottle occasionally to ensure proper mixing.
Allow the serum to cool to room temperature, aliquot into
50-mL tubes, and store at −20°C.
2. The dissociation procedure works on any age retina (E18–
P12); purification using rats older than P12 has not been veri-
fied using this protocol (but see ref. (13)).
3. The papain should be obtained from Worthington.
4. Do not use L15 as it inhibits papain.
5. During the dissociation procedure, never expose the cells to
glutamate, aspartate, or glutamine nor allow cells to be cooled
lower below room temperature to avoid toxic effects on gan-
glion cells.
6. Ovomucoid itself does not inhibit the papain but probably a
contaminant; the source of the ovomucoid matters.
7. In step 4 of the tissue dissociation, tissue pieces may be allowed
to settle by gravity rather than using centrifugation.
8. Dishes used for panning must be Petri plastic, not tissue cul-
ture plastic treated. Falcon is a good brand, and others may
work but should be tested first.
9. The time of the entire panning procedure can be significantly
shortened by decreasing the time of all panning plate cell incu-
bations by half without much loss in yield or purity.
10. When solutions are removed from the panning dishes during
washes, they must be instantly replaced with fresh solution so
that cells do not dry out.
11. Do not wash too vigorously, or you will wash off the RGCs.
12. Do not resuspend the final cell pellet in DMEM, because there
is no BSA—you may lose cells on the wall of the pipette tip
from nonspecific sticking.
13. DMEM should not be used, as the osmolarity is 335. Combined
with the B27 supplement and other additions indicated, the
final osmolarity of 360 becomes cytotoxic. Neurobasal osmo-
larity is 225, becomes about 260 with the various supple-
ments/additions.
14. The B27 supplement seems to go off quickly once opened; it
is recommended to buy the small size and replace the bottle of
medium frequently.
 11 Rodent Retinal Ganglion Cell Cultures 127

15. Do not use F12 culture medium or combination with F12, as

F12 contains amounts of iron which are cytotoxic for RGCs.
16. MTT is toxic. Handle with gloves; do not inhale, touch, or
swallow.
Panning Tips
17. For panning, it is preferable to use antibodies that recognize
surface antigens that are not cleaved by the papain or trypsin
used to prepare the cell suspension. For this reason, antibodies
to glycolipids (e.g., galactocerebroside) or gangliosides (e.g.,
A2B5) are ideal.
18. In some cases, when the panning antigen is a protein, the
enzymes used do not completely cleave the antigen of interest,
and so enzymes can still be used (e.g., RAN-2 and Thy1). In
many cases, however, the enzymes completely destroy the
antigen. It is still possible to pan using antibodies to this antigen,
if the cell suspension is allowed to recover at 37°C for 1 h.
19. Always pan at room temperature, never at 37°C. The higher
temperature allows activation of cell adhesion mechanisms and
can result in adherence of contaminating cell types.
20. Always use at least two panning dishes; the first should be coated
with an antibody to deplete an unwanted contaminating cell
type or with an irrelevant antibody in order to make sure that
microglia/macrophages are depleted (about 6% of cells in the
developing optic nerve). They will not be successfully eliminated
by panning for them on a dish that is not antibody coated.
21. An alternative for removing microglia/macrophages is to just
coat the first dish with the lectin BSLI (Bandeiraea simplicifolia
lectin I; this lectin also binds to brain endothelial cells).
22. When coating panning dishes, the first antibody (the goat anti-
rabbit or anti-mouse) should always be affinity purified. Because
unrelated proteins like BSA will occupy most binding sites on
the Petri dish, monoclonal supernatants (which generally con-
tain 10% FCS) cannot be put straight onto the dish unless you
precoat with an affinity-purified anti-mouse antibody. However,
an affinity-purified antibody (or a lectin) to the cell surface
antigen of interest can be put onto the dish directly.
23. For panning, the second antibody (the one that actually binds
to the cell surface antigen of interest) should be a monoclonal
supernatant whenever possible. The reason for this is that the
only mouse antibody type in a supernatant is the one of inter-
est. Using a polyclonal antiserum risks that there will be many
irrelevant antibodies to occupy (anti-rabbit) sites on the
panning dish. Likewise, ascites fluid may contain numerous
host-derived antibodies and will also occupy (anti-mouse) sites
on the panning dish. In some cases, ascites from IgM-secreting
128 S.D. Skaper

hybridoma cells can be used if an anti-mouse IgM, mu-chain-

specific antibody is used to first coat the dish, as there do not
seem to be many irrelevant IgM antibodies in ascites.
24. If there is no alternative and a polyclonal antiserum or ascites
must be used, then it is still possible to use this antibody for
panning. In this case, however, the panning dish must be
coated with only one antibody (the anti-rabbit or anti-mouse
antibody), and the cell suspension should be incubated with
the rabbit or mouse antibody directly. In this way, the wanted
antibodies will bind to the cell surface and the unwanted ones
can be eliminated by spinning the cell suspension down and
discarding the supernatant (and preferably washing the sus-
pension one more time).
25. The most delicate step in panning is removing the tightly
adhering purified cells from the panning dish. A saturating
amount of antibody can be utilized on the first panning dishes
used for depletion of contaminants from the cell suspension;
however, the concentration of antibody on the final panning
dish is crucial and must be titrated carefully. Cells that are too
tightly bound will be difficult to release viably, while if too
loosely bound, they will wash off (= poor yield). This is particularly
crucial if the antigen is not destroyed by trypsin, as antibodies
are not well cleaved by trypsin. In general, an optimal concen-
tration of antibody causes about half of the adherent cells to
be phase dark and flat (tightly adherent) and the other half
to be phase bright and roundish (less adherent). This can be
determined by a titration experiment in which the yield of
viable cells is optimized; typically, supernatants on the final
dish are diluted 1:20 and ascites about 1:2,000.
26. In general, if the antigen used to purify cells on the final pan-
ning dish is not trypsin sensitive, then the antibody used needs
to be an IgM rather than an IgG. The IgG-bound cells are dif-
ficult to release while retaining viability, perhaps because of its
high affinity for the antigen. If the only antibody available is an
IgG and the antigen is not trypsin sensitive, it is still possible to
purify the cells. In this case, the final dish must be coated with
either protein A or protein G (or a mixture of both) rather
than an anti-mouse IgG antibody. Protein A and protein G are
easily cleaved by trypsin, and the cells are easily released.

References
1. David S, and Aguayo AJ (1981) Axonal elon- in the CNS: implications for therapy. Regen
gation into peripheral nervous system “bridges” Med 3, 907–923
after central nervous system injury in adult rats. 3. Mansour-Robaey S, Clarke DB, Wang YC,
Science 214, 931–933 Bray GM, and Aguayo AJ (1994) Effects of
2. Gervasi NM, Kwok JC, and Fawcett JW (2008) ocular injury and administration of brain-
Role of extracellular factors in axon regeneration derived neurotrophic factor on survival and
 11 Rodent Retinal Ganglion Cell Cultures 129

regrowth of axotomized retinal ganglion cells. 12. Unoki K, and LaVail MM (1994) Protection of
Proc Natl Acad Sci USA 91, 1,632–1,636 the rat retina from ischemic injury by brain-
4. Mey J, and Thanos S (1993) Intravitreal injec- derived neurotrophic factor, ciliary neu-
tions of neurotrophic factors support the sur- rotrophic factor, and basic fibroblast growth
vival of axotomized retinal ganglion cells in factor. Invest Ophthalmol Vis Sci 35,
adult rats in vivo. Brain Res 602, 304–317 907–915
5. Peinado-Ramon P, Salvador M, Villegas-Perez 13. Ma CHM, and Taylor JSH (2010) Trophic
MP, and Vidal-Sanz M (1996) Effects of axo- responsiveness of purified postnatal and adult
tomy and intraocular administration of NT-4, rat retinal ganglion cells. Cell Tissue Res 339,
NT-3, and brain-derived neurotrophic factor 297–310
on the survival of adult rat retinal ganglion 14. Barres BA, Silverstein BE, Corey DP, and Chun
cells. A quantitative in vivo study. Invest LL (1988) Immunological, morphological,
Ophthalmol Vis Sci 37, 489–500 and electrophysiological variation among reti-
6. Clarke DB, Bray GM, and Aguayo AJ (1998) nal ganglion cells purified by panning. Neuron
Prolonged administration of NT-4/5 fails to 1, 791–803
rescue most axotomized retinal ganglion cells 15. Zhang XM, Li Liu DT, Chiang SW, Choy KW,
in adult rats. Vision Res 38, 1,517–1,524 Pang CP, Lam DS, et al (2010) Immunopanning
7. Carmignoto G, Maffei L, Candeo P, Canella R, purification and long-term culture of human
and Comelli C (1989) Effect of NGF on the retinal ganglion cells. Mol Vis 16,
survival of rat retinal ganglion cells following 2,867–2,872
optic nerve section. J Neurosci 9, 1,263–1,272 16. Bader DR, MacLeish PR, and Schwartz EA
8. Lenzi L, Coassin M, Lambiase A, Bonini S, (1978) Responses to light of solitary rod pho-
Amendola T, and Aloe L (2005) Effect of toreceptors isolated from tiger salamander
exogenous administration of nerve growth fac- retina. Proc Natl Acad Sci USA 75,
tor in the retina of rats with inherited retinitis 3,507–3,511
pigmentosa. Vision Res 45, 1,491–1,500 17. Huettner JE, and Baughman RW (1986) Primary
9. Cui Q, Yip HK, Zhao RC, So KF, and Harvey culture of identified neurons from the visual cor-
AR (2003) Intraocular elevation of cyclic AMP tex of postnatal rats. J Neurosci 6, 3,044–3,060
potentiates ciliary neurotrophic factor-induced 18. Wysocki LJ, and Sato VL (1978) “Panning”
regeneration of adult rat retinal ganglion cell for lymphocytes: a method for cell selection.
axons. Mol Cell Neurosci 22, 49–61 Proc Natl Acad Sci USA 75, 2,844–2,848
10. Leaver SG, Cui Q, Plant GW, Arulpragasam A, 19. Brewer GJ, Torricelli JR, Evege EK, and Price
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1,328–1,341 20. Mosmann T (1983) Rapid colorimetric assay
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Blagburn J, and Blanco RE (2005) Fibroblast proliferation and cytotoxicity assays. J Immunol
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ganglion cells by activating the extracellular Varon S (1986) An automated colorimetric
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Neurochem 93, 1,422–1,433 Brain Res 25, 191–198
 Chapter 12

Culture of Purified Glial Cell Populations from Optic Nerve

Stephen D. Skaper

Abstract
Glial cells play a key role in nervous system function, providing neurotrophic factor support to neurons as
well as taking part in two-way neuron-glia signaling (e.g., neurotransmitter release). White matter-derived
glia are important in certain neurodegenerative diseases involving axonal loss, for example in multiple scle-
rosis. Here we describe procedures for the preparation and culture of mixed nerve cells from postnatal rat
optic nerve, followed by protocols which can serve for the purification of individual populations of glia
from this tissue, namely O2A progenitors and oligodendrocytes, and astrocytes and astrocyte precursors.

Key words: Optic nerve, O2A progenitors, Oligodendrocytes, Astrocytes, Astrocyte precursors,
Immunopanning, Trophic factors, Differentiation, Cell culture

1. Introduction

Recent findings suggest that glial cells, though lacking the excitability
usually associated with most neurons, may be more actively involved
in brain function than has been previously thought. Collectively,
these findings indicate that glial cells can sense, and potentially
respond to, a large array of neuronal signals. Because glial cells are
intimately associated with most neurons, neurobiologists need to
consider the possible significance of active neuronal-glial signaling.
Although a variety of glial preparations have been studied, the
basic findings are common and are exemplified by studies of rat
optic nerve glia (1–3). Cultures of cells isolated from postnatal
optic nerves contain four types of macroglial cells that can be distin-
guished by their distinct antigenic phenotypes and morphologies:
type 1 astrocytes, type 2 astrocytes, oligodendrocytes, and a progeni-
tor cell, termed the O2A progenitor (4–6). The O2A progenitors
are developmentally bipotential, giving rise to both oligodendro-
cytes and type 2 astrocytes in vitro (7, 8). O2A progenitors persist

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_12, © Springer Science+Business Media, LLC 2012

131
132 S.D. Skaper

in adult animals (9, 10) in large numbers (11). These cell types are
not thought to comprise all kinds of central nervous system glia;
rather, they may be restricted to the kinds of glia present in white
matter. As such, they may be of considerable utility in the study of
white matter disease processes affecting the visual system. For
example, multiple sclerosis is characterized by optic nerve lesions
(12). This chapter details the preparation of mixed optic nerve cells
from postnatal or embryonic rat tissue, and serves as the “point of
departure” for subsequent methodology dealing with the purifica-
tion of oligodendrocytes, O2A progenitors, astrocytes, and astrocyte
precursor cells of optic nerve origin.

2. Materials

2.1. Equipment 1. Stereo dissecting microscope (backlighting of stage is pre-

and Labware ferred) with fiber optic light source.
2. Laminar flow cabinet for dissections.
3. Laminar flow biological safety cabinet (CL2).
4. Humidified, water-jacketed culture incubator at 37°C and 5%
CO2/95% air.
5. Water bath set at 37°C.
6. Dissecting tools (Fine Science Tools).
7. Benchtop centrifuge to accommodate 15- and 50-mL tubes.
8. 15- and 50-mL polypropylene plastic centrifuge tubes (sterile).
9. 12-mL test tubes, sterile.
10. 6-mL flat bottom tubes (Bijou), sterile.
11. 1.5-mL Eppendorf tubes.
12. 10-cm ∅ sterile tissue culture dishes.
13. 6- and 10-cm ∅ sterile Petri culture dishes.
14. #21- and #23-guage hypodermic needles.
15. 5- and 10-mL syringes.
16. Nitex mesh, 20 mm (Tetko HC3-20).
17. 0.22- and 0.45-mm filters (Millipore).
18. Neubauer hemocytometer (Fisher Scientific).
19. Glass coverslips (12 mm ∅ #1, Menzel-Gläser, Menzel GmbH,
Braunschweig, Germany) (see Note 1).
20. 24-well tissue culture plates.
21. 96-well poly-D-lysine-treated tissue culture plates (BD
Biosciences).
 12 Culture of Purified Glial Cell Populations from Optic Nerve 133

2.2. Reagents 1. Earle’s balanced salt solution (EBSS) (Invitrogen).

2. Dulbecco’s phosphate buffered saline (D-PBS) (Invitrogen).
3. Minimal essential medium with Earle’s salts (MEM) (Invitrogen).
4. L-15 medium with L-glutamine and L-amino acids (Invitrogen).
5. Dulbecco’s modified Eagle’s medium (DMEM), containing
4.5 g/L glucose, L-glutamine, and pyruvate (Invitrogen).
6. DMEM (American Type Culture Collection, ATCC).
7. RPMI-1640 medium (ATCC).
8. Neurobasal-A medium (Invitrogen).
9. B27 supplement, 50×, with antioxidants (Invitrogen).
10. Fetal calf serum (Invitrogen) (see Note 2).
11. Penicillin/streptomycin, 10,000 U/mL penicillin + 10,000 mg/mL
streptomycin (100× stock), sterile, for cell culture (Invitrogen).
12. L-Glutamine (200 mM stock), sterile, for cell culture
(Invitrogen).
13. Sodium pyruvate (100 mM stock), sterile, for cell culture
(Sigma).
14. Collagenase Type 1-S (Sigma).
15. Papain (Worthington (Lorne)).
16. DNAse, Type I (Sigma).
17. Trypsin, 2.5% solution, sterile, for cell culture (Sigma).
18. Trypsin (0.25%)-EDTA, sterile, for cell culture (Invitrogen).
19. Poly-D-lysine (MW 70,000–150,000), sterile, for cell culture
(Sigma).
20. Bovine serum albumin (BSA), essentially fatty acid and globu-
lin free (Sigma).
21. Ovomucoid (trypsin inhibitor from chicken egg white, type
III-O, free of ovoinhibitor) (Sigma).
22. L-Cysteine hydrochloride (Sigma).
23. Insulin, from bovine pancreas (Sigma).
24. Transferrin, human, partially iron saturated, tested for cell cul-
ture (Sigma).
25. Progesterone (Sigma).
26. Putrescine (Sigma).
27. Sodium selenite (Sigma).
28. T3 (3,3¢,5-triiodo-L-thyronine, sodium salt), cell culture tested
(Sigma).
29. Thyroxine (T4) (Sigma).
30. Affinity purified goat anti-mouse IgG + IgM (H + L) (Jackson
ImmunoResearch).
134 S.D. Skaper

31. Affinity-purified goat anti-mouse IgM, mu-chain-specific

(Jackson ImmunoResearch).
32. A2B5 IgM monoclonal antibody, clone 105 (Millipore).
33. RAN-2 IgM hybridoma TIB-119™ (ATCC).
34. Anti-galactocerebroside, clone mGalC1 antibody (Millipore).
35. Thy 1.1 IgM hybridoma TIB-103™ (name T11D7e2) (ATCC).
36. Anti-neuroepithelial C5 antibody (Prof. Ben Barres, Stanford
University).
37. N-acetyl cysteine (Sigma).
38. Ciliary neurotrophic factor, human, recombinant (CNTF)
(Peprotech).
39. Platelet-derived growth factor, human, recombinant
(PDGF-AA) (Peprotech).
40. 8-CPT-2¢-O-Me-cAMP (Merck Biosciences).
41. Isobutylmethylxanthine (IBMX) (Sigma).
42. Forskolin (Sigma).
43. Trypan blue stain 0.4%, for cell culture (Invitrogen).
44. Dimethyl sulfoxide, for cell culture (DMSO) (Sigma).

2.3. Stock Solutions 1. Low ovomucoid solution (10×). Dissolve 300 mg BSA and
300 mg ovomucoid in 20 mL D-PBS; adjust pH to 7.4. Pass
through 0.45-mm and then 0.22-mm filters attached to a 30-mL
syringe. Store in 1 mL aliquots at −20°C.
2. High ovomucoid solution (10×). Dissolve 2 g BSA and 2 g ovo-
mucoid in 20 mL D-PBS; adjust pH to 7.4. Pass through
0.45-mm and then 0.22-mm filters attached to a 30-mL syringe.
Store 0.6 mL aliquots at −20°C (see Note 3).
3. Forskolin (1,000×). Add 2.4 mL DMSO to a 10-mg bottle of
forskolin (=5 mM). Make 100 mL aliquots and store at −20°C.
The final concentration of solvent should not exceed 0.1% in
the culture medium.
4. N-acetyl cysteine (1,000×). Dissolve 50 mg N-acetyl cysteine in
10 mL of double-distilled water. Sterilize by passing through a
0.22-mm filter. Make 20 mL aliquots and store at −20°C.
5. IBMX (100 mM). Dissolve 22 mg IBMX in 10 mL D-PBS.
Sterilize by passing through a 0.22-mm filter. Make 40 mL aliquots
and store at −20°C.
6. Tris pH 9.5 (50 mM). Dissolve 12.1 g Trizma base in 200 mL
double-distilled water. Adjust pH to 9.5 with HCl.
7. Sato 100× stock solution. To 20 mL of Neurobasal-A medium
add the following (final concentrations in brackets): 200 mg
transferrin [100 mg/mL), 200 mg BSA [100 mg/mL], 5 mL of
 12 Culture of Purified Glial Cell Populations from Optic Nerve 135

stock progesterone solution (stock is 2.5 mg/100 mL ethanol)

[60 ng/mL or 0.2 mM], 32 mg putrescine [16 mg/mL],
200 mL of stock sodium selenite (stock is 40 mg in 1 mL of
0.1 N NaOH plus 10 mL Neurobasal-A) [40 ng/mL], 10 mL
of stock T3 (stock is 8 mg in 1 mL of 0.1 N NaOH) [30 ng/mL],
and 10 mL of stock T4 (stock is 8 mg in 1 mL of 0.1 N NaOH)
[40 ng/mL]. Aliquot the above 100× stock solutions (200 mL)
and store at −20°C. The ethanol stocks for progesterone, sodium
selenite, T3, and T4 should be prepared fresh each time a new
batch of 100× Sato medium is made.

2.4. Sato Serum-Free 1. To 20 mL of Neurobasal-A medium add 200 mL Sato 100× stock
Culture Medium solution, 400 mL 50× B27 supplement, 200 mL sodium
pyruvate 100× stock, 200 mL glutamine 100× stock, 200 mL
of 100× penicillin/streptomycin stock (stored at −20°C),
20 mL N-acetyl cysteine 1,000× stock.
2. Next add 200 mL of 100× insulin stock (stored at 4°C; stock
must be prepared every 4–6 weeks—do not freeze). The 100×
insulin stock (0.5 mg/mL) is prepared by adding 2 mg of insulin
to 4 mL of tissue culture grade water containing 20 mL of 1 N
HCl. Filter the stock through a 0.22-mm filter and store at 4°C.
3. Filter the above medium through a 0.22-mm filter. Before fil-
tering the medium, filter about 5 mL of Neurobasal-A medium
to rinse away traces of detergent in the filter. The quality of the
filter matters—Millex-GV from Millipore is best. Falcon plastic
is preferable, along with Monoject syringes.
4. Recommended trophic factors for mixed optic nerve cultures:
Insulin (5 mg/mL) plus CNTF (10 ng/mL). Do not filter the
medium once the growth factors are added due to the risk of
their sticking to the filter. In addition, forskolin (5 mM) can be
included in the medium, as it promotes survival of mixed and
purified cultures.

3. Methods

3.1. Preparing an Optic 1. Prepare suspensions using papain only when they will be
Nerve Cell panned or when older nerves are being dissociated. The pro-
Suspsension Using cedures described here are modified from (12). Otherwise
Papain use the simpler trypsin/collagenase protocol (described
below—Subheading 3.2). Use sterile technique in all steps
(see Notes 4–6).
2. Dissect optic nerves and optic chiasm from postnatal day (P)
P6 to P10 rats (if you intend to collect oligodendrocytes use
P9–P10 animals to obtain a larger yield). Remove the head and
136 S.D. Skaper

skull and scoop the brain out—the optic nerves will be lying on
the base of the skull. Cut the nerves behind the chiasm and
also behind each eye and collect the nerves into a 35-mm Petri
dish containing 2 mL of HEPES-buffered MEM (pH 7.3–7.4)
(can also use D-PBS containing CaCl2 and MgCl2). Expect a
final yield of about 16,000 O2A cells per animal, and base your
rat usage on this (i.e., desired final number of cells).
3. Cut each nerve with a fine scissors or scalpel into as many pieces
as possible (at least 5–10 per nerve). Transfer the MEM (or
D-PBS containing CaCl2 and MgCl2) and nerve pieces to a
bijoux or polystyrene 12-mL test tube.
4. About 10 min before use, add 160 U of papain to 5 mL of
MEM/HEPES plus DNase stock 1:100 (stock is 0.4%) in a
bijou or polystyrene 12-mL test tube. Dissolve the papain by
placing this solution in a 37°C water bath for a few minutes.
Just before use, add L-cysteine (1 mg) to activate the papain
solution, adjust pH to 7.4 (as judged roughly by the color of
the phenol red pH indicator in the MEM, with 1 N NaOH).
Filter the final papain solution through a 0.22-mM filter. Be
sure to rinse the filter first with about 5 mL of MEM solution
to remove any traces of detergent in the filter.
5. Using a Pasteur pipette, remove the MEM/HEPES from the
vial containing the tissue and add the papain solution. Incubate
at 37°C for 75 min for P7 nerves, 90 min for P12 nerves, and
45 min for P0 nerves. Every 15 min gently agitate the tube.
6. Prepare 10 mL of the ovomucoid inhibitor solution: 9 mL of
MEM plus 1 mL of the “LOW” ovomucoid 10× stock solution
(containing 15 mg of BSA and 15 mg of ovomucoid per mL of
MEM) plus 1:100 (100 mL) of the DNase stock solution.
7. Use a Pasteur pipette to gently remove the papain solution.
Wash the optic nerve pieces gently by adding 1 mL of inhibitor
solution, allow the optic nerve pieces to settle, and then remove
the ovomucoid solution with a Pasteur pipette.
8. Serially triturate the nerve pieces in the ovomucoid inhibitor
solution, 1 mL at a time, with a 2.5-mL syringe and #21-gauge
needle × 2 passes, followed by a #23-gauge needle × up to 8
passes. That is, add 1 mL, pass the pieces through the needle 1
or 2 times, let the pieces settle, collect the suspension above
the pieces with a Pasteur pipette into a 10-mL test tube. Add
another mL of inhibitor solution, triturate again, and so on. In
this way, cells successively removed from the nerve pieces are
not repetitively passed through the needle (see Note 7).
9. If the optic nerve pieces fail to completely fall apart, it is likely
that enzyme concentration was too low; enzyme incubation
was too short; and enzyme lot was too old. Now, cap the tube
and gently invert once or twice to mix the cells.
 12 Culture of Purified Glial Cell Populations from Optic Nerve 137

10. Filter the cell suspension through a 20-mm nylon mesh. Nitex
mesh from Tetko works well. Sterilize the mesh before use
(UV exposure for a few minutes is sufficient), prewet the mesh
on the bottom side, then form a cone with the filter on top of
a test tube, and pass 1 mL of MEM or inhibitor solution
through it before using a Pasteur pipette to transfer the cell
suspension through the filter.
11. Transfer a 100-mL sample to a 1.5-mL eppendorf tube for cell
counting (see Chapter 3).
12. Collect the cells by centrifuging at 200 × g for 10 min.
13. Gently remove the supernatant by aspiration. Using a 1-mL
Pipetman, resuspend the cell pellet in another ovomucoid solu-
tion containing 5.4 mL MEM plus 0.6 mL “High” ovomu-
coid 10× stock (contains 60 mg of BSA and 60 mg of
ovomucoid, but NO DNase). To resuspend, first add 1 mL of
this ovomucoid solution, resuspend with the 1-mL pipetman,
then add the remaining 5 mL of the ovomucoid solution, cap
tube, and gently invert once or twice to mix (see Note 8).
14. Centrifuge as in step 11, and aspirate supernatant. Use a 200-
mL pipetman to gently remove the residual solution, leaving
only the cell pellet.
15. Resuspend the cells in Sato serum-free medium at 106 cells per
mL. Yields are typically about 100,000 cells per pair of P7 optic
nerves.
16. Take a 100-mL aliquot for a cell count (see Chapter 3). Spin
down the rest of the cells and resuspend in about 7 mL of L15
or other air buffered solution containing insulin (0.5 mg/mL).
A 100× insulin stock is prepared by dissolving 2 mg of insulin
into 4 mL of H2O containing 20 mL of 1 N HCl (can be stored
at 4°C for 6 weeks). Insulin at high concentration activates
insulin-like growth factor-1 receptors on O2A progenitors and
oligodendrocytes (13), and is an essential survival factor for
O2A cells and oligodendrocytes (14, 15).

3.2. Preparing an Optic 1. Dissect and mince nerves in a 35-mm dish containing EBSS
Nerve Cell Suspension (see Note 9).
Using Trypsin and 2. Prepare enzyme solution containing 4 mL EBSS, 5 mg colla-
Collagenase genase, and 200 mL trypsin stock (stock is 2.5% in EBSS or
D-PBS and is stored at −70°C). Filter through a 0.22-mm filter.
Add DNase 1:100 from stock (stock is 0.4% in EBSS, stored at
−20°C).
3. Add nerves to trypsin solution in a bijou (or polystyrene 12-mL
test tube) and incubate for 20 min in a water bath at 37°C.
4. After this time add another 200 mL trypsin stock and incubate
again for 20 min. (incubate 20 min for P7 nerves and 10–15 min
138 S.D. Skaper

for P0 nerves). During all incubations agitate gently every

10 min.
5. Remove by aspiration the trypsin-containing supernatant, leav-
ing optic nerves behind.
6. Serial triturate with 2 mL of 10% fetal calf serum in DMEM
(#21 needle for two passes, #23 needle for several more, until
all nerves are dissociated).
7. Centrifuge at 200 × g for 10 min.
8. Resuspend in Sato serum-free medium and culture.

3.3. Adult Optic Nerve 1. Dissect (6–8 adult female rats) nerves into D-PBS, and cut
Dissociation each of the nerves into 15 pieces.
(Collagenase 2. Add collagenase (333 U/mL) in 4 mL of D-PBS and pass
and Trypsin) through a 0.22-mM filter. Incubate nerve pieces with this
solution at 37°C for 1 h.
3. Add 4 mL of trypsin (30,000 U/mL) in D-PBS; incubate at
37°C for 20 min.
4. Discard the trypsin solution and add 4 mL of 15,000 U/mL
trypsin in D-PBS with 0.27 mM EDTA; incubate at 37°C for
20 min.
5. Add an equal volume of 20% fetal calf serum in D-PBS (with
Ca2+, Mg2+) containing 0.08% DNase. Incubate at 37°C for
10 min.
6. Triturate with a 1-mL pipette (2×) before triturating with 21#
needle and 23# needle in low ovomucoid.
7. Wash in high ovomucoid as usual.

3.4. Preparation of 1. Dissociate the nerves with 0.125% trypsin in 4 mL D-PBS con-
Embryonic Optic Nerve taining 50 mL of 4% DNase stock.
Cells Using Trypsin 2. Incubate at 37°C for 30 min.
3. Triturate in 10% fetal calf serum buffer: (1 mL fetal calf serum,
9 mL D-PBS, 100 mL 4% DNase stock) using #21- and #23-
gauge needles as described above in Subheading 3.3.
4. Centrifuge at 200 × g for 10 min.
5. Resuspend in Sato serum-free medium and culture as desired.

3.5. Purification To purify O2A progenitors, an optic nerve cell suspension is pre-
Procedure for O2A pared by a papain dissociation procedure (modified from (16)) and
Progenitors passed sequentially over three antibody-coated “panning” (17)
dishes (papain must be used and not trypsin, to maximize cell yield
and to avoid destroying the RAN-2 surface antigen). First, a RAN-
2-coated dish is used which depletes the suspension of macrophages
(which stick to the first antibody-coated dish they are placed on),
meningeal cells, and type-1 astrocytes. In addition, about 20% of the
 12 Culture of Purified Glial Cell Populations from Optic Nerve 139

O2A progenitors adhere to the RAN-2 dish. The nonadherent cells

are then transferred to a galactocerebroside-coated dish, to which
only oligodendrocytes adhere. Lastly, the remaining nonadherent
cells are transferred to an A2B5 dish. Only O2A progenitors adhere
to this dish. The O2A progenitors can be cultured directly on this
dish or can be removed with trypsin for culture on coverslips.
Provided all three panning steps are used, O2A progenitors can be
purified to near-complete homogeneity. If the RAN-2 dish is omit-
ted, a purification of 99.5% is achieved (1 in 200 cells have a flat
morphology and are vimentin immunoreactive, identifying them as
meningeal or type-1 astrocyte contaminants). If the galactocerebro-
side dish is omitted, the A2B5 dish will be contaminated by newly
formed oligodendrocytes which are still A2B5 immunopositive.

3.5.1. Preparation 1. Incubate two 10-cm ∅ Petri dishes overnight at 4°C in 10 mL

of Panning Dishes of 50 mM Tris (pH 9.5) containing 10 mg/mL of affinity-
purified goat anti-mouse IgG (H + L), and a third dish with
affinity-purified goat anti-mouse IgM, mu-chain-specific.
Affinity-purified antibodies must be used, so that irrelevant
proteins do not saturate most of the binding sites on the dish
(this is also the reason why the primary antibodies cannot be
used as a single layer on panning dishes) (see Note 10).
2. Before the papain dissociation, rinse each Petri dish 3 or 4
times with D-PBS.
3. Add the primary antibody in a D-PBS/BSA (0.2%, v/v) solu-
tion for at least 1 h. Dish 1: RAN-2 supernatant (1 mL) plus
D-PBS/BSA (4 mL). Dish 2: galactocerebroside antibody
(5 mg/mL) diluted in 4 mL of PBS/BSA. Dish 3: A2B5 anti-
body (5 mg/mL) diluted in 4 mL of PBS/BSA. In this case,
the third Petri dish can also be coated with goat anti-mouse
IgG (H + L), as there will be no irrelevant IgGs to compete
with the A2B5 for binding sites.
4. Rinse each dish 3 times with D-PBS.
5. Add enough D-PBS to coat the dish; remove the PBS just
before use.

3.5.2. Incubation 1. Transfer the cell suspension to the RAN-2 antibody-coated

of the Cell Suspension Petri dish(es) and leave for 30 min (rock gently after 15 min
on the Panning Dishes to ensure access of all cells to the panning surface area) (see
Note 11).
2. Transfer the nonadherent cells to the galactocerebroside
antibody-coated Petri dish for 45 min (agitate gently every
15 min). Only oligodendrocytes will stick to this plate, so if
you want pure oligodendrocytes wash and save this plate
(see below). This step depletes the suspension of virtually
all oligodendrocytes.
140 S.D. Skaper

3. Transfer the nonadherent cells to the A2B5 antibody-coated

Petri dish and incubate for 45 min (agitate gently every 15 min)
(see Note 12).
4. Wash off nonadherent cells from the A2B5 dish with MEM/
HEPES (about 10 mL per wash) or other air-buffered solution
about 5 times (follow progress under microscope), being care-
ful not to let the adherent cells dry out. During these washes,
periodically rock (agitate) the dish. This last step is critical, as
too much rocking will wash some of the O2A cells off the dish,
while too little will leave contaminating nonadherent cells.

3.5.3. Removing O2A Cells 1. Prepare 4 mL of EBSS (preequilibrated as in step 2 below);

from the A2B5 Dish with add 0.2 mL of 2.5% trypsin.
Trypsin 2. Remove the MEM from the A2B5 dish with adherent O2A
progenitors and rinse once with EBSS which has been pree-
quilibrated in a 6-cm ∅ dish in the CO2 incubator for several
hours (so that its pH is ok).
3. Remove the EBSS wash and add the 4 mL of EBSS/trypsin
(0.125% trypsin final concentration).
4. Incubate in a 37°C incubator for exactly 10 min.
5. Squirt the adherent cells off with a 1-mL pipetman. Be careful
not to overdo this action—check under the microscope to
make sure that the cells were released.
6. Transfer the 4 mL of trypsin/cell suspension to a 15-mL tube
containing 10 mL of 30% fetal calf serum (3 mL of fetal calf
serum plus 7 mL of DMEM). This is the most critical step in
the entire protocol; if the cells are undertrypsinized (most
likely because the batch of trypsin has gone off) they will be
removed from the plate but they will not survive past the first
24 h in culture (see Note 13).
7. Take a 100-mL aliquot for a cell count (see Chapter 3).
8. Centrifuge the cell suspension for 15 min at 200 × g.
9. Traces of insulin or serum can be washed out by resuspending
the cells and washing one last time in L15 or MEM containing
0.5% BSA. It is essential to include this much BSA in any fur-
ther washes; at this point the purified cells will not survive
washing without it.
10. Culture the O2A progenitors on poly-D-lysine-coated glass
coverslips (12 mm ∅; at least 5,000 cells/coverslip) in 96-well
plates in Sato medium, 100 mL, containing PDGF-AA at 5 ng/mL.
See Subheading 3.9.1 for coating of coverslips.
11. Expected yield: 15,000–20,000 O2A cells per P8 rat.

3.6. Adult O2A Panning The panning procedure for adult O2As is very similar to that for
Procedure perinatal O2A progenitors. There are a few differences, as noted
 12 Culture of Purified Glial Cell Populations from Optic Nerve 141

below. Apart from these, follow the original panning procedure for
perinatal O2A progenitors for the rest of the procedure. The number
of dishes and amounts of reagents are adequate for eight adult rats.
1. Prepare adult optic nerve suspension with collagenase and
trypsin (instead of papain).
2. Prepare 3–10-cm ∅ Petri plastic panning dishes: 2 RAN-2
dishes and 1 A2B5 dish (instead of RAN-2, galactocerebroside,
and A2B5 dishes): Add to each dish 12 mL of the Tris pH 9.5
buffer plus 40 mL of the affinity-purified antibody (anti-mouse
IgG + IgM antibody for the RAN-2 dish, and anti-mouse IgM
for the A2B5 dish).
3. Incubate the cell suspension on the panning dish as follows:
RAN-2 dish, 30 min; A2B5 dish, 45 min.
4. To remove the purified O2A cells from the A2B5 dish with
trypsin, use trypsin from Gibco (instead of Sigma).
5. To determine the cell yield, 10 mL (instead of 100 mL) of cell
suspension is directly dropped on a culture dish (three separate
drops for averaging). Let the cells settle down to the bottom of
the dish. Count the total cell number in the drop. Yield = num-
ber of cells in the drop × 100 × number of mL.

3.7. Purifying The simplest way is to let the purified O2A progenitors differenti-
Oligodendrocytes ate into oligodendrocytes by culturing for 2 days in survival factors
Instead of O2A (CNTF, insulin, forskolin) but not mitogens. To use the purified
Progenitors oligodendrocytes on the galactocerebroside dish, use O1 instead
of galactocerebroside antibodies to prepare this panning dish (O1
is an IgM, galactocerebroside is an IgG; it is easier to remove cells
from IgM-coated dishes).

3.8. Purification The following protocols are similar to the protocols for purifying
of Astrocytes and oligodendrocyte lineage cells. Only the modifications are described
Astrocyte Precursor below.
Cells from Rat Optic
Nerve

3.8.1. Astrocyte 1. Dissociate the minced optic nerves with 160 U papain in 5 mL
Purification from D-PBS with DNase and L-cysteine, and incubate at 37°C for
Perinatal Optic Nerves 45 min (P1), 60 min (P4), or 75 min (P8).
2. Triturate the tissue as for purification of O2A progenitors.
3. For panning, coat the first dish with a mouse monoclonal
MRC-OX7 anti-Thy1.1 antibody ((5 mg/mL) diluted in 4 mL
of PBS/BSA for a 10-cm ∅ dish) to deplete macrophages and
fibroblasts.
4. Coat the second dish with the mouse monoclonal anti-
A2B5 antibody (for P1). For purification from P4 or P8 optic
142 S.D. Skaper

nerves, the second dish is also coated with the monoclonal

anti-galactocerebroside antibody (to remove oligodendrocytes).
5. Coat the third and last dish with a mouse monoclonal anti-
neuroepithelial C5 antibody. The astrocytes will bind to the
C5 dish. Astrocytes can then be cultured directly on the C5
dish, or removed with trypsin.

3.8.2. Purification of 1. Dissociate the nerves with 80 U of papain in 5 mL of D-PBS

Astrocyte Precursor Cells with DNase and L-cysteine, at 37°C for 30 min.
from Embryonic Day 17 2. Triturate the tissue as for purification of O2A progenitors.
Optic Nerves
3. For panning, coat the first dish with Thy1.1 supernatant
(1 mL) plus 4 mL of PBS/BSA for a 10-cm ∅ dish.
4. Coat the second dish with a mouse monoclonal anti-neuroep-
ithelial C5 antibody. Astrocyte precursor cells will bind to the
C5 dish. The A2B5 antibody dish is omitted because the
astrocyte precursor cells are A2B5 positive, as are the O2A
progenitors.
5. Astrocyte precursor cells can then be cultured directly on C5
dish, or removed with trypsin.

3.9. Culturing Optic 1. Soak the desired quantity of 12-mm ∅ glass coverslips in 70%
Nerve Cells ethanol (prepared using MilliQ water) for at least 24 h prior to
use. Rinse with clean 70% ethanol and store in 70% ethanol
3.9.1. Coating Glass
until use.
Coverslips with
Poly-D-Lysine 2. Use sterile forceps to remove coverslips and place these lying
flat, but not overlapping each other, in a 10-cm ∅ Petri dish
containing autoclaved MilliQ water. Rinse several times with
this water to remove ethanol.
3. Add enough poly-D-lysine solution (dilute the 1 mg/mL poly-
D-lysine stock, which should be stored at −20°C, to 10 mg/mL
in autoclaved MilliQ water) to cover the slips (see Note 14).
4. Incubate the coverslips with poly-D-lysine for a minimum of
30–60 min.
5. Using a sterile forceps, individually pick up each coverslip, rinse
3 times with sterile MilliQ water (6-cm ∅ Petri dish works fine
for this), and place a single coverslip in each well of a 24-well
tissue culture plate.
6. Remove by aspiration any residual water in each well and allow
the coverslips to air dry in a laminar flow hood.

3.9.2. Plating Optic Nerve 1. Transfer 20 mL of the cell suspension (resuspended in Sato serum-
Cell Suspensions free medium, as described in preparation of the cell suspension)
onto the center of each poly-D-lysine-coated coverslip.
2. Incubate the multiwell plate with coverslips in a 5% CO2/95%
air incubator (37°C) for 15 min to allow the cells to adhere.
 12 Culture of Purified Glial Cell Populations from Optic Nerve 143

3. Add 500 mL of plating medium to each well, and return the

plate(s) to the CO2 incubator.

3.10. Culturing Purified 1. Culture medium: To 20 mL of DMEM add 200 mL of Sato

Adult O2A Progenitors 100× stock, 400 mL of 50× B27 supplement, 200 mL of 100×
penicillin/streptomycin stock, 200 mL of 100× insulin stock.
2. Biotin (10 ng/mL final concentration in medium, starting from
a 1,000× stock in D-PBS) enhances long-term survival of puri-
fied adult O2A cells, and its addition is highly recommended.
3. Addition of N-acetyl cycteine is strongly recommended to
enhance survival. N-acetyl cycteine acts a slow release form of
L-cysteine. The DMEM contains cystine but no L-cysteine; this
seems to be crucial for the cells to maintain high intracellular
glutathione levels.
4. Peptide factors: PDGF-AA (10 ng/mL), CNTF (10 ng/mL),
8-CPT-2¢-O-Me-cAMP (125 mM) (or 10 mM forskolin and
0.1 mM IBMX). 8-CPT-2¢-O-Me-cAMP is a cAMP agonist,
while forskolin activates adenylylcyclase and IBMX inhibits
cyclic AMP phosphodiesterase. These agents act to elevate
intracellular levels of cyclic AMP, which promotes survival of
O2A progenitors.
5. To plate cells on coverslips, preplate cells (3,000–5,000 cells in
20–30 mL) on each poly-D-lysine-coated coverslip and let the
cells settle for 10 min before adding 500 mL of medium.
6. To plate cells on dish, dilute cells to about 30,000/1 mL, plate
100 mL on a poly-D-lysine-coated 6-cm ∅ dish and immediately
spread the medium drop with cells evenly on the dish using a
glass Pasteur pipette that had been bent at a 90° angle by heating.
Allow the cells to settle in the 37°C incubator for 10–15 min
before adding 2.5 mL of medium.

4. Notes

1. The source of glass is very important for the coverslips: German

suppliers are highly recommended, in particular the one listed
here.
2. Heat inactivation of fetal calf serum is recommended to destroy
heat-labile complement. Thaw the bottle of serum in advance,
using a 37°C water bath. Next, immerse the serum bottle in
the water bath after reequilibrating to 56°C and leave for
30 min. Swirl the bottle occasionally to ensure proper mixing.
Allow the serum to cool to room temperature, aliquot into
50-mL tubes and store at −20°C.
144 S.D. Skaper

3. If you encounter difficulty dissolving the BSA and ovomucoid

for the “HIGH” ovomucoid stock solution, it’s ok also to prepare
a 5× stock and freeze down aliquots of 1.2 mL.
4. Fresh papain should be obtained every month.
5. Never freeze the papain stock solution.
6. Never prepare papain in L15 medium since it inhibits the
papain.
7. The quality of the syringe plastic matters, as plastic stabilizers
can reach out and cause toxicity, especially when using serum-
free medium.
8. Never shake tubes containing a cell suspension.
9. Hank’s balanced salt solution (lacking calcium and magne-
sium) can be used in place of EBSS.
10. Rabbit antisera and IgG ascites can generally not be used for
panning procedures, as irrelevant IgGs contributed by these
would compete with the A2B5 for binding sites. This is a com-
mon mistake in immunopanning.
11. If optic nerves from more than 25 animals are used, transfer
the cells to another RAN-2 panning dish for 20 min. The
RAN-2 dishes eliminate meningeal and astrocyte contamina-
tion of the final, A2B5 dish. However, it does not deplete the
suspension of all astrocytes, as many astrocytes in the optic
chiasm (but not nerve) are RAN-2 negative.
12. If you want to recover live O2A cells from the A2B5 dish, the
A2B5 concentration must be titered so that it is low enough
to easily remove the cells with trypsin, but not so low that cells
don’t adhere strongly enough to remain once nonadherent
cells are washed from the dish.
13. O2A progenitors from the A5B5 dish can be cultured directly
on this dish, if there is no need to culture on particular surfaces
(e.g., coverslips) where detachment and cell counting is called
for.
14. Only use poly-D-lysine which is cell culture grade.

References

1. Barres BA, Chun LLY, and Corey DP (1988) white matter glia: the type-l astrocyte. Neuron
Ion channel expression by white matter glia: I. 5, 527–544
Type 2 astrocytes and oligodendrocytes. Glia 4. Raff MC, Abney ER, Cohen J, Lindsay R, and
1, 10–30 Noble M (1983) Two types of astrocytes in
2. Barres B.A, Koroshetz WJ, Swartz KJ, Chun cultures of developing rat white matter: differ-
LLY, and Corey DP (1990) Ion channel ences in morphology, surface gangliosides, and
expression by white matter glia: the 02A glial growth characteristics. J Neurosci 3, 1289–l 300
progenitor cell. Neuron 4, 507–524 5. Miller RH, and Raff MC (1984) Fibrous and
3. Barres BA, Koroshetz WJ, Chun LLY, and protoplasmic astrocytes are biochemically and
Corey DP (1990) Ion channel expression by developmentally distinct. J Neurosci 4, 585–592
 12 Culture of Purified Glial Cell Populations from Optic Nerve 145

6. Miller RH, ffrench-Constant C, and Raff MC 13. Zhao M, Wan ZL, Whittaker L, Xu B, Phillips
(1989) The macroglial cells of the rat optic NB, Katsoyannis PG, et al (2009) Design of an
nerve. Annu Rev Neurosci 12, 517–534 insulin analog with enhanced receptor binding
7. Raff MC, Miller RH, and Noble M (1983) A selectivity: rationale, structure, and therapeutic
glial progenitor cell that develops in vitro implications. J Biol Chem 284, 32178–32187
into an astrocyte or an oligodendrocyte 14. Pang Y, Zheng B, Fan LW, Rhodes PG, and
depending on culture medium. Nature 303, Cai Z (2007) IGF-1 protects oligodendrocyte
390–396 progenitors against TNF a-induced damage by
8. Raff MC, Williams BP, and Miller RH (1984) activation of PI3K/Akt and interruption of the
The in vitro differentiation of a bipotential glial mitochondrial apoptotic pathway. Glia 55,
progenitor cell. EMBO J 3, 1857–l864 1099–1107
9. ffrench-Constant C, and Raff MC (1986) The 15. Wilczak N, Chesik D, Hoekstra D, and De
oligodendrocyte-type 2 astrocyte cell lineage Keyser J (2008) IGF binding protein altera-
is specialized for myelination. Nature 323, tions on periplaque oligodendrocytes in multi-
335–338 ple sclerosis: implications for remyelination.
10. Wolswijk G, and Noble M (1989) Identification Neurochem Int 52, 1431–1435
of an adult-specific glial progenitor cell. 16. Huettner JE and Baughman RW (1986)
Development 105, 387–400 Primary culture of identified neurons from the
11. Barres BA (1991) New roles for glia. J Neurosci visual cortex of postnatal rats. J Neurosci 6,
11, 3685–3694 3044–3060
12. Green AJ, McQuaid S, Hauser SL, Allen IV, 17. Williams DL, Lo JL, and Amborski JF (1986)
and Lyness R (2010) Ocular pathology in mul- Enrichment of T lymphocytes from bovine periph-
tiple sclerosis: retinal atrophy and inflammation eral blood mononuclear cells using immuno-
irrespective of disease duration. Brain 133, affinity depletion technique (“panning”). Vet
1591–1601 Immunol Immunopathol 11, 199–204
 Chapter 13

Isolation and Culture of Rat Cone Photoreceptor Cells

Stephen D. Skaper

Abstract
In retinal diseases characterized by photoreceptor degeneration, the main cause of clinically significant
vision loss is cone, rather than rod, loss. Photoreceptor apoptosis can be affected by the availability and/
or delivery of neurotrophic proteins, and cultures of photoreceptors are valuable for studying these pro-
cesses. In the present study, a technique was designed to purify cones to make it possible to screen for
neuroprotective molecules. The present chapter describes a technique for preparing cultures of purified rat
retina cone photoreceptors based upon panning with peanut agglutinin lectin, which selectively binds to
cones. In addition, we describe a protocol for the purification and culture of retinal pigmented epithelial
cells from postnatal rat.

Key words: Cone photoreceptors, Retina, Pigmented epithelial cells, Rat, Cell culture, Neurotrophic
factors

1. Introduction

Photoreceptors of the retina are highly specialized neurons that

transduce light stimuli to membrane potential changes signaled to
second-order neurons. These cells are essential for normal vision,
but they degenerate in a number of conditions, including genetic
diseases such as retinitis pigmentosa (1), environmental insults
such as light damage (2), and as a result of normal aging (3, 4).
Although most of the mutations responsible for retinitis pigmen-
tosa in humans and animal models affect rod photoreceptor–
specific genes, rod apoptosis is followed by secondary cone
degeneration. Photoreceptor rescue or neuroprotection are topics
of great current interest that are necessary for formulating thera-
peutic approaches (5, 6).
Nonmammalian vertebrates have the remarkable ability to
replace neurons lost through damage. Fish, and to a limited extent
birds, replace lost neurons by the dedifferentiation of Müller glia

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_13, © Springer Science+Business Media, LLC 2012

147
148 S.D. Skaper

to a progenitor state followed by the replication of these neuronal

progenitor cells (7). Recent studies have investigated whether
regeneration can be stimulated in the mouse and rat retina. Several
groups have reported that at least some types of neurons can be
regenerated in the mammalian retina in vivo or in vitro and that
the regeneration of neurons can be stimulated using growth
factors, transcription factors, or subtoxic levels of excitatory amino
acids. For example, basic fibroblast growth factor supports survival
of cultured rat (8) and porcine (9) photoreceptors and delays pho-
toreceptor degeneration in inherited retinal dystrophy in rats (10).
Ciliary neurotrophic factor alone (11, 12) or in combination with
brain-derived neurotrophic factor (13) promotes photoreceptor
development and survival. An analog of ciliary neurotrophic factor
(Axokine) delayed photoreceptor loss in a feline model of heredi-
tary retinal degeneration (14).
Retinal pigmented epithelial (RPE) cells are melanin-containing
cells that constitute a monolayer between the neural retina and the
choroid. RPE cells play a key role in maintaining the normal func-
tion of retina and can express several neurotrophic factors such as
platelet-derived growth factor, epidermal growth factor, vascular
endothelial growth factor, and pigment-derived epithelial factor
(15). In addition, RPE cells secrete glial cell line–derived neu-
rotrophic factor and brain-derived neurotrophic factor (BDNF)
(16). Delivery of pigment epithelium–derived factor is protective
in experimental animal models of retinal degeneration involving
photoreceptors (17–19). The identification of other cone survival
factors and screening for factors supporting their survival requires
the availability of purified cultures of mammalian cones. The following
chapter describes a detailed protocol for the culture of cone pho-
toreceptors from rat retina based on a peanut agglutinin (PNA)
lectin–panning procedure that allows their selective isolation. The
utility of the technique benefits from the fact that PNA lectin binds
to cone photoreceptors from various species in both normal and
pathologic conditions. In addition, the preparation of RPE cells
from rat is described.

2. Materials

2.1. Equipment 1. Stereo dissecting microscope (backlighting of stage is pre-

and Labware ferred) with fiber optic light source.
2. Laminar flow cabinet for dissections.
3. Laminar flow biological safety cabinet (CL2)
4. Humidified, water-jacketed culture incubator at 37°C and 5%
CO2/95% air.
5. Water bath set at 37°C.
 13 Isolation and Culture of Rat Cone Photoreceptor Cells 149

6. Dissecting tools (Fine Science Tools).

7. Bench centrifuge to accommodate 15- and 50-mL tubes.
8. 15- and 50-mL polypropylene plastic centrifuge tubes (sterile).
9. 10-cm ∅ sterile tissue culture dishes (any supplier)
10. 3.5-cm ∅ and 10-cm ∅ sterile petri plastic culture dishes (any
supplier).
11. Glass coverslips (12 mm ∅ #1, Menzel-Gläser, Menzel GmbH,
Braunschweig, Germany) (see Note 1)
12. 96-well poly-D-lysine-treated tissue culture plates (BD
Biosciences).
13. 0.22-μm filters (Millipore).
14. Neubauer hemocytometer (Fisher Scientific).

2.2. Reagents 1. Dulbecco’s phosphate-buffered saline, pH 7.4, sterile (DPBS)

(Invitrogen).
2. Water, sterile, tissue culture grade.
3. L-15 medium, with L-glutamine and L-amino acids (Invitrogen).
4. Neurobasal-A medium (Invitrogen).
5. B27 supplement, 50×, with antioxidants (Invitrogen).
6. Fetal calf serum (FCS) (Invitrogen) (see Note 2).
7. Penicillin/streptomycin (100× stock), sterile, for cell culture
(Invitrogen).
8. Gentamicin (50 mg/mL stock), sterile, for cell culture (Sigma).
9. Papain (Worthington (Lorne)).
10. DNase, type I (Sigma).
11. Trypsin, 2.5% solution, sterile, for cell culture (Sigma).
12. Collagenase (Sigma).
13. Poly-D-lysine (MW 30,000–70,000), sterile, for cell culture
(Sigma).
14. Metrizamide (Sigma).
15. Merosin, human (Millipore).
16. Goat antirabbit IgG directed against PNA lectin (Sigma).
17. PNA lectin (affinity-purified) (Sigma).
18. Affinity-purified goat antimouse IgG + IgM (H + L) (Jackson
ImmunoResearch).
19. CD90/Thy1 antibody (MRC OX-7) (Abcam).
20. Bovine serum albumin, essentially fatty acid and globulin free,
(BSA) (Sigma).
21. Ovomucoid (trypsin inhibitor from chicken egg white, type
III-O, free of ovoinhibitor) (Sigma).
150 S.D. Skaper

22. L-Cysteine.
23. Insulin, from bovine pancreas (Sigma).
24. Transferrin, human.
25. Progesterone.
26. Putrescine.
27. Sodium selenite.
28. T3 (3,3¢,5-triiodo-L-thyronine, sodium salt), cell culture–
tested (Sigma).
29. Sodium pyruvate (100 mM stock), sterile, for cell culture (Sigma).
30. L-Glutamine (200 mM stock), sterile, for cell culture
(Invitrogen).
31. BDNF, human, recombinant (Peprotech).
32. CNTF, human, recombinant (CNTF) (Peprotech).
33. Forskolin.
34. N-acetyl cysteine.
35. Phenol red (0.5%), sterile, for cell culture (Sigma).

2.3. Stock Solutions 1. 10× EBSS stock solution without Ca2+, Mg2+, and bicarbonate.
1.16 M NaCl (68 g/L), 54 mM KCl (4 g/L), 10 mM
NaH2PO4·H2O (1.4 g/L), 10 mM D-glucose (10 g/L), and
0.05% phenol red (10 mL/L of 0.5% solution). Filter-sterilize
and store at 4°C.
2. D-Glucose, 30% (w/v). Dissolve 15 g of D-glucose in 50 mL
double-distilled water.
3. NaHCO3, 1 M. Dissolve 4.2 g of NaHCO3 in 50 mL double-
distilled water.
4. MgSO4, 100 mM. Dissolve 1.23 g of MgSO4·7H2O in 50 mL
double-distilled water.
5. EDTA, 0.5 M. Dissolve 7.3 g of EDTA in 50 mL double-dis-
tilled water.
6. EBSS-papain solution with Mg2+, EDTA, and bicarbonate.
Combine 20 mL of 10× EBSS solution, 2 mL of 100 mM
MgSO4, 2.4 mL of 30% D-glucose, 0.4 mL of 0.5 M EDTA,
and 5.2 mL of 1 M NaHCO3. Bring to a final volume of
200 mL with double-distilled water.
7. Borate buffer 0.15 M, pH 8.4. Dissolve 28.6 g of sodium borate
(Na2B4O7·10H2O) in 500 mL double-distilled water (pH will
be ~9.2). Adjust pH to 8.4 with 5 N HCl. Filter-sterilize and
store at 4°C (up to 6 months).
8. Low ovomucoid solution. Mix together 9 mL DPBS, 1 mL 10×
low ovomucoid stock (15 mg each BSA and ovomucoid/mL
DPBS), and 200 μL 0.4% DNase stock (made by dissolving
 13 Isolation and Culture of Rat Cone Photoreceptor Cells 151

4 mg/mL of DNase in L-15 medium; filter-sterilize, aliquot,

and store at −20°C). Check pH and adjust to 7.4 if necessary.
Filter-sterilize the final solution.
9. High ovomucoid solution. Mix together 4.8 mL DPBS and
1.2 mL 6× high ovomucoid stock (50 mg each BSA and ovo-
mucoid/mL DPBS). Filter-sterilize.
10. Forskolin (1,000×). Add 2.4 mL dimethyl sulfoxide to a 10-mg
bottle of forskolin (=5 mM). Make 100 μL aliquots and store
at −20°C. The final concentration of solvent should not exceed
0.1% in the culture medium.
11. N-acetyl cysteine (1,000×). Dissolve 60 mg N-acetyl cysteine in
1 mL Neurobasal-A medium. Filter-sterilize, make 20 μL
aliquots, and store at −20°C.
12. Metrizamide (prepare day before experiment). Prepare a 20%
(1 g per 5 mL) metrizamide solution in L-15 medium. Prepare
a 40% (2 g in 5 mL) metrizamide solution in DPBS. Filter each
of the above solutions using a 0.22-μm filter attached to a
5-mL syringe. Prepare 5 mL of each solution (will keep up to
1 month at 4°C).
13. Sato 100× stock solution. To 20 mL of Neurobasal-A medium,
add the following: 200 mg transferrin (100 μg/mL), 200 mg
BSA (100 μg/mL), 5 μL of stock progesterone solution (stock
is 2.5 mg/100 μL ethanol) (final = 60 ng/mL or 0.2 μM),
32 mg putrescine (16 μg/mL), 200 μL of stock sodium sele-
nite (stock is 40 mg in 1 mL of 0.1 N NaOH plus 10 mL
Neurobasal-A) (final = 40 ng/mL), and 10 μL of stock T3
(stock is 8 mg in 1 mL of 0.1 N NaOH) (final = 30 ng/mL).
Aliquot the above 100× stock solutions (200 μL) and store at
−20°C. The ethanol stocks for progesterone, sodium selenite,
and T3 should be prepared fresh each time a new batch of
100× Sato medium is made.

2.4. High-Insulin Sato 1. To 20 mL of Neurobasal-A medium, add 200 μL Sato 100×

Culture Medium stock solution, 100 μL 50× B27 supplement, 200 μL sodium
pyruvate 100× stock, 200 μL glutamine 100× stock, 200 μL of
100× penicillin/streptomycin stock (stored at −20°C), and
20 μL N-acetyl cysteine 1,000× stock.
2. Next, add 200 μL of 100× insulin stock (stored at 4°C; stock
must be prepared every 4–6 weeks—do not freeze). The 100×
insulin stock (0.5 mg/mL) is prepared by adding 2 mg of insu-
lin to 4 mL of tissue culture grade water containing 20 μL of
1 N HCl. Filter the stock through a 0.22-μm filter and store at
4°C.
3. Filter the above medium through a 0.22-μm filter. Before fil-
tering the medium, filter about 5 mL of Neurobasal-A medium
to rinse away traces of detergent in the filter. The quality of the
152 S.D. Skaper

filter matters—Millex-GV from Millipore—is best. Falcon plas-

tic is preferable, along with Monoject syringes.
4. Before using the medium, add neurotrophic factors (BNDF,
20 μL of a 5-μg/mL stock; CNTF, 20 μL of a 1-μg/mL stock)
and forskolin (5 μM; prepare 1,000× stock in dimethyl sulfox-
ide so that final concentration of solvent does not exceed 0.1%)
to promote cell survival.

2.5. Coating of Culture Merosin only:

Dishes or Coverslips
1. Prepare the merosin coating solution as follows: To 5 mL of
(Prepare Day Before
Neurobasal-A medium, add 10 μL merosin and 50 μL of 100×
Experiment) penicillin/streptomycin stock solution.
2. If using coverslips, first wash 3 times with sterile water.
3. For coating, use 0.4 mL merosin per cm2 surface area (e.g.,
0.5 mL merosin solution per well of a 24-well plate and 0.1 mL
per well of a 96-well plate).
4. Coverslips, if used, can be sterilized by dipping in 70% ethanol
and briefly passing through a flame (see Note 3).
5. Incubate with merosin solution overnight at 37°C.
Poly-D-lysine only:
1. Dissolve a 5-mg bottle of poly-D-lysine in 5 mL borate buffer.
Add 200 μL of this poly-D-lysine stock (1 mg/mL) to 20 mL
tissue culture grade sterile water (see Notes 4 and 5).
2. For coating, use 0.2 mL poly-D-lysine per cm2 surface area.
3. Incubate with poly-D-lysine at room temperature (20–22°C)
for 60 min.
4. Remove poly-D-lysine solution and rinse wells 3 times with
sterile water.
Merosin ± Poly-D-lysine:
1. Coat first with poly-D-lysine, then add merosin, and incubate
overnight.

2.6. Coat Panning 1. Prepare 50 mM Tris by dissolving 0.79 g of Tris in 100 mL

Dishes with Second double-distilled water and adjusting pH to 9.5 with 5 N
Antibody and PNA NaOH.
(Prepare Day Before 2. To a 10-cm ∅ petri dish, add 10 mL of 50 mM Tris, pH 9.5
Experiment) containing 10 μg PNA/mL (PNA dish).
3. To a second 10-cm ∅ petri dish, add 10 mL Tris to OX-7 dish
at 10 μg/mL (Thy-1 dish).
 13 Isolation and Culture of Rat Cone Photoreceptor Cells 153

4. To a third 10-cm ∅ petri dish, add 30 μL of affinity-purified

goat antimouse IgG + IgM (2.4 mg/mL).
5. Incubate all dishes on a flat surface (to ensure uniform coating)
at 4°C overnight.

3. Methods

3.1. Cone 1. Rinse PNA dish 4 times with DPBS (mark side to pour) and
Photoreceptors rinse very well. Store in DPBS until ready to use (in the cold).
3.1.1. Complete 2. Add 1 mL OX-7 + 0.2 mL 4% BSA (40 mg/mL DPBS) + 3 mL
Preparation of Antibody DPBS to a 10-cm ∅ OX-7 dish.
Dishes (Day of Experiment) 3. Incubate the OX-7 dish at room temperature for 2 h on a flat
surface.
4. Rinse the OX-7 dish 3 times with DPBS and keep in DPBS
until needed (in the cold).

3.1.2. Equilibrate EBSS 1. Aliquot 4 mL EBSS (lacking calcium and magnesium) into one
and Sterilize Mesh Filter universal 10-mL tube (bijou) and 6 mL EBSS into another
tube.
2. Leave them to equilibrate in the 37°C 5%CO2/95% air incuba-
tor by leaving caps on loose. These solutions should change
color.
3. Expose the nitex nylon mesh (70-μm pore size) filter to UV
light under the vertical flow culture hood 20 min per side.

3.1.3. Dissociation 1. Dissect out postnatal day P17 mouse or rat retinas. Store
of Retina minced retinas in 35-mm petri dish in DPBS.
2. Equilibrate EBSS-papain (1 mM EDTA) buffer with
5%CO2/95% O2 for 15 min in a 37°C water bath.
3. Dissolve 100 U papain in 5 mL preequilibrated papain buffer.
Add 30 μL of a 2.4-mg/mL L-cysteine solution made up in
DPBS. Sterilize with a 0.22-μm filter. Remove the DPBS and
add retinas to papain by pouring. Add 200 μL of 0.4% DNase
and place in the 37°C bath.
4. Digest tissue for 90 min; gently agitate the tissue every
15 min.
5. Add DNase when needed (cells get sticky and do not settle).
This usually requires 400 μL of 0.4% DNase added over the
90-min period.
6. While waiting, make the following solutions: Panning buffer
containing 15 mL DPBS, 15 μL 100× insulin stock (see high-
insulin Sato medium), and 75 μL of 4% BSA.
154 S.D. Skaper

7. Remove the supernatant (step 5 tissue digestion) by aspiration.

8. Wash the tissue with 4 mL of low ovomucoid solution and
allow the cells to settle. Remove the supernatant and discard.
9. Add 2 mL of low ovomucoid solution and triturate gently 5
times with a 1-mL pipetman.
10. Let cells settle, then transfer the supernatant to a new centri-
fuge tube.
11. Again, add 2 mL of low ovomucoid solution and triturate as
before. In subsequent triturations, use slightly more force until
all cells are in suspension.
12. Centrifuge cells at 200 × g for 15 min at room temperature;
discard the supernatant.
13. Resuspend the cell pellet in high ovomucoid solution and cen-
trifuge at 200 × g for 15 min.
14. Remove the supernatant and resuspend the cell pellet in 1 mL
of panning buffer. Bring the volume to 10 mL.
15. Close the centrifuge tube tightly and incubate in a 37°C water
bath for 90 min to let cells recover.
16. Wet the nitex mesh with leftover panning buffer and build a
filter funnel using forceps.
17. Filter the cells with a 1-mL pipette and bring the volume up to
15 mL.

3.1.4. Panning 1. Remove the DPBS from the OX-7 dish.

2. Add the cell suspension to the dish, keeping this on a flat
surface.
3. Wait 30 min, rocking vigorously after 15 min (but avoid splash-
ing the medium out of the dish). Check for cell adhesion under
the microscope.
4. Remove the DPBS from the PNA dish. First, shake the dish
hard, then transfer the supernatant from the OX-7 dish by
pouring. Use a pipette to remove the remaining cells.
5. Incubate the OX-7 dish for 30 min, shaking gently every 15 min.
6. During this time, make the following: FCS buffer 10 mL
Neurobasal-A medium plus 3 mL heat-inactivated FCS (see
Note 2) and trypsin solution (4 mL preequilibrated EBSS plus
200 μL 2.5% trypsin). Filter-sterilize both solutions.
7. Discard the supernatant from the PNA dish and wash gently
5–7 times with DPBS to remove all nonadhering cells. Do this
by adding 10 mL of DPBS, swirling, and dumping the solu-
tion. Check under the microscope to ensure that all nonadher-
ent cells have been removed.
8. Add 6 mL of preequilibrated EBSS to the dish and discard
quickly.
 13 Isolation and Culture of Rat Cone Photoreceptor Cells 155

9. Add 4 mL of trypsin solution (from step 6) and incubate

10 min in the 37°C incubator.
10. Squirt cells off the dish quickly with a 1-mL pipette (working
around the dish once), then add 2 mL of FCS buffer (from
step 6), and transfer the solution to a 15-mL centrifuge tube.
11. Add 5 mL of FCS buffer to the plate and again pipette once
around. Add the cells recovered from this step to the 15-mL
centrifuge tube in step 10.
12. Invert the centrifuge tube and remove 100 μL to count cells.
13. Centrifuge the tube with cells at 200 × g for 15 min and resus-
pend the cell pellet in high-insulin Sato medium to give a den-
sity of 106 cells/mL.
14. Plate the cells according to the experimental design. If the final
surface coating is merosin, first rinse 3 times with Neurobasal-A
medium before cell plating.

3.2. Culture 1. Cut off anterior portion of eye (make sure to remove all of
of RPE Cells the iris). Remove the lens, vitreous, cornea, and retina.
3.2.1. RPE Dissociation 2. Remove the RPE by pulling the RPE/choroid out of the eye
socket with forceps and store in Ca2+/Mg2+—free DPBS.
Remove the optic nerve.
3. Equilibrate the EBSS-papain (1 mM EDTA) solution with 5%
CO2/95% O2 for 15 min in a 37°C incubator.
4. Dissolve 100 U of papain and 333 U/mL of collagenase in
5 mL preequilibrated papain buffer. Next, add 0.8 mg of
L-cysteine to this solution.

5. Sterilize the solution in point 4 using a 0.22-μm filter. Remove

the DPBS and add retinas to papain by pouring in. Next, add
200 μL 0.4% DNase.
6. Digest the tissue in 37°C bath for 90 min with gentle agitation
of the tissue every 15 min.
7. Remove the supernatant by aspiration, being careful not to
draw up the digested tissue.
8. Wash the tissue with 4 mL of the low ovomucoid solution and
let cells settle.
9. Remove supernatant and discard.
10. Add 2 mL of the low ovomucoid solution. Triturate gently
with a long (9-in.) sterile Pasteur glass pipette using 5 up-and-
down strokes.
11. Let cells settle, remove the supernatant, and transfer the cells
to a new centrifuge tube.
12. Add again 2 mL of low ovomucoid solution and repeat the
trituration step.
156 S.D. Skaper

13. In subsequent trituration steps, use slightly more force until

the RPE is released from the choroid and cells are in
suspension.
14. Choroid does not dissociate and so should be removed and
discarded.
15. Centrifuge the cells at 200 × g for 15 min at 25°C.
16. Discard the supernatant and resuspend the cells in high ovo-
mucoid solution.
17. Centrifuge at 200 × g for 15 min.
18. Remove the high ovomucoid solution but leaving 2 mL to
resuspend the cells.

3.2.2. Purification 1. Using a 15-mL polystyrene centrifuge tube, layer metrizamide

of RPE Cells (6 mL) with 40% on the bottom.
2. Carefully add the 2 mL of high ovomucoid solution contain-
ing cells (see Subheading 3.1, step 18) to the top of the metri-
zamide and centrifuge at 500 × g for 15 min at 20–22°C.
3. Prepare a 4% BSA solution (0.4 g BSA in 10 mL L-15) and
filter-sterilize. This will be used as a cushion for the 40% metri-
zamide layer (step 5 below).
4. Using a hypodermic syringe and needle, carefully remove
about 1 mL of the cell suspension between the 40% layer of
metrizamide and the high ovomucoid solution.
5. Resuspend the 1 mL of metrizamide cell suspension in 6 mL of
L-15.
6. To a fresh 15-mL centrifuge tube, to which has been added 1 mL
of 4% BSA solution, now place the cell suspension from step 5.
7. Centrifuge at 200 × g for 15 min (this step may be difficult
because the metrizamide is very dense).
8. Remove the supernatant and resuspend cells in high-insulin
Sato culture medium.
9. Count cells (see Chap. 3) and plate at a density of 50,000–
100,000 cells cm2.

4. Notes

1. The source of glass is very important for the coverslips:

German suppliers are highly recommended, in particular the
one listed here.
2. Heat inactivation of FCS is recommended to destroy heat-labile
complement. Thaw the bottle of serum in advance, using a 37°C
water bath. Next, immerse the serum bottle in the water bath
 13 Isolation and Culture of Rat Cone Photoreceptor Cells 157

after reequilibrating to 56°C and leave for 30 min. Swirl the

bottle occasionally to ensure proper mixing. Allow the serum
to cool to room temperature, aliquot into 50-mL tubes, and
store at −20°C.
3. When flame-sterilizing cover glasses, it is recommended to
“wave” the cover glass through the air immediately after flam-
ing. Because of their fragile nature, heat buildup may cause
frequent shattering of the glass—hence the need to dissipate
the heat.
4. Poly-D-lysine is very hygroscopic and difficult to weigh. It is
advisable to purchase the material as a 5-mg quantity and then
dissolve the entire amount in borate buffer. The solutions can
be stored at 4°C for up to 6 months.
5. Working solutions of poly amino acids should be prepared in
sterile tissue culture plastic polypropylene tubes. Take care:
The poly amino acid will adhere to glass surfaces and also to
polystyrene plastic.

References
1. Sancho-Pelluz J, Arango-Gonzalez B, progressive photoreceptor degeneration in RCS
Kustermann S, Romero FJ, van Veen T, rats. Eur J Neurosci 22, 1057–1072
Zrenner E, et al (2008) Photoreceptor cell 8. Fontaine V, Kinkl N, Sahel J, Dreyfus H, Hicks
death mechanisms in inherited retinal degen- D (1998) Survival of purified rat photorecep-
eration. Mol Neurobiol 38, 253–269 tors in vitro is stimulated directly by fibroblast
2. LaVail MM, Yasumura D, Matthes MT, Lau- growth factor-2. J Neurosci 18, 9662–9672
Villacorta C, Unoki K., Sung C-H, et al (1998) 9. Traverso V, Kinkl N, Grimm L, Sahel J, Hicks
Protection of mouse photoreceptors by sur- D (2003) Basic fibroblast and epidermal
vival factors in retinal degenerations. Invest growth factors stimulate survival in adult
Ophthalmol Vis Sci 39, 592–602 porcine photoreceptor cell cultures. Invest
3. Gao H, Hollyfield JG (1992) Aging of the Ophthalmol Vis Sci 44, 4550–4558
human retina: differential loss of neurons and 10. Faktorovich EG, Steinberg RH, Yasumura D,
retinal pigment epithelial cells. Invest Matthes MT, LaVail MM (1990) Photoreceptor
Ophthalmol Vis Sci 33, 1–17 degeneration in inherited retinal dystrophy
4. Curcio CA, Allen KA, Kalina RE (1993) delayed by basic fibroblast growth factor.
Aging of the human photoreceptor mosaic: Nature 347, 83–86
evidence for selective vulnerability of rods in 11. Fuhrmann S, Kirsch M, Hofmann HD (1995)
central retina. Invest Ophthalmol Vis Sci 34, Ciliary neurotrophic factor promotes chick
3278–3296 photoreceptor development in vitro.
5. Karl MO, Reh TH (2010) Regenerative medi- Development 121, 2695–2706
cine for retinal diseases: activating endogenous 12. Kassen SC, Thummel R, Campochiaro LA,
repair mechanisms Early changes in synaptic Harding MJ, Bennett NA, Hyde DR (2009)
connectivity following progressive photorecep- CNTF induces photoreceptor neuroprotection
tor degeneration in RCS rats. Trends Mol Med and Müller glial cell proliferation through two
16, 193–202 different signaling pathways in the adult zebrafish
6. Wenzel A, Grimm C, Samardzija M, Remé CE retina. Exp Eye Res 2009 88, 1051–1064
(2005) Molecular mechanisms of light-induced 13. Caffé AR, Söderpalm AK, Holmqvist I, van
photoreceptor apoptosis and neuroprotection Veen T (2001) A combination of CNTF and
for retinal degeneration. Prog Retin Eye Res BDNF rescues rd photoreceptors but changes
24, 275–306 rod differentiation in the presence of RPE in
7. Cuenca N, Pinilla I, Sauvé Y, Lund R (2005) retinal explants. Invest Ophthalmol Vis Sci 42,
Early changes in synaptic connectivity following 275–282
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14. Chong NH, Alexander RA, Waters L, Barnett Inhibition of nuclear translocation of apopto-
KC, Bird AC, Luthert PJ (1999) Repeated sis-inducing factor is an essential mechanism of
injections of a ciliary neurotrophic factor ana- the neuroprotective activity of pigment epithe-
logue leading to long-term photoreceptor sur- lium-derived factor in a rat model of retinal
vival in hereditary retinal degeneration. Invest degeneration. Am J Pathol 173, 1326–1338
Ophthalmol Vis Sci 40, 1298–1305 18. Miyazaki M, Ikeda Y, Yonemitsu Y, Goto Y,
15. Cepeda IL, Flores J, Cornfeldt ML, O’Kusky Kohno R, Murakami Y, et al (2008) Synergistic
JR, Doudet DJ (2007) Human retinal pigment neuroprotective effect via simian lentiviral vec-
epithelial cell implants ameliorate motor defi- tor-mediated simultaneous gene transfer of
cits in two rat models of Parkinson disease. J human pigment epithelium-derived factor and
Neuropathol Exp Neurol 66, 576–584 human fibroblast growth factor-2 in rodent
16. Ming M, Li X, Fan X, Yang D, Li L, Sheng models of retinitis pigmentosa. J Gene Med
Chen S, et al (2009) Retinal pigment epithelial 10, 1273–1281
cells secrete neurotrophic factors and synthe- 19. Miyazaki M, Ikeda Y, Yonemitsu Y, Goto Y,
size dopamine: possible contribution to thera- Sakamoto T, Tabata T, et al (2003) Simian len-
peutic effects of RPE cell transplantation in tiviral vector-mediated retinal gene transfer of
Parkinson’s disease. J Trans Med 2009, 7:53 pigment epithelium-derived factor protects
doi:10.1186/1479-5876-7–53 retinal degeneration and electrical defect in
17. Murakami Y, Ikeda Y, Yonemitsu Y, Onimaru Royal College of Surgeons rats. Gene Ther 10
M, Nakagawa K, Kohno R, et al (2008) 1503–1511
 Chapter 14

Culture of Rat Retina Pigmented Epithelial Cells

Stephen D. Skaper

Abstract
Retinal pigment epithelium cells play a key role in maintaining the normal function of retina and can
express several neurotrophic factors, which support the neurosensory retina and may also provide trophic
signals to the host dopaminergic neurons. The following chapter describes a protocol for the purification
and culture of retinal pigmented epithelial cells from postnatal rat.

Key words: Pigmented epithelium, Retina, Cell culture, Neurotrophic factors

1. Introduction

Retinal pigmented epithelial (RPE) cells are melanin-containing

cells that constitute a monolayer between the neural retina and the
choroid. In RPE cells, tyrosine is catalyzed by tyrosinase to L-dopa
that is polymerized to form melanin (1). L-dopa in RPE cells is
converted into dopamine in the terminals of nigrostriatal dopamin-
ergic neurons and provides dopamine to the nigrostriatal system
directly after RPE cells are transplanted (2, 3). RPE cells play a key
role in maintaining the normal function of retina and can express
several neurotrophic factors such as platelet-derived growth factor,
epidermal growth factor, vascular endothelial growth factor, and
pigment-derived epithelial factor (PEDF) (4), which support the
neurosensory retina and may also provide trophic signals to the
host dopaminergic neurons. In addition, RPE cells secrete glial cell
line–derived neurotrophic factor and brain-derived neurotrophic
factor (BDNF) (3), which may contribute to the beneficial effects
of RPE cells transplantation in an animal model of Parkinson’s
disease. In this context, RPE-conditioned medium protects cultured
primary dopaminergic neurons against rotenone- and 6-hydroxy-
dopamine-induced cell loss (3).

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_14, © Springer Science+Business Media, LLC 2012

159
160 S.D. Skaper

PEDF is a 50-kDa secreted glycoprotein and a noninhibitory

member of the serine protease inhibitor gene family and was origi-
nally discovered in conditioned media from RPE cells but since has
been identified throughout the central nervous system (5). PEDF
has neuroprotective properties for various types of neurons including
cerebellar granule neurons (6), retinal neurons (5), hippocampal
neurons (7), spinal cord motor neurons (8), striatal (enkephalinergic)
neurons (9), and mesencephalic (dopaminergic) neurons (9, 10).
PEDF upregulation induced by memantine, an N-methyl-D-
aspartate receptor antagonist, is involved in increased proliferation
of hippocampal progenitor cells (11).These observations raise the
possibility that application of PEDF may be helpful in designing
new therapeutic strategies for neurodegenerative diseases such as
amyotrophic lateral sclerosis, Parkinson’s disease, Huntington’s
disease, Alzheimer’s disease, and brain ischemia (12). The following
chapter provides a protocol for the culture of RPE cells from rat.

2. Materials

2.1. Equipment 1. Stereo dissecting microscope (backlighting of stage is

and Labware preferred) with fiber optic light source.
2. Laminar flow cabinet for dissections.
3. Laminar flow biological safety cabinet (CL2).
4. Humidified, water-jacketed culture incubator at 37°C and 5%
CO2/95% air.
5. Water bath set at 37°C.
6. Dissecting tools (Fine Science Tools).
7. Bench centrifuge to accommodate 15- and 50-mL tubes.
8. 10-cm ∅ sterile tissue culture dishes.
9. Glass coverslips (12 mm ∅ #1, Menzel-Gläser, Menzel GmbH,
Braunschweig, Germany) (see Note 1).
10. 96-well poly-D-lysine-treated tissue culture plates (BD
Biosciences).
11. 15- and 50-mL polypropylene plastic centrifuge tubes (sterile).
12. 0.22-mm filters (Millipore).
13. Neubauer hemocytometer (Fisher Scientific).

2.2. Reagents 1. Dulbecco’s phosphate-buffered saline, pH 7.4, sterile (DPBS)

(Invitrogen).
2. Water, sterile, tissue culture grade.
3. L-15 medium, with L-glutamine and L-amino acids (Invitrogen).
4. Neurobasal-A medium (Invitrogen).
 14 Culture of Rat Retina Pigmented Epithelial Cells 161

5. B27 supplement, 50×, with antioxidants (Invitrogen).

6. Fetal calf serum (Invitrogen) (see Note 2).
7. L-Cysteine.
8. Insulin, from bovine pancreas (Sigma).
9. Transferrin, human.
10. Progesterone.
11. Putrescine.
12. Sodium selenite.
13. T3 (3,3¢,5-triiodo-L-thyronine, sodium salt), cell culture tested
(Sigma).
14. Penicillin/streptomycin (100× stock), sterile, for cell culture
(Invitrogen).
15. Gentamicin (50 mg/mL stock), sterile, for cell culture (Sigma).
16. Papain (Worthington (Lorne)).
17. Collagenase (Sigma).
18. DNase (type I) (Sigma).
19. Sodium pyruvate (100 mM stock), sterile, for cell culture
(Sigma).
20. L-Glutamine (200 mM stock), sterile, for cell culture
(Invitrogen).
21. Bovine serum albumin, essentially fatty acid and globulin free,
(BSA) (Sigma).
22. Ovomucoid (trypsin inhibitor from chicken egg white, type
III-O, free of ovoinhibitor) (Sigma).
23. Poly-D-lysine (MW 30,000–70,000), sterile, for cell culture
(Sigma).
24. Metrizamide (Sigma).
25. Merosin, human (Millipore).
26. BDNF, human, recombinant (Peprotech).
27. Ciliary neurotrophic factor, human, recombinant (Peprotech).
28. Forskolin.
29. N-acetyl cysteine.
30. Phenol red (0.5%), sterile, for cell culture (Sigma).

2.3. Stock Solutions 1. 10× EBSS stock solution without Ca2+, Mg2+, and bicarbonate.
1.16 M NaCl (68 g/L), 54 mM KCl (4 g/L), 10 mM
NaH2PO4·H20 (1.4 g/L), 10 mM D-glucose (10 g/L), and
0.05% phenol red (10 mL/L of 0.5% solution). Filter-sterilize
and store at 4°C.
162 S.D. Skaper

2. D-Glucose, 30% (w/v). Dissolve 15 g of D-glucose in 50 mL

double-distilled water.
3. NaHCO3, 1 M. Dissolve 4.2 g of NaHCO3 in 50 mL double-
distilled water.
4. MgSO4, 100 mM. Dissolve 1.23 g of MgSO4·7H2O in 50 mL
double-distilled water.
5. EDTA, 0.5 M. Dissolve 7.3 g of EDTA in 50 mL double-
distilled water.
6. EBSS-papain solution with Mg2+, EDTA, and bicarbonate.
Combine 20 mL of 10× EBSS solution, 2 mL of 100 mM
MgSO4, 2.4 mL of 30% D-glucose, 0.4 mL of 0.5 M EDTA,
and 5.2 mL of 1 M NaHCO3. Bring to a final volume of
200 mL with double-distilled water.
7. Borate buffer 0.15 M, pH 8.4. Dissolve 28.6 g of sodium borate
(Na2B4O7·10H2O) in 500 mL double-distilled water (pH will
be ~9.2). Adjust pH to 8.4 with 5 N HCl. Filter-sterilize and
store at 4°C (up to 6 months).
8. Low ovomucoid solution. Mix together 9 mL DPBS, 1 mL 10×
low ovomucoid stock (15 mg each BSA and ovomucoid/mL
DPBS), and 200 mL 0.4% DNase stock (made by dissolving
4 mg/mL of DNase in L-15 medium; filter-sterilize, aliquot,
and store at −20°C). Check pH and adjust to 7.4 if necessary.
Filter-sterilize the final solution.
9. High ovomucoid solution. Mix together 4.8 mL DPBS and
1.2 mL 6× high ovomucoid stock (50 mg each BSA and ovo-
mucoid/mL DPBS). Filter-sterilize.
10. Forskolin (1,000×). Add 2.4 mL dimethyl sulfoxide to a 10-mg
bottle of forskolin (=5 mM). Make 100 mL aliquots and store
−20°C. The final concentration of solvent should not exceed
0.1% in the culture medium.
11. N-acetyl cysteine (1,000×). Dissolve 60 mg N-acetyl cysteine in
1 mL Neurobasal-A medium. Filter-sterilize, make 20 mL
aliquots, and store at −20°C.
12. Metrizamide (prepare day before experiment). Prepare a 20%
(1 g/5 mL) metrizamide solution in L-15 medium. Prepare a
40% (2 g in 5 mL) metrizamide solution in DPBS. Filter each
of the above solutions using a 0.22-mm filter attached to a
5-mL syringe. Prepare 5 mL of each solution (will keep up to
1 month at 4°C).
13. Sato 100× stock solution. To 20 mL of Neurobasal-A medium,
add the following: 200 mg transferrin (100 mg/mL), 200 mg
BSA (100 mg/mL), 5 mL of stock progesterone solution (stock
is 2.5 mg/100 mL ethanol) (final = 60 ng/mL or 0.2 mM),
32 mg putrescine (16 mg/mL), 200 mL of stock sodium selenite
 14 Culture of Rat Retina Pigmented Epithelial Cells 163

(stock is 40 mg in 1 mL of 0.1 N NaOH plus 10 mL

Neurobasal-A) (final = 40 ng/mL), and 10 mL of stock T3
(stock is 8 mg in 1 mL of 0.1 N NaOH) (final = 30 ng/mL).
Aliquot the above 100× stock solutions (200 mL) and store at
−20°C. The ethanol stocks for progesterone, sodium selenite,
and T3 should be prepared fresh each time a new batch of
100× Sato medium is made.

2.4. High-Insulin Sato 1. To 20 mL of Neurobasal-A medium, add 200 mL Sato 100×

Culture Medium stock solution, 100 mL 50× B27 supplement, 200 mL sodium
pyruvate 100× stock, 200 mL glutamine 100× stock, 200 mL of
100× penicillin/streptomycin stock (stored at −20°C), and
20 mL N-acetyl cysteine 1,000× stock.
2. Next, add 200 mL of 100× insulin stock (stored at 4°C; stock
must be prepared every 4–6 weeks—do not freeze). The 100×
insulin stock (0.5 mg/mL) is prepared by adding 2 mg of insulin
to 4 mL of tissue culture grade water containing 20 mL of 1 N
HCl. Filter the stock through a 0.22-mm filter and store at 4°C.
3. Filter the above medium through a 0.22-mm filter. Before filtering
the medium, filter about 5 mL of Neurobasal-A medium to
rinse away traces of detergent in the filter. The quality of the
filter matters—Millex-GV from Millipore—is best. Falcon plastic
is preferable, along with Monoject syringes.
4. Before using the medium, add neurotrophic factors (BNDF,
20 mL of a 5 mg/mL stock; ciliary neurotrophic factor, 20 mL
of a 1 mg/mL stock) and forskolin (5 mM; prepare 1,000×
stock in dimethyl sulfoxide so that final concentration of
solvent does not exceed 0.1%) to promote cell survival.

2.5. Coating of Culture 1. Prepare the merosin coating solution as follows: To 5 mL of

Dishes or Coverslips Neurobasal-A medium, add 10 mL of merosin and 50 mL of
(Prepare Day Before 100× penicillin/streptomycin stock solution.
Experiment) 2. For coating, use 0.4 mL merosin per cm2 surface area.
3. Coverslips, if used, can be sterilized by dipping in 70% ethanol
and briefly passing through a flame (see Note 3).
4. Incubate with merosin solution overnight at 37°C.
5. Poly-D-lysine coating solution: Dissolve a 5-mg bottle of poly-
D-lysine in 5 mL borate buffer. Add 200 mL of this poly-D-
lysine stock (1 mg/mL) to 20 mL of tissue culture grade sterile
water (see Notes 4 and 5).
6. For coating, use 0.2 mL poly-D-lysine per cm2 surface area.
7. Incubate with poly-D-lysine at room temperature (20–22°C)
for 60 min.
164 S.D. Skaper

3. Methods

3.1. Dissociation 1. Cut off anterior portion of eye (make sure to remove all of the
of RPE iris). Remove the lens, vitreous, cornea, and retina.
2. Remove the RPE by pulling the RPE/choroid out of the eye
socket with forceps and store in Ca2+/Mg2+—free DPBS.
Remove the optic nerve.
3. Equilibrate the EBSS-papain (1 mM EDTA) solution with 5%
CO2/95% O2 for 15 min in a 37°C incubator.
4. Dissolve 100 U of papain and 333 U/mL of collagenase in
5 mL preequilibrated papain buffer. Next, add 0.8 mg of
L-cysteine to this solution.

5. Sterilize the solution in point 4 using a 0.22-mm filter. Remove

the DPBS and add retinas to papain by pouring in. Next, add
200 mL 0.4% DNase.
6. Digest the tissue in 37°C bath for 90 min with gentle agitation
of the tissue every 15 min.
7. Remove the supernatant by aspiration, being careful not to
draw up the digested tissue.
8. Wash the tissue with 4 mL of the low ovomucoid solution and
let cells settle.
9. Remove supernatant and discard.
10. Add 2 mL of the low ovomucoid solution. Triturate gently
with a long (9-in.) sterile Pasteur glass pipette using five up-
and-down strokes.
11. Let cells settle, remove the supernatant, and transfer the cells
to a new centrifuge tube.
12. Add again 2 mL of low ovomucoid solution and repeat the
trituration step.
13. In subsequent trituration steps, use slightly more force until the
RPE is released from the choroid and cells are in suspension.
14. Choroid does not dissociate and so should be removed and
discarded.
15. Centrifuge the cells at 200 × g for 15 min at 25°C.
16. Discard the supernatant and resuspend the cells in high
ovomucoid solution.
17. Centrifuge at 200 × g for 15 min.
18. Remove the high ovomucoid solution but leaving 2 mL to
resuspend the cells.

3.2. Purification of RPE 1. Using a 15-mL polystyrene centrifuge tube, layer metrizamide
(6 mL) with 40% on the bottom.
 14 Culture of Rat Retina Pigmented Epithelial Cells 165

2. Carefully add the 2 mL of high ovomucoid solution containing

cells (see Subheading 3.1, step 18) to the top of the metriz-
amide and centrifuge at 500 × g for 15 min at 20–22°C.
3. Prepare a 4% BSA solution (0.4 g BSA in 10 mL L-15) and
filter-sterilize. This will be used as a cushion for the 40% metri-
zamide layer (step 5 below).
4. Using a hypodermic syringe and needle, carefully remove
about 1 mL of the cell suspension between the 40% layer of
metrizamide and the high ovomucoid solution.
5. Resuspend the 1 mL of metrizamide cell suspension in 6 mL
of L-15.
6. To a fresh 15-mL centrifuge tube, to which has been added
1 mL of 4% BSA solution, now place the cell suspension from
step 5.
7. Centrifuge at 200 × g for 15 min (this step may be difficult
because the metrizamide is very dense).
8. Remove the supernatant and resuspend cells in high-insulin
Sato culture medium.
9. Count cells (see Chap. 3) and plate at a density of 50,000–
100,000 cells cm2.

4. Notes

1. The source of glass is very important for the coverslips:

German suppliers are highly recommended, in particular the
one listed here.
2. Heat inactivation of fetal calf serum is recommended to destroy
heat-labile complement. Thaw the bottle of serum in advance,
using a 37°C water bath. Next, immerse the serum bottle in
the water bath after reequilibrating to 56°C and leave for
30 min. Swirl the bottle occasionally to ensure proper mixing.
Allow the serum to cool to room temperature, aliquot into
50-mL tubes and store at −20°C.
3. When flame-sterilizing cover glasses, it is recommended to
“wave” the cover glass through the air immediately after
flaming. Because of their fragile nature, heat buildup may cause
frequent shattering of the glass—hence the need to dissipate
the heat.
4. Poly-D-lysine is very hygroscopic and difficult to weigh. It is
advisable to purchase the material as a 5-mg quantity and then
dissolve the entire amount in borate buffer. The solutions can
be stored at 4°C for up to 6 months.
166 S.D. Skaper

5. Working solutions of poly amino acids should be prepared in

sterile tissue culture plastic polypropylene tubes. Take care:
The poly amino acid will adhere to glass surfaces, and also to
polystyrene plastic.

References

1. Aroca P, Urabe K, Kobayashi T, Tsukamoto K, 7. DeCoster MA, Schabelman E, Tombran-Tink

and Hearing VJ (1993) Melanin biosynthesis J, and Bazan NG (1999) Neuroprotection by
patterns following hormonal stimulation. J Biol pigment epithelial-derived factor against gluta-
Chem 268, 25650–25655 mate toxicity in developing primary hippocam-
2. Watts RL, Raiser CD, Stover NP, Cornfeldt pal neurons. J Neurosci Res 56, 604–610
ML, Schweikert AW, Allen RC, et al (2003) 8. Bilak MM, Corse AM, Bilak SR, Lehar M,
Stereotaxic intrastriatal implantation of human Tombran-Tink J, and Kuncl RW (1999)
retinal pigment epithelial (hRPE) cells attached Pigment epithelium-derived factor (PEDF)
to gelatin microcarriers: a potential new cell protects motor neurons from chronic glutamate-
therapy for Parkinson’s disease. J Neural mediated neurodegeneration. J Neuropathol Exp
Transm Suppl 65, 215–227 Neurol 58, 719–728
3. Ming M, Li X, Fan X, Yang D, Li L, Sheng 9. McKay BS, Goodman B, Falk T, and Sherman
Chen S, et al (2009) Retinal pigment epithelial SJ (2006) Retinal pigment epithelial cell trans-
cells secrete neurotrophic factors and synthe- plantation could provide trophic support in
size dopamine: possible contribution to thera- Parkinson’s disease: results from an in vitro
peutic effects of RPE cell transplantation in model system. Exp Neurol 201, 234–243
Parkinson’s disease. J Trans Med 2009, 7:53 10. Falk T, Zhang S, and Sherman SJ (2009)
doi:10.1186/1479-5876-7-53 Pigment epithelium derived factor (PEDF) is
4. Cepeda IL, Flores J, Cornfeldt ML, O’Kusky neuroprotective in two in vitro models of
JR, and Doudet DJ (2007) Human retinal pig- Parkinson’s disease. Neurosci Lett 458, 49–52
ment epithelial cell implants ameliorate motor 11. Namba T, Yabe T, Gonda Y, Ichikawa N, Sanagi
deficits in two rat models of Parkinson disease. T, Arikawa-Hirasawa E, et al (2010) Pigment
J Neuropathol Exp Neurol 66, 576–584 epithelium-derived factor up-regulation induced
5. Tombran-Tink J, Barnstable CJ (2003) PEDF: by memantine, an N-methyl-D-aspartate receptor
a multifaceted neurotrophic factor. Nat Rev antagonist, is involved in increased proliferation
Neurosci 4, 628–636 of hippocampal progenitor cells. Neuroscience
6. Yabe T, Wilson D, and Schwartz JP (2001) 167, 372–383
NFkappaB activation is required for the neuro- 12. Yabe T, Sanagi T, and Yamada H (2010) The
protective effects of pigment epithelium- neuroprotective role of PEDF: implication for
derived factor (PEDF) on cerebellar granule the therapy of neurological disorders. Curr Mol
neurons. J Biol Chem 276, 43313–43319 Med 10, 259–266
 Chapter 15

Mammalian Growth Cone Turning Assays Identify Distinct

Cell Signalling Mechanisms That Underlie Axon Growth,
Guidance and Regeneration
Andrew J. Murray, Andrew G. Peace, Steven J. Tucker,
and Derryck A. Shewan

Abstract
The cell signalling mechanisms underlying mammalian central nervous system axon growth and guidance
change during development, such that axons that establish appropriate connectivity in the embryo fail to
regenerate after injury to the adult nervous system. The growth cone turning assay has been used in
Xenopus neurons to elucidate mechanisms of axon guidance during development. Here, we describe how
we have adapted this assay for rat dorsal root ganglion neurons to study the influence of extracellular
secreted factors causing growth cone attraction and repulsion. Additionally, we describe how this method
can be combined with small interfering RNA and cDNA transfections to manipulate protein expression in
growth cones, and fluorescence resonance energy transfer to monitor the activity of signalling pathways in
live neurons. This assay provides the unique ability to manipulate and visualise the internal status of growth
cone signalling whilst challenged with extracellular chemotropic signalling molecules, and can be used to
develop strategies to promote axon regeneration in the mature mammalian central nervous system.

Key words: Axon guidance, Regeneration, Growth cone, Turning assay, Fluorescence resonance
energy transfer, Cell signalling, cAMP

1. Introduction

The growth cone is the motile tip of a growing axon responsible

for integrating signals from the extracellular environment and
controlling the rate and direction of axonal growth. The growth
cone senses its molecular environment and makes directional
growth decisions according to both the spatial expression of extra-
cellular repulsive and attractive guidance cues, and the internal
status of growth cone signalling molecules that ultimately mediate

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_15, © Springer Science+Business Media, LLC 2012

167
168 A.J. Murray et al.

cytoskeletal regulation. When axons are severed in the mature

nervous system, the distal tip of the severed axon quickly forms a
growth cone and attempts to mount a regenerative response.
However, a variety of growth-inhibiting molecules present in the
mature nervous system blocks axon regrowth (1).
Since the development of a reliable method for characterising
growth cone turning to gradients of secreted factors (2), numerous
insights have been gained into the intracellular and extracellular
mechanisms of growth cone guidance. These include descriptions
of how manipulating signalling pathways within growth cones
alters the attractive and repulsive responses to extracellular
factors (3, 4). These studies into growth cone turning during
development have been mainly carried out in spinal and retinal
neurons in embryos of the South African clawed-toed frog
Xenopus laevis. However, the study of responses to extracellular
factors by developing or regenerating mammalian neurons
requires a number of adaptations because, curiously, mammalian
(or even zebrafish) neurons temporarily halt neurite outgrowth
when exposed to temperatures higher than ambient room tem-
perature. Here, we describe a growth cone turning assay that can
be used to study the responses of mature mammalian neurons to
gradients of extracellular secreted factors. We have previously
used this assay to examine cyclic nucleotide-mediated signalling
pathways and their potential to initiate growth cone attraction (5, 6)
and switch repulsion to attraction when stimulated in the pres-
ence of secreted molecules such as myelin associated glycoprotein
and netrin-1 (7). We describe how this assay can be used to
manipulate growth cone intracellular signalling pathways with
pharmacological agents and, additionally, demonstrate how the
growth cone turning assay can be evolved to combine chemotro-
pic observations with efficient transfection of mammalian dorsal
root ganglion (DRG) neurons with small interfering RNA or
cDNA, further enhancing the repertoire of manipulations avail-
able through protein knockdown, knockout or over-expression.
Furthermore, we use fluorescence resonance energy transfer
(FRET) to visualise signalling pathways activated in growth cones
in real time, gaining unique insights into the mechanisms that
control axon growth and guidance. We have used this method to
study the responses of rat DRG growth cones to extracellular
guidance molecules as well as dissect intracellular signalling
pathways involved in DRG growth cone turning, but we envisage
that this could be easily employed to study turning responses in
a variety of mammalian neurons.
 15 Mammalian Growth Cone Turning Assays Identify Distinct Cell… 169

2. Materials

2.1. Dorsal Root 1. Bottenstein and Sato’s fluid (BSF). Ham’s F12 culture medium
Ganglion Neuron (Invitrogen) supplemented with 16 μg/mL putrescine,
Culture 100 μg/mL transferrin, 0.3% bovine serum albumin (BSA),
100 U/mL penicillin/streptomycin, 0.06 μg/mL progesterone,
0.16 μg/mL sodium selenite, 20 μg/mL insulin (all Sigma-
Aldrich) and 0.1 μg/mL nerve growth factor (NGF) (Serotec)
(see Note 1).
2. Low-walled, glass bottom culture dishes (MatTek).
3. Poly-L-lysine (70,000–150,000 mol. weight) (PLL) (Sigma-
Aldrich) and appropriate growth substratum, for example
laminin (Invitrogen).
4. 15% BSA dissolved in Ham’s F12 and stored in 2 mL aliquots
in 15-mL centrifuge tubes (Sterilin) at −20°C.
5. 37°C cell culture incubator with 5% CO2/95% air and class I
tissue culture hood.
6. Haemocytometer for counting cell numbers.
7. Trypsin inhibitor/DNAse. Hank’s balanced salt solution
(HBSS; Invitrogen) containing 0.002% trypsin inhibitor,
0.025% DNAse and 1% BSA (all Sigma-Aldrich).

2.2. DRG Neuron 1. Amaxa nucleofector machine (Lonza AG).

Transfection 2. Nucleofector solution (Lonza AG).
3. Nucleofection cuvettes and Pasteur pipettes (Lonza AG).
4. Appropriate concentration of siRNA or transfection quality
DNA.

2.3. Growth Cone 1. Glass pipettes (Intracell) pulled on a Sutter Pipette Puller to
Turning Assays have a 1 μm bore width.
2. Picospritzer III with a TSS10 dual pulse stimulator (both
Intracell) connected to a compressed air supply and micropi-
pette holder held by a micromanipulator.
3. Inverted phase contrast microscope with heated stage and CO2
supply and camera, connected to a computer equipped with
live imaging software. If CO2 is not available, HEPES buffer
can be used as an alternative.
4. ImageJ software for analysis of growth cone turning, available
freely from http://rsbweb.nih.gov/ij/.
170 A.J. Murray et al.

2.4. FRET Analysis 1. cDNA constructs designed to encode particular signalling pro-
teins with appropriate FRET-paired fluorophores.
2. Leica AF6000LX imaging system or equivalent FRET-capture
imaging system.
3. Leica application software or equivalent for analysis of FRET
time-lapse experiments.

3. Methods

DRG neurons represent an important model for nervous system

regeneration. They are unique among peripheral neurons in that
they project one axonal branch to the periphery and another into
the central nervous system during development, but when injured
in the adult, only the peripheral branch can regenerate whilst the
central branch is inhibited at the boundary with the spinal cord
(8). The procedure described here is based on the protocol devel-
oped for Xenopus neurons (2). A major advantage of the Xenopus
system is that neurons develop at room temperature (15–20°C).
Mammalian neurons, however, require a temperature of 37°C and
a CO2-buffered atmosphere.
One major problem with examining the growth of mammalian
neurons is the poor growth rate of axons when exposed to an
atmosphere at 37°C. This is likely due to the heated microscope
stage causing rapid evaporation of the culture medium and a lack
of 5% CO2 in the atmosphere. The lack of CO2, in the short term,
can be overcome with the addition of 15 μM HEPES to the culture
medium to buffer the pH under normal atmospheric conditions
(9). To overcome the exposure of the culture medium to the heated
atmosphere, we overlay the medium with a thin layer of oil, pre-
venting evaporation and gas exchange. Figure 1a shows that the
growth rate of DRG neurites is comparable in a sealed culture dish
at 37°C and culture medium overlayed with oil. Typical responses
of DRG growth cones to a gradient of NGF are shown in Fig. 1b.

3.1. Preparing Growth 1. Prepare 10 μg/mL PLL in sterile distilled H2O and gently
Substrata pipette 500 μL of this solution onto each central glass well of
MatTek low-walled 50-mm petri dishes under aseptic condi-
tions and leave overnight.
2. The next day, remove the PLL and allow the dishes to dry
completely in a tissue culture hood (see Note 2).
3. Prepare a solution of 2 μg/mL L-laminin in F12 medium and
gently pipette 500 μL into the central well of each MatTek dish.
4. Place dishes in a 37°C incubator for at least 2 h, but ideally
overnight. Other extracellular matrix substrates, such as
fibronectin, may also be applied in this way.
 15 Mammalian Growth Cone Turning Assays Identify Distinct Cell… 171

Fig. 1. (a) Growth rates of DRG neuron neurites in a sealed culture dish, exposed to a heated (37°C) atmosphere and
‘sealed’ with a layer of oil. Note that the oil layer allows similar levels of neurite outgrowth to that seen in a sealed tissue
culture dish. (b) Typical responses of DRG neuron growth cones to a gradient of NGF emitted from the micropipette. The
bias of neurites growing in the direction of the gradient indicates an attractive response. (c) Position of the axon/growth
cone (left ) compared to the micropipette (right ). (d) Image from (c) showing the angle of the growth cone and distance
from the micropipette.

3.2. Preparation Here, we describe the culture of dissociated adult DRG neurons,
of Dissociated DRG as we believe they represent a good model for studying axon regen-
Neurons for Growth eration. However, we have also used this assay with DRG neurons
Cone Turning Assays at other developmental stages to study intrinsic cell signalling
changes in developing neurons (5, 7), and the procedure can be
used to study other mammalian neuronal types (10):
1. Dissect the spinal column from a sacrificed rat and remove as
much connective tissue and muscle as possible, then use cuticle
nippers to remove dorsal vertebral plates to expose the spinal
cord from the dorsal side.
2. Locate the DRGs lateral to the spinal cord on either side at each
vertebral segment, carefully cut the dorsal roots and place the
DRGs in warm F12 culture medium in a 35-mm petri dish.
3. Trim the roots from the DRGs and incubate the ganglia in F12
medium containing 0.125% collagenase for 1 h at 37°C in a 5%
CO2/95% air incubator.
4. Transfer the ganglia/collagenase to a 15-mL centrifuge tube
containing 10 mL F12 medium to wash out the collagenase.
Remove all F12 solution leaving the ganglia behind and add
172 A.J. Murray et al.

2 mL of warm BSF and gently triturate the ganglia using a

sterile glass Pasteur pipette followed by a Gilson P1000 pipette
(see Note 3). Then add a further 2 mL BSF medium.
5. Allow the larger debris to settle to the bottom of the tube and
carefully transfer 1 mL of the solution to 15-mL centrifuge
tubes each containing 2 mL 15% BSA, slowly pipetting the
solution down the side of the tube so that the cell suspension
sits on top of the BSA cushion (see Note 4). Centrifuge the
tubes at 100 × g for 5 min. Myelin and fibrous debris will stay
at the media/BSA interface, and neurons will pellet at the bot-
tom of the tube.
6. Gently remove the tubes from the centrifuge and carefully
remove the F12/BSA without disturbing the pellet of neurons
at the bottom of each tube. Gently re-suspend the pellet in
nucleofector solution (100 μL per transfection; pool the pel-
lets from all tubes used) (see Note 5).
7. Add the appropriate concentration of DNA plasmid or siRNA
duplex, transfer to a transfection cuvette and pulse in the
nucleofector machine set to programme G13. Immediately
after transfection, take a small sample (around 5 μL) to count
on a haemocytometer and add the rest to 900 μL of BSF to a
1.5-mL Eppendorf tube (see Note 6).
8. Count the neurons on the haemocytometer and dilute the dis-
sociated neurons to 2,500 per mL in BSF. Remove low-walled
culture dishes from the CO2 incubator and remove the laminin
solution from each well (see Note 7). Add 500 μL of the diluted
cell suspension to the central well of each MatTek dish, being
careful not to spill the suspension outside of the coverslip.
Carefully return the dishes to the incubator and leave overnight.
9. The following morning, gently add 4.5 mL of BSF medium to
each culture dish and check for signs of neurite outgrowth (see
Note 8).

3.3. Set-up of 1. Using a Sutter Pipette Puller, pull 10-cm borosilicate glass
Picospritzer and pipettes with filaments, O.D. 1.0 mm, I.D. 0.5 mm (Intracell),
Equipment for Growth so that they have a bore width of 1 μm (see Note 9).
Cone Turning Assays 2. Connect a compressed air supply to a Picospritzer III under
the control of a TSS10 dual pulse stimulator (or equivalent) to
deliver air pulses at a frequency of 2 Hz for 20 ms duration, at
a pressure of 3 psi and a delay of 0.1 ms.
3. Using Eppendorf Microloader pipette tips and a P20 Gilson
(or equivalent) pipette, fill the tip of the glass micropipette
with the reagent to be assayed (4 or 5 μL will suffice), at a
1,000-fold greater concentration to that required at the growth
cone (2), and connect to the Picospritzer pipette holder
attached to a micromanipulator.
 15 Mammalian Growth Cone Turning Assays Identify Distinct Cell… 173

4. Preheat the microscope chamber to 37°C and equilibrate to

5% CO2. If CO2 is not available, add 15 mM HEPES buffer to
the neuronal culture medium (see Note 10).
5. Remove the culture dishes from the incubator and gently over-
lay the medium with a thin (2–3 mL) layer of vegetable oil that
has been pre-warmed to 37°C. Make sure that the oil covers
the entire surface and does not mix with the medium (see Note
11). Vegetable oil does not seem to adversely affect neuronal
cultures, but other types of oil, for example mineral oil, may be
effective.
6. If using any pharmacological reagents to manipulate the intra-
cellular signalling pathways in the neurons, bath apply to the
culture medium at least 30 min before beginning an assay
(although this depends on the agent being used).
7. Using phase optics, identify a growth cone to be analysed and
position it so it is visible on the computer screen. Position the
micropipette so that it is 100 μm from the growth cone at an
angle of 45° (see Fig. 1; Note 12).
8. Position the micropipette so that it is on or just above the sur-
face of the culture dish.
9. Take an image of the growth cone/pipette.
10. Switch on the Picospritzer/stimulator to begin the assay. Leave
running for 30–60 min, depending on the typical rate of neu-
rite outgrowth.
11. After this time, take a second image of the growth cone posi-
tion. Elevate the micropipette and reposition the culture dish
for the next growth cone (see Note 13).

3.4. Analysis of 1. Using ImageJ (NIH), measure the start angle of growth cone
Growth Cone Turning with respect to the micropipette by drawing a line through the
centre of the growth cone from the most proximal segment
of the neurite (this angle should be ~45° to the horizontal
position of the micropipette, from either above or below the
horizontal; see Fig. 1c, d).
2. Repeat step 1 with the image from the end of the assay (30 or
60 min); measure the angle between the final position of the
growth cone, its original position and the original direction of
growth at time zero. A final position of the growth cone on the
pipette side of the original 45° direction of growth represents
growth cone attraction to the gradient, whilst a final position
on the other side of the original 45° direction of growth repre-
sents growth cone repulsion.
3. Measure the distance that the growth cone has advanced
during the assay. If a growth cone has not advanced at least
10 μm, we consider it unresponsive because filopodia can
174 A.J. Murray et al.

advance such distances without growth cone advance, and

therefore we do not include such growth cones in our data.

3.5. FRET Combined It is assumed that growth cone turning is achieved by biased expression
with an Applied of intracellular signalling molecules leading to unilateral assembly
Gradient or disassembly of the cytoskeleton. We can now directly measure
protein activity across a growth cone when challenged with a
molecular gradient by combining the turning assay with FRET.
When a gradient is applied, changes in the distribution of FRET
across the growth cone indicate whether the extracellular gradient
activates or inhibits intracellular signalling proteins, revealing an
asymmetrical distribution of activity.
1. Plate DRG neurons transfected with appropriate FRET cDNA
constructs onto MatTek dishes coated as described in
Subheading 3.1, but use 5–10 μg/mL laminin to enhance
numbers of adherent transfected cells.
2. Analyse cells 24–48 h post-transfection using a FRET-capturing
system (e.g. see Subheading 2.4) using an inverted-stage
microscope set-up as described in Subheading 2.3.
3. Ensure FRET system and filters are properly configured for the
FRET probes being used and that appropriate correction
images are taken. These include untransfected, donor only and
acceptor only controls to correct for cross-talk between fluo-
rescent channels. Using FRET analysis software, these can be
used to generate correction factors in line with the algorithm
used by Wouters et al. (11).
4. Care should also be taken to avoid bleaching of the fluoro-
phores during time-lapse imaging. Control experiments should
be conducted to optimise exposure times, fluorescent light
intensity and frequency of image capture.
5. Select a growth cone that is flat, spread and contains lamellipodia
and filopodial protrusions. Care must also be taken to choose
a growth cone emitting suitable levels of fluorescence. Rotate
the dish so that the growth cone is facing the side of the
stage with the micromanipulator, at a 45° angle.
6. Having completed set-up and bleaching control measurements,
time-lapse experiments should be conducted by capturing
consecutive images in three separate channels: the donor chan-
nel (donor fluorophore excitation wavelength and donor fluo-
rophore emission capture), the FRET channel (donor fluorophore
excitation wavelength and acceptor fluorophore emission
capture) and the emission channel (acceptor fluorophore exci-
tation wavelength and acceptor fluorophore emission capture).
These channels are combined by the software to create a
pseudocolour sensitised FRET emission channel (FRET SE),
 15 Mammalian Growth Cone Turning Assays Identify Distinct Cell… 175

which quantifies the degree of FRET across the specimen being

examined. In the case of using an applied gradient, a phase
contrast image should also be taken to visualise the tip of the
micropipette.
7. Load the micropipette with the desired protein, agonist or
pharmacological reagent as described in Subheading 3.2.
8. Take an image (three FRET channels + one differential inter-
ference contrast/phase), indicating the start of the time
lapse (t0).
9. Lower the micropipette into position under differential inter-
ference contrast/phase optics, placing the tip 100 μm from the
growth cone.
10. Repeat step 8 at the end of the time course. In our experimental
set-up, 5 min (t5) is sufficient to detect a change in the distri-
bution of the FRET sensor, although different time lapses may
be suitable depending on protein analysed or gradient used.
However, growth cones change morphology and orientation
over longer time points, and selecting similar regions of interest
may become more challenging.
11. Raise the micropipette and select a new growth cone.

3.6. Analysis of FRET 1. Regions of interest (ROIs) are outlined within the growth
Distribution cone for both the start and end point images. One ROI is
drawn outlining 50% of the surface area of the growth cone on
the side facing the pipette and the other is drawn outlining
50% of the growth cone on the side further from the pipette,
with the junction at which they meet bisecting the central axis
of the growth cone in line with the neurite shaft immediately
proximal to the growth cone.
2. A ratiometric value for the distribution of FRET across the
growth cone is obtained for each image using the formula:
NEAR SIDE FRET SE value/FAR SIDE FRET SE value.
A value <1 suggests the protein is less active on the near side of
the growth cone, whilst a value of >1 suggests the protein is
more active on the near half of the growth, i.e. the side exposed
to the gradient.
3. The procedure should be repeated for at least 15 growth cones,
with values for t0 and t5 plotted on a cumulative distribution
graph (see Fig. 2). Statistical comparison can be performed
comparing t0 and t5 values; a significant mean increase at t5
indicates a near side bias of signalling protein activity caused by
the gradient, whereas a significant mean decrease at t5
indicates a far side bias. Over longer time periods, this bias in
distribution of signalling molecule activity can be related to
growth cone attraction or repulsion.
176 A.J. Murray et al.

Fig. 2. Detection of asymmetric signalling activity within growth cones exposed to a gradient of the cAMP agonist,
Sp-cAMPS. (a) Near/Far ratio of FRET emission (abscissa) indicating the distribution of B-Raf FRET activity across embry-
onic rat DRG growth cones plotted against the percentage of growth cones that display that ratio or less (ordinate axis) at
the beginning of the experiment (t0, dark circles) and 5 min after application of the gradient (t5, light circles), with each
point representing one growth cone. The mean ratio for each group is indicated by corresponding symbols above the
abscissa. High values represent higher B-Raf activity on the side closest to the pipette, and values less than 1 represent
higher B-Raf activity on the side further from the pipette. Sp-cAMPS clearly instigates higher B-Raf activity on the side of
the growth cone towards the gradient. (b, c) Grayscale pictures of pseudocoloured FRET images at t0 (b) and t5 (c), with
darker colours indicating low FRET and lighter colours indicating high FRET. Arrow in (c) indicates the direction of the
applied gradient. Scale bars represent 10 μm. *P < 0.05, student’s t test.

4. Notes

1. BSF without insulin and NGF is stable at 4°C for several weeks.
After supplementing medium with NGF/insulin, BSF can be
stored for approximately 2 weeks at 4°C.
2. The PLL solution should be completely dried from the well
before applying the growth substrate, as liquid PLL is toxic to
cells.
3. Be very careful not to damage the neurons during the tritura-
tion step. Generally, a few passes using the glass Pasteur pipette
and Gilson P1000 pipette are sufficient.
4. 15% BSA can be prepared in advance and stored at −20°C. It
must be thawed in a 37°C water bath and mixed carefully
before use so that the BSA is evenly distributed.
5. If you are planning to carry out growth cone turning assays on
untransfected neurons, re-suspend the neuron pellet in
100 μL trypsin inhibitor/DNAse, before quickly adding
900 μL of BSF medium and moving on to step 8.
6. The optimum concentration of siRNA or plasmid DNA needs
to be established for each study. The transfection stage should
be completed as quickly as possible, ideally in less than 2 min,
 15 Mammalian Growth Cone Turning Assays Identify Distinct Cell… 177

as the nucleofector solution is toxic with prolonged exposure.

Following transfection, care should be taken not to agitate
the culture as the neurons are particularly fragile at this stage.
7. A thin film should be visible over the glass coverslip if the
laminin has adhered to the substrate. Be careful to not let
the coverslip dry out between removing the laminin solution
and adding the cell suspension.
8. Mature DRG neurons that have been transfected with either
siRNA or plasmid DNA using the Amaxa nucleofector machine
typically require 48 h to produce pronounced neurite out-
growth. This time period also allows for significant knockdown
of target proteins using siRNA (5) and expression of fluores-
cently tagged proteins used for FRET analysis (7).
9. An accurate bore width is very important as slight deviations
in the diameter of the glass pipette can dramatically alter the
gradient. A stage micrometre can be used to check bore width.
10. Without CO2, but with HEPES buffered medium, we rou-
tinely see good neurite outgrowth for at least 6 h.
11. We have tested various types of oil for their ability to ‘seal’ the
culture medium and prevent evaporation and gas exchange
whilst still allowing the micropipette to pass through without
becoming blocked.
12. It is important to carry out turning assays with the pipette angled
both above and below the growth cone at the start of the assay.
Growth cones have been reported to turn in a clockwise direc-
tion, even in the complete absence of extracellular cues, due to
the interactions of myosin Va and myosin Vb with actin (12).
Therefore, carrying out equal numbers of assays from above and
below the pipette avoids any bias due to this natural turning.
13. Due to the small bore width of the pipette, it is easily blocked
by proteins and chemicals, and therefore we routinely check
that the pipette is still ejecting at the beginning and end of each
assay by aligning the pipette with small items of unattached
debris that can be seen hovering above the coverslip surface.
Solutions containing BSA are particularly prone to blockage.

References

1. Filbin MT (2003) Myelin-associated inhibitors 4. Shewan D, Dwivedy A, Anderson R, Holt CE

of axonal regeneration in the adult mammalian (2002) Age related changes underlie switch in
CNS. Nat Rev Neurosci 4, 703–713 netrin-1 responsiveness as growth cones
2. Lohof AM, Quillan M, Dan Y, Poo MM (1992) advance along the visual pathway. Nat Neurosci
Asymmetric modulation of cAMP activity induces 5, 955–962
growth cone turning. J Neurosci 12, 1253–1261 5. Murray AJ, Shewan DA (2008) Epac medi-
3. Song H, Poo MM (1999) Signal transduction ates cAMP-dependent axon growth, guidance
underlying growth cone responses to diffusible and regeneration. Mol Cell Neurosci 38,
factors. Curr Opin Neurobiol 9, 355–363 578–588
178 A.J. Murray et al.

6. Murray AJ, Peace AG, Shewan DA (2009) acid (HEPES) as a tissue culture buffer. Proc
cGMP promotes neurite outgrowth and growth Soc Exp Biol Med 130, 305–310
cone turning and improves axon regeneration 10. Erskine L, Reijntjes S, Pratt T, Denti L,
on spinal cord tissue in combination with Schwarz Q, Vieira JM, Alakakone B, Shewan
cAMP. Brain Res 1294, 12–21 D, Ruhrberg C (2011) VEGF signaling
7. Murray AJ, Tucker SJ, Shewan DA (2009) through neuropilin 1 guides commissural axon
cAMP-dependent axon guidance is distinctly crossing at the optic chiasm. Neuron 70,
regulated by Epac and protein kinase A. J 951–965
Neurosci 29, 15434–15444 11. Wouters FS, Verveer PJ, Bastiaens PIH (2001)
8. Golding J, Shewan D, Cohen J (1997) Imaging biochemistry inside cells. Trends Cell
Maturation of the mammalian dorsal root entry Biol 11, 203–211
zone—from entry to no entry. Trends Neurosci 12. Tamada A, Kawase S, Murakami F, Kamiguchi
20, 303–308 H (2010). Autonomous right screw rotation
9. Shipman C Jr (1969) Evaluation of of growth cone filopodia drives neurite turning.
4-(2-hydroxyethyl)-1-piperazineethanesulfonic J Cell Biol 188, 429–441
 Chapter 16

Culture of Dissociated Sensory Neurons from

Dorsal Root Ganglia of Postnatal and Adult Rats
Davina E. Owen and Julie Egerton

Abstract
The development of new therapeutics for management of pain is likely to become much more mechanism
based, and therefore, we need a more thorough understanding of the different pain development path-
ways. The afferent fibers of sensory neurons, with their cell bodies in the dorsal root ganglia (DRG), are
thought to be key in pain mechanisms. DRG neurons can be prepared from embryonic, postnatal, or adult
tissue. Embryonic preparations have the advantage of higher cell yields and greater proportion of neurons,
but they are dependent on neurotrophins for the first week of culture. In contrast, dissociated postnatal
and adult DRG sensory neurons offer the possibility to study mature neurons that may better resemble the
in vivo characteristics of these cells. Here, we describe the dissociation of DRG sensory neurons from
postnatal and adult rats. DRG are dissected and dissociated using a prolonged trypsin/collagenase treat-
ment, followed by mechanical separation of the neurons. We have routinely prepared them for electro-
physiological studies by the methods outlined in this chapter and describe some of the pitfalls that we have
encountered, with hints of how to overcome them.

Key words: Dorsal root ganglia, Dissociated cell culture, Growth factors, Rat, Sensory neurons,
Adult, Postnatal

1. Introduction

Dorsal root ganglion (DRG) neurons extend processes that are

located both in the peripheral (PNS) and central nervous systems
(CNS), grow in both environments during development, and
subsequently are used for the study of aspects of both systems.
Sensory neurons that originate in the DRG are unique in that,
instead of the typical axon-dendrite structure of other neurons,
they are unipolar neurons with a single axon stem bifurcating into
a peripheral branch that goes to the distal tissues and a central
branch that goes to spinal cord. Many components of the PNS and
CNS may be affected by pathologies leading to neuropathic or

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_16, © Springer Science+Business Media, LLC 2012

179
180 D.E. Owen and J. Egerton

inflammatory pain, but the sensory neurons are often affected early
and more severely. They are a key component of the nociceptive
pathway, and as such, we need to have a better understanding of
their role, in their response to and generation of inflammatory
mediators, and the control of their excitability in the development
of chronic pain.
The use of dissociated neurons, in contrast to acute prepara-
tions or slice cultures, means that a defined mechanism can be
more easily studied. The isolated neuron’s environment can easily
be manipulated in vitro to try and reflect the in vivo environment
or even the disease state. DRG neurons are very accessible and easy
to harvest in embryonic, young, and adult animals, and they survive
well in culture. Typically, DRG neurons are cultured from rats,
mice, or chicken embryos, and in most cases, one can obtain many
neurons from a single animal. For example, a complete preparation
from an adult rat yields ~40 DRGs or ~100,000 neurons. The yield
from an average age pup is ~500,000 neurons.
Postnatal DRG cultures can be used as a screening tool as the
yields are considerably greater and they grow well on multiwell
plate formats, which makes them open to fluorometric imaging
plate reader/calcium imaging (1), neurite outgrowth, cell health
assays, and cocultures. Unlike embryonic DRG neurons, postnatal
and adult DRG neurons do not need nerve growth factor (NGF)
or serum for survival (2), although the presence of NGF does affect
their phenotype. For example, NGF regulates the capsaicin sensi-
tivity of adult DRG neurons (3). The protocol in this chapter,
which involves dissociation of rat DRGs using a prolonged trypsin/
collagenase treatment, followed by mechanical separation of the
neurons, has been modified from Lindsay (2) and adapted for opti-
mum electrophysiological experimentation.

2. Materials

2.1. Postnatal DRG 1. Hank’s balanced salt solution (HBSS) (calcium-/magnesium-

Neurons free, Invitrogen).
2. 0.05% Trypsin-EDTA (Invitrogen) (see Note 1).
3. 0.1% Collagenase Type 1A-S (Sigma). Dilute 1 mL of 0.5%
stock diluted in 4 mL HBSS. The 0.5% stock solution is pre-
pared by dissolving 50 mg of collagenase in sterile HBSS. This
can be divided into 1 mL aliquots and stored at −20°C for at
least 6 months.
4. Human β-NGF (R&D Systems). Reconstitute a 100-μg vial of
β-NGF in 2 mL of phosphate buffered saline (pH 7.4)
(PBS) + 0.1% bovine serum albumin (BSA) to yield a 0.05 μg/mL
 16 Culture of Dissociated Sensory Neurons from Dorsal Root… 181

stock. Filter-sterilize, aliquot into 50-μL portions, and store at

−20°C for at least 6 months.
5. N2 supplement (100×, Invitrogen).
6. Penicillin/streptomycin solution (“Pen/Strep”), 100×
(10,000 U/mL penicillin and 10,000 μg/mL streptomycin)
(Invitrogen).
7. 7.5% BSA solution (Invitrogen).
8. Mouse laminin, 1 mg/mL (Sigma). Prepare a working solu-
tion of 5 μg/mL by diluting 50 μL of the supplier’s stock solu-
tion in 10 mL of PBS.
9. Poly-L-lysine hydrobromide (Sigma P2636): Dilute in sterile
distilled water to give a working solution of 100 μg/mL.
10. Dissociation medium: Prepare Dulbecco’s modified Eagle’s
medium (DMEM; high glucose, with sodium pyruvate,
Invitrogen) containing 10% (v/v) heat-inactivated fetal calf serum
(FCS) and 50 U/mL penicillin + 50 μg/mL streptomycin.
11. Preparation of substrate for plating should be started the day
before. Coat tissue cell culture dishes/plates/coverslips with
100 μg/mL poly-L-lysine, leave overnight at room tempera-
ture, and wash off well with sterile H2O. Next, coat the cell
culture dishes/plates/coverslips with 5 μg/mL laminin, and
leave at 37°C (for at least 50 min before plating cells). Remove
all the laminin just before plating DRG cells (see Note 2).
12. Growth medium: Supplement 50 mL of DMEM to contain
50 ng/mL β-NGF (add 50 μL from a 50 μg/mL stock), N2
supplements (500 μL from 100× stock), 0.05% BSA (334 μL
from 7.5% BSA solution), 50 U/mL penicillin, and 50 μg/mL
streptomycin (5 mL of 100× stock)—note that this medium is
serum-free.

2.2. Adult DRG 1. Nutrient Mixture F-12 (with phenol red; Invitrogen).
Neurons 2. Ultroser G (Biosepra); reconstitute with 20 mL distilled water,
filter-sterilize, and add fresh to culture medium to 4% w/v.
3. Poly-DL-ornithine hydrobromide (Sigma P8638): Prepare a
25 mg/mL stock solution in distilled water and filter-sterilize.
Next, prepare a working solution of 500 μg/mL poly-DL-ornithine
by adding 200 μL of the 25 mg/mL stock to 10 mL of sterile
water.
4. Collagenase (Type IV, Sigma): Prepare a 6.25 mg/mL stock
solution (0.625% w/v) in F-12 medium (no phenol red) by
dissolving 50 mg in 8 mL medium and filter-sterilize. Further
dilute the collagenase to 0.625 mg/mL (0.0625% w/v) by
adding 200 μL of the 6.25 mg/mL stock to 1.8 mL of F-12
medium.
182 D.E. Owen and J. Egerton

5. BSA (essentially fatty acid–free, Sigma): Prepare a 15% (w/v)

solution by dissolving 1.5 g in 10 mL of F-12 medium and
filter-sterilize. It is best to make this on the day that DRG cells
are prepared for culture.
6. Mouse nerve growth factor (mNGF), 7.5S murine (Promega):
Prepare a 100 μg/mL stock solution in F-12 medium, filter-
sterilize, and store as 30 μL aliquots at −20°C. Next, dilute
5 μL of the mNGF stock solution in 5 mL of F-12 medium to
give 100 ng/mL mNGF.
7. Growth medium: To 500 mL of nutrient mixture F-12, add
5 mL of “Pen/Strep” stock (100 U/mL) penicillin and
100 μg/mL streptomycin, 5 mL of 100× N2 supplement, and
20 mL of 100% Ultroser G (4% w/v final).
8. The plating substratum should be started before preparation of
the dissociated cells. Coat tissue cell culture dishes/plates/cov-
erslips with 500 μg/mL poly-DL-ornithine and leave for at least
30 min at room temperature under the culture cabinet. Wash
twice with sterile distilled water and air-dry uncovered in the
cabinet. Next, coat the cell culture dishes/plates/coverslips
with freshly prepared laminin solution (5 μg/mL) and incubate
at 37°C for at least 3.5 h. Aspirate off the laminin solution
under a culture cabinet and allow to dry uncovered in the cabi-
net for at least 10 min prior to seeding with DRG cells.

2.3. Apparatus 1. Dissecting microscope preferably in a laminar flow cabinet.

2. Laminar flow hood.
3. Dissecting instruments (Fine Science Tools).
4. Humidified tissue culture incubator at 37°C and 5% CO2.
5. Water bath set at 37°C.
6. Bench centrifuge to spin 15-mL falcon tubes.

3. Methods

3.1. Postnatal DRG 1. Sterilize dissection instruments by treating with 70% v/v etha-
Neurons nol and allow to dry in the dissection cabinet.
3.1.1. Dissection 2. Collect the DRG from 7- to 9-day-old rat pups (Sprague
Dawley). Cull the rat pups following Schedule 1 (cervical
dislocation followed by decapitation) according to Home
Office protocols.
3. Remove the tail and make an incision through the skin
following the line of the spinal column from the head to the
tail. Peel the skin away and trim away the adipose tissue from
the shoulders.
 16 Culture of Dissociated Sensory Neurons from Dorsal Root… 183

4. Using a small pair of sharp pointed scissors (Stevens tenotomy,

straight/sharp scissors) make an incision through the whole
length of the dorsal column only. This is achieved by putting
the lower blade of the scissors into the spinal column and cut-
ting through the dorsal spinal vertebrae from the head to the
tail to reveal the spinal cord.
5. The vertebrae should be trimmed on one side to just above the
level of the row of DRG (see Note 3). Remove the spinal cord
by scooping it out from underneath starting at the neck.
Remove vertebrae from the other side of the column again to
just above the level of the DRG which can now be seen.
6. Remove the DRGs one at a time using forceps (Inox 5) which
are placed behind the cell body easing it out of the pocket it
sits in. Use the forceps or small scissors in the other hand to cut
off the roots as close to the cell body as possible. Transfer the
DRGs to a 60-mm ∅ petri dish containing cold HBSS (on ice).
Continue in this way until all the DRGs have been collected
from each pup. It should be possible to collect 30–35 DRGs
(maybe 40 with more experience and care when exposing the
DRGs) from each pup.
7. Clean the DRG in the cold HBSS to remove meninges and
excess roots that may still be attached. Transfer the clean gan-
glia to another 60-mm petri dish containing 5 mL of trypsin-
EDTA. The trypsin-EDTA should be only partially defrosted
to keep the ganglia as cold as possible.

3.1.2. Dissociation 1. Incubate the ganglia in the trypsin-EDTA for 15–20 min at
37°C.
2. Add 5 mL DMEM (10% FCS) to the dish with ganglia and
transfer to a 15-mL tube. Leave the DRG to settle to the bot-
tom or centrifuge at 200 × g for 3 min.
3. Carefully remove the supernatant using a 5-mL Stripette (do
not use vacuum aspiration).
4. Add 0.1% collagenase solution (1 mL of 0.5% stock plus 4 mL
HBSS) to the ganglia and incubate for 90 min at 37°C.
5. Add 5 mL DMEM (10% FCS) and centrifuge at 200 × g for
3 min.
6. Carefully remove the supernatant using a 5-mL Stripette (do
not use vacuum aspiration).
7. Add 1 mL DMEM (10% FCS) and mechanically dissociate the
ganglia by triturating no more than 6–10 times using a 1-mL
pipette tip.
8. Dilute the cell suspension (point 7) to 10 mL with DMEM
(10% FCS) and plate onto a 100-mm ∅ tissue culture dish for 1 h
at 37°C. The nonneuronal cells and large-diameter DRG neurons
184 D.E. Owen and J. Egerton

will stick to the dish, whereas the small-/medium-diameter

neurons will float in the medium. The longer the incubation, the
more likely that the small/medium diameter neurons will stick
to the dish, in which case, wash the dish 1–2 times with the
medium in the dish (see Note 4).
9. Collect the medium (containing unattached neurons) and
transfer to a 15-mL tube. Centrifuge at 200 × g for 5 min.
10. Remove the medium using a Stripette and resuspend the cell
pellet with 1 mL serum-free DMEM (50 ng/mL β-NGF, N2
supplements, and 0.05% BSA) using a 1-mL pipette tip to
break up cell pellet (see Notes 5 and 6).
11. Count cells with a hemocytometer (see Note 7).
12. Plate cells directly into growth medium at a density appropriate
to the experimental application. Examples of some plating den-
sities for different applications are given below as a guide:

96-well plates 30,000 cells per well in 100 μL growth

medium
384-well plates 20,000 cells per well in 50 μL growth
medium
Electrophysiology 200,000 cells per 22-mm coverslip/35-mm
dish in 2 mL medium, or 100,000 cells
(in 0.5 mL) spotted onto the coverslip
only and incubate for 1 h to allow
neurons to attach before flooding the
culture dish with 2 mL medium
Immunocytochemistry 100,000 cells per coverslip/35-mm dish in
2 mL medium
Single cell imaging 70,000–100,000 cells in 50–100 μL spotted
onto a coverslip, incubate for 1 h to allow
neurons to attach before flooding the
culture dish with 2 mL medium

13. Contaminating glia that may still be present in the cultures can
be reduced by adding the antimitotic cytosine β-D-arabinoside
to a final concentration of 1 μM after 24 h in culture. This is
conveniently done by preparing a stock solution of 1 mM cyto-
sine β-D-arabinoside in water, filter-sterilizing, and storing as
aliquots at −20°C. This can be added 1:1,000 to the cultures.

3.2. Adult DRG 1. Sterilize dissection instruments by treating with 70% v/v etha-
Neurons nol, and allow to dry in the dissection cabinet.
3.2.1. Dissection 2. Weigh the rats and then sacrifice by exposure to a rising con-
centration of CO2. Confirm the death of each animal by expos-
ing the heart and severing one of the main cardiac blood
vessels. Perform exsanguination by placing each rat on its side
 16 Culture of Dissociated Sensory Neurons from Dorsal Root… 185

on absorbent bench coat (this minimizes the amount of blood

in the spinal column) (see Note 8).
3. In the dissection cabinet, remove the head of the rat to gain
access to the spinal column. Make an incision through the skin
following the line of the spinal column from the head to the
tail and peel back the skin. Use a pair of scissors to remove any
excess tissue surrounding the dorsal column.
4. Using a pair of pointed bone cutters (Littauer bone cutters,
straight), cut through each side of the dorsal vertebrae, remove
each vertebrae away to expose the spinal cord, and move in the
head to tail direction taking care not to damage the spinal cord.
Carefully pick up and lift out the spinal cord using a pair of
small standard forceps (standard pattern) while cutting the
DRG nerve branches close to the spinal cord (this will mini-
mize the loss of any DRG).
5. Remove the DRG one at a time using forceps (Inox 5) which
are placed behind the cell body, easing it out of the pocket it
sits in. Use the forceps or small scissors in the other hand to cut
off the roots as close to the cell body as possible. Transfer the
DRG to a 60-mm petri dish containing cold HBSS (on ice).
Continue in this way until all the DRGs have been collected from
each rat. It should be possible to collect 30–35 DRGs from each
rat. 40 DRGs per rat may even be achievable once the operator
is skilled and careful when exposing the cord.
6. Trim roots and nonneuronal tissue from each set of ganglia
with fine forceps and fine spring-loaded scissors (treated with
70% ethanol) by removing the roots and any attached non-
DRG tissue under the dissecting microscope. Place the trimmed
DRG into new 35-mm dishes containing 1.8 mL fresh HBSS.

3.2.2. Dissociation 1. Add 200 μL of 6.25 mg/mL collagenase (0.0625% final con-
centration) to the medium containing the DRG and swirl gen-
tly to mix. Incubate the ganglia for 2–3 h at 37°C, 5% CO2.
2. Using a 5-mL Stripette, carefully transfer the ganglia to a 15-mL
conical-bottomed centrifuge tube and wash twice with 10 mL
prewarmed growth medium to remove the collagenase.
3. Remove the medium by aspiration and resuspend the ganglia
in 2 mL growth medium. Triturate the DRG approximately 20
times through a flame-polished Pasteur pipette (smallest hole
possible) to obtain a single cell suspension.
4. Slowly drip the cell suspension onto a cushion of 2 mL 15%
BSA in growth medium in a 15-mL centrifuge tube and centri-
fuge at 200 × g at room temperature for 10 min to remove
nonneuronal cells and debris.
5. Carefully remove the supernatant by aspiration until approximately
50 μL remains covering the cell pellet. Resuspend the cell pellet
186 D.E. Owen and J. Egerton

in 1 mL of prewarmed growth medium containing mNGF

(1 μL mNGF for every 1 mL medium) using a 1-mL pipette.
Transfer the cell dissociate to a sterile 5-mL tube ready for
seeding.
6. Count cells with a hemocytometer (see Note 7).
7. Plate cells directly into growth medium at a density appropri-
ate to the experimental application. It is our experience that
the number of neuronal cells from adult DRG preparations is
much lower than those achieved from postnatal rats. Only a
small number of wells of a 96-well plate would be possible, and
so, we would conclude that adult DRGs are not suitable for
higher-throughput formats. For electrophysiology, we have
used 12 mm ∅ coverslips and plated cells at 2,000 cells per
coverslip.
8. Contaminating glia that may still be present in the cultures can
be reduced by adding the antimitotic cytosine β-D-arabinoside
to a final concentration of 1 μM after 24 h in culture.

4. Notes

1. We purchase 0.5% (10×) trypsin-EDTA and dilute with calcium-

and magnesium-free PBS. It is crucial that the trypsin-EDTA
is kept at <4°C before use and in the absence of calcium and
magnesium; otherwise, the trypsin will become inactive, and
the dissociation, incomplete. This reduces the quality of the
final cell preparation.
2. We have utilized both commercially available poly-L-lysine–
coated plates and coverslips and ones that we have made. It
seems the cells are slightly better for some applications on the
homemade substrates, but this should be determined for your
application.
3. The DRG cannot be seen at this time, so it is wise to cut a little
higher until greater confidence is achieved. More vertebrae can
always be trimmed off once the spinal cord has been removed
and the DRG can be seen.
4. This plating step can be omitted if it is not necessary to remove
the contaminating glial cells from your preparation. The qual-
ity of the neurons may well be improved by eliminating this
step, as the preparation time is reduced and the neuronal cells
do not have an increased exposure to FCS. The presence of
glial cells in the preparation may well produce trophic factors
to support the DRG neurons and make the in vitro preparation
more comparable to in vivo. The nonneuronal cells can easily
be left in the preparation for the purposes of electrophysiology
 16 Culture of Dissociated Sensory Neurons from Dorsal Root… 187

or single cell injection, for example, where the operator should

be able to distinguish morphologically between neuronal and
nonneuronal cell types.
5. If the DMEM stated in the methods is not used and a different
recipe DMEM substituted, this may affect the expression/
function of receptors and ion channels. In our experience, the
removal of pyruvate from the medium causes loss of calcium
responses to capsaicin. It is possible to culture both postnatal
and adult DRG neurons in Neurobasal medium plus B27 sup-
plement (Invitrogen). A comparison may be necessary to
establish if cellular functionality is affected.
6. The addition of growth factors to the growth medium should
be investigated according to the functionality required. For
example, brain-derived growth factor (20 ng/mL) addition to
postnatal DRG neurons is required for purinergic P2X 2/3
receptor–responsive cells in place of NGF.
7. Automated cell counters such as Cedex may not be suitable for
counting DRG neurons as the small neurons may be too small
for such devices to detect. Also, most automated devices take
large amounts of sample to complete a count, which is not usu-
ally practical with DRG preparations. New-generation auto-
mated cell counters which use much smaller sample volumes
and have higher resolution for cell detection could be com-
pared with manual hemocytometer counts to check their
accuracy.
8. In practice, 2–4 adult rats can be dissected per session per per-
son due to time constraints; cell viability and responsiveness
decrease if dissection time is prolonged.

References

1. Wood JN, Winter J, James IF, Rang HP, Yeats but are not required for survival of adult
J, and Bevan S (1988) Capsaicin-induced sensory neurones. J Neurosci 8, 2394–2405
ion fluxes in dorsal root ganglion cells in culture. 3. Winter J, Forbes CA, Sternberg J, and Lindsay
J Neurosci 8, 3208–3220 RM (1988) Nerve growth factor (NGF) regu-
2. Lindsay RM (1988) Nerve growth factors lates adult rat cultured dorsal root ganglion neu-
(NGF, BDNF) enhance axonal regeneration ron responses to capsaicin. Neuron 1, 973–981
 Chapter 17

Culture and Proliferation of Highly Purified Adult

Schwann Cells from Rat, Dog, and Man
Kirsten Haastert-Talini

Abstract
This chapter presents fast and easy protocols to obtain highly purified cultures of proliferating adult rat,
canine, and human Schwann cells. Cell preparation from predegenerated adult sciatic nerves combined
with the use of melanocyte growth medium supplemented with forskolin, fibroblast growth factor-2, pituitary
extract, and heregulin as selective, serum-free culture medium and two methods for a consecutive cell-
enrichment step are described. Our protocols result in approximately 90% pure Schwann cell cultures (or
higher). The average time to obtain highly purified in vitro cultures of adult Schwann cells is 21 days.

Key words: Adult schwann cells, Rat, Human, Dog, Enrichment, In vitro

1. Introduction

The culture of adult Schwann cells from laboratory animals and

humans is of high relevance for therapeutic approaches, e.g., trans-
plantation of these cells to promote central or peripheral nervous
system regeneration. Furthermore, such cultures could be of
interest to investigate cell–cell interactions between neurons and
Schwann cells or stem cells and Schwann cells in cell replacement
approaches. The culture of highly purified Schwann cells could
further be of interest with regard to developmental myelination or
demyelination and remyelination in peripheral neuropathies.
To study the role of Schwann cells in peripheral neuropathies
in vitro or the effects of adult Schwann cell transplantation in
regenerative medicine in vivo, it is needed to culture highly purified
adult Schwann cell populations. The number of contaminating
fibroblasts which might induce bias of any study result should be
reduced as much as possible. Numerous publications exist on

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_17, © Springer Science+Business Media, LLC 2012

189
190 K. Haastert-Talini

the purification and in vitro culture of adult Schwann cells from

different species (1–15). We established techniques for isolation
and high enrichment of rat (16, 17), dog (18, 19), and human
(17, 20) adult Schwann cells. The use of fresh tissue results in poor
primary cell yields; therefore, we recommend predegenerated tissue
as source for adult Schwann cells. Predegeneration of peripheral
nerve tissue allows Wallerian degeneration to take place. In response
to nerve injury, Schwann cells switch from a myelinating to a non-
myelinating phenotype, and together with macrophages, they
remove necrotic tissue and myelin debris. Furthermore, Schwann
cells proliferate to form bands of Büngner which help the regrowing
axons to elongate their growth cones in the direction of the
denervated targets (21–23). Thus, predegeneration specifies the
induction of Schwann cell dedifferentiation and proliferation prior
to tissue dissociation; predegeneration in vitro additionally
promotes fibroblast outgrowth from the cultured tissue (16, 18).
Our methods are based on cell harvest from predegenerated
peripheral nerve tissue, 20-h dissociation of tissue, culture of cells
in serum-free melanocyte growth medium (MGM) supplemented
with Schwann cell mitogens, and either an easy and fast washing
step (“cold jet”) or an immunopurification step to reduce contami-
nating fibroblasts from mixed peripheral nerve cell cultures. The
medium composition was basically determined in adult rat Schwann
cell cultures (16) and later adopted for culturing adult Schwann
cells from canine and human origins. We use MGM instead of
Dulbecco’s modified Eagle’s medium (DMEM) because in our
hands, the use of DMEM was inductive to fibroblast overgrowth.
The same holds true for the use of 5% or 10% fetal calf serum
(FCS) in the medium; in our hands, only serum-free culture condi-
tions successfully suppressed overgrowth of Schwann cells by fibro-
blasts. The mitogens used in our culture medium are commonly
used in other protocols as well. We used to produce the fibroblast
growth factor-2 (FGF-2) in our lab, but the commercially available
FGF-218kD exerts the same beneficial effects. We found the use of
mitotic toxins like arabinoside-C not useful in enrichment of adult
rat and human Schwann cells because it inhibited growth of both
fibroblasts and Schwann cells. However, for cell cultures from
canine origin, it was more selective for the suppression of fibroblast
growth. Final removal of contaminating fibroblasts from rat and
human cultures is based on different attachment properties of cul-
tured adult rat and human peripheral nerve cells. In these cultures,
fibroblasts grow mainly flat on the cell culture surfaces, and the
adult Schwann cells predominantly grow on top of the fibroblasts.
The “cold jet” technique was modified from an enrichment method
to obtain Schwann cell–free cultures of dorsal root ganglion
neurons (24) and includes a washing step. A technique we also use
in neonatal rat Schwann cell enrichment is the removal of fibroblast
via magnetic bead–based cell separation (25). While this technique
 17 Culture and Proliferation of Highly Purified Adult Schwann Cells… 191

resulted in a reduction of the cell yield for adult rat and human
Schwann cell cultures, it was more effective than “cold jet” in the
purification of adult canine Schwann cells.

2. Materials

2.1. In Vitro 1. DMEM, high glucose without L-glutamine (Gibco BRL).

Predegeneration 2. Penicillin/streptomycin (Gibco BRL).
3. Amphotericin (Gibco BRL).
4. Collagen G (Biochrom).
5. Melanocyte Growth Medium, MGM (plus manufacturer’s
supplements) (PromoCell).
6. Scalpels (No. 21, Fine Science Tools).
7. Dumont forceps (Dumont No. 5, Fine Science Tools).
8. Vannas microscissors (No. 15003-08, Fine Science Tools).
9. Stereo microscope (Stemi SV 6, Carl Zeiss AG).
10. FCS (gold, Gibco BRL).

2.2. Dissociation of 1. Distilled water (aqua ad iniectabila, Ampuwa® Fresenius Kabi,

Predegenerated Tissue Germany).
and Initial Seeding 2. Poly-L-ornithine hydrobromide (PORN), 30–70 kDa; (Sigma-
Aldrich) stock solution: boric acid/NaOH at 0.15 M each,
adjusted to pH 8.4 by using HCl: 0.1 N, store at +4°C for
weeks (at −20°C for months).
3. Laminin (Becton Dickinson), store in aliquots at −80°C.
4. Poly-L-lysine hydrobromide (PLL, 70–150 kDa) (Sigma-Aldrich;
0.5 mg/mL in distilled water, store at +4°C for weeks).
5. Collagenase (Type IV, 160 U/mg, Gibco BRL).
6. Dispase (Roche Diagnostics).
7. DNase I (Roche Diagnostics).
8. Hank’s Balanced Salt Solution, (without Mg and Ca (HBSS),
Gibco, Invitrogen).
9. Trypan blue solution (Gibco BRL).
10. Forskolin (FK, 7-deacetyl-7-[O-(N-methylpiperazino)-g -
butyryl]-, dihydrochloride, Calbiochem).
11. FGF-218kD (PeproTech).
12. Bovine pituitary extract (BPE-26, PromoCell).
13. Recombinant human neuregulin 1-b1/heregulin-b1 epidermal
growth factor (EGF) domain (rhuHRG, [rHRG-b1176–246]),
E. coli (R&D Systems).
14. Bovine serum albumin (BSA, Fraction V, Sigma-Aldrich).
192 K. Haastert-Talini

2.3. Removal 1. Cytosine arabinoside (Sigma-Aldrich).

of Fibroblasts/ 2. Dynabeads® sheep anti-rat IgG (Dynal Biotech, Invitrogen).
Enrichment of Adult
3. Rat anti-canine Thy1-antibody (anti-Cdw 90 canine, Serotec).
Schwann Cell Cultures
4. Dynal® MPC-S (example for magnet, Dynal Biotech, Invitrogen).
5. Trypsin/EDTA (0.5 g/L trypsin, 0.2 g/L EDTA; Gibco BRL).
6. Phosphate-buffered salt solution (PBS, pH 7.1).
7. Roto-Shake Genie® (Scientific Industries Inc.).

2.4. Analysis 1. 4% Paraformaldehyde in PBS.

of Schwann 2. Blocking buffer: 5% BSA in PBS or 3% goat serum (Gibco,
Cell Purity by Invitrogen) in PBS.
Immunocytochemistry 3. Primary antibody for rat Schwann cells: mouse anti-rat
p75low-affinity nerve growth factor receptor (p75LNGFR) antibodies (from a
hybridoma cell line in our lab; dilute 1:5 in blocking buffer,
antibody concentration 35 mg/mL (16)).
4. Primary antibody for human Schwann cells: mouse anti-human
p75LNGFR antibodies (Accurate Chemical & Scientific
Corporation, USA; dilute 1:20 in blocking buffer (20)).
5. Primary antibody for rat and human Schwann cells: rabbit anti-
cow S100 calcium-binding protein antibodies (DakoCytomation;
dilute 1:200 in blocking buffer/0.1% Triton-X-100 (Sigma-
Aldrich) (16, 20)).
6. Primary antibody for canine Schwann cells: rabbit anti-human
p75LNGFR (dilute 1:200 in blocking buffer, p75 pAb Promega).
7. Primary antibody for rat fibroblasts: mouse anti-rat Thy1-
antibodies (from a hybridoma cell line in our lab; dilute 1:3 in
blocking buffer, antibody concentration 20 mg/mL (16)).
8. Primary antibody for canine fibroblasts: rat anti-canine Thy1-
antibody (anti-Cdw 90 canine, dilute 1:1,000 in blocking
buffer), Serotec.
9. Secondary antibodies: Cy3-conjugated (dilute 1:400 in block-
ing buffer; Jackson ImmunoResearch) or goat anti-rabbit Alexa
555 (dilute 1:500 in blocking buffer, Invitrogen) to visualize
primary antibody binding in fluorescence microscopy.
10. 4¢,6¢-Diamidino-2-phenylindole (DAPI, dilute 1:1,000 in
PBS; Sigma-Aldrich) for nuclear counterstaining.

3. Methods

3.1. In Vitro 1. Harvest fresh peripheral nerve tissue and transfer it into
Predegeneration DMEM + 1% Pen/Strep (see Note 1).
2. Depending on the nerve anatomy, the procedure to remove
the epineurium with the help of Vannas microscissors and
 17 Culture and Proliferation of Highly Purified Adult Schwann Cells… 193

Dumont (No. 5) forceps is slightly different. The epineurium

of rat nerves can be stripped off as whole, while isolated nerve
fascicles have to be pulled out of the connective tissue from
canine and human peripheral nerve tissue (see Note 2). To
facilitate this step, the nerve tissue could be cut in segments of
1 cm in length.
3. Collect the epineurium-free nerve tissue in DMEM + 1% Pen/
Strep (e.g., 10 mL in a 15-mL tube).
4. Transfer the preparation to a sterile work bench and wash the
tissue with DMEM.
5. For in vitro predegeneration, place groups of four to five
1-cm segments into the wells of an uncoated 6-well plate
(see Note 3).
6. Cover the tissue segments with 1.5–2.0 mL of 10% FCS in
culture medium: MGM (+ manufacturer supplements) + 2 mM
forskolin + 5 mg/mL BPE-26 + 10 ng/mL FGF-218kD + 1% Pen/
Strep (see Note 4).
7. Over the next 7–10 days, exchange the predegeneration
medium every second day (see Note 5).

3.2. Dissociation of For initial seeding of adult Schwann cells, the culture surfaces have
Predegenerated Tissue to be coated to enhance cell adherence.
and Initial Cell Seeding

3.2.1. Cell Culture Surface 1. Twenty-four hours before initial seeding, remove the PORN
for Adult Rat and Human stock solution from the +4°C storage and dilute 1:10 with ice-
Schwann Cell Cultures cold sterile distilled water (final concentration 1 mg PORN/
mL). Keep the PORN solution on ice and remove an aliquot
of laminin from −80°C and thaw it on ice.
2. Mix the ice-cold PORN solution with the laminin to give a
final concentration of 6-mg laminin/mL PORN.
3. For coating, cover the cell culture wells thoroughly (e.g.,
6-well plate: 1.5 mL/well) with ice-cold PORN-laminin solu-
tion and incubate for 24 h at room temperature (see Note 6).
4. Wash the wells with either distilled water or MGM and keep
them covered with either solution to avoid drying.

3.2.2. Cell Culture Surface Use PLL to coat plates for canine Schwann cell cultures.
for Adult Canine Schwann
1. Two hours before initial seeding, remove the prepared PLL
Cell Cultures
solution from the +4°C storage and thoroughly cover the cell
culture wells with it.
2. Incubate for 1 h, then wash the wells with either distilled water
or MGM and keep them covered with either solution to avoid
drying.
194 K. Haastert-Talini

3.2.3. Dissociation of 1. Prepare the solution for enzymatic dissociation as follows:

Predegenerated Nerve DMEM + 10% FCS + 1% Pen/Strep + 0.125% collage-
Tissue nase + 1.25 U/mL dispase (1.5–2 mL/predegeneration well
on 6-well plate).
2. Remove the medium from predegeneration dishes and use a
sterile scalpel to cut the nerve tissue into short segments of
2–4 mm in length.
3. Add dissociation solution to each predegeneration well and
incubate the tissue for 2 h in a 37°C and 5% CO2 incubator.
4. Transfer the solutions and tissue residue into an adequate
number of 15-mL tubes (one tube per predegeneration well).
Use an equal volume of HBSS to wash the wells and to reduce
the enzymatic activity.
5. Mechanically separate the tissue residue into single cells by
gentle pipetting on and off the suspension through fire-polished
glass pipettes until the solution becomes optically homogeneous.
6. Centrifuge the single-cell solution at 235 × g for 5 min at +21°C.
7. Remove the supernatant and suspend the pellet in DMEM.
Repeat the centrifugation (see Note 7).

3.2.4. Initial Seeding 1. Dilute the cell pellet in culture medium (see Note 8). To
enhance the seeding efficiency, add 1% BSA to the culture
medium for the first 24 h (see Note 9).
2. It is important to realize that seeding efficiency, initial survival
rate, and proliferation rate of primary adult Schwann cells
depend crucially on optimal cell densities in culture (26).
Therefore, count the number of viable cells prior to seeding,
e.g., after 1:2 dilution of a 10-mL cell suspension aliquot in
trypan blue (will stain dead/dying cells blue).
3. Plate primary adult peripheral nerve cells at a density of 106 living
(trypan blue-negative) cells/well on a 6-well plate (35 mm2).
4. The next day, change the medium to culture medium without
BSA and culture the primary peripheral nerve cells for 2–5 days
at +37°C and 5% CO2 (see Note 10).

3.3. Enrichment The fact that the adult Schwann cells grow mainly on top of the
of Adult Schwann Cell more flattened and more securely adherent fibroblasts allows
Cultures/Removal separation of the two cell types by an easy washing step.
of Fibroblasts 1. Remove the medium and slowly add 1 mL of ice-cold PBS and
3.3.1. Enrichment of Adult immediately aspirate for 2–3 times in order to flush the well
Rat and Human Schwann (6-well plate) and loosen the Schwann cells.
Cells 2. Apply a stream of 1-mL ice-cold culture medium, using a blue
1-mL Gilson tip and pipette on and off for several times,
detaching the Schwann cells from the underlying fibroblast
 17 Culture and Proliferation of Highly Purified Adult Schwann Cells… 195

layer. Especially those parts of the wells with high cell densities
have to be rinsed. To reduce the risk of loosening also fibroblasts,
monitor the detachment using a phase-contrast microscope.
3. Transfer the suspension of floating cells, mainly Schwann cells,
to a 15-mL tube (see Note 11) and centrifuge (235 × g for
5 min at +21°C).
4. Resuspend the cell pellet in culture medium, determine the
viable cell number with trypan blue staining (see above), and
seed the enriched adult Schwann cells into freshly prepared
PORN-laminin-coated wells.

3.3.2. Removal of For enrichment of adult canine Schwann cells, “cold jet” does not
Fibroblasts from Adult work efficiently; however, these cells can be immunopurified.
Canine Schwann Cell
1. Wash magnetic Dynabeads® sheep anti-rat IgG in 0.1% BSA in
Cultures
PBS (10-mL beads in 500-mL 0.1% BSA). Initially, the
Dynabeads have to be fluffed up and then mixed with the
washing solution inside an eppendorf cup (1.5 mL).
2. Bring the magnet close to the wall of the cup; this draws the
magnetic Dynabeads toward the magnet, allowing the superna-
tant to be easily removed. Repeat the procedure 2 more times.
3. To couple the Dynabeads with rat anti-canine Thy1-antibody
(dilute 1:300 in MGM), add 10 mL of the washed Dynabeads
to 300 mL of the antibody solution in another eppendorf cup.
4. Securely seal the vial with parafilm, put it into an ice-filled
50-mL tube, and rotate it around its horizontal axis 45 min at
+4°C (e.g., place Roto-Shake Genie® in +4°C room). This
step facilitates equal coating of the beads with the antibodies.
5. Wash the antibody-coupled beads 3 times with 0.1% BSA as
before.
In parallel, all cells are enzymatically detached from the wells.
6. Remove the medium and incubate the cells with trypsin/
EDTA for 1 min at +37°C (within the CO 2 incubator),
followed by further incubation at room temperature and
observation under phase-contrast microscopy.
7. As soon as all cells are detached, add culture medium/collagenase
IV (dilute 1:1), pipette the cells on and off, and finally, transfer
them into a 15-mL tube for centrifugation later on.
8. Again, wash each well with 500-mL culture medium; add these
washes to the cell suspension.
9. Centrifuge at 235 × g for 5 min at +21°C.
10. Resuspend the cell pellet in culture medium/DNase I (dilute
10:1) and centrifuge again.
11. Resuspend the final cell pellet in culture medium (1 × 105
cells/300 mL).
196 K. Haastert-Talini

Then, incubate the cell suspensions with anti-Thy1-coupled

magnetic beads to allow the beads to bind to the Thy1 (CD90)
surface antigens on fibroblasts (10 mL beads + 300 mL cell
suspension).
12. Carefully mix beads and cell suspension within an eppendorf
cup, securely seal the vial with parafilm, put into an ice-filled
50-mL tube, and rotate it around its horizontal axis for 45 min
at +4°C.
13. Bring the magnet close to the wall of the cup; this draws the
fibroblasts, which are now covered by magnetic Dynabeads,
toward the magnet, and the supernatant, including the enriched
Schwann cells, can easily be collected.
14. Determine the number of viable cells by trypan blue staining
and seed the enriched adult Schwann cells into freshly pre-
pared PORN-laminin-coated wells.
15. After the cells have grown to 90% confluency (about 10 days),
perform a second immunopurification, as above.

3.3.3. Conditions for After final purification of adult Schwann cells, the cultures can be
Propagation of Adult split whenever they reach confluency. For this, the cells are detached
Schwann Cell In Vitro with the use of trypsin/EDTA and reseeded on freshly prepared
PORN-laminin-coated culture dishes. Appropriate cell densities
for seeding enriched adult Schwann cells (purity > 95%) are as
follows:
● Adult rat Schwann cells: 5 × 104 cells/cm2.
● Adult canine and human Schwann cells: 2 × 105/cm2.
We do not include a proliferation step in our protocol as we
found proliferation rates to be enhanced in adult human Schwann
cells by permanently supplementing fresh heregulin to each
medium exchange (20). Elevated proliferation rates were induced
in adult rat Schwann cell cultures just by passaging the cells with
the “cold jet” technique (16). Adult canine Schwann cells show a
proliferation rate under the given conditions, which does not
require any further culture supplement.

3.4. Analysis Although phase-contrast microscopy may give an impression of

of Schwann cell culture purity with the flatter morphology of fibroblasts and a
Cell Purity by clearer nucleus-soma relation in Schwann cells, it is necessary to
Immunocytochemistry check for Schwann cell purity of cultures before and after enrichment.
Immunocytochemistry should be used to rigorously determine
culture purity. For this purpose, sister cultures should be grown on,
e.g., 24-well plates and analyzed (see Note 12). Adult Schwann cells can
be identified by staining either for intracellular calcium-binding
protein S100 or for cell surface p75LNGFR (16–20). Nuclear counter-
staining with DAPI will visualize S100- or p75LNGFR-immunonegative
 17 Culture and Proliferation of Highly Purified Adult Schwann Cells… 197

Fig. 1. Highly purified human Schwann cell culture characterized by immunocytochemistry against the intracellular
calcium-binding protein S100 (a, b) or the cell surface molecule p75LNGFR (c). Nuclear counterstain with DAPI visualizes
S100- or p75LNGFR-immunonegative cells, e.g., fibroblasts, which additionally display darker and bigger nuclei in DAPI staining
(indicated by white arrows). Arrow heads in (a) indicate bright nuclei of dying cells.

cells, e.g., fibroblasts. Figure 1 illustrates a highly purified human

Schwann cell culture, characterized by immunoreactivity against
S100 (see Fig. 1a, b) and p75LNGFR (see Fig. 1c). Fibroblasts even-
tually display a weak staining but could additionally be identified
by their multipolar, flattened morphology with darker and larger
nuclei in DAPI-staining, while adult Schwann cells show a typical
bipolar or tripolar morphology and smaller nuclei.
1. Remove the culture medium and fix the cells with 4% paraform-
aldehyde (15 min at room temperature).
2. Remove the fixative and incubate the cells with blocking buffer
(30 min at room temperature).
3. Add a single type of primary antibody (see Note 13) and
incubate overnight in the dark at +4°C.
4. Wash the cells with blocking buffer.
5. Incubate with secondary antibody for 2 h at room temperature
(in the dark!).
6. Counterstain cell nuclei with DAPI (15 min at room tempera-
ture, in the dark!).
7. Wash the cells with PBS and keep them covered with PBS for
analysis under the inverted fluorescence microscope.

4. Notes

1. Due to logistical circumstances, human nerve tissue (residues

of sural nerve transplants harvested during reconstructive surgery
in vivo, donor age (13–60 years), male or female (20)) or dog
nerve tissue (donor animals euthanized because of infaust
prognosis with no peripheral neuropathies (18, 19)) could be
stored at +4°C for a maximum of 20 h.
198 K. Haastert-Talini

2. This work has to be done under the stereo microscope because

it is of high importance that exclusively nerve fascicles are
harvested, those appear dense and never hollow-like, e.g., blood
vessels.
3. For predegeneration of canine nerve samples, it is eventually of
advantage to precoat the cell culture surface with 2 mL collagen
G (1:1 in MGM + manufacturer supplements). Therefore, the
mixture is allowed to solidify for 1 h at +37°C. Attachment of
the epineurium-free nerve fascicles to the collagen coating is
facilitated by incubation of the tissue in dry wells for 15 min at
+37°C.
4. For canine tissue, also 1% amphotericin should be added to the
predegeneration culture medium.
5. The medium has to be removed very carefully, and before adding
new medium, the “dry” plates could eventually be incubated
for 15 min at +37°C to enhance tissue adherence on the cul-
ture surfaces.
6. To avoid evaporation and drying of the coating, the well plate
has to be covered and sealed with parafilm (eventually an addi-
tional wrap with aluminum foil could also help).
7. In case there are cell clusters or tissue clumps remaining, gentle
pipetting could also be performed with culture medium + col-
lagenase, (0.125%) again followed by centrifugation and
another optional pipetting with a 10:1 mixture of culture
medium and DNase before final centrifugation. However, it is
most important to treat the cells with absolute care. One must
avoid triturating already separated cells; therefore, all single-
cell solution supernatants need to be collected and diluted to
reduce enzymatic activity within the solution. Furthermore,
unnecessary centrifugation should be avoided.
8. The right volume of culture medium to be added to the final
cell pellet has to be carefully calculated (e.g., 0.5 mL/tissue
dissociated in 1 well on a 6-well plate), as one needs to avoid
additional centrifugation prior to initial seeding of the cells.
9. For culturing cells of human origin, 10 nM of fresh rhuHRG
(heregulin) has to be added to the culture medium to increase
the proliferation rate whenever the medium is exchanged.
10. For culturing cells of canine origin, 1 mM cytosine arabinoside
should be added from day 2 to 6 in order to reduce the number
of fibroblasts.
11. If necessary, “cold jet” can be repeated once or twice with
culture medium.
12. In a 24-well plate, a volume of 150 mL of buffer or antibody in
buffer is sufficient for incubation. During incubation, the wells
 17 Culture and Proliferation of Highly Purified Adult Schwann Cells… 199

should be covered to avoid evaporation and the plates should

at best be wrapped in aluminum foil for darkness.
13. It is advised to use only single staining against Schwann cells or
fibroblasts.

Acknowledgments

In the first place, I wish to thank all persons who helped to estab-
lish and to advance the described techniques: Dr. Christina Mauritz,
Dipl. Biol.; Maike Wesemann; Sukhada Chaturvedi, PhD; Dr. Peer
Seef; Dr. Ruth Schmitte; and Silvana Taubeler-Gerling. Many
thanks also to Prof. Dr. Claudia Grothe for creating a supportive
working atmosphere. Special thanks go to our clinical colleagues
and their patients for providing us with human and canine nerve
tissue samples: Prof. Dr. Cordula Matthies; Prof. Dr. Götz Penkert;
Dr. Veronika Stein, PhD; Dr. Henning Schenk, PhD; Dr. Cornelia
Flieshardt; and Prof. Dr. Andrea Tipold.

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modified canine Schwann cells-In vitro and dent regulation of human Schwann cell prolif-
in vivo evaluation of their suitability for peripheral eration. Glia 30, 165–177
 Chapter 18

Use of PC12 Cells and Rat Superior Cervical Ganglion

Sympathetic Neurons as Models for Neuroprotective
Assays Relevant to Parkinson’s Disease
Cristina Malagelada Grau and Lloyd A. Greene

Abstract
Cellular models composed of primary neuronal cultures or neuron-like cell lines are commonly used to
study neuron cell death and to test the neuroprotective properties of specific compounds. Cellular models
are easily accessible, permitting dissection and modulation of signaling pathways involved in neuron death.
For example, drug or shRNA delivery is more straightforward since there is no blood–brain barrier to
cross. However, since these models have their limitations, any important findings should ultimately be
verified with animal models and human samples. Here, we describe two cellular models that can be used
as a highly informative and easy to use starting point for testing potential neuroprotective drugs for
Parkinson’s disease: PC12 cells and sympathetic neuronal cell cultures. We describe in detail the protocols
needed to apply these models to study neuroprotection in the context of Parkinson’s disease.

Key words: PC12, Parkinson’s disease, 6-Hydroxydopamine, 1-Methyl-4-phenylpyridinium, Nerve

growth factor

1. Introduction

Tumor-derived cell lines have been extensively used as models to

study pathological conditions. The features of such proliferating
cells—homogenous and available in large numbers and easy to
grow and to transfect or to infect with viral particles—make them
highly suitable to screen drugs, manipulate genes, and explore sig-
naling pathways. For neurodegeneration studies, one of the most
used and cited culture systems has been the PC12 cell line which
was derived from a transplantable rat pheochromocytoma (1). These
cells have the machinery to synthesize, release, take up, and store
catecholamines, the major species of which is dopamine. A notable
feature of PC12 cells is that they respond to nerve growth factor
(NGF). In response to NGF, PC12 cells are converted from proliferating

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_18, © Springer Science+Business Media, LLC 2012

201
202 C. Malagelada Grau and L.A. Greene

chromaffin-like cells to nondividing sympathetic-neuron-like cells

that extend axons and become electrically excitable (2, 3). As will
be described here, generating and using PC12 cells for neurode-
generation experiments is a relatively simple process.
With respect to their use for studying neurodegeneration,
PC12 cells are sensitive to mitochondrial toxins that mimic Parkinson’s
disease (PD), such as 6-hydroxydopamine (4), 1-methyl-4-
phenylpyridinium (MPP+) (5), rotenone (6, 7), or paraquat (8).
These toxins induce cellular degeneration within 24–48 h after
exposure. The cells are also reported to undergo degeneration and
death in response to overexpression of mutant forms of synuclein
that are associated with familial PD (9).
These properties make PC12 an uncomplicated and convenient
in vitro model to study causes and possible treatments for PD.
However, one must keep in mind that, as with all models, PC12
cells have their limitations. Any result obtained with this tumor cell
line does not assure that the same finding will hold in in vivo mod-
els or in the human disease. Therefore, any hypotheses arising from
PC12 cell studies should be tested also in primary neuronal cell
cultures and in vivo models and, ultimately, wherever possible, in
PD itself (10).
One type of primary culture used to study PD is composed of
neurons derived from dissociated rodent superior cervical sympathetic
ganglia. Like dopaminergic neurons in the substantia nigra, nora-
drenergic sympathetic neurons of PD patients develop Lewy bodies
and undergo degeneration (11, 12). From an experimental point
of view, sympathetic neurons have the advantage that they are rela-
tively easy to obtain and culture (13), are a homogeneous popula-
tion, and also succumb to PD mimetics such as 6-hydroxydopamine
(6-OHDA) and MPP+ (5, 14, 15). They are also useful to assess
the role of specific genes by knockdown or by overexpression via
transfection or viral infection (13, 16). In addition, they are the
primary neuronal counterparts of NGF-treated PC12 cells and as
such represent the next step in progressing from experiments in a
cell line to cultures of primary neurons.
In this chapter, we will first guide the reader through the process
of culturing PC12 cells and sympathetic neurons. We will then
describe how to use these cultures to monitor cell viability when
testing compounds and treatments for their capacity to protect
against PD mimetics.

2. Materials

All work should be carried out in a cell culture hood using sterile
technique. It is recommended to disinfect surfaces with 70% etha-
nol before and after the proceedings.
 18 Use of PC12 Cells and Rat Superior Cervical Ganglion… 203

2.1. Coating Plastic 1. Rat tail collagen (Roche). Stock solution in 0.2% acetic acid in
Culture Ware sterile double distilled or deionized water. Working dilution
Substrates 1:15 in sterile double distilled or deionized water.

2.2. Coating Glass 1. Poly-D-lysine (Millipore) molecular weight >300 kDa. Dilute
Coverslip Substrates stock solution (1 mg/mL) in sterile double distilled water to
the final working concentration of 50 μg/mL.
2. Rat tail collagen (Roche). Stock solution in 0.2% acetic acid in
sterile double distilled or deionized water. Working dilution
1:15 in sterile double distilled or deionized water.

2.3. PC12 Cells 1. Complete medium: RPMI 1640 cell culture medium contain-
ing 10% heat inactivated horse serum (Sigma) (see Note 1), 5%
fetal bovine serum, and penicillin/streptomycin (50
units/50 μg of each per mL).
2. Differentiation medium: RPMI 1640 cell culture medium
containing 1% heat-inactivated horse serum and penicillin/
streptomycin (50 units/50 μg of each per mL) and 50 ng/mL
recombinant human or murine NGF (commercially available
from suppliers such as Alomone). This should be added from
1,000× stock just before use.
3. Freezing medium: complete medium with 10% dimethyl sul-
foxide (DMSO).

2.4. Sympathetic 1. Differentiation medium.

Neuron Cultures 2. Uridine/fluorodeoxyuridine: a 500-μM stock solution in ster-
ile double distilled or deionized water.
3. Trypsin (Invitrogen): 0.25% (w/v) in phosphate buffered
saline (PBS) (sterile and without EDTA).
4. RPMI 1640 cell culture medium.
5. One tube with 15 mL RPMI 1640 cell culture medium + NGF
(50 ng/mL) without serum and other additives.
6. One tube with 25–30 mL of RPMI1640 cell culture medium
without serum and other additives.
7. One bucket of ice.
8. Two insulin needles.
9. One polystyrene support for dissection (5 × 5 × 1 cm). Wrapping
the support in aluminum foil makes sterilization with 70%
ethanol much more easier.
10. 70% Ethanol.
11. Two micro dissecting tweezers (Roboz).
12. One dissecting tweezers (Roboz).
13. One dissecting scissors (Roboz).
14. One regular scissors (Roboz).
204 C. Malagelada Grau and L.A. Greene

2.5. PD Toxin 1. 10-mM 6-OHDA (Tocris) stock in sterile double distilled or

Treatments deionized water
2. 100-mM MPP+ (Sigma) stock in sterile double distilled or
deionized water

2.6. Viability Assay 1. 10× nuclei buffer (100 mL): cetyldimethyl-ethanolammonium

bromide (5 g), NaCl (0.165 g), glacial acetic acid (2.8 mL),
10% Triton X-100 (50 mL), 1 M MgCl2 (2 mL), 10× PBS
(10 mL), and H2O (35.2 mL). The working dilution is 1× in
distilled water (17).
2. Neubauer hemacytometer chamber with coverslip.

3. Methods

We will start by describing how to grow PC12 cells and how to

treat them with NGF. We will then discuss how to prepare rat sym-
pathetic neuron cultures. We also provide instructions on how to
perform treatments of the cultures with PD mimetic toxins that
induce cell death, and finally, we will suggest several options to
assess cell viability.

3.1. Growing PC12 To coat the plates, pipette the necessary amount of collagen (1:15)
Cells (see Note 2) to uniformly cover the surface. For a 10-cm culture dish, 1 mL is
sufficient, and this volume can be accordingly adjusted for culture
3.1.1. Coating Plasticware
dishes/wells of different sizes. Alternatively, one can add a larger
volume and then remove the excess with a sterile pipette. Allow the
plates to dry uncovered under a tissue culture hood for at least an
hour. After drying, plates can be stored at room temperature,
wrapped in aluminum foil or in the initial plastic bags, for 2–3
weeks (see Notes 3 and 4).

3.1.2. Thawing and Plating 1. If PC12 cells are received or stored in a frozen state (−80°C or
PC12 Cells in liquid nitrogen), they will be in complete medium and 10%
DMSO. Thaw the vial at 37°C in a water bath. This step should
be done quickly to diminish the toxicity of DMSO.
2. Prepare a sterile falcon tube with 10 mL of warm (37°C)
complete medium.
3. Add the thawed content of the cryotube to the 10 mL of
complete medium.
4. Mix well and centrifuge the cells for 5 min at 240 × g at room
temperature in a table top centrifuge.
5. Remove the supernatant (containing DMSO) and add 10 mL
of fresh complete medium to resuspend the cell pellet.
6. Mix well by trituration in a pipette and plate the contents in a
10-cm collagen-coated tissue culture plate. This step also serves
 18 Use of PC12 Cells and Rat Superior Cervical Ganglion… 205

to break up cell clumps. Shake the plate well, back and forth
and left to right, to spread the cells uniformly. Avoid shaking
circularly or otherwise the cells will tend to concentrate at the
center of the plate.
7. Once the cells are plated, renew the culture medium every 2–3 days.
Approximately 2/3 of the medium should be exchanged.
Maintain a volume of 5–8 mL of medium.

3.2. Subculturing It is recommended to subculture the cells when 90–95% conflu-

PC12 Cells ence is reached. One plate can be split in two or three plates.
1. Before detaching the cells from the plasticware, aspirate some
of the medium from the plate to reach a volume of 3–5 mL.
This avoids potential splashing of the medium out of the plate
during cell detachment.
2. Incline the plate at about 30°, and with a plastic sterile pipette,
eject a few milliliters of medium over the cell monolayer. Cells
will detach with the pressure exerted by the medium.
3. After applying medium in this way to the entire surface, tritu-
rate the cells several times with the pipette to break up cell
clumps. Split the resulting cell suspension into two or three
plates (see Notes 5 and 6).
4. Bring the total volume per plate to 5–8 mL of complete
medium.

3.3. Freezing PC12 1. Detach cells from the plates.

Cells 2. Centrifuge the cell suspension at 242 × g for 5 min.
3. Resuspend the pellet from one plate in 2 mL of complete
medium containing 10% DMSO.
4. Pipette the suspended cells into cryotubes and place them in
an isopropanol-freezing container.
5. Place the container with the cells in a −80°C freezer. The iso-
propanol container will freeze the cryotubes about 1°C/min
(see Note 7). The cryotubes can be stored at −80°C for up to
several months and transferred to liquid nitrogen for longer
periods of storage.

3.4. Treating PC12 PC12 cells dramatically change their phenotype when they are
Cells with NGF exposed to NGF. They exit the cell cycle, project long neurite-like
processes, and take on many properties of differentiated sympathetic
neurons, including synthesis, storage, and release of catecholamines
(principally dopamine).
1. Detach PC12 cells from the plate as described in
Subheading 3.2.
2. Dilute the cell suspension with differentiating medium
(RPMI1640, 1% horse serum, and 50 ng/mL NGF). Typically,
206 C. Malagelada Grau and L.A. Greene

one confluent culture of PC12 cells can be used to seed 5–20

cultures of the same size for NGF treatment. Note that the
NGF stock (50 μg/mL) should be stored at 4°C and should
be freshly added to RPMI 1640/1% horse serum medium as
needed.
3. Plate the cells on the appropriate collagen-coated plasticware.
4. Exchange ¾ of the culture medium every 2–3 days with dif-
ferentiating medium plus fresh NGF to a final dilution of
50 ng/mL.
5. The cell should begin to extend neurites within 24 h and will
continue to do so over the next 7–10 days.

3.5. Cultivating From one rat litter of 12–14 pups, a 24- or 48-well plate can be
Sympathetic Neurons obtained. The cell density can be adjusted as desired by counting
from Rat Superior the cells at the end of the dissection. The steps below describe the
Cervical Ganglia preparation of cultures from one litter of newborn rat pups. A video
of the dissection and culture procedure can be found at (13).
1. Soak all the dissection tools in 70% ethanol for at least 10 min.
2. Spray or wipe down with 70% ethanol all the surfaces of the
dissecting hood, the microscope, and the fiber optic lights.
Also, disinfect with 70% ethanol the small polystyrene foam
support wrapped in aluminum foil. Put a sterile piece of gauze
over the support.
3. Prepare two 10-cm plates with 5 mL of RPMI or PBS. These
will be used to rinse the tools during the dissection to wash off
any adhering tissue.
4. Place the lids of the 10-cm plates face up in the hood and pipette
onto them one or two drops of RPMI for each pup that will be
dissected. After dissection, the ganglia will be transferred to
these drops for cleaning under the dissecting microscope.
5. Put the tubes with RPMI on ice.
6. Keep the pups in a clean box. Dissect one pup at a time.
7. Spray the pup for dissection with 70% ethanol and immediately
decapitate it using a pair of sharpened small dissecting scissors.
Remove the head closer to the shoulders than to the jaw so as
to avoid cutting through the ganglia.
8. Place the head on the polystyrene support facing up.
9. With the fine scissors, cut the skin of the neck. Pin the skin
flaps with the insulin needles to the polystyrene support. This
will hold the head in place and prevent the skin from folding
over the field of dissection.
10. Pipette RPMI onto the exposed tissue to remove blood cells
and debris and to prevent drying.
 18 Use of PC12 Cells and Rat Superior Cervical Ganglion… 207

11. Under the dissecting microscope, with two pairs of fine-tip

tweezers, remove the salivary glands and connective tissue.
12. Remove the sternocleidomastoid muscle on the side of dissec-
tion to have a better view of the carotid artery. The carotid
branches into the external and the internal carotids. The
sympathetic ganglion is right beneath the branch point of
the artery. With one set of tweezers, pull the lower end of the
carotid up, and with the other, remove the translucent tear-
shaped ganglion attached to the vessel, that is, the sympathetic
ganglion. Close to the external carotid, there is another rounder
and smaller translucent ganglion. This is the nodose ganglion.
Be careful not to confuse the two ganglia.
13. Put the ganglion in one of the RPMI1640 drops on the culture
plate lid.
14. Repeat the dissection to remove the second ganglion on the other
side of the neck.
15. Under the dissecting microscope, clean the ganglia by teasing
away any remains of the carotid and surrounding connective
tissue. This will diminish contamination of the cultures by
nonneuronal cells.
16. Transfer the cleaned ganglia to the 15-mL tube with RPMI
plus NGF on ice. Collect all ganglia in this way until the dissec-
tion of all the pups is finished.
17. Centrifuge the ganglia at 168 × g for 3 min. Take care because
the ganglia tend to attach to the wall of the tube.
18. Remove the supernatant carefully and add 1 mL of 0.25%
trypsin in PBS (sterile). Incubate at 37°C for 30 min.
19. To neutralize the trypsin after the 30-min incubation, add
10 mL of RPMI medium with serum. Centrifuge at 168 × g for
3–5 min, remove the supernatant, and add 2 mL of RPMI with
1% horse serum and 50 ng/mL NGF.
20. Triturate the ganglia for 40–50 strokes in a glass Pasteur pipette
with a fire-polished tip. A second Pasteur pipette with a nar-
rower fire-polished tip can be used for the final 15 strokes.
21. Plate the cells (normally two ganglia per well in a 24-well plate)
at the desired density, adding more RPMI with 1% horse serum
and NGF to bring to the appropriate volume for plating.
22. Twenty-four hours later, add uridine/fluorodeoxyuridine to
the wells to reach a final concentration of the antimitotic of
10 μM (see Note 8).
23. Change the medium of the cultures every 2–3 days. Add
10-μM uridine/fluorodeoxyuridine to the fresh medium for
the first two medium changes.
24. Use the cultures between 7 and 15 days after plating.
208 C. Malagelada Grau and L.A. Greene

3.6. Toxin Treatments Both NGF-differentiated PC12 cells and sympathetic neurons will
be ready to use after at least 7 days following plating.
1. Prepare stock solutions of the toxins. Use stock concentrations
to minimize the volume of solution added to the cultures.
2. Change the medium right before any treatment.
3. If a specific compound is tested in conjunction with the toxins,
pretreatment should be done at least 30 min to 1 h before add-
ing the toxin. This is to permit entry into the cells. Always
include controls of sister cultures treated with the vehicles used
for delivery of toxin or tested agent. If the vehicle is DMSO,
the final amount should be no more than 0.1% of the final vol-
ume in the culture well.
4. To treat neuronal PC12 cells with (see Notes 9–12):
– 6-OHDA: a stock of 10 mM is recommended to treat the
cells at a final concentration of 50–100 μM.
– MPP+: a stock of 100 mM is recommended to treat the
cells at a final concentration of 500 μM–1 mM.
5. To treat rat sympathetic neurons, prepare stocks as above, but
the final concentrations of the toxins should be much lower:
5–10 μM for 6-OHDA and 50–100 μM for MPP+ (see Notes
9–12).
6. After treatments, wait for the desired time to assess the cul-
tures. If viability is monitored, 24 and/or 48 h are suggested.
7. For analyzing the samples by Western immunoblotting, choose
shorter incubation times.

3.7. Viability To quantify viable cells, there are several methods to choose from.
Assessment However, 6-OHDA and MPP+ with their capability to inhibit
mitochondrial complexes can interfere with tetrazolium salt assays,
leading to an overestimation of cell death. We routinely use a
method with a detergent solution that dissolves the cell plasma
membrane and leaves the nuclear membrane intact (17). This per-
mits counting of the numbers of surviving cells in the cultures.
With a Neubauer hemacytometer chamber, healthy round nuclei
can be counted under a phase microscope and distinguished from
the smaller, darker dead or degenerating nuclei.
The procedure is as follows:
1. Aspirate to fully remove the medium.
2. Add 0.5 mL of detergent solution to the wells (in a 48-well
plate, add accordingly for other-size dishes or wells) and mix
well (see Note 13).
3. Place approximately 10 μL of the nuclear suspension under the
coverslip of the Neubauer chamber.
4. Count at least 100 nuclei (see Note 14).
 18 Use of PC12 Cells and Rat Superior Cervical Ganglion… 209

5. Cell survival can be expressed as the percentage of viable cells in

experimental cultures compared with sister control cultures.
For transfection experiments, cell viability (after treatment)
can be assessed by counting living green fluorescent protein-positive
cells in strips under a fluorescence microscope. If the transfection
rate is low (as in superior cervical ganglion cultures), counting all
the labeled cells in the culture is recommended.

4. Notes

1. If horse serum needs to be inactivated, thaw the bottle and put

it in a water bath at 56°C for 30 min.
2. RPMI medium requires an atmosphere enriched in CO2 to
maintain the proper pH. Therefore, an incubator should be
used that maintains at 7.5–8% CO2 atmosphere.
3. Coating glass substrates (such as coverslips or glass slide cul-
tures) for immunofluorescence. To perform confocal micros-
copy on either neuronal PC12 cells or superior cervical ganglion
neurons, it is necessary to culture them on glass. This requires
coating the surface with a substrate that permits cell attach-
ment. To coat the glass, first incubate overnight in a solution
containing 50 μg/mL of poly-D-lysine. Following this, wash
with sterile distilled water, coat with collagen, and let dry as
described above.
4. Do not irradiate collagen plates with UV. This damages colla-
gen polymers and decreases cell-binding capability.
5. It is easier to carry out initial experiments (e.g., concentration-
response curves) with neuronal PC12 cells, and afterward, to
confirm the main results with sympathetic neurons. It is also
useful to use 48-well plates to maximize the number of repli-
cate cultures. Routinely, PC12 cells are grown in 10-cm dishes.
When using differentiated PC12 cells, the type of plates used
for experiments can be adapted to optimize use. For instance,
if the experiment is testing viability, it is better to use 24- or
48-well plates to permit multiple (at least 4) replicates. On the
other hand, if the object is to collect protein extracts, it is bet-
ter to use 6-well plates or even 10-cm dishes.
6. PC12 cells tend to detach from the plasticware easily, and even
more so, if the plate has been coated with collagen more than
3 weeks earlier. If such detachment starts and the cells are not
dying, try to replate them on a fresh collagen coated plate.
This problem is common in neuronal PC12 cells after transfec-
tion: the older the coating, the more they will detach after
transfection.
210 C. Malagelada Grau and L.A. Greene

7. If you do not have an isopropanol container, you can place the

cryotubes on ice for 10 min and then freeze them at −80°C.
8. Uridine and deoxyuridine stocks should be prepared and stored
in separated tubes.
9. Toxins as 6-OHDA or MPP+ are generally used to mimic PD
in vitro. The dopamine transporter specifically takes up both
toxins. Once inside the cells, these toxins deplete catecholamines
and inhibit mitochondrial oxidative metabolism. Therefore,
their toxicity range will depend on the density of the dopamine
transporters on the cells and also on the cellular density itself.
For example, identical concentrations of 6-OHDA or MPP+
will be more toxic in low-density cultures than in more dense
ones. The concentrations listed here are intended to cause
40–60% cell death. It is important to work within this range for
testing neuroprotective compounds. Also, excess concentra-
tions of the toxins will drive nonapoptotic as well as apoptotic
cell death.
10. These toxins inhibit mitochondrial complexes and deplete cells
of ATP. In doing so, they also generate damaging reactive oxy-
gen species.
11. Both 6-OHDA and MPP+ should be freshly prepared just
before use. Both are light sensitive, and therefore, their solu-
tion tubes should be wrapped in aluminum foil.
12. Always wear gloves when weighing out and working with these
toxins.
13. For viability assays that involve counting nuclei, resuspend the
contents of the well with a pipette before withdrawing 10 μL
for placement in the Neubauer chamber. Nuclei tend to sink to
the bottom of the well. Cultures to which the nuclear counting
solution has been added can be stored for several days. In this
case, seal the plate with Parafilm to prevent evaporation and
store it at 4°C for 5 days at maximum.
14. As noted above, at least 100 nuclei should be counted per cul-
ture for viability assays. If the numbers of nuclei are excessive,
more lysis buffer can be added to dilute the nuclear suspen-
sion. Keep track of the volume in final calculations of cell
numbers.

Acknowledgments

This work was supported by grants from the NIH-NINDS,

American Parkinson’s Disease Association, and Parkinson’s Disease
Foundation.
 18 Use of PC12 Cells and Rat Superior Cervical Ganglion… 211

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 Chapter 19

Compartmented Chambers for Studying

Neurotrophic Factor Action
Stephen D. Skaper

Abstract
Neurotrophic factors released by target tissues maintain the survival and differentiation of innervating
neurons. The manner by which these target-derived neurotrophic proteins communicate with innervating
neurons has been actively pursued for over three decades. The present chapter describes a technique for
preparing and maintaining compartmented chambers for culturing neurons derived from either superior
cervical ganglia (sympathetic neurons) or dorsal root ganglia (sensory neurons). This system recapitulates
the selective stimulation of neuron terminals that occurs in vivo following release of target-derived
neurotrophins.

Key words: Campenot cultures, Superior cervical ganglia neurons, Dorsal root ganglia neurons, Cell
culture, Neurotrophic factors, Retrograde transport

1. Introduction

Many neurons, including sympathetic and sensory neurons, depend

for their survival on retrograde signals to their cell bodies generated
by nerve growth factor (NGF) or other neurotrophins at their axon
terminals (1). Apoptosis resulting from the loss of retrograde NGF
signaling contributes to the elimination of excess and misconnected
neurons during development and to the death of neurons during
the course of neurodegenerative diseases (2). Possible mechanisms
of retrograde signaling include (1) retrograde transport of signaling
endosomes, carrying NGF bound to activated TrkA, (2) retrograde
transport of signaling molecules downstream of TrkA, and (3) retro-
grade propagation of a phosphorylation signal without transport
of signaling molecules. Two or more retrograde signaling mecha-
nisms may also exist to regulate neuronal cell survival, including
recent evidence that withdrawal of NGF from distal axons produces

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_19, © Springer Science+Business Media, LLC 2012

213
214 S.D. Skaper

side compartments

teflon divider

collagen scratches
center compartment

culture dish

distal neurites cell bodies/ distal neurites

proximal neurites

Fig. 1. Schematic diagram of Campenot chamber culture setup for isolation of distal neuronal
axons. A 35-mm petri dish is divided into three chambers by a Teflon divider (7.5 mm high)
that is sealed to the floor of the dish (a collagen-coated coverslip) with silicone grease.
The floor of the narrow (1 × 5 mm) central compartment in which neurons are plated is
transected by a series of parallel scratches which guide the growing neurites into the left
and right side compartments. The lower portion of the diagram shows an illustrative
enlargement of the neurons on a single track. Compartmented cultures isolate cell bodies
and proximal axons and distal axons of neurons. See text for further details.

a retrograde apoptotic signal, which is transported to the cell bodies,

where it initiates the apoptotic program, leading to the death of
the neuron (3).
How target-derived neurotrophins communicate to the cell
body of innervating neurons has been actively researched for more
than three decades. In order to study retrograde transport, a culture
system was originally devised by Robert Campenot, in which cell
bodies are isolated from their axons (Fig. 1) (4). The technique of
preparing these compartmented chambers for culturing sensory
and sympathetic neurons recapitulates the selective stimulation of
neuron terminals that occurs in vivo following release of target-
derived neurotrophins. Since there is fluidic isolation between the
compartments, treatments can be applied separately to cell bodies/
proximal axons or distal axons, and cell bodies/proximal axons and
distal axons can be separately harvested and analyzed. Distal axons
can be axotomized, and the neurons can be studied while their
axons regenerate. Compartmented cultures of rodent superior
cervical ganglion (SCG) sympathetic neurons have been used in
investigations of neurotrophin retrograde transport (5) and signaling
(6–9), axonal transport, and axonal protein and lipid biosynthesis
(10–14). Compartmented cultures of sensory neurons from
 19 Compartmented Chambers for Studying Neurotrophic Factor Action 215

rodent dorsal root ganglia (DRG) have been used in investigations

of neurotrophin retrograde signaling (15, 16) and anterograde
transport and axonal secretion of brain-derived neurotrophic
factor (17), among other applications. Compartmented chamber
cultures can support other cell types including retinal ganglion
neurons (18), cortical neurons (19), and spinal cord neurons (20).
Retrograde signaling events that require long-range microtubule
dependent retrograde transport have important implications for
the treatment of neurodegenerative disorders (21, 22). This
chapter describes a procedure for preparing Campenot chambers
and their use, modified from their original descriptions (4, 10).

2. Materials

2.1. Equipment 1. Stereo dissecting microscope (backlighting of stage is pre-

and Labware ferred) with fiber optic light source
2. Laminar flow biological cabinet (CL2)
3. Humidified, water-jacketed culture incubator at 37°C and 5%
CO2/95% air
4. Water bath set at 37°C
5. 0.5-mL microfuge tubes
6. 15- and 50-mL conical centrifuge tubes
7. 35-mm ∅ sterile tissue culture dishes
8. 100-mm ∅ glass petri dishes
9. 50-mL syringes

2.2. Compartmented 1. Teflon dividers (Camp 10, Tyler Research, Edmonton, Alberta,
Chamber Components Canada) (see Note 1)
2. Pin rake (Camp-PR, Tyler Research)
3. Grease loader (Camp-GLSS, Tyler Research)
4. Corning high-vacuum grease (Fisher Scientific)
5. 23-gauge Luer Lock connector (Fisher Scientific)
6. Right-angled hemostatic forceps (Fine Science Tools)

2.3. Reagents 1. Tissue culture grade water, sterile (Invitrogen)

2. Dulbecco’s modified Eagle’s medium (DMEM) (+4.5 g/L
glucose, L-glutamine, pyruvate) (Invitrogen)
3. RPMI-1640 medium (Invitrogen)
4. N2 supplement (100×) (Invitrogen)
5. Horse serum (Invitrogen) (see Note 2)
6. Fetal calf serum (Invitrogen) (see Note 2)
216 S.D. Skaper

7. Collagen solution, type 1 from rat tail, for cell culture, sterile
filtered (Sigma-Aldrich)
8. 7.5% bovine serum albumin (BSA) solution (Invitrogen)
9. Penicillin/streptomycin, 5,000 U/mL penicillin + 5,000 μg/mL
streptomycin (100× stock), sterile, for cell culture (Invitrogen)
10. Cytosine β-D-arabinofuranoside (Ara-C) (Sigma-Aldrich)
11. N2-methylcellulose 400CPS (Xenex Laboratories, Coquitlam,
BC, Canada)
12. Brain-derived neurotrophic factor, recombinant human
(PeproTech)
13. NGF, recombinant human (PeproTech)
14. NOCHROMIX® (Sigma-Aldrich)

2.4. Culture Media 1. Plating medium for newborn rat DRG neurons: Supplement
and Other Solutions 50 mL of DMEM to contain N2 supplements (500 μL
from 100× stock), 0.05% BSA (334 μL from 7.5% BSA
solution), 50 U/mL penicillin, and 50 μg/mL streptomy-
cin (5 mL of 100× stock). NGF is added as appropriate
(see Subheading 3.2).
2. Plating medium for newborn rat SCG neurons: Supplement
50 mL of RPMI-1640 to contain 50 ng/mL NGF (add
50 μL from a 50 μg/mL stock), 1% heat-inactivated horse
serum, and 50 U/mL penicillin and 50 μg/mL streptomycin
(5 mL of 100× stock). NGF is added as appropriate (see
Subheading 3.2).
3. N2-methylcellulose: Weigh out 0.6 g of methylcellulose and
place it in a 250-mL bottle, add a stir bar, and autoclave it for
20 min on dry (from this point, all work must be sterile). Next,
add 200 mL of serum-free medium (RPMI-1640 or DMEM)
supplemented with N2 components and stir in a cold room
until it dissolves. Aliquot into 15-mL tubes and store at −20°C.
To facilitate routine use, prepare also 1-mL aliquots and store
at −20°C.
4. Ara-C is prepared as a 10-mM stock solution in phosphate-
buffered saline, filter-sterilized, and stored as aliquots at −20°C
for up to 6 months. Once thawed, the aliquot tube may be
kept at 4°C for 4 weeks. Where used, Ara-C is diluted in
culture medium to a final concentration of 1 μM.

2.5. Preparation 1. Dilute the stock collagen solution to 1 mg/mL using 0.001 N
of Collagen HCl as diluent (alternative, tissue culture grade water can be
Substratum used). Add 1.5 mL of the diluted collagen solution per 35-mm
tissue culture plate and leave overnight in the CO2 incubator at
37°C. Then, remove the dishes from the incubator, aspirate to
collagen solution, and leave the dishes under the biological
 19 Compartmented Chambers for Studying Neurotrophic Factor Action 217

safety cabinet with the tops removed to allow to dry. These

may be utilized for the current experiment or stored at 4°C for
1 month.

3. Methods

3.1. Setting Up the 1. Fill a 50-mL syringe with Corning vacuum grease. Use the
Compartmented syringe to fill the grease loader, wrap it in foil, and autoclave
Chambers (To Be for 45 min.
Done 1–2 Days Before 2. Make a scratch in the middle of a collagen-coated 35-mm ∅
Preparing Cells) dish with an outward motion. Scratching the substratum
removes the collagen and exposes the bare plastic beneath the
surface of the dish floor, which is a poor substratum for growth
cone attachment. This results in axon growth being confined
to the collagen-coated tracks formed between the scratches.
Axons are then directed to grow to the left and right where
they encounter the silicone grease barriers, grow under the
silicone grease, and emerge into the distal compartments (see
Note 3).
3. Place a drop of N2-methylcellulose solution on the middle of
the scratch. This prevents silicone grease from adhering directly
to the substratum in this zone when the Teflon divider is seated
(see Note 4). Set dish(es) aside until the divider is greased.
4. Attach a 23-gauge Luer Lock connector to the grease loader.
Grip the Teflon divider with a pair of 90° angle hemostatic
forceps and hold it horizontally with the divider facing up
under a binocular microscope. Outline the divider with grease.
Each time the adapter is placed at a new starting point, insert
the adapter into the grease from the previous step to assure a
continuous track of grease.
5. Once grease is applied to the entire divider, remove the lid
from one of the prepared 35-mm dishes, pick up the dish, and
quickly invert along the axis parallel with the scratches so that
the N2-methylcellulose droplet does not run.
6. While viewing the divider under the microscope, hold the dish
in position over the divider, oriented so that the scratches will
cross under the barriers between the proximal and distal com-
partments and placed so that the dish is resting gently on the
silicone grease Press down on the bottom of the dish with a
pair of tweezers. Make sure to press on the inside of the divider
at the four corners (see Note 5).
7. Using the pair of hemostatic forceps, turn the dish over and
release the divider (placing the dish on the work surface while
218 S.D. Skaper

the divider remains clamped in the forceps can cause the

position of the divider to shift) (see Note 6).
8. Place the dish with the divider under the microscope and focus
on the bottom of the middle compartment. With the grease
loader, make a small barrier on the floor of the dish at the
opening of the proximal compartment. This is to avoid that
cells placed in the middle compartment leak out (see Note 7).
9. After having set up several cultures, place the appropriate
medium (for SCG or DRG cultures) in each of the side com-
partments and place in an incubator in which the cells will be
maintained. Allow the cultures to sit for several hours and then
check for leakage. If medium has leaked into the middle
compartment, then the culture is unusable (see Note 8).
10. Prior studies have tested the ability of these cultures to prevent
diffusion between the axon and the cell body compartment
(5, 23, 24). This can easily be tested by adding low concentra-
tions of a dye such as trypan blue to one compartment only,
and look for diffusion of the dye. There should be little or no
diffusion visible within 24 h (see Note 9).

3.2. Culturing of SCG The culture media formulations used are those described in the
or DRG Neurons relevant chapters of this volume, detailing protocols for preparing
rodent SCG or DRG cell cultures. The original formulation devel-
oped for rat SCG neurons uses rat serum (25), which is expensive
to obtain commercially and expensive and labor-intensive to prepare
in the laboratory. This formulation is not used here but, as pointed
out by Campenot and colleagues (10), some conditions may produce
diminished axon growth and may reduce the ability of axons to
cross into distal compartments. As recommended (10), methylcel-
lulose culture media are supplemented with methylcellulose, which
thickens the medium and reduces the shearing forces caused by
fluid movements that can perturb attachment of neurons to the
substratum. It facilitates wetting of the collagen before seating the
Teflon divider, and it prevents neurons from settling in the syringe
during plating.
1. Day 1: Replace culture medium in the side (distal) compartments
with medium containing 100 ng/mL NGF plus + Ara-C. Add
100,000 cells to center (proximal) compartment, using medium
containing 10 ng/mL NGF plus Ara-C (see Note 10).
2. Day 2: Add medium containing 10 ng/mL NGF plus Ara-C to
the outside of the Teflon divider until the medium flows over
the grease barrier and exchanges fluid with the center compart-
ment (see Note 11).
3. Day 3: Replace medium in the side compartments with medium
containing 100 ng/mL NGF but omitting Ara-C; replace
medium in the space outside of the Teflon divider with medium
containing 10 ng/mL NGF but omitting the Ara-C.
 19 Compartmented Chambers for Studying Neurotrophic Factor Action 219

4. Day 6: Replace medium in the side compartments with medium

containing 1 ng/mL NGF plus Ara-C and the surrounding
space (outside Teflon divider) with NGF-free medium + Ara-C
(see Note 12).
5. Day 7 onward: Use for experimentation.

3.3. Analysis With experience, construction of the culture dishes should require
of Neurons about 3 h for 48 cultures, while preparing the neurons requires
3–4 h. Compartmented cultures from both SCG and DRG pro-
vide enough cellular material for biochemical analyses such as
immunoblotting, and cell bodies/proximal axons can be harvested
separately from distal axons. Morphological analyses have their
limitations, in that the presence of the Teflon divider produces
menisci in the surface of the culture medium that interfere with the
light path in inverted, phase-contrast microscopy. This can reduce
the contrast, making visualization of the neurons difficult, espe-
cially in the center compartment and close to the barriers in the
distal compartments. The menisci distort the ring of light produced
by the phase condenser so that it is not circular and therefore
cannot be aligned on the target annulus in the phase telescope.
Campenot and colleagues have solved this problem by constructing
a rotating shield that blocks ~80% of the ring of light, allowing
only a segment of the ring of light to pass through (10). Also, they
have modified the mechanical support of the light source/
condenser to increase the range of movement for adjustment in
the XY plane. These modifications make it possible to rotate the
shield and move the light source/condenser such that the segment
of the light ring that is not blocked by the shield projects nearly
completely onto the target annulus, which results in a reasonable
phase-contrast image. As the distortion arising from the meniscus
varies with location in the culture dish, adjustments must be made
whenever the field of view is moved. Also, the adjustments produce
better phase-contrast images at higher magnification, as the curva-
ture of the segment of the meniscus that must be compensated is
smaller, and therefore more uniform, when the field of view is
smaller. The need for this modification varies for different micro-
scopes. Tyler Research Corporation has provided Campenot and
colleagues with a modification for their Nikon Diaphot inverted
microscope. It is suggested that investigators intending to perform
such analyses liaise with Tyler for such modifications.
Recent studies have used microfluidic chambers instead of
these compartmented chambers. Microfluidic barriers are created,
which consist of narrow channels through which axons grow, such
that the extracellular space is kept to a minimum (26, 27).
Movement of substances across the barrier is controlled by estab-
lishing a flow through the extracellular space in the channels driven
by a pressure gradient produced by maintaining different fluid
220 S.D. Skaper

levels in the compartments on each side of the barrier. As the

culture substratum is on glass, this permits the use of inverted
confocal microscopy (unlike traditional compartmented chambers),
and shorter axons are compatible with this approach. A disadvan-
tage is that the amount of cellular material is too small for conven-
tional analysis of proteins and lipids.

4. Notes

1. All dividers are machined from virgin Teflon and will last
for years if properly maintained. Many other types of dividers
are available or can be produced relatively quickly by the
company in response to an order. You can also provide a
drawing that they will cut to your specifications (biomedical@
tylerresearch.com).
2. Heat inactivation of fetal calf serum (and horse serum) is
recommended to destroy heat-labile complement. Thaw the
bottle of serum in advance, using a 37°C water bath. Next,
immerse the serum bottle in the water bath after re-equilibrating
to 56°C and leave for 30 min. Swirl the bottle occasionally to
ensure proper mixing. Allow the serum to cool to room
temperature, aliquot into 50-mL tubes, and store at −20°C.
3. The width of the collagen tracks is critical for axons to cross
into the distal compartments. The optimal track width for rat
sympathetic neuron cultures is about 200 μm (10). Extremely
narrow tracks are not conducive to axonal growth, and if tracks
are too wide, the growth cones have enough room to make
U-turns away from the barrier.
4. Do not let the medium used for wetting the collagen run
outside the area of the scratches before the divider is seated, as
this can interfere with sealing of the perimeter of the divider to
the dish. It is suggested to leave one or two tracks at each edge
dry, to reduce the risk of the droplet escaping beyond the
scratches (10). As the droplet does not extend the full distance
along the tracks, evaporation can leave a deposit on the sub-
stratum coinciding with the edge of the droplet across the
tracks, and which may interfere with axon growth. Construct
each culture dish and add medium covering the full length of
the tracks as soon as possible after placing the droplet.
5. It is important to press firmly enough so that the grease makes
a complete seal with the dish but if too much pressure is added,
the axons will not cross into the side compartments.
6. The dividers can be re-used after each experiment but must
first be cleaned. Remove the divider from the plate, wipe off all
19 Compartmented Chambers for Studying Neurotrophic Factor Action 221

of the remaining grease, and place in NOCHROMIX® sulfuric

acid solution. NOCHROMIX® is a crystalline, inorganic
oxidizer. Mix one pouch (3 oz.) with 3.7 L of concentrated
sulfuric acid. Exercise caution and wear personal protective
equipment. Soak dividers for at least 1 h (new dividers should
also be prepared starting at this step). After removing from the
acid, rinse five times with double-distilled water, boil for
20 min, and repeat the boil-rinse steps for a total of three cycles
(NOCHROMIX-sulfuric acid can be reused until it discolors).
Allow the dividers to dry under a laminar flow hood, place in
glass 100-mm petri dishes, wrap in aluminum foil, and auto-
clave. Store at room temperature until ready to use.
7. To prevent the original N2-methylcellulose solution droplet
from drying out, place two drops of distal compartment
medium on the scratched region of the substratum in each
distal compartment, making sure that the scratched region is
completely covered.
8. When first learning this technique, it is recommended to set
up extra cultures to replace any that leak (most likely to occur
during the initial trials period).
9. Because cultures are ordinarily maintained with higher levels of
medium in the left and right compartments than in the dish
perimeter, dishes in which there is a leak between compart-
ments display a decrease in the level of culture medium in the
distal compartment and an increase in culture medium in the
dish perimeter. As the dishes are usually constructed a day in
advance, leaking dishes are normally identified before plating
the neurons by the appearance of culture medium in the dish
perimeter. One can also check for leaks by omitting phenol red
from the distal compartment medium when assembling
cultures and checking for the appearance of phenol red in
distal compartments.
10. Ara-C is provided in proximal compartments containing the
cell bodies for the initial 5–7 days after plating to inhibit growth
of Schwann cells and other glia. It is not included in the
medium supplied to distal compartments, as these compart-
ments are not exposed to cell suspension at plating.
11. When changing the medium, it is important to aspirate the
liquid from the top of each side compartment. Also, never
change the medium from the middle compartment itself, only
from the surrounding space (outside the Teflon divider) and
let it flow over the grease barrier into the center.
12. After 5–7 days, the distal axons of the neurons are sufficiently
established in the distal compartments so that the NGF can be
withdrawn from the proximal compartments containing the
cell bodies and proximal axons, and neuronal survival is sup-
ported solely by the NGF supplied to the distal axons.
222 S.D. Skaper

References

1. Campenot RB (2009) NGF uptake and 15. Heerssen HM, Segal RA (2002) Location,
retrograde signaling mechanisms in sympa- location, location: a spatial view of neurotro-
thetic neurons in compartmented cultures. phin signal transduction. Trends Neurosci 25,
Results Probl Cell Differ 48, 141–158 160–165
2. Levi-Montalcini R (1987) The nerve growth 16. Watson FL, Heerssen HM, Bhattacharyya A,
factor 35 years later. Science 237, 1154–1162. Klesse L, Lin MZ, Segal RA (2001) Neurotrophins
3. Oppenheim, R.W. (1991) Cell death during use the Erk5 pathway to mediate a retrograde sur-
development of the nervous system. Annu Rev vival response. Nat Neurosci 10, 981–988
Neurosci 14, 453–501 17. Ng BK, Chen L, Mandemakers W, Cosgaya
4. Campenot RB (1977) Local control of neurite JM, Chan JR (2007) Anterograde transport
development by nerve growth factor. Proc Natl and secretion of brain-derived neurotrophic
Acad Sci USA 74, 4516–4519 factor along sensory axons promote Schwann
5. Ure DR, Campenot RB (1997) Retrograde cell myelination. J Neurosci 27, 7597–7603
transport and steady-state distribution of 18. Underhill SM, Goldberg MP (2007) Hypoxic
125I-nerve growth factor in rat sympathetic injury of isolated axons is independent of iono-
neurons in compartmented cultures. J Neurosci tropic glutamate receptors. Neurobiol Dis 25,
17, 1282–1290 284–290
6. Campenot RB (2009) NGF uptake and retro- 19. Hayashi H, Campenot RB, Vance DE, Vance
grade signaling mechanisms in sympathetic JE (2007) Apolipoprotein E-containing lipo-
neurons in compartmented cultures. in Results proteins protect neurons from apoptosis via a
and Problems in Cell Differentiation, Vol. 48 Cell signaling pathway involving low-density lipo-
Biology of the Axon (ed. Koenig, E.) pp 141–158 protein receptor-related protein-1. J Neurosci
(Springer, Berlin/Heidelberg) 27, 1933–1941
7. Zweifel LS, Kuruvilla R, Ginty DD (2005) 20. Sonderegger P, Fishman MC, Bokoum M,
Functions and mechanisms of retrograde Bauer HC, Neale EA, Nelson PG (1984) A few
neurotrophin signalling. Nat Rev Neurosci 6, axonal proteins distinguish ventral spinal cord
615–625 neurons from dorsal root ganglion neurons. J
8. Ginty DD, Segal RA (2002) Retrograde neu- Cell Biol 98, 364–368
rotrophin signaling: Trk-ing along the axon. 21. Eide FF, Lowenstein DH, Reichardt LF (1993)
Curr Opin Neurobiol 12, 268–274 Neurotrophins and their receptors–current
9. Campenot RB, MacInnis BL (2004) Retrograde concepts and implications for neurologic dis-
transport of neurotrophins: fact and function. J ease. Exp Neurol 121, 200–214
Neurobiol 58, 217–229 22. Ginty DD, Segal RA (2002) Retrograde neu-
10. Campenot RB, Lund K, Mok SA (2009) rotrophin signaling: Trk-ing along the axon.
Production of compartmented cultures of rat Curr Opin Neurobiol 12, 268–274
sympathetic neurons. Nat Protoc 4, 23. Campenot RB (1979) Independent control of
1869–1887 the local environment of somas and neurites.
11. Posse De Chaves EI, Vance DE, Campenot RB, Methods Enzymol 58, 302–307
Kiss RS, Vance JE (2000) Uptake of lipopro- 24. Heerssen HM, Pazyra MF, Segal RA (2004)
teins for axonal growth of sympathetic neurons. Dynein motors transport activated Trks to pro-
J Biol Chem 275, 19883–19890 mote survival of target-dependent neurons. Nat
12. Bertrand J, Winton MJ, Rodriguez-Hernandez Neurosci 7, 596–604
N, Campenot RB, McKerracher L (2005) 25. Mains RE, Patterson PH (1973) Primary cul-
Application of Rho antagonist to neuronal cell tures of dissociated sympathetic neurons. I.
bodies promotes neurite growth in compart- Establishment of long-term growth in culture
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ganglion cell axons in the optic nerve of adult Biol 59, 329–345
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support of neuronal survival without retro- idic culture platform for CNS axonal injury,
grade transport of nerve growth factor. regeneration and transport. Nat Methods 2,
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 Chapter 20

Preparation and Culture of Adrenal Chromaffin Cells

Natalia Domínguez, Miriam Rodríguez, J. David Machado,
and Ricardo Borges

Abstract
Cultured chromaffin cells have been used for almost 40 years in the study of different cell functions using
biochemical, electrophysiological, pharmacological, and toxicological approaches. Chromaffin cells are
essentially secretory cells that are used to model sympathetic neurons or neuroendocrine cells. In this
chapter, we describe the most common methods currently used to isolate and culture chromaffin cells from
the animals used most commonly: cows, rats, and mice. We also provide some advice on the use of these
cells in the laboratory.

Key words: Adrenal, Chromaffin cells, Culture, Collagenase, Papain

1. Introduction

Cell culture techniques are now routinely applied to modern

laboratory research, chromaffin cells being no exception. Since the
first seminal description of their isolation from mammalian glands
(1, 2), the procedure has continually evolved. The improvements
in tissue culture procedures and advances in the materials used,
notably the highly purified enzymes currently available, has stan-
dardized isolation protocols and improved their reproducibility.
In this chapter, we provide the “recipes” for culturing cow, rat,
and mouse chromaffin cells, describing the standard protocols that
we use.

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_20, © Springer Science+Business Media, LLC 2012

223
224 N. Domínguez et al.

2. Materials

2.1. General Supplies, All procedures require standard culture room facilities with a
Solutions, and Culture laminar flow cabin, a Bunsen burner, a water bath set at 37°C
Medium (preferably with agitation), and a 37°C incubator with a water-
saturated atmosphere containing 5% CO2. An inverted microscope
with phase contrast and a low vibration clinical centrifuge are also
required. To plate chromaffin cells onto glass coverslips, we strongly
recommend high-quality glass Marienfeld (Lauda-Königshofen,
Germany). Other required materials and basic solutions include:
1. Sterile plastic Pasteur pipettes.
2. Syringes and 0.22-mm syringe filters.
3. Adjustable volume pipettes with sterile tips.
4. 15-mL conical centrifuge tubes (or 50-mL tubes for culturing
cow cells).
5. Hemocytometer (Neubauer).
6. 24-well culture plates.
7. Ca2+- and Mg2+-free Locke’s balanced salt solution: 154 mM
NaCl, 5 mM KCl, 3.6 mM NaHCO3, 5 mM HEPES, and
11 mM glucose.
8. Trypan blue: 4 mg/mL in Locke’s solution.
9. Culture medium: We always use standard Dulbecco’s modified
Eagle’s medium mixed 1:1 with Ham’s F12 and antibiotics to
culture chromaffin cells. The medium is completed by adding
10% fetal calf serum (Lonza, Basel, Switzerland, DE14-801 F,
see Note 1).
10. All the solutions used are sterilized by filtering through 0.22-
mm syringe filters and are supplemented to contain penicillin G
(100 IU/mL) and gentamicin sulfate (40 mg/L).

2.2. Materials 1. Sterile surgical material required: forceps, scissors, two scalpel
for Preparation blades, and one haemostatic clamp.
of Bovine Chromaffin 2. 2× 100 mm Ø Petri dishes.
Cells
3. Glass beakers: 2× » 30 mL, 1× » 100 mL, and 1× » 250 mL (for
waste).
4. Two »35-mm Ø glass funnels.
5. Standard cotton gauzes, »200-mm and »90-mm pore-size nylon
meshes.
6. 5-mL syringes and 0.22-mm sterilizing syringe filters.
7. For gradient purification, a refrigerated centrifuge capable of
reaching »8,000 × g is also required, along with two sterile
capped 30-mL transparent tubes suitable for centrifugation.
 20 Preparation and Culture of Adrenal Chromaffin Cells 225

8. Collagenase IA.
9. Bovine serum albumin fraction V (BSA).
10. Deoxyribonuclease I (DNAase I).
11. Renografin® (ER Squibb & Sons, New Brunswick, NJ, USA)
or Urografin® (Schering España, Madrid, Spain) prepared at
15% in sterile pure water (this solution is isotonic).

2.3. Materials for 1. Rats over 4 weeks of age.

Preparation of Adult 2. Stereo microscope (×20 magnification).
Rat Chromaffin Cells
3. Petri dishes (35 mm Ø).
4. Collagenase IA.
5. BSA.
6. DNAase I.
7. Hyaluronidase I-S.

2.4. Materials 1. Animals of less than 4 weeks of age.

for Preparation of 2. Stereo microscope (×20 magnification).
Young Rat or Mouse
3. Petri dishes (35 mm Ø).
Chromaffin Cells
4. Papain (Worthington Lakewood, NJ).

3. Methods

General Guidelines. Cells are very sensitive to low pressure and as

such, strong pipette suction and strong centrifugation should be
avoided. Similarly, do not use the brake function on the centrifuge.
Standard guidelines for working in culture rooms should be followed,
such as wearing gloves and keeping a well-ordered hood to main-
tain laminar flow conditions. Unnecessary material in the working
space will increase air turbulence inside the hood. As we primarily
use chromaffin cells for secretion experiments, we have tailored the
procedure to produce healthy secretory cells. However, it is possible
that the reader may find alternative protocols designed for other
purposes, although the maintenance of a healthy secretory machinery
usually ensures that the cell is healthy and suitable for any experi-
mental purposes.

3.1. Chromaffin Any of the methods described below for chromaffin cell isolation
Cell Preparation and culture are appropriate for amperometry, patch clamping, or
for Single-Cell fluorescent microscopy. The following is a rule of thumb for
Experiments amperometric studies using chromaffin cells of any species:
1. The isolation procedure should aim primarily to obtain healthy
cells rather than a high yield. We recommend reducing the
226 N. Domínguez et al.

concentration of the enzymes used for digestion in the papers

cited below by 15% and minimizing the mechanical disruption
during tissue digestion. For bovine chromaffin cells, we use a
modified version of the original protocol using a bolus of
collagenase injected through the adrenal vein, rather than per-
fusion (3, 4). For rat cells, we adhere to the conditions described
by Gilabert (5), and for mouse cells, we use a modified version
of the procedure described by Sorensen et al. (6).
2. Cells should be plated at low density (»20,000–50,000 per cm2).
3. Replace culture medium every 48 h using serum-free
medium.
4. Although cells can be maintained for over a week after plating,
optimal secretion usually occurs within 24 h of plating. However,
cells may be used within a few hours.

3.2. Coverslips Glass coverslips can be sterilized by briefly flaming both sides using
and Adherent Support a Bunsen burner before placing them in culture plates. If it is nec-
Sterilization essary to coat them with a substrate (see Note 2), place a drop of
0.01% poly-D-lysine solution (Sigma, Catalog P-1024, prepared in
water) on each coverslip. Leave it for 20 min and then wash three
times with sterile pure water. Sterilize the coverslip by exposing it
to UV light in the fume hood for 30 min and use the treated glass
within 1 week of preparation.

3.3. Cell Viability Although many automatic systems for cell counting now exist, a
and Counting standard Neubauer hemocytometer is still a valuable and cheap tool
to count cells and to get a general idea of their viability. We prepare
a 1:9 dilution of cells by mixing 20 mL of cell suspension + 100 mL
of Locke’s solution + 80 mL of trypan blue staining solution (see
above). Guidelines for the correct use of hemocytometers have
been published elsewhere (7).

3.4. Procedure Glands may be obtained from the local abattoir. Choose only intact
for Bovine glands to avoid contamination and discard those with visible signs
Chromaffin Cells of internal blood coagulation. Do not remove the fat at the abattoir,
as it provides protection against contamination. The glands do not
require any special care for their transportation to the laboratory if
the time from sacrifice to culturing is less than 1 h. In the case of
longer intervals, it is advisable to inject 3–4 mL of Locke’s solution
into the glands and transport them in a plastic bag on ice. Other
protocols for bovine cell culture preparation have been published
elsewhere (4, 8–14).
Once in the culture room, wearing gloves spray the glands
with 60% ethanol under the laminar flow cabinet, making sure
that the solution does not enter the adrenal vein, and remove the
surrounding fat from the glands. The general procedure to mini-
mize contamination is to proceed progressively from a nonsterile
 20 Preparation and Culture of Adrenal Chromaffin Cells 227

(transport containers, external fat) to a sterile environment. As such,

it is advisable to flame the surgical instruments periodically. Once
the glands are clean of surrounding fat and connective tissue, spray
again with ethanol and remove all the material used and discard the
tissue from the hood. Clean the surface of the hood with ethanol.
The procedure described below is intended for two glands but can
be easily adapted for more glands. In addition, Fig. 1 can be printed
and posted in the culture room as a brief guide to the procedure:
1. Prepare the enzymatic solution. Each gland will require 20 mL
of a solution containing 1.5–2 mg/mL of collagenase IA and
twice this amount (3–4 mg/mL) of BSA dissolved in Locke’s
solution. These calculations are aimed for a commercial a colla-
genase with an activity of 300–400 IU/mg, and should be
adjusted when the enzyme has a different activity (see Note 3).
The addition of 30 mg/mL of DNAase I increases the yield of
the isolation procedure as it prevents cell aggregation that results
from the presence of free DNA. However, given the usually high
yield of bovine cultures, this step is not critical. Sterilize the solu-
tion by filtering through a 0.22-mm syringe filter into a 50-mL
conical tube and place it capped in the water bath at 37°C.
2. Place the glands in a sterile Petri dish and inject each gland
twice with 3–4 mL of warm (37°C) Locke’s solution. Place the
glands in a 100-mL glass beaker, keeping the vein orifice facing
up. Cover the beaker with a dry sterile Petri dish and incubate
for 5–10 min in the water bath.
3. Inject the glands with 3–4 mL of enzyme solution and return
the glands to the beaker. Try to avoid accumulation of liquid
in the bottom of the beaker which could contaminate the gland
(see Fig. 1a).
4. After 20 min, repeat step 3. Exercise care when performing the
injections as the gland has been partially digested and may rupture
with excessive pressure.
5. After 20 min, the glands should be noticeably softer, indicating
the success of the digestion. If this is not the case, repeat the
injection and check again after 10 min. It is important to keep
the collagenase incubation time to a minimum to avoid over-
digestion of the chromaffin tissue.
6. Using a scalpel and clean scissors, open the gland longitudi-
nally (along its length). It should appear as shown in Fig. 1b,
with the cream-colored section representing the medulla. Make
several cuts along the medulla with small scissors, and use forceps
to collect the material and transfer it to a clean Petri dish.
Do not collect any purple material that contains the cortical
cells. This procedure should be performed swiftly.
7. Using two scalpel blades, mince the tissue into small pieces and
transfer the material to a 50-mL conical tube using a plastic
228 N. Domínguez et al.

Fig. 1. Procedure to isolate and culture bovine chromaffin cells (see text for guidelines).
 20 Preparation and Culture of Adrenal Chromaffin Cells 229

Pasteur pipette. Add the rest of the fresh collagenase solution

and incubate this tissue at 37°C (see Fig. 1c). Every 5–10 min,
carefully dissociate the material by triturating with 5–6 strokes
of a Pasteur pipette, always avoiding the production of bubbles
(see Fig. 1d). The total incubation time should not exceed
20–25 min.
8. Place a piece of sterile cotton gauze onto a funnel and filter the
dissociated material to remove the bulk debris, collecting the
cells in two 50-mL conical tubes. Distribute the filtrate between
both tubes and wash the gauze with Locke’s solution to maxi-
mize the yield and to dilute the collagenase (see Fig. 1e).
9. Centrifuge at »300 × g for 5 min, discard the supernatant, and
resuspend the pellet by gently tapping it against the hood
bench. Fill the tubes with fresh Locke’s solution and repeat the
centrifugation.
10. Discard the supernatant, resuspend the cells, and pass them
through a 200-mm nylon mesh. Clean the mesh by rinsing with
fresh Locke’s solution to produce a final volume of 10 mL (see
Fig. 1g).
11. The cells obtained are contaminated with debris, ruptured
cells, and red and cortical cells. For many purposes, they are fit
for culturing and may be diluted into culture medium at this
stage. However, we prefer to purify the chromaffin cells, as
outlined below (see steps 12–18).
12. Place 20 mL of 15% Renografin solution in two 30-mL centri-
fuge tubes of (see Note 4).
13. Mix 10 mL of cell suspension with 10 mL of Renografin to
produce a final Renografin concentration of 7.5%.
14. Very slowly and carefully, add the cell suspension onto the sur-
face of the 15% Renografin solution using a plastic Pasteur
pipette. This is the most critical step! If the interface between
the two Renografin concentrations is broken, the discontinu-
ous gradient is compromised, and most of the chromaffin cells
will enter the pellet (see Fig. 1h).
15. After equilibrating the tubes by weight, centrifuge at 7,500 × g
at »18°C for 20 min (see Fig. 1i).
16. The chromaffin cells will appear in the interface of the gradient
and can be easily recovered with a plastic Pasteur pipette (see
Fig. 1j).
17. To remove the Renografin, resuspend the cells in Locke’s
solution in a 50-mL conical tube and centrifuge at »300 × g
for 5 min (see Fig. 1k).
18. Resuspend the pellet by tapping the tube against the work
surface of the hood and add 10 mL of culture medium (see
Note 1). As the medium contains calcium, it is important to
230 N. Domínguez et al.

avoid unnecessary manipulation of the cell suspension to prevent

aggregation.
19. Pass the cell suspension through a 90-mm nylon mesh (see
Fig. l).
20. Count and plate the cells accordingly (see Fig. 1m, n). Although
the described yield in the past was as large as 80–100 × 106 cells
per gland, nowadays it is preferable to reduce this number
to obtain healthier cells. Thus, 10–20 × 106 cells per gland is a
suitable yield. As a rule of thumb, we recommend the follow-
ing densities:
– For biochemical and secretory studies in 24-well plates:
500,000 cells/well in 1–2 mL of complete medium.
– For single-cell experiments on 12 mm Ø glass coverslips:
50,000 cells/well in 1 mL of complete medium. Bovine
chromaffin cells do not usually require an adherent
substrate (collagen, polylysine, etc.).
– For collecting cells by scraping from 35 mm Ø Petri dishes:
3–4 × 106 cells/well in 3 mL of complete medium.
21. Change the medium using serum-free medium every 2–3 days,
depending on the cell density (see Note 1).

3.5. Procedure Animals older than »4 weeks produce a lower yield of cells than
for Adult Rat young rats. This is also observed with mice:
Chromaffin Cells
1. Prepare the dissociation solution (3–4 mL) and maintain it at
room temperature: collagenase type I (250–350 IU/mL),
3 mg/mL BSA, 0.15 mg/mL DNAase I, and 0.15 mg/mL
hyaluronidase I-S in Locke’s buffer.
2. Sacrifice the animals according to institutionally approved ethi-
cal procedures. Place the animal on its back and spray the
abdomen with 70% ethanol.
3. To access the abdominal cavity, perform an incision through
the abdominal wall, cutting the skin along the linea alba and
pulling it out to either side, using scissors to separate the skin
from underlying tissue. The abdominal cavity should tear open,
exposing the organs. Locate the two kidneys with the adrenal
glands on top, which should be readily visible (see Fig. 2a).
4. Remove the glands with fine-curved forceps and place them in
ice-cold Locke’s buffer.
5. Under a stereo dissection microscope coupled to a cold-light
source, remove the adipose tissue surrounding the gland with
a stainless steel scalpel blade and decapsulate the adrenal glands.
Next, remove the adrenal cortex to isolate the medullar tissue
and cut this into four pieces using a scalpel. Keep the tissue wet
throughout with cold Locke’s buffer.
 20 Preparation and Culture of Adrenal Chromaffin Cells 231

Fig. 2. Procedure to isolate and culture mouse chromaffin cells (see text for guidelines).

6. Recover the adrenal tissue using a sterile plastic pipette and

transfer it to a 15-mL conical tube. Add 3 mL of the dissociation
solution and incubate for 25–30 min at 37°C. The solution
should be gently triturated every 10 min through a 1 mL
pipette tip. During the last 5 min, the solution should be
continuously triturated using a 100-mL pipette, until it becomes
turbid.
7. Add cold Locke’s buffer to a final volume of 10 mL to stop the
enzyme reaction.
232 N. Domínguez et al.

8. Place the tube containing the digested tissue in the rotor of a

precooled (4°C) refrigerated centrifuge (where possible) and
spin at 100 × g for 10 min.
9. Discard the supernatant by decanting and resuspend the pellet
with 0.8 mL of prewarmed medium.
10. Add one or two drops on each poly-D-lysine-coated coverslip
and place them in the incubator for 1 h to allow the cells to
settle.
11. Carefully refill the culture dish with 1–1.5 mL of medium and
return the plates to the incubator.
12. Replace the medium after 24 h and every 48 h thereafter (see
Note 1).

3.6. Procedure We use animals of 2–4 weeks of age. Given the small amount of
for Young Mouse collagen in these adrenal tissues, papain is used instead of collagenase,
or Rat Chromaffin producing a considerably higher cell yield. As the quantity of starting
Cells material from the adrenal glands is very small, this procedure is
intended for single-cell measurements. We usually use 12-mm Ø
glass coverslips placed in 24-well plates, although other sizes can
be used. Figure 1 can be printed and posted in the culture room as
a brief guide to the procedure:
1. Prepare the papain solution in Locke’s buffer (12–18 IU in
200 mL, sufficient for four glands), and store it at 37°C.
2. Sacrifice the animal according to institutionally approved ethical
procedures, place it on its back, and spray the abdomen with
70% ethanol. Open the abdomen and remove both adrenal
glands by pulling the glands away with an angled forceps (see
Fig. 2a).
3. Under a stereo microscope (×20 magnification), carefully
remove the adrenal capsule as much as possible of the cortical
tissue (see Fig. 2b, c). Work on a sheet of clean filter paper
soaked with Locke’s solution. This helps to avoid drying and
overheating of the tissue by the light. Transfer the medullary
tissue to a sterile Petri dish (35 mm Ø) containing 1 mL of
sterile ice-cold Locke’s solution (see Fig. 2d).
4. Transfer the pieces to a 15-mL conical tube with 200 mL of
papain solution and leave them for 15–20 min at 37°C without
shaking. The incubation time should be adjusted according to
the age of the animals, as younger animals will require shorter
digestion.
5. Wash the tissue with 800 mL of fresh Locke’s solution.
6. Remove as much of the liquid as possible, taking particular
care as the tissue is almost digested, and then add 200 mL of
complete medium (see Note 1).
7. Disaggregate the tissue by triturating it several times, first
through a 1 mL pipette tip and then through a 100-mL pipette
 20 Preparation and Culture of Adrenal Chromaffin Cells 233

tip, until the suspension becomes turbid. The degree of cell

dissociation can be assessed by taking 10 mL of the suspension
and observing under the inverted microscope. It is not recom-
mended to completely dissociate the tissue, as excessive manip-
ulation may damage the cells.
8. Take 20 mL of this cell suspension to count the number of cells
in a Neubauer hemocytometer (with experience, cell counting
usually becomes unnecessary).
9. Plate the cells by placing a drop of the cell suspension on
12 mm-Ø poly-D-lysine-coated coverslips and allow the cells
to settle for 20 min in the incubator before adding 1 mL of
medium.
10. Return the plates to the incubator.
11. The cells should be used within the first 3 days after culturing.
Where necessary, change the medium (serum-free) every 48 h.

4. Notes

1. The use of a 1:1 mix of Dulbecco’s modified Eagle’s medium

with Ham’s F12 medium helps cells to maintain their round
shape. This simple trick was suggested by Dr. O. Humberto
Viveros and produces excellent results. We prepare 1 L of this
mix from powdered medium and after adding antibiotics and
filtering, we divide the contents between two bottles. Ten per-
cent (v/v) fetal calf serum is added to one bottle (complete
medium), while the second is kept serum-free (incomplete
medium). The serum-free medium lasts longer than the com-
plete, and it is used to change the medium after 48 h.
2. Other adherent substrates have been used, including collagen,
poly-DL-ornithine, laminin, or fibronectin (3). We use round
glass coverslips of 12 mm Ø that fit readily into 24-well culture
plates. Cell adhesion improves notably when the glass is pretreated
with these substrates, although we do not use any substrate
for bovine cells unless they are to be used for immunocy-
tochemistry or electron microscopy.
3. A few decades ago, the quality of commercial enzymes and sera
varied considerably from one batch to another. During the
1980s, some particularly active batches of collagenase produced
over 100 × 106 cells/gland, possibly due to contamination with
other proteases. Although some variation occurs within different
batches of these products, the controls applied by manufacturers
nowadays have greatly reduced this problem.
4. Several strategies have been described for purifying chromaffin
cells from other contaminants and debris. One is differential
plating, which is based on the low adherence of chromaffin
234 N. Domínguez et al.

cells when compared to fibroblasts or endothelial cells. Although

this method gained some popularity, it is currently only
performed by few, if any, researchers. Discontinuous gradients
of Ficoll® or Percoll® are used by some groups for cell purifica-
tion, and we have used this system in the past. However, we find
that Renografin produces a purer population of chromaffin
cells that are enriched in adrenaline.

Acknowledgments

We are indebted to the contributions of our predecessors working

in the field of cell culture. This work was supported by the Spanish
Ministry of Science and Innovation (MICINN, Grants # BFU
BFU2007-64963, CSD2008-00005 and BFU2010-15822) and
the Canary Islands Government (GC, Grants # C2008/01000239).
N.D. is a recipient of an FPU scholarship from MICINN, M.R.
received a grant from CajaCanarias, and J.D.M. is the holder of a
“Ramón y Cajal” contract from the MICINN.

References
1. Kloppenborg PW, Island DP, Liddle GW, culture of bovine chromaffin cells. Nat Protoc
Michelakis AM, and Nicholson WE (1968) A 2, 1248–1253
method of preparing adrenal cell suspensions 9. Krause W, Michael N, Lubke C, Livett BG, and
and its applicability to the in vitro study of adre- Oehme P (1996) Catecholamine release from
nal metabolism. Endocrinology 82, 1053–1058 fractionated chromaffin cells. Eur J Pharmacol
2. Hochman J, and Perlman RL (1976) Catecho- 302, 223–228
lamine secretion by isolated adrenal cells. 10. Livett BG, Boksa P, Dean DM, Mizobe F, and
Biochim Biophys Acta 421, 168–175 Lindenbaum MH (1983) Use of isolated chro-
3. Livett BG (1984) Adrenal medullary chromaffin maffin cells to study basic release mechanisms.
cells in vitro. Physiol Rev 64, 1103–1161 J Auton Nerv Syst 7, 59–86
4. Moro MA, Lopez MG, Gandia L, Michelena P, 11. Fenwick EM, Fajdiga PB, Howe NB, and Livett
and Garcia AG (1990) Separation and culture BG (1978) Functional and morphological char-
of living adrenaline- and noradrenaline- acterization of isolated bovine adrenal medullary
containing cells from bovine adrenal medullae. cells. J Cell Biol 76, 12–30
Anal Biochem 185, 243–248 12. Almazan G, Aunis D, Garcia AG, Montiel C,
5. Gilabert JA (2004) Necessary conditions to main- Nicolas GP, and Sanchez-Garcia P (1984)
tain rat adrenal chromaffin cells in primary culture. Effects of collagenase on the release of
In Cell Biology of the Chromaffin cell. Borges, R [3 H]-noradrenaline from bovine cultured
& Gandia, L. Eds. La Laguna. pp 269–274. web- adrenal chromaffin cells. Br J Pharmacol 81,
pages.ull.es/users/isccb12/TheBook.htm 599–610
6. Sorensen JB, Nagy G, Varoqueaux F, Nehring 13. Bader MF, Ciesielski-Treska J, Thierse D, Hesketh
RB, Brose N, Wilson MC, and Neher E (2003) JE, and Aunis D (1981) Immunocytochemical
Differential control of the releasable vesicle study of microtubules in chromaffin cells in cul-
pools by SNAP-25 splice variants and SNAP- ture and evidence that tubulin is not an integral
23. Cell 114, 75–86 protein of the chromaffin granule membrane.
7. Strober W (2001) Trypan blue exclusion test J Neurochem 37, 917–933
of cell viability. Curr Protoc Immunol Appendix 14. Baker PF, and Knight DE (1981) Calcium
3, Appendix 3B control of exocytosis and endocytosis in bovine
8. O’Connor DT, Mahata SK, Mahata M, Jiang adrenal medullary cells. Philos Trans R Soc Lond
Q, Hook VY, and Taupenot L (2007) Primary B Biol Sci 296, 83–103
 Chapter 21

Indirect Immunofluorescence Staining

of Cultured Neural Cells
Massimo Barbierato, Carla Argentini, and Stephen D. Skaper

Abstract
Immunofluorescence is a technique allowing the visualization of a specific protein or antigen in cells or
tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein
isothiocyanate. There are two major types of immunofluorescence staining methods: (1) direct immunofluo-
rescence staining in which the primary antibody is labeled with fluorescence dye and (2) indirect immuno-
fluorescence staining in which a secondary antibody labeled with fluorochrome is used to recognize a
primary antibody. This chapter describes procedures for the application of indirect immunofluorescence
staining to neural cells in culture.

Key words: Immunocytochemistry, Fluorescence, Microscopy, Immunoreactivity, Glia, Cell culture

1. Introduction

Fluorescence can be described as a luminescent light produced by

certain molecules which, excited by a particular wavelength of
radiation, emit a longer wavelength of light energy before return-
ing to the equilibrium state (1). In biological terms, fluorescence is
probably the most powerful contrast method available to visualize
cell structures. However, there are some limits: chemical reactions
reduce the lifetime of fluorophores, and thereby the visualization
process and some samples can exhibit autofluorescence or photo-
bleaching (2–5). Immunofluorescence is a specific example of immu-
nocytochemistry/immunohistochemistry in which fluorochromes
are linked to antibody. It is a powerful and versatile technique for
the optical detection and localization of proteins, glycans, lipids,
and small biological and nonbiological molecules in cells and tissues.
A critical component of this technique is the fluorescence microscope,

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_21, © Springer Science+Business Media, LLC 2012

235
236 M. Barbierato et al.

Fig. 1. Schematic diagram illustrating the principles of fluorescence microscopy. Basic

components of an epifluorescence inverted microscope. In this type of microscope, a
mercury or xenon low-pressure arc lamp generates a beam of full spectrum light. The
excitation wavelength light is selected by a proper excitation filter, and once redirected to
the specimen by a dichroic mirror, it passes through the objective lens. In this case, the
objective needs to illuminate and image the sample. Fluorescent light passes an emission
filter in order to select the proper wavelength and is then reflected by a mirror to the
eyepiece and/or to a CCD camera (often a peltier-cooled CCD camera) to display on a
monitor and storage images. Modern microscopes collect light by a cube, in which there
are emission filters, a dichroic mirror, and a barrier filter, to prevent the exciting wave-
lengths from reaching the eyepiece and the CCD camera. Moreover, fluorescence micro-
scopes often have a visible light lamp to permit immunohistochemical work or studies
with living cells. 1, visible light lamp; 2, condenser; 3, sample; 4, visible light; 5, fluorescent
light; 6, lens; 7, UV mercury/xenon arc lamp; 8, UV light; 9, excitation filter; 10, excitation
light; 11, dichroic mirror; 12, emission filter; 13, mirror; 14, eyepiece; 15, digital camera
(or film camera) and storage or display computer system.

in which light is emitted from a UV lamp (usually a low-pressure

mercury lamp) with peaks at around 253.7 and 185 nm. The UV
light is filtered in order to excite different fluorochromes, while
other filters are used to detect the resulting fluorescence (see
Fig. 1). Like classical optical microscopy, conventional fluores-
cence methods have a limit of resolution due to diffraction and
lens aberrations (Abbe law) (6, 7). Sharp points well below this
limit appear blurry. In microscopy, diffraction is more important
than lens aberration and results from the numerical aperture (NA),
the type of glass of lens, and the light wavelength. In this case, the
Rayleigh criterion states that two point sources are just resolved
when the principal diffraction maximum of the Airy disk of one
21 Indirect Immunofluorescence Staining of Cultured Neural Cells 237

point falls in the first minimum of the other. Diffraction through a

condenser-objective system of a microscope is described by
R = 0.61λ / ((NAcondenser + NAobjective) / 2),
where R is the angular distance, l is the light wavelength, and NA
is the numerical aperture. The factor 0.61 is derived from calculation
of the position of the first dark ring surrounding the central Airy
disk of the diffraction pattern, in agreement with the Bessel function.
NA is described by
NA = η sin α,
where h is the refractive index of the medium between the lens and
the sample and a is half of the lens angle. The lowest value of R,
for l = 546 nm (green light) and NA 1.25 (like the most common
immersion lens), is approximately 220 nm at best.
Popular fluorescence microscopes are epifluorescence micro-
scopes, suitable for fluorescent or phosphorescent organic and
inorganic samples. In this type of microscope, UV light is diverted
by a dicroic mirror and passes through the objective lens in the
opposite direction. A filter interposed between the light source and
the optic system selects the right wavelength to excite the sample.
Before reaching the eyepiece, the light beam passes through a filter
that shields it from reflected UV light. The sample image can often
be detected by a digital camera for storage or display on a computer
monitor in real time. Cameras can be cooled for long exposures. In
the confocal microscope, fluorescently labeled samples are scanned
point by point by a beam. There are essentially three types of confocal
microscopes: confocal laser scanning, spinning-disk (Nipkow disk)
confocal, and programmable array. All confocal microscopes scan a
sample on various levels and then assemble a three-dimensional
image. The resolution is higher than that obtained with an epifluo-
rescence microscope, and all image parameters, including color
balance, can be adjusted freely. However, recently, super-resolution
nanoscopy techniques have broken Abbe’s limit, with resolution
approaching ten nanometers. These new methods include [fluores-
cence] phosphoactivation localization microscopy/stochastic
optical reconstruction microscopy, total internal reflection fluorescence
microscopy, stimulated emission depletion, structured illumination
microscopy, and multimodality microscopes (8, 9).
Immunofluorescence techniques can be either direct (or primary)
or indirect (or secondary antibody). In direct immunofluorescence,
the antibody, which is chemically linked to a fluorophore, recognizes
the target molecule and binds to it, resulting in direct detection.
In contrast, indirect immunofluorescence uses two antibodies:
the first (primary antibody) is specific for the target molecule, while
the second (secondary) recognizes the species in which the primary
antibody was raised. Because an antibody consists of a variable
238 M. Barbierato et al.

region (which recognizes the antigen) and an invariant region, one

can generate several primary antibodies that recognize various
antigens (with different variable regions), with all sharing the same
invariant region. For this reason, only the secondary antibody is
tagged to a fluorochrome or to biotin. In the latter case, one needs
to add a fluorophore linked to the streptavidin that recognizes biotin.
Direct immunofluorescence uses a single antibody for labeling and
detection, has a relatively fewer steps, and reduces cross-reactivity
or nonspecificity. Although the indirect fluorescence antibody
(IFA) protocol is more complex and time-consuming, it offers up
to 20-fold greater signal sensitivity. In addition, difficult-to-make
dye-coupled antibodies can be reused in multiple experiments.
Moreover, the secondary antibodies can bind to multiple sites on
the primary antibody and thus produce a brighter signal since more
dyes are brought to a single location. On other hand, IFA is more
subject to cross-reactivity and aspecificity and requires more con-
trols to avoid cross reactivity between primary and secondary
antibodies. This chapter will detail a protocol that is useful in the
application IFA to the characterization of neural cells in culture.

2. Materials

2.1. Apparatus 1. 1-L beakers

and Labware 2. 1-L Cylinder
3. Micropipettes (20–1,000 mL)
4. Stirrer hotplate
5. Forceps
6. 12-mm-diameter glass microscope slides (Thermo Fischer
Scientific)
7. Staining boxes or 24-well plates
8. Analytical balance
9. Microscope, Leica DMI4000 B (Leica Microsystems GmbH)
(or comparable model)
10. Digital camera, Leica DFC 480 (Leica Microsystems GmbH)
(or comparable model)

2.2. Reagents 1. Paraformaldehyde (PFA) (Sigma-Aldrich)

2. Poly-L-lysine hydrobromide, mol wt 70,000–150,000 (Sigma-
Aldrich)
3. Bovine serum albumin (BSA) (Sigma-Aldrich)
4. Gelatin
5. Chrome alum (potassium aluminum sulfate)
 21 Indirect Immunofluorescence Staining of Cultured Neural Cells 239

6. Goat serum (Sigma-Aldrich)

7. Glial fibrillary acidic protein (GFAP) primary antibody
(MAB3402, Millipore)
8. Alexa Fluor® 555 goat anti-mouse secondary antibody
(Invitrogen)
9. 4¢,6-Diamidino-2-phenylindole (DAPI) (Sigma-Aldrich)
10. Citifluor mounting medium (Citifluor Ltd., UK)
11. Mowiol 4-88 (Merck KGaA, Germany)
12. Fluoromount-G (Southern Biotech, USA)
13. Vectashield (Vector Laboratories)

2.3. Solutions 1. Tris, 200 mM, pH 8.5: Dissolve 24.22 g of Tris base in 800 mL
of distilled deionized water in a beaker. Stir the mixture, and
adjust pH to 8.5 by adding concentrated HCl under a chemical
fume hood. Adjust the volume to 1 L by adding distilled deion-
ized water. Sterilize by autoclaving (see Note 1).
2. Phosphate buffer (PBS), 10×: 1.369 M NaCl (80 g/L), 26.827 mM
KCl (2 g/L), 100 mM Na2HPO4 (14.2 g/L, pH > 9.5), and
14.697 mM KH2PO4 (2 g/L). Add NaOH (1 N) to adjust pH
to 7.2 ± 0.2 at 25°C. Use diluted 10 times (1×).
3. 4% PFA in PBS: Warning—see Note 2. Dissolve 4 g of PFA in
50 mL of distilled deionized water in a beaker, under a chemi-
cal fume cupboard. Add 1 mL of 1 N of NaOH. On a stirring
hotplate, stir and heat (~65°C) the solution until the PFA is
completely dissolved, always under a chemical fume cupboard.
Add 10 mL of 10× PBS and allow the solution to cool at room
temperature. Check the pH (after cooling) using pH papers
(never the pH meter!), and, in case, adjust to pH 7.4 (at 25°C)
using 1 M HCl (~1 mL). Adjust the volume to 100 mL with
distilled deionized water. Filter the solution through a 0.45-
mm membrane filter (it is possible that some particles of the
power of PFA do not solubilize). The solution can be stored at
−20°C for several months. However, it is better to aliquot the
prepared stock solution. After use, do not freeze/thaw again.
Once thawed, the solution can be kept for up to 1 week at
+4°C. After this time, discard the aliquot.
4. Mowiol: Mowiol is a mounting medium. Weigh 6 g of Mowiol
4-88 and mix in a solution of 6 mL of glycerol and 6 mL of
distilled deionized water. Shake for 2 h at room temperature.
Add 12 mL of 200 mM Tris pH 8.5, and heat at 50°C for
about 3 h. Shake occasionally. Filter the solution through a
0.45-mm membrane filter, aliquot, and store at −20°C for 2
months (or at +4°C for 2 weeks).
5. “Antibody” buffer: 150 mM NaCl (2.25 g/200 mL), 50 mM
Tris base (1.5 g/200 mL), 1% (w/v) BSA (2 g/200 mL),
240 M. Barbierato et al.

100 mM L-lysine (3.6 g/200 mL), and 0.04% azide (add

2 mL/200 mL of a 4% stock in water). Adjust pH to 7.4 (use
pH paper) adding 1 N HCl (about 6 mL). This solution can be
stored at 4°C indefinitely.

2.4. Coating Glass 1. Heat 400 mL of distilled water to 60°C on stirring hotplate.
Microscope Slides 2. Dissolve 5 g of gelatin.
with Gelatin
3. Add 200 mg of chrome alum and cool.
4. Dip slides individually and allow to air dry by leaning them
vertically against a test tube rack.
5. Store in a slide box 4°C until used.

2.5. Coating Glass 1. Dissolve 5 mg lyophilized powder of poly-L-lysine hydrobro-

Microscope Slides mide, mol wt 70,000–150,000 in 10 mL of sterilize distilled
with Poly-L-Lysine deionized water.
2. Add the poly-L-lysine solution to coverslips in a staining box or
in a 24-well plate to cover the entire surface (1 mL should be
sufficient).
3. Place the staining box or the plate at 37°C, in a cell culture
CO2 incubator, for at least 2 h.
4. Discard the poly-L-lysine solution and allow the surfaces to
dry.
5. Store the box or the plate at +4°C for few months. The poly-
L-lysine solution can be stored at +4°C for several months.

3. Methods

3.1. Coverslip Transfer 1. Using a coarse pair of forceps (keep cell side face up), transfer
to Staining Box coverslips from the 24-well plate to a staining box (a homemade
box with 24 flat-topped pedestals glued to its bottom to support
coverslips and keep them raised above the bottom which will
have some water for humidification). The box should also have
a top to prevent evaporation and keep slides humidified.
2. In the following steps, all volumes are 50–100 mL unless
otherwise specified. However, you can use as little as 25 mL if
necessary.

3.2. Fixation and 1. Place coverslips in the 4% PFA solution for 10 min at room
Permeabilization temperature, or 30 min at +4°C (except for bromodeoxyuri-
dine staining or other fixative-sensitive antigens, in which case
use 60–90 s maximum) (see Note 3). PFA cross-links proteins
so that they do not solubilize when the cells are permeabilized
(see below). Otherwise, fix in methanol or ethanol for 15 min
 21 Indirect Immunofluorescence Staining of Cultured Neural Cells 241

at +4°C or in an acetone solution (acetone-methanol 1:1 or

acetone-acetic acid 2:1) for at list 10 min (until acetone dries)
at room temperature. Alcohol and acetone solutions, in contrast
to PFA, work by precipitating proteins and allow permeabiliza-
tion without detergents (step 2 below).
2. After the above fixation step, pick up the coverslip with forceps
and rinse four times in PBS/0.05% Triton X-100. This step
can be combined with Subheading 3.3 by adding the Triton
(0.1%) to the goat serum solution (see Note 4).

3.3. Block of 1. Incubate the coverslips for 1 h at room temperature or over-

Nonspecific Binding night at +4°C in a buffer consisting of serum of the secondary
antibody species diluted 1:10 to 1:100 in PBS or in “antibody”
buffer. The serum contains high levels of antibodies and pro-
teins which bind noncovalently to nonspecific sites and also
covalently to reactive aldehyde groups created by the PFA.
Without this step, the primary antibody would bind both to
these reactive aldehyde groups and the nonspecific binding
sites and create high levels of background staining (see Note
5). A less expensive nonspecific binding site block can be
achieved by incubation in a 3% BSA/PBS solution.
2. After the blocking step, drain the coverslip (it is not necessary
to rinse in PBS).

3.4. Primary Antibody 1. Incubate the coverslip in the primary antibody solution for 2 h
Incubation at room temperature or overnight at 4°C (see Note 6). To
prepare this solution, dilute the primary antibody with 2%
serum in PBS or in “antibody” buffer. The best dilution
depends on the concentration of primary antibody and the
density of the antigen. Generally supernatants are used neat or
diluted up to 1:10. Ascites are used at 1:100 to 1:1,000, as are
polyclonal antisera. (The optimal dilution can be determined
in a separate experiment.) (see Note 7).
2. Rinse the coverslip five times in PBS.

3.5. Secondary 1. Incubate the coverslip in the secondary antibody solution for
Antibody Incubation 1–2 h. To prepare this solution, dilute the secondary antibody
with 2% serum in PBS or “antibody” buffer. The dilution
depends on the lot and concentration of the secondary anti-
body, the density of the antigen, and, to some extent, the con-
centration and the antigen affinity of the primary antibody that
was used.
2. A separate experiment can be used to establish the proper
antibody dilution. As a rule of thumb, the secondary antibodies
will generally be used at a final concentration of 20 mg/mL.
We suggest using a dilution of about 1:100 (1:500–1:1,000)
for secondary antibodies. For single-label staining, use fluorescein
242 M. Barbierato et al.

(e.g., FITC) or rhodamine (e.g., TRITC) dyes. It is better to

use stabilized fluorochromes, but they are more expensive
because they are patented, although generally they are brighter
and less sensitive to pH than common dyes. For a double-label
staining, use a combination of antibody labeled with different
fluorochromes. In these cases, check that the emission curves
do not overlap or that the overlap is as limited as possible.
Remember that hereafter all steps need to be carried out under
low light conditions to avoid fluorophore bleaching.
3. Rinse three times in PBS.
4. Incubate the coverslips with a counterstain solution to stain
nuclei (or other cell compartments). It is important to ascertain
the total number of cells on the coverslip, in particular if all
cells do not express the antigen. For nuclei, use a DNA stain
like DAPI. DAPI binds strongly to DNA of live and fixed cells
(it easily crosses intact cell membranes). When bound to dou-
ble-stranded DNA or RNA, DAPI is excited by light at 358 nm
and emits at 461 or 500 nm, respectively (blue). However, its
emission spectrum overlaps with green fluorescent dyes (like
fluorescein) or red fluorescent dyes (like Texas Red), but this
can be easily averted using an appropriate filter set. Other fluores-
cent dyes, like Hoechst dyes, can be used in place of DAPI.
Intercalating DNA compounds like DAPI or Hoechst can
exert mutagenic properties, and appropriate care should be
exercised in their handling and disposal.
5. See Note 8.
6. Mount coverslips in glycerol mounting medium. However, as
glycerol does not harden, you must handle slides carefully in
order not to move them. If a mounting medium which hard-
ens is desired, use Mowiol or a mounting medium such as
Fluoromount-G or Citifluor mounting medium (or Vectashield)
by inverting the coverslip onto a drop of the mounting medium
on a 25 × 75-mm glass microscope.
7. Allow to air dry for 10 min, and clean around the edges before
examining under the microscope.
8. Slides can be stored for several months in the dark at 4°C. If
using glycerol or Mowiol as mounting medium, it is better to
seal by nail polish in order to prevent drying during storage.
9. See Note 9.

3.6. Immunostaining 1. Never perform immunostaining without appropriate controls.

Controls Negative controls must be set up for every labeling experiment;
often other controls are also required.
2. Negative controls: When the primary antibody is omitted or
replaced with an irrelevant primary antibody of the same type
and concentration, be certain that there is no staining.
 21 Indirect Immunofluorescence Staining of Cultured Neural Cells 243

3. Positive controls: If no specific staining is observed, it is essential

to demonstrate that the labeling protocol used would have
worked where than antigen is known to be expressed.
4. Preabsorption controls: Particularly, when staining with anti-
bodies to a peptide or other antigen available in purified form,
it is important to show that preabsorption of the primary
antibody with a 100 (or greater) molar excess of the purified anti-
gen eliminates staining.
5. Double-label controls: When carrying out double labeling, show
that the secondary antibodies each recognize specifically only
the appropriate primary antibody. You should show also that
the filter set used is able to discriminate several fluorochromes
with minimal overlap of the emission spectra.

3.7. Troubleshooting 1. No staining:

– Fixative sensitivity (wrong fixative used)
– Antibodies not working (e.g., contaminated by microor-
ganisms or freeze–thaw leading to misfolded antibodies)
– Wrong secondary antibody used (e.g., anti-mouse antibody
to detect a rabbit primary)
2. High background staining:
– Failure to block nonspecific binding (either too little goat
serum or too short an incubation). Change the type of
serum or use a 3% BSA solution.
– Secondary antibody concentration too high.
– Primary antibody concentration too high.
– Secondary antibody may specifically detect antigens on the
tissue stained.
– Primary or secondary antibody may be binding to Fc
receptors expressed by some types, particularly macrophages
or microglia.
3. Specific staining is weak:
– Primary antibody concentration is too low.
– Secondary antibody concentration is too low.
– Fluorophore has been exposed to light during the staining.
– Density of antigen is low (see Note 10).

3.8. Staining The procedure below is intended as a “generic” illustration and

of Cortical Astrocyte should be adapted to the cells and antigen of interest.
Cultures
1. Transfer coverslips to the staining box with a coarse pair of
forceps.
244 M. Barbierato et al.

2. Fix with 4% PFA (about 100 mL per coverslip) for 10 min at

room temperature. Wash three times with PBS.
3. Permeabilize the coverslips with 50% goat serum/0.1% Triton
for 30 min.
4. Incubate with primary antibody for 1 h at room temperature.
Prepare 1 mL of the GFAP antibody solution in an Eppendorf
tube. GFAP is a rabbit polyclonal antibody which should be
diluted in the BSA/Tris buffer to 1:1,000. After 1 h, rinse the
coverslips three times with PBS.
5. Secondary antibody incubation for 1 h at room temperature.
Prepare 1 mL of each antibody solution in an Eppendorf
tube. Use FITC-anti-rabbit at 1:500 (dilute into BSA/Tris).
After the 1 h staining, rinse each coverslip three times with
PBS.
6. Mount in Citifluor mounting medium on a microscope slide.
Gently blot excess mounting medium. You can use nail polish
to seal. Allow slips to air dry for 30 min before examining on
the microscope.
7. See Fig. 2 for a GFAP stained culture.

Fig. 2. Immunostaining of cortical astrocytes for GFAP. Astrocytes cultured from 2-day-old
neonatal rat cortex on poly-L-lysine-coated coverslips were fixed with paraformaldehyde.
Staining was performed using a mouse anti-rat GFAP monoclonal primary antibody, followed
by an Alexa Fluor® 555 goat anti-mouse secondary antibody. Images were captured
using a Leica DMI 4000B fluorescence microscope equipped with a Leica DFC480
camera, analyzed, and stored using Leica Application Suite software (version 2.8.1).
Magnification: ×400.
 21 Indirect Immunofluorescence Staining of Cultured Neural Cells 245

4. Notes

1. Caution: The pH of the Tris solution is temperature-dependent

and decreases about 0.03 pH unit for each increase of 1°C.
2. PFA is a potential carcinogen. It is a skin and eye irritant and is
poisonous if ingested. Handle only under a fume cupboard
and wear gloves. PFA waste cannot be disposed of down the
drain and must be collected in a special waste container labeled
clearly and only used to collect this waste.
3. For intracellular antigens such as GFAP, you can use instead a
10 min fixation in −20°C acid-alcohol (5% glacial acetic acid
plus 95% ethanol) or in 100% methanol; whenever alcohol fixa-
tion is used, the Triton permeabilization step can be skipped.
4. Goat serum is used because the fluorochrome-conjugated sec-
ondary antibodies are raised in goats; this minimizes any
unwanted binding of the secondary antibody to the goat anti-
bodies that bind to the nonspecific binding sites.
5. Do not permeabilize cells when staining for surface-expressed
antigens, particularly for galactocerebroside (a glycolipid) or
A2B5 (a ganglioside)—these lipid antigens are solubilized by
Triton.
6. Incubation with primary antibody can be extended from
60 min to overnight at +4°C if necessary.
7. Typical supernatant antibody concentration is 1–10 mg/mL,
and ascites concentrations are 1–10 mg/mL, as are polyclonal
antisera. Thus, typical final primary concentrations are about
1–10 mg/mL. (When the best concentration is not known
and a guess must be made, use the supernatant undiluted, the
ascites at 1:100, and polyclonals at 1:500. However, beware
of the “prozone” effect—especially with IgM monoclonal
antibodies.)
8. This is optional: Postfix in acid-alcohol for 10 min—particu-
larly helpful for high-density cultures to stabilize the antibody
binding and to decrease the likelihood that the cells will detach
during coverslipping. This would not hurt (unless using phy-
coerythrin-coupled secondaries, which it destroys) and gener-
ally improves the appearance. Rinse three times in PBS.
9. Citifluor media, like AF1 and AF2, are mixtures of PBS and
glycerol and contain a proprietary substance that helps retard
fluorescein bleaching but is not so helpful for rhodamine
or Texas Red bleaching. Vectashield works for both.
N-paraphenylenediamine (0.1%) is useful for FITC bleaching
too (but is carcinogenic). N-propylgallate (0.4% or higher for
confocal studies) retards rhodamine and Texas-Red bleaching
but does not help for FITC. Note that FITC has a steep pH
246 M. Barbierato et al.

optimum, being maximally bright between pH 8 and 9.

Outside this range, its intensity is many times lower.
10. If using a monoclonal antibody, consider using a polyclonal if
possible or move to an immunostaining technique with a higher
amplification: biotin-conjugated secondary antibodies followed
by streptavidin-FITC or an immunoenzymatic technique such
as peroxidase methods (secondary antibody conjugated to
horseradish peroxidase or use the avidin-biotin-peroxidase
complex or peroxidase anti-peroxidase methods).

References
1. Lakowicz JR (2006) Principles of fluorescence Rhodamine 6G by one- and two-photon
spectroscopy (3rd edition). Springer, New York, induced confocal fluorescence microscopy.
USA Chemphyschem. 6, 791–804
2. Monici M (2005) Cell and tissue autofluores- 6. Abbe E (1873) Archiv fuer Mikroskopische
cence research and diagnostic applications. Anatomie und Entwicklungs mechanik. 9,
Biotechnol Annu Rev 11, 227–256 413
3. Heikal AA (2010) Intracellular coenzymes as nat- 7. Liang R (2010) Optical design for biomedical
ural biomarkers for metabolic activities and mito- imaging. Spie Press, Washington USA
chondrial anomalies. Biomark Med 4, 241–263 8. Toomre D, and Bewersdorf J (2010) A new
4. Benson DM, Bryan J, Plant AL, Gotto AM Jr, wave of cellular imaging. Annu Rev Cell Dev
and Smith LC (1985) Digital imaging fluores- Biol 26, 285–314
cence microscopy: spatial heterogeneity of 9. Petibois C (2010) Imaging method for elemen-
photobleaching rate constant in individual tal, chemical, molecular, and morphological
cells. J Cell Biol 100, 1309–1323 analyses of single cells. Anal Bioanal Chem
5. Eggeling C, Volkmer A, and Seidel CA 397, 2051–2065
(2005) Molecular photobleaching kinetics of
 Chapter 22

Neurite Outgrowth Assessment Using High

Content Analysis Methodology
Nicholas M. Radio

Abstract
High content analysis of neurite outgrowth enables the rapid and comprehensive phenotypic assessment
of individual neurons in a multiwell format amenable to high throughput assays. The resulting data are
considered “high content” because multiple measurements of neuronal outgrowth and morphometric
data are calculated from hundreds of individual cells within each image. This approach has been widely
adopted by the pharmaceutical industry to accelerate neurological drug discovery and in vitro safety assess-
ment. High content technology utilizes automated fluorescent and/or brightfield microscopy for image
acquisition. The acquired images are then quantified using mathematical algorithms to measure pertinent
neurobiological morphometric information, including neurite length, count, and extent of branching for
each cell within the images. Furthermore, evaluation of the individual cell-level measurements enables the
detection of subpopulations of cellular responders not apparent when examining well-level averages. Using
this technology, neurite outgrowth can be quantified in each well, derived from hundreds of cell measure-
ments in a 96-well microplate in approximately 30 min.

Key words: Neurite outgrowth, Immunofluorescence, High content analysis, High content screening,
Neuronal differentiation, In vitro neurotoxicity, PC12 cells

1. Introduction

Neurite outgrowth is a critical cellular event underlying the devel-

opment and functionality of the nervous system and has been
widely studied to evaluate neuronal health and assessment of neu-
ronal differentiation (1). Neurite outgrowth is a process that results
from the differentiation of precursor cells to a neuronal phenotype
and the initiation of sheetlike lamellipodia that ultimately condense
into short processes (2). As the cells mature, the processes will
increase in length and complexity (see Fig. 1). In some neuronal
cell models, including primary cells, neurons will become polarized
by developing a single long axon and several secondary processes

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_22, © Springer Science+Business Media, LLC 2012

247
248 N.M. Radio

Fig. 1. Early events in neurite outgrowth. (a) Diagram of early events in neurite outgrowth illustrating a cell body with lamel-
lipodia, the development of minor processes (tipped with a growth cone), and transformation of the processes into an axon
and dendrites. (b) Hoffman modulation contrast photomicrographs of neurite outgrowth in PC12 cells 2 h, 48 h, and 72 h
after treatment with 100 ng/mL NGF to induce differentiation. Reproduced with permission from Elsevier (5).

shorter in length. The growth of axonal and dendritic processes

(collectively called neurites) is a critical determinant of neuronal
connectivity, and disruption of this process can induce human cog-
nitive deficits (3, 4). For example, deficits in the regulation of the
dendritic cytoskeleton affect the functionality of dendrites and syn-
apses believed to underlie some cases of mental retardation (4).
Additionally, novel compounds that facilitate neurite outgrowth
formation can potentially accelerate treatment of a wide variety of
neurological diseases and traumas that result in nerve injury, includ-
ing stroke, Parkinson’s, and Alzheimer’s diseases.
There are a number of well-characterized neuronal cell models
that have been used to characterize chemical effects on neurite
outgrowth (5). Of these models, pheochromocytoma (PC12) cells
are a rodent noradrenergic cell line that has been widely utilized in
neurobiological studies to examine chemical perturbations of neurite
outgrowth (6). Following exposure to 10–100 nanograms per mil-
liliter (ng/mL) nerve growth factor (NGF), PC12 cells differenti-
ate into a sympathetic-like neuron and develop extensive neuritic
processes. Unlike primary neuronal cell cultures, neurites produced
from PC12 cells do not give rise to definitive axons or dendrites
(7). Many studies have utilized PC12 cells to evaluate the effect of
environmental compounds on neurite outgrowth (8, 9). More
recently, a PC12 cell clone, Neuroscreen-1™ (NS-1) cells, has
been used as an in vitro model for neurite outgrowth evaluation (1).
 22 Neurite Outgrowth Assessment Using High Content Analysis Methodology 249

In comparison with the parent PC12 cell line, NS-1 cells are less
prone to cellular aggregation, allowing for the evaluation of neu-
rite outgrowth in individual cells, while retaining many of the
properties of PC12 cells (1). Previous studies using more tradi-
tional, manually derived methodologies demonstrate the ability of
a cell model to detect changes in neurite outgrowth; they were
mostly restricted to a single chemical analysis due to throughput
limitations (8, 9). For large volume chemical screening, fully auto-
mated techniques amendable to high-throughput analysis are nec-
essary to facilitate the rapid assessment of compound libraries.
Advances in automated microscopy combined with algorithm-
based quantification metrics have evolved into a cell-based mea-
surement approach called high content analysis. High content
analysis integrates quantitative microscopy with automated tech-
nology controlling image acquisition, quantified cell-level data
analysis, and data collection (10, 11). High content platforms are
designed to track phenotypic changes at the individual cell level in
multiwell plates using fluorescent labels and/or bright field imag-
ing. As a result of individual cell assessment, high content approaches
enable the detection of subtle heterogeneous effects not apparent
in average population responses (12). Furthermore, the increased
throughput abilities of high content combined with the microplate
formats enable the use of comprehensive dose-response curves to
characterize a concentration-dependent profile. For example, anal-
ysis in a 96-well plate enables a 12-point dose-response curve for
eight different compounds (see Fig. 2).

1 2 3 4 5 6 7 8 9 10 11 12

A -NGF -9 - 8.5 -8 - 7.5 -7 - 6.5 -6 - 5.5 -5 - 4.5 -4

B -NGF -9 - 8.5 -8 - 7.5 -7 - 6.5 -6 - 5.5 -5 - 4.5 -4

C -NGF -9 - 8.5 -8 - 7.5 -7 - 6.5 -6 - 5.5 -5 - 4.5 -4

D +NGF -9 - 8.5 -8 - 7.5 -7 - 6.5 -6 - 5.5 -5 - 4.5 -4

E +NGF -9 - 8.5 -8 - 7.5 -7 - 6.5 -6 - 5.5 -5 - 4.5 -4

F +NGF -9 - 8.5 -8 - 7.5 -7 - 6.5 -6 - 5.5 -5 - 4.5 -4

G BIS -9 - 8.5 -8 - 7.5 -7 - 6.5 -6 - 5.5 -5 - 4.5 -4

H BIS -9 - 8.5 -8 - 7.5 -7 - 6.5 -6 - 5.5 -5 - 4.5 -4

Fig. 2. Ninety-six-well plate chemical exposure format for neurite outgrowth evaluation. NS-1 cells are exposed to an
11-point dose-response curve (semilogarithmic concentration range from 1 nM to 100 μM) from eight separate chemicals
(Rows A–H ). Column 1 serves as a control column, with three replicates each of undifferentiated NS-1 cells (−NGF), cells
incubated with 100 ng/mL NGF (+NGF), and cells incubated with 100 ng/mL NGF coadministered with 3 μM bisindole-
maleamide-I (BIS-I) to serve as an internal pharmacologic control to evaluate expected neurite outgrowth inhibition. All
treatments contain 0.1% DMSO.
250 N.M. Radio

The complex nature of the regulation of neurite outgrowth

provides a wide range of potential targets for chemical perturba-
tion. Chemically mediated interference with gene expression,
membrane receptors, ion channels, or intracellular signaling can
affect neurite initiation and growth. For example, neurite out-
growth is supported by a set of extracellular cues, including trophic
factors, extracellular matrix molecules, and activity-dependent
depolarization (13). Because of these many potential molecular
targets, the use of neurite outgrowth as a “nodal indicator” of neu-
ronal cell health and differentiation status has recently been inves-
tigated using high content methodologies. This approach may be
more suitable for hazard identification and screening for chemical-
induced changes in neurite outgrowth when the site of chemical
action is unknown. These studies have employed a variety of cel-
lular models, including cell lines, primary cultures, and stem cell
models. Furthermore, over the last 2 years, the algorithms have
evolved to examine additional neurobiological end points captured
in punctate detection, including synaptogenesis (14). Taken
together, high content analysis allows the comprehensive evalua-
tion of neurite outgrowth in an efficient manner amenable to
screening assays, as will be detailed in this chapter.

2. Materials

2.1. Cell Culture 1. NS-1 cells, a PC12 subclone (Thermo Fisher Scientific), are
Reagents maintained at 37°C in a 95% humidified incubator containing
5% CO2.
2. NS-1 cells are cultured in RPMI 1640 medium (Lonza
BioWhittaker, Walkersville, MD), supplemented with 10%
equine serum (HyClone, Logan, UT), 5% heat-inactivated fetal
bovine serum (HyClone), 1% L-glutamine (Lonza BioWhittaker),
1% penicillin/streptomycin (Lonza BioWhittaker), and 100 ng/
mL NGF (Sigma-Aldrich). After adding all components of the
medium, adjust the pH to 7.4 with HCl (see Note 1).
3. Human recombinant NGF (Sigma-Aldrich) is supplied as a
0.1 mg lyophilized powder. Dilute the powder with 500 μL of
0.1% bovine serum albumin in phosphate buffered saline
(PBS). Aliquot the 500 μL volume into 10-μL × 50-μL vials
and store at −80°C until time of use. On the day of use,
prepare NGF-supplemented medium (200 ng/mL) by adding
a 50 μL aliquot to 50 mL of RPMI medium.
 22 Neurite Outgrowth Assessment Using High Content Analysis Methodology 251

4. Trypsin-Versene (EDTA) solution (Lonza BioWhittaker),

stored at −20°C.
5. Culture cells on BD BioCoat® Collagen Type IV, 75-cm2
culture flasks with vented cap (BD Biosciences).
6. Use Falcon 15- or 50-mL sterile conical tubes (BD Biosciences)
for cell transfer.
7. Transparent 96-well polystyrene microplates that have been
precoated with collagen IV (BD Biosciences) are used for cell
plating. Store plates at 4°C (see Note 2).
8. Bisindolemaleamide-I (Bis-I) (EMD, Darmstadt, Germany), a
potent protein kinase C-inhibitor, is used as a pharmacologic
positive control for neurite outgrowth inhibition and is dis-
solved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and
stored in single use aliquots at −20°C. The final concentration
to selectively inhibit neurite outgrowth without affecting cel-
lular viability is 3.0 μM.

2.2. Immuno- 1. Paraformaldehyde (Electron Microscopy Sciences, Hatfield

cytochemistry PA) is prepared at a 4% (v/v) final concentration in fresh PBS.
Hoechst 33342 (Thermo Fisher Scientific) is included in the
fixative solution at a concentration of 5.0 μg/mL to selectively
label cell nuclei.
2. PBS 10× stock (Invitrogen). Prepare 1× working solution by
dilution of one part 10× with nine parts water. Store 1× and
10× solutions at 4°C.
3. Immunocytochemical staining buffer (ISB): prepare 10× stock
containing 9 mM CaCl2, 26.8 mM KCl, 14.7 mM KH2PO4,
4.9 mM MgCl2, 1.4 mM NaCl, 80.6 mM Na2HPO4, 0.1%
saponin, 5% BSA, and 0.5% NaN3. Prepare a 1× working solu-
tion by dilution of one part 10× with nine parts water. Store 1×
and 10× solutions at 4°C.
4. A mouse anti-βIII-tubulin primary antibody (1:800) (Thermo
Fisher Scientific) is used to label cell bodies and neurites.
Dissolve the antibody in ISB and combine with a DyLight
Fluor 488-conjugated goat anti-mouse secondary antibody
(1:500) (Thermo Fisher Scientific) dissolved in ISB.
5. Optical adhesive film seal (Thermo Fisher Scientific) is used for
sealing plates.

2.3. Image Acquisition 1. Automated image acquisition is performed using the ArrayScan
and Analysis VTI high content imaging platform (Thermo Fisher Scientific).
2. Neurite outgrowth analysis is performed using the Neuronal
Profiling bioapplication (Thermo Fisher Scientific).
252 N.M. Radio

3. Methods

3.1. Cell Culture 1. A single passage number of NS-1 cells (i.e., ten after receipt
from supplier) should be used in every experiment to avoid
potential interpassage variability (15).
2. Grow NS-1 cells in T-75 flasks and trypsinize for use when
they reach 70–80% confluency (see Note 3).
3. To dislodge cells, aspirate the growth medium from the T-75
culture flask.
4. Add 3.0 mL of prewarmed (37°C) Trypsin-Versene solution to
the T-75 culture flask.
5. Gently tilt the flask so that the Trypsin-Versene solution covers
the cell surface. The first addition of Trypsin-Versene serves as
a wash step to remove the existing medium/serum and allows
for a smaller volume of Trypsin-Versene to be added.
6. After covering the cell surface of the flask with the 3.0 mL of
Trypsin-Versene, immediately aspirate the Trypsin-Versene
solution.
7. Add 1.0 mL of prewarmed (37°C) Trypsin-Versene solution to
the T-75 culture flask. Gently tilt the flask so that the Trypsin-
Versene solution covers the cell surface.
8. Incubate the cells in the Trypsin-Versene solution for 5 min at
37°C.
9. Add 9.0 mL of prewarmed complete medium directly to the
growth surface of the flask to dislodge the cells. Add the
medium to the growth surface of the flask several times to
ensure the cells are dislodged.
10. Transfer the cells into a 15-mL conical tube suitable for
centrifugation.
11. Centrifuge the cells at 300 × g for 5 min.
12. After centrifugation, remove the supernatant containing the
Trypsin-Versene solution and resuspend the pellet of cells in
3 mL of complete medium (see Note 4).
13. Perform a cell count using a hemocytometer to calculate the
cell density of the harvested cells.
14. Subdivide the harvested cells into two groups: −NGF-treated
cells (3 wells total) and +NGF-treated cells (93 wells total).
For each group, dilute the cells so the final density is 2,000
NS-1 cells per 90 μL. For the −NGF-treated cells, dilute the
cells in growth medium. For +NGF-treated cells, dilute the
cells in medium containing 100 ng/mL NGF.
15. Add 90 μL per well of the harvested cell suspension (containing
2,000 NS-1 cells) into a transparent, 96-well collagen IV–coated
 22 Neurite Outgrowth Assessment Using High Content Analysis Methodology 253

microplate. Ensure the cells are homogeneously mixed by

triturating five times before plating the cells.
16. Leave the cells undisturbed on a solid bench top for 30 min
following cell plating before moving the cells to the incubator.
This will facilitate an even cell seeding for optimal neurite
identification.
17. Wait a total of 2 h to allow the cells sufficient time to adhere to
the bottom of the wells.
18. Add to the cells 10 μL of the 10× intermediate concentration
of the test compound(s) of interest. The positive control to
inhibit neurite outgrowth, Bis-I should be prepared at a 10×
intermediate concentration of 30 μM. Negative control wells
should only be treated with DMSO vehicle at a 10× concentra-
tion of 1.0%. After compound addition to the cells, the final
volume is now 100 μL, with the positive control group exposed
to 3 μM Bis-I and the negative control group exposed to 0.1%
DMSO (see Note 5).
19. Expose cells to the compounds of interest and return to the
incubator (37°C, 5% CO2) for 96 h; no medium exchange is
necessary.

3.2. Immuno- 1. Prepare a 2× fixative solution containing 8% paraformaldehyde

cytochemistry and 10 μg/mL Hoechst 33342 in 1× PBS. Prewarm the fixa-
tive to 37°C before adding to the cells. Prewarming the fixative
is critical to maintain the cell and neurite integrity.
2. Add 100 μL of the 2× fixative solution to the existing 100 μL
of cell medium for a final concentration of 4% paraformalde-
hyde and 5 μg/mL Hoechst 33342. Add the fixative solution
using a low-velocity fluid dispension to the side of the wells.
Incubate for 20 min.
3. During the fixation, prepare 1:800 solution of the mouse anti-
βIII-tubulin primary antibody in 1× ISB.
4. Following the 20-min fixative incubation, carefully aspirate the
paraformaldehyde and Hoechst 33342–containing solution.
5. Fill the wells with 100 μL of ISB, and then drain each well with
gentle aspiration. Wash two additional times with 100 μL of
1× ISB (see Note 6).
6. Aspirate the ISB buffer and label the cell bodies and processes
with 50 μL of mouse anti-βIII-tubulin primary antibody
(1:800 dilution) for 1 h.
7. Aspirate the primary antibody and wash three times with
100 μL of 1× PBS.
8. Aspirate the 1× PBS buffer and label with 50 μL of DyLight™
488-conjugated goat anti-mouse secondary antibody (1:500
dilution) for 1 h. Protect from light.
254 N.M. Radio

9. Aspirate the secondary antibody and wash three times with

100 μL of 1× PBS, leaving the buffer from the final wash in the
plate.
10. Seal the plate with a plate seal. Store the plate at 4°C in the
dark until use. Plates can be safely stored in this manner for at
least 6 months.

3.3. Image Acquisition 1. The following instructions are based on use of the ArrayScan
VTI high content platform in conjunction with the iDEV™
Assay Development Workflow software in conjunction with
the Neuronal Profiling bioapplication. The method describes
automated measurement of neurite outgrowth in differenti-
ated cells in 96-well plates stained using the immunocytochem-
ical procedure described above. The assay parameters cited in
this method are associated with an optimized image analysis
protocol for measuring neurite outgrowth in differentiated
NS-1 cells. The notes associated with this method provide
guidance for adjustment of default settings and assay parameters
for adapting the protocol to measure neurite outgrowth in
other neuronal cell types (i.e., other cell lines, primary neu-
ronal cultures, etc.). Bold text refers to a menu or heading in
the Neuronal Profiling bioapplication. Italicized text refers to
an option or value input by the end-user (see Note 7).
2. Turn on the ArrayScan VTI HCS Reader, open the vHCS:Scan
Software, select the iDEV™ Assay Development Workflow mode,
and log in.
3. From the Select Protocol menu, click on the Change button
to select the NeuronalProfiling.V4 bioapplication.
4. Proceed to the Configure Acquisition parameters: Select the
×10 imaging Objective from the associated drop-down menu.
Set the Camera Configuration to an option that is compatible
with the ArrayScan system in use. Set Acquisition Camera
Mode to Standard. Set the number of channels to 2. The field
dimensions using these settings are 660.48 by 660.48 μm.
5. Specify the correct fluorophores to be utilized in the Image
Formation subbox: Use the drop-down menu to select XF100
– Hoechst as the Channel 1 dye and XF100 – FITC as the
Channel 2 dye.
6. In the XF100 – Hoechst Channel 1, select Fixed from the
Exposure Type drop-down menu. Set the Initial Exp Time
(s) to 0.01. In the XF100 – FITC Channel 2, select Fixed from
the Exposure Type drop-down menu. Set the Initial Exp
Time (s) to 0.03. These exposure values serve as initial expo-
sure settings that will used during initial image acquisition.
Acquired images deemed representative to the investigator
should be used to finalize exposure times, so nuclear images
22 Neurite Outgrowth Assessment Using High Content Analysis Methodology 255

use 25% of the camera’s dynamic range and the cell body/
neurite uses 55–65% (see Note 8).
7. Select Autofocus from the Autofocus Camera Mode drop-
down and specify Autofocus interval 3 fields (see Note 9).
8. Click on a representative undifferentiated (−NGF) control well
from the plate layout map. In cellular nuclei Channel 1, select
the Acquire Image button and then select Autofocus. Set the
final exposure value interactively by clicking the Autoexpose
button. Repeat this process for Channel 2 for cell body and
neurite image acquisition. Once suitable images are acquired
for both channels of the negative control well, select Save
Field so the image set is available for subsequent analysis.
9. Click a representative differentiated (+NGF) control well from
the plate layout map. Acquire images for both the nuclear
(Channel 1) and cell body/neurite (Channel 2) fluorophores.
Select Save Field so the image set is available for analysis. Select
Next to proceed to Configure Assay Parameters.
10. Within the Protocol Optimization Task List, proceed to the
Image Preprocessing step. Select the Object Type as Bright
for both Channels 1 and 2. Specify and enable the LowPassFilter
as the Background Removal Method for both Channels 1
and 2. The Value for both channels should be determined by
measuring the diameter of typically large valid objects (nucleus
in Channel 1 and cell body in Channel 2). The diameter of
these objects can be measured by using the status tray at the
bottom of the window. Due to a greater chance of cell body
size heterogeneity, double the diameter of the measured cell
body size.
11. Proceed to the Nucleus Identification Ch1 step. Enable
Smoothing, select the Uniform Method, and specify a
Value of 1.
12. For the Field Nucleus Identification Ch1, enable
Thresholding, select the Fixed Method, and determine a
Value by determining a difference (“delta”) between the
brightest background pixels and the dimmest foreground fluo-
rescence. Use half of this delta for a starting Value, and adjust
as needed, to produce accurate borders on the nucleus. Enable
Segmentation, selecting the Shape Method, and enter a
Value corresponding to half the diameter of a typical nucleus.
Object Cleanup should also be enabled to clean up the object
mask and remove small objects from identification.
13. Proceed to the Nucleus Validation Ch1 step. To be included
in analysis, any identified nucleus can be validated based on
size, shape, and fluorescence intensity measurements. Enable
the Nucleus.BorderObject.Ch1, Nucleus.Area.Ch1, and
Nucleus.ShapeLWR.Ch1 selection features. To exclude
256 N.M. Radio

aggregated cells and noncellular particles from analysis, specify

the following ranges for each of these features: Nucleus.Area.
Ch1: minimum 20, maximum 400 pixels; Nucleus.ShapeLWR.
Ch1: minimum 1.0, maximum 3.0.
14. Proceed to the Cell Body Identification Ch2 step. Do not
enable Smoothing.
15. For the Field Cell Body Identification Ch2, enable
Thresholding, select the Fixed Method, and repeat the
instructions listed in step 20 for determining an appropriate
thresholding value to delineate neuron detection relative to
image background. Enter a value of 10 for the Minimum Cell
Body Nucleus Overlap to specify the minimum percentage
overlap between the cell body and the nucleus required to
associate the cell body with the nucleus. Enable Segmentation
and select the Use Seeds Method. Next, specify a Cell Body
Demarcation of 2 to specify the degree in pixels in which the
cell body excludes neurites. The main purpose of this parame-
ter is to eliminate neurites identified as part of Cell Bodies.
Increasing values should retract the Cell Body overlays extend-
ing down neurites. Enable the Mask Modification with 3 so
neurite origins are clear.
16. Proceed to the Cell Body Validation Ch2 step. Enable
the Cellbody.BorderObject.Ch2, Cellbody.Area.Ch2, and
CellbodyLWR.Ch2 selection features. To exclude aggregated
cells and noncellular particles from analysis, specify the follow-
ing ranges for each of these features: Cellbody.Area.Ch2:
minimum 10, maximum 1,000 pixels; CellbodyLWR.Ch2:
minimum 1.0, maximum 3.0.
17. Proceed to the Neurite Identification Ch2, enable
Smoothing, select the Uniform Method, and specify a Value
of 1.
18. For the Field.Neurite.Detection Ch2 Assay Parameters,
enable Detection, select the Binomial Method, enter a Value of
2, and enter a Neurite Identification Modifier value of −0.97.
19. Under the Field.Neurite.Identification Ch2, enter a
Direction Length value of 2, a Point Resolution value of 1,
and do not enable the Rejection of Multiply-traced Neurite
(see Note 10). If increased sensitivity to neurite detection is
desired, enable the Aggressive Tracing feature. Enter a value
of 0 for Trace Within Cell Body ZOI.
20. Proceed to the Neurite Validation Ch2 step, enable the
Neurite.Length.Ch2, and enter a minimum and maximum
value range of 0 and 1,000, respectively.
21. Modification of the Neuronal Profiling bioapplication is now
complete. Using these settings, a representative composite
22 Neurite Outgrowth Assessment Using High Content Analysis Methodology 257

Fig. 3. Differentiated NS-1 cells (100 ng/mL NGF, 96 h) image acquired with a ×10 objec-
tive lens and a ×0.63 coupler using the ArrayScan VTI. (a) Nuclei are stained blue with
Hoechst 33342 (5 ng/mL), and cell bodies and neurites are stained green using an anti-
βIII-tubulin primary antibody (1:800) and DyLight™ 488-congjugated antibody (1:500).
The resulting image was analyzed using the Neuronal Profiling bioapplication (b) for mor-
phometric data including cell body area, number of neurites, neurite length, and branch
point for each cell measured.

image of the NS-1 cell and applied algorithm overlay can be

generated as shown in Fig. 3.
22. Click on Next to proceed to the Subpopulation Characte-
rization task. Within the Event Subpopulations field, define
the Type 1 Event so that NeuriteTotalLengthCh2 or
NeuriteTotalCountCh2 are designated. Two additional sub-
population events can be specified if desired by the user. Within
the Scan Limits field, enable the Min Objects for Well to 200
objects per well. Alternatively, this value can be adjusted
according to the statistical requirements of the user.
23. Click on Next to proceed to the Select Features to Store task.
Specify all available features to be stored for the Cell Features,
Field Features, and Well Features. Select the MEAN_
NeuriteTotalLengthCh2 as the Default Well Feature and
set the Lower Extent as 1 and the Upper Extent as 20 μm.
24. Click on Go to Scan, enter a Plate ID (if not using a bar-
code), such as the scientist’s initials, experiment date, and
plate number (i.e., NMR12.11.2010Plate1). Enter a Plate
Name, such as a description of the assay contained on the
plate (i.e., Bis-I dose-response curve in NS-1 cells). Additional
notes can be electronically recorded if desired within the Scan
Comments field.
25. Press the Play icon to start the high content scan of the assay
plate.
258 N.M. Radio

Fig. 4. Effects of neurite outgrowth inhibitors on NS-1 cells. NS-1 cells were plated at 2,000 cells per well in 100 ng/mL
NGF and evaluated for either total neurite length (closed circle) or viability (open square) following 96-h exposure to Bis-I.
Inset shows representative images of either control (0.1% DMSO) or 3 μM Bis-I-treated cells following the 96-h exposure.
Data are expressed as percent of the 0.1% DMSO control and are presented as means ± standard deviation from six total
wells analyzed across two independent experiments. Treatments that are Significantly different from control for total
neurite length (asterisk) or viability (multiplication sign) (one-way ANOVA followed by Dunnett’s test, p < 0.05). Reproduced
with permission from Oxford University Press (1).

26. The time requirements for the immunofluorescent labeling of

the cells are approximately 4 h. Time requirement for the
image acquisition (three fields per well) and analysis per 96-well
plate is approximately 30 min.
27. Typical results of neurite outgrowth in NS-1 cells exposed to a
dose response of Bis-I are represented in Fig. 4.

4. Notes

1. Store the L-glutamine and penicillin/streptomycin mixture as

10 mL aliquots at −20°C. On the day of use, thaw and add to
1 L of RPMI medium. Store the supplemented medium at 4°C
until use.
2. Alternatively, plates can be manually coated with collagen IV
substrate. While this method is less expensive, quality control
issues pertaining to batch-to-batch plate preparation is gener-
ally avoided when using precoated plates.
3. Approximately 1.5 million NS-1 cells are harvested per 75%
confluent T-75 flask. The viability (as assessed by trypan blue
exclusion is at least 95%).
4. For parent PC12 cell models, it is critical that the cells be
trypsinized and triturated three times through a 20-G needle
prior to plating to avoid excessive cell clumping.
 22 Neurite Outgrowth Assessment Using High Content Analysis Methodology 259

5. Statistically significant decreases in total neurite length will be

evident when final DMSO solvent concentrations ³1.0% (v/v)
are used.
6. Regardless of manual or automatic-mediated additions, post-
fixative wash steps are potentially significant causes of cell loss
and neurite integrity disruption. To minimize these effects,
ensure that all wash additions are employed at low velocity and
added indirectly to the sides of the well.
7. For detailed descriptions of terminology and assay parameters
associated with this method, the reader is referred to the
Thermo Scientific ArrayScan VTI HCS Reader User Guide
and the Neuronal Profiling V3.5 Bioapplication Guide.
8. Exposure times in each fluorescent channel are determined by
presampling of untreated control wells. Exposure times are cal-
culated in a manner that utilizes a predetermined percentage of
the dynamic range (0–4096) of the 12-bit CCD-camera
(Target parameter in AutoExposure options). Pixel satura-
tion should be avoided in order to avoid imposing an artificial
ceiling on the fluorescent intensity values recorded by the
instrument. For neurite outgrowth, it is recommended that
the dynamic range of the camera (Target parameter) be set to
between 55% and 65%.
9. The Autofocus interval determines the points within a well
where a focus adjustment is performed. Conservative sug-
gested autofocus intervals based upon the magnification of
image acquisition are 4 fields using ×5, 3 fields using ×10, 2
fields using ×20, and 1 field using ×40 magnification.
10. The Reject Multiply Traced Neurites parameter concerns what
happens in the event of neurites contacting several cell bod-
ies. If Rejection of Multiply-traced neurites is selected, then
multiply traced neurites will be rejected from analysis. If this
feature is not selected, then one of the multiple neurite copies
will be kept and the rest will be rejected from analysis. The
preserved neurite is therefore assigned to a single cell body.

Acknowledgments

The author wishes to thank Theresa Freudenrich, Joshua

Harrill, and William Mundy for their advice and technical
assistance, as well as Neil Durso and Theresa Freudenrich for
their comments and suggestions on an earlier version of this
book chapter.
260 N.M. Radio

References
1. Radio N, Breier J, Shafer T, Mundy W (2008) inhibition the site of action? Toxicol Appl
Assessment of chemical effects on neurite out- Pharmacol 160, 217–230
growth in PC12 cells using high content 9. Parran D, Mundy W, Barone S Jr (2001)
screening. Tox Sci 105, 106–118 Effects of methylmercury and mercuric chlo-
2. Craig A, Banker G (1994) Neuronal polarity. ride on differentiation and cell viability in PC12
Annu Rev Neurosci 17, 267–310 cells. Toxicol Sci 59, 278–290
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of postnatal neurobiological development: imaging: Data as far as the eye can see. Nat
Implication for human development. Dev Methods 2, 547–555
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4. Ramakers G (2002) Rho proteins, mental neuroscience. Nat Rev Neurosci 9, 779–788
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Trends Neurosci 25, 191–199 Johnston P (2010) Early safety assessment
5. Radio N, Mundy W (2008). Developmental using cellular systems biology yields insights
neurotoxicity testing in vitro: models for assess- into mechanisms of action. J Biomol Screen
ing neurite outgrowth. Neurotoxicology 29, 15, 783–797
361–376 13. Neely M, Nicholls J (1995) Electrical activity,
6. Vaundry D, Stork P, Lazarovici P, Eiden L growth cone motility and the cytoskeleton. J Exp
(2002) Signaling pathways for PC12 cell dif- Biol 198, 1433–1446
ferentiation: Making the right connections. 14. Harrill J, Robinette B, Mundy W (2011) Use
Science 296, 1648–1649 of high content image analysis to detect chemical-
7. Greene L (1977) A quantitative bioassay for induced changes in synaptogenesis in vitro.
nerve growth factor (NGF) activity employing Toxicol In Vitro 25, 368–387
a clonal pheochromocytoma cell line. Brain 15. Heumann R, Valet G, Maison D, Kemper J,
Res 133, 350–353 Reiser G, Hamprecht B (1977) Influence of
8. Das K, Barone S (1999) Neuronal differentia- the time in culture on cellular and neuronal
tion in PC12 cells is inhibited by chlorpyrifos properties of neuroblastoma x glioma hydrid
and its metabolites: Is acetylcholinesterase cells. J Cell Sci 27, 141–155
 Chapter 23

Dissociated Cell Culture for Testing Effects of Carbon

Nanotubes on Neuronal Growth
William Lee and Vladimir Parpura

Abstract
Cell culture has emerged as an important research method for studying the effects of carbon nanotubes
(CNTs) on primary neurons. We describe the procedure for preparation of dissociated mixed cell culture
from postnatal rat hippocampi. Based on morphological criteria and specific neuronal cell markers, neurons
can be selected within this mixed cell culture and studied. We present the procedure for the assessment
of neuronal cell morphology based on intracellular fluorescence of the vital dye calcein that accumulates
in live neurons. This procedure encompasses fluorescence imaging and measurement of the following
parameters: neurite number, total neurite length, mean neurite length, number of growth cones, number
of branches, and number of branches per neurite. These combined cell culture and fluorescence microscopy
approaches can be successfully used for assessment of the effects that CNTs, as water-soluble agents, have
on neuronal cell growth and neurite outgrowth.

Key words: Carbon nanotubes, Cell culture, Imaging, Morphology, Neurons, Growth, Neurite
outgrowth

1. Introduction

One of the challenges in the field of biomedical engineering and

regenerative medicine is to employ materials that could help restore
neural function at the site of injury, which may stem from traumatic
insults or neurodegenerative diseases in the central nervous system
(CNS) (1). Many experimental approaches aim to explore the
effects of biocompatible materials in the form of injectable com-
pounds and/or scaffolds to spur neuronal cell growth and neurite
outgrowth (2). Carbon nanotubes (CNTs), as water-soluble agents
or as cell-permissive scaffolds/substrates, are emerging as a mate-
rial that holds great promise in such applications [for a review see
ref. (3)].

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_23, © Springer Science+Business Media, LLC 2012

261
262 W. Lee and V. Parpura

A prerequisite for any medical use of CNTs in vivo is to

understand their effects and interactions with the cell type(s) of
interest. Here, we describe the procedure in which we use a
dissociated cell culture system to characterize the effect of CNTs,
as water-soluble agents, on modulation of neuronal cell growth
and neurite outgrowth (4–7). The advantage of using a dissociated
cell culture model over slice culture or in vivo models is the ability
to control for experimental conditions in a well-defined manner.
We describe the procedure for preparing rat hippocampal
mixed dissociated cell cultures, which is followed by the descrip-
tion of a procedure using the fluorescent vital dye, calcein, to reveal
neuronal cell morphology. Lastly, we describe the procedure for
imaging and analysis of neuronal cell morphology to assess the
effects that CNTs exert on these neural cells.

2. Materials

2.1. Coverslip 1. Polyethyleneimine (PEI) (500 mg/mL; Sigma-Aldrich): Dilute

Preparation the commercially available 50% w/v PEI solution 1:10 in sterile
water to yield a 50 mg/mL solution. Sterile filter the solution
through a 0.2-μm nylon filter (see Notes 1 and 2).
2. Round glass coverslips (see Note 3), plastic Petri dishes
(35 × 10 mm and 100 × 15 mm), circular sterile filter paper
(90 mm in diameter, qualitative; Whatman), and UV steriliza-
tion lamp (see Note 4).

2.2. Carbon Nanotubes 1. Single-walled carbon nanotubes (SWNTs), water-soluble. We

have been using custom-made (provided by Robert C. Haddon,
University of California, Riverside, CA) chemically functional-
ized SWNTs with polyethylene glycol (PEG) or poly-m-
aminobenzene sulfonic acid (PABS) (see Fig. 1) as previously
described (7, 8). These water-soluble materials are commer-
cially available (Carbon Solutions, Inc., Riverside, CA; cat. no.
P7-SWNT and P8-SWNT for PEG and PABS functionalized
SWNTs, respectively).

2.3. Cell Culture 1. 0- to 2-day-old Sprague-Dawley rat pups (see Note 5).
2. Dissection tools (from Fine Science Tools): 10-mm angled
spring scissors, 2.5-mm angled spring scissors, Graefe curved
forceps (serrated), fine Dumont forceps (#55), and a blunt
curved glass probe (see Note 6). Dissection tools encased
within a Petri glass dish (Pyrex brand, 150 × 15 mm) (see Note 7)
need to be sterilized prior to use (autoclave; 121°C; 20 psi;
45 min).
23 Dissociated Cell Culture for Testing Effects of Carbon Nanotubes… 263

Fig. 1. Water-soluble CNTs used to investigate their effects on neuronal growth. Image of
two vials containing functionalized water-soluble SWNTs, SWNT-PEG (left ) and SWNT-
PABS (right ) at a stock concentration of 2 mg/mL, which appears as black liquid.

3. Hanks’ balanced salt solution (HBSS) (500 mL; Invitrogen).

Store at room temperature (20–24°C).
4. Minimal essential medium (MEM) (without phenol red; cat.
no. 51200-038, Invitrogen). Store in the refrigerator (4°C).
5. D-glucose stock solution (2 M): Dissolve 18.02 g of D-glucose
in MEM and adjust to a final volume of 50 mL. Make 1 mL
aliquots and store at −20°C until use.
6. 10 mM HEPES/HBSS: Dissolve 1.1915 g of HEPES in
500 mL of HBSS and adjust the pH to 7.35 with 5 M NaOH.
Filter-sterilize through a Corning 0.22-μm cellulose acetate
(CA) membrane filter (Mfg. no. 4306) and store at room
temperature.
7. Papain solution: 20 IU/mL papain, 0.2 mg/mL L-cysteine,
100 IU/mL penicillin, 100 μg/mL streptomycin, 20 mM
D-glucose, and 10 mM HEPES in HBSS. Calculate the volume
needed to dissolve 100 mg of papain (Sigma-Aldrich) to yield
20 IU/mL (see Note 8). Knowing the final volume needed,
calculate the appropriate amount of D-glucose (1:100 dilution
from a 2 M D-glucose stock solution), penicillin/streptomycin
(1:100 dilution penicillin-streptomycin stock solution,
10,000 IU/mL to 10,000 μg/mL, respectively; Invitrogen),
and L-cysteine. Add these ingredients into an appropriate
amount of 10 mM HEPES/HBSS to make a calculated volume
of solution. Adjust the pH to 7.35 with NaOH and sterile filter
264 W. Lee and V. Parpura

through a 0.22-μm CA membrane filter. Add 100 mg of papain

to the solution. Put the mixture on a stirring plate until the
papain dissolves. Make 1 mL aliquots and store at −20°C
until use.
8. Trypsin inhibitor (100 mg/mL) stock solution: Make this
solution in a laminar flow hood with aseptic techniques by
adding 10 mL of 10 mM HEPES/HBSS to a vial containing
1 g of Type II-O trypsin inhibitor (Sigma-Aldrich). Let the
bottle sit for approximately 2 min to fully dissolve all of the
contents of the bottle. Then, make 500 μL aliquots in 1.5-mL
sterile microcentrifuge tubes and store at −20°C until use.
9. Mito-MEM containing MEM supplemented with 2 mM
L-glutamine, 20 mM D-glucose, 1 mM sodium pyruvate,
100 IU/mL penicillin, 100 μg/mL streptomycin, 14 mM
sodium bicarbonate, 0.1% (v/v) Mito + serum extender, and
5% (v/v) fetal bovine serum. Make this medium solution in a
laminar flow hood. To make 200 mL of this cell culturing
medium, mix 181.8 mL of MEM with 2 mL each of the fol-
lowing stock solutions: penicillin/streptomycin, D-glucose
stock, sodium pyruvate (100 mM; Invitrogen), and L-glutamine
(200 mM; Invitrogen). Add 200 μL of Mito + serum extender
(Collaborative Biomedical Products, cat no. 50006) and
235.2 mg of sodium bicarbonate. After all the above supple-
ments have been mixed and thoroughly dissolved (see Note 9),
sterile filter the solution through a 0.22-μm pore CA mem-
brane filter and add 10 mL of sterile fetal bovine serum (see
Note 10) to make a total volume of 200 mL. Store at 4°C.

2.4. Cell Imaging 1. An imaging chamber with a circular recess at its bottom to
accommodate for mounting of a coverslip (see Note 11). Seal
the coverslip to the bottom of the chamber using Dow
Corning® High Vacuum Grease (see Note 12).
2. External solution: Prepare a saline solution containing 140 mM
NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, and 10 mM
HEPES in water (pH = 7.4). Filter through a 0.2-μm filter and
store at 4°C. Immediately prior to use, warm it to room
temperature and add 5 mM D-Glucose (22.5 mg of glucose
per 25 mL of solution) to complete the external solution.
3. Calcein-AM stock solution: To make stock solution of calcein
acetoxymethyl (AM) ester (Invitrogen), add 50 μL of dry
dimethyl sulfoxide (DMSO) (see Note 13) to an individual vial
containing 50 μg of calcein-AM to yield a 1 μg/μL stock
solution. Store aliquots (1–10 μL) at −20°C.
4. Pluronic acid stock solution (25% w/v): Warm dry DMSO to
37°C. Weigh out 50 mg of pluronic acid F-127 (Invitrogen)
and add 200 μL of dry DMSO to it in a microcentrifuge tube
 23 Dissociated Cell Culture for Testing Effects of Carbon Nanotubes… 265

to make a ~25% w/v solution. Vortex, then incubate the mixture

at 37°C until it completely dissolves, which may take 30–60 min.
Store at room temperature wrapped in aluminum foil. At lower
temperatures, pluronic acid may precipitate out of solution and
should be heated in a water bath again to redissolve it into
solution. Prepare this stock solution fresh once a month.
5. An inverted microscope equipped with differential interference
contrast and epifluorescence illumination, 60× oil immersion
objective, and a standard fluorescein isothiocyanate (FITC)
filter set for imaging calcein.
6. A camera for image acquisition and a shutter inserted in the
fluorescence light path; both devices are computer/software
interfaced.
7. Image analysis software.

3. Methods

3.1. Coverslip 1. Sterile technique needs to be used for coverslip preparation,

Preparation which should be done in a laminar flow hood. One hippocampus
can provide dissociated cells for about 20 coverslips. Place ten
coverslips onto a circular sterile filter paper (90 mm in diameter)
inlayed into an inverted lid of a sterile 10-cm Petri dish with
maximal spacing between them. Prepare multiple sets (four
sets of ten coverslips for one rat pup dissection) pending on the
number of rat pups used. Sterilize the coverslips and filter paper
using UV lights (see Note 3).
2. Mix 100 μL of 50 mg/mL PEI stock solution with 4.9 mL of
sterile water in a 15 mL plastic tube to obtain a 1 mg/mL PEI
solution.
3. Apply 100 μL of 1 mg/mL PEI solution to each coverslip with
care to avoid spilling the liquid off of the top of the coverslip
and onto the filter paper. Cover inverted lids with their
bottoms facing up. After the PEI solution has been on the
coverslip for 3 h, aspirate the solution and wash the top of the
coverslip two times with autoclaved/sterile water and allow
the water to rest on top of the coverslip for 3 more hours.
Change the water and incubate it on the coverslip for 3 h; repeat
this step. After completion of two incubation periods, aspirate
the water and air-dry them inside the laminar flow hood.
4. Using a stainless steel tool such as a 5-mm flat-head screw
driver, with the tip heated to glow by a Bunsen burner, emboss
crossed grooves at the bottom of 35 × 10-mm Petri dishes to
generate four segments. Sterilize dishes with UV light. Using
tweezers, place one coverslip with the PEI-coated surface up
into each segment of the dish (see Notes 14 and 15).
266 W. Lee and V. Parpura

3.2. Cell Culture The described procedures below, performed on rats, must be
approved by the Institutional Animal Care and Use Committee
prior to performing them. Hippocampal dissociated cell cultures
are prepared from 0- to 2-day-old Sprague-Dawley rats using pre-
viously described procedures; this cell culture approach yields a
mixed cellular population of neurons, astrocytes, and microglia,
and it has been used to study neuronal cell morphology (4–7, 9).
Since excellent photographs and video clips of rodent hippocampal
dissections are described elsewhere (10, 11), we only briefly and
textually address the isolation of hippocampi.

3.2.1. Preparatory Tasks 1. Prior to beginning a primary cell culture, place 50 μL of mito-MEM
onto each coverslip inlayed within 35-mm Petri dishes using
sterile technique in a laminar flow hood. Incubate dishes with
their lids on overnight inside the 95% air/5% CO2 atmosphere
incubator at 37°C. This step represents a convenient way to
allow the coverslip surface to wet properly and consequently
allow good cellular adhesion. If shorter lead time is desired, we
also have good results with cultures when medium was applied
onto PEI-coated coverslips ~4 h prior to application of dissoci-
ated cells; this is roughly ~2.5 h before the initiation of a the
dissection and culturing procedure (see Subheading 3.2.2).
2. Prepare the dissection area. Wear surgical gloves which should
be disinfected by spraying with rubbing alcohol (70% isopro-
panol) as needed. Take great care to clean the area (including
the dissecting scope, light source, and table top) thoroughly
with rubbing alcohol, which reduces the likelihood of contam-
ination. Place a terry cloth towel on the surface of the dissecting
area. This will protect the surface of the dissecting table from
debris; it will also prevent accidental sliding of dishes and
tools/beakers. Place two Kimwipe™ sheets at the bottom of
each of the two 200-mL beakers. This will protect tips of
dissections tools. Fill beakers with 70% isopropanol up to the
75 mL mark. In one beaker, place your gross dissecting tools
(10-mm angled spring scissors, Graefe curved serrated forceps,
and blunt curved glass probe), while in the other, place your
fine (Dumont forceps #55 and 2.5-mm angled spring scissors)
dissecting tools, with their tips (covered with pipette tips)
immersed in alcohol. Remove protecting pipette tips and shake
alcohol off any tool prior to its use on animal tissue.
3. Attend the media, solutions, and dishes. (a) Place a bottle of
fresh mito-MEM medium in an incubator to warm; this will
take ~3 h. Open the lid (unscrew ~½ of turn), but do not
remove it. This will allow pH equilibration. Medium can be
used for up to 2 weeks; however, the fresher the better. (b)
Also, remove a tube of papain solution (previously prepared in
1 mL aliquots) from a freezer and place in a hood to thaw.
 23 Dissociated Cell Culture for Testing Effects of Carbon Nanotubes… 267

Once thawed, place it in an incubator. (c) Fill a 50-mL

centrifuge tube with fresh HBSS, which will be the source of
HBSS for the following steps. Some of this HBSS may end up
being unused and, if so, it will be subsequently discarded. Be
sure to flame both the tube and the bottle before and after
removing the HBSS, a practice that needs to be used will all
culturing media/solutions. Place 5 mL of HBSS (from a
50-mL centrifuge tube) into a 15-mL centrifuge tube, which
will be used to collect hippocampi to be cultured. Also, add
1 mL of HBSS into 35-mm culture dishes (2 per brain). Place
the 15-mL centrifuge tube and the dishes on ice for later use.
In addition, fill a 10-mL syringe (no needle) with HBSS.
4. Take a small transfer box with the lid (approximate box dimen-
sions are 15 × 9 × 4 cm; with the bottom of the box covered
with 3–4 layers of Kimwipe sheets) to the animal room and
retrieve 1–2 pups to be dissected (see Note 16).

3.2.2. Dissection 1. Begin at a dissecting bench. Use sterile technique. Remove the
and Culturing pup from the transfer box, and spray its skin with alcohol.
Holding the pup’s front legs with your index finger and thumb,
position the pup with its back turned toward you. Using 10-mm
curved dissecting scissors, sever the spine/spinal cord at the
base of the skull to euthanize the pup. Then, make an incision
just under the skin and almost completely around the circum-
ference of the head. Peel the skin back to expose the skull.
Inserting the scissors through the hole created during the
euthanasia, cut the skull around the entire circumference of the
head. Be careful to keep the tips of the scissors pointed out so
as not to damage the brain beneath.
2. Lift the skull “cap” from the head using the Graefe forceps.
This should expose the brain. There may be a substantial amount
of blood bathing the brain at this point. Wash this blood
away using a few milliliters of HBSS from the 10-mL syringe.
While continuing to hold the pup, use the blunt probe to
dislodge the olfactory bulbs. This can be accomplished by
gently scooping (using the head of the hockey stick like glass
probe) the anterior end of the brain. Again using the blunt probe,
gently scoop the posterior end of the brain. This will separate
the brain from the spinal cord and allow it to be removed to a
culture dish containing 1 mL of cold HBSS.
3. Place the entire brain into a culture dish containing HBSS.
Separate the brain into two hemispheres by using the heel of a
fine dissecting scissors to “glide” from the midpoint of the cere-
bral cortex anteriorly through the front of the cortex between
the olfactory bulbs. With fine forceps, gently pry the hemispheres
apart to reveal the thalamus below. Again using the forceps,
pinch the remaining tissue between each hemisphere and the
thalamus to free them.
268 W. Lee and V. Parpura

4. Remove each hemisphere to another culture dish, medial side

up, containing fresh HBSS. Use forceps to grip the meninges
and peel them away from the cortex. It is not necessary to
remove all the meninges, just those over the tissue of interest.
In addition, the choroid plexus may cover a portion of the
hippocampus and may need to be removed.
5. The hippocampus will appear in the shape of the letter “C” at
the posterior end of each hemisphere. The “C” begins near the
dorsal surface rostrally and curves ventrally and caudally. The
outer edge of the hippocampus is continuous with the rest of
the cortex. The inner edge remains free with the lateral ventricle
lying beneath it. Make an incision along the outer edge of the
hippocampus. Incisions must also be made at the anterior and
posterior ends of the hippocampus. This should completely
free the tissue from the cortex. Using forceps, transfer the hippo-
campi to a 15-mL tube containing cold HBSS. Repeat steps
1–5 for the second pup (if dissecting 2 pups at the sitting) (see
Note 16).
6. Wash the tissue by decanting the HBSS from the centrifuge
tube and adding 5 mL of fresh HBSS.
7. Replace the HBSS with papain solution and leave for 1 h at
37°C in a 95% air/5% CO2 incubator to enzymatically treat the
tissue.
8. Decant the papain solution from the tube and again wash the
tissue with HBSS.
9. Acutely prepare trypsin inhibitor solution (10 mg/mL) by
adding 0.5 mL of 100 mg/mL trypsin inhibitor stock to
4.5 mL of 10 mM HEPES/HBSS. To stop the enzymatic
action of papain, apply a trypsin inhibitor solution to the tissue
for 5 min at room temperature.
10. Remove the trypsin inhibitor and wash the tissue with 5 mL of
HBSS.
11. Decant HBSS and add 1.0 mL of warm culture mito-MEM
medium per hippocampus.
12. Gently triturate (~40–60 strokes) the tissue through a 5-mL
serological glass pipette until no visible clumps remain. Add
warm culture medium so that the final dilution is 1.0 mL per
hippocampus.
13. Plate the dissociated cells onto glass coverslips inlayed in
35-mm dishes at 50 μL per each coverslip, precoated with PEI
and primed with 50 μL of medium.
14. Place the dishes with their lids on into an incubator for 3 h to
allow cells to settle and attach to the substrate.
 23 Dissociated Cell Culture for Testing Effects of Carbon Nanotubes… 269

15. Wash debris from each dish after the 3 h. Fill each dish with
1 mL of mito-MEM. Remove this mito-MEM with suction
and replace it with 1.0 mL of mito-MEM. For cultures that
examine the effect of water-soluble SWNTs, replace the dish
with 1.0 mL of mito-MEM with appropriate concentration of
SWNTs dissolved in the medium.
16. Maintain the cultures in an incubator (37°C; 95% air/5% CO2
atmosphere) for 3 days to allow for adequate growth, at which
point neuronal cell morphology can be assessed.

3.3. Cell Imaging 1. Inside a laminar flow biosafety hood, place a dish taken from
the 37°C incubator. Transfer one coverslip from a culture dish
3.3.1. Calcein Loading
into a 35-mm Petri dish containing loading solution, com-
posed of 1 μg/mL of calcein-AM and 0.025% of pluronic F-127
in external solution. Incubate the coverslip for 15 min at room
temperature (see Note 17). To transfer a coverslip, use a sterile
fine forceps with the tip flamed using an alcohol burner. Return
the Petri dish containing unused coverslips in culture media
back to the 37°C incubator immediately after transfer of an
experimental coverslip (see Note 18).
2. After the end of the 15-min incubation period in loading
solution, transfer the coverslip into another 35-mm Petri dish
containing 1 mL of external solution to allow for the dye to
de-esterify for 15 min.

3.3.2. Imaging 1. Prepare a clean imaging chamber for attachment of the cover-
and Analysis slip. After placing the chamber up-side down, apply a streak of
sealing grease at the recess of the chamber. Using sterile forceps,
take the coverslip containing cells out of external solution and
place it centered onto the chamber recess with the cell-side facing
toward the recess and lightly press it with forceps to loosely
adhere the coverslip to the grease. Flip the chamber so that the
open bath faces up, while the coverslip is at the bottom. Press
the chamber down against a Kimwipe™ to seal the coverslip
(see Note 19). Add ~400 μL of external solution (room tem-
perature) into the chamber. Check for leaks (see Notes 20 and
21). Aspirate the external solution and replace it with 400 μL
of external solution.
2. We use an inverted microscope (Nikon TE300) equipped with
differential interference contrast and epifluorescence illumina-
tion (xenon arc lamp, 100 W) to image the cells (see Note 22).
To visualize the calcein-loaded cells, we use a 60× plan apochro-
matic oil immersion objective (numerical aperture, 1.4; Nikon)
and a standard FITC filter set. We use a cooled charge-coupled
device camera (CoolSNAP HQ; Photometrics, Tucson, AZ)
270 W. Lee and V. Parpura

Fig. 2. Calcein-loaded neurons grown on PEI-coated glass coverslips and treated with chemically functionalized water-
soluble SWNTs added to the culture medium. Fluorescence images of live neurons, accumulating the vital stain, calcein.
Neurons grown on PEI-coated glass coverslips (control, sham treated) can be treated with CNTs (each at 1 μg/mL), either
SWNT-PABS (middle) or SWNT-PEG (right ) to affect their growth characteristics [consult the original work (7) for details].
Arrows indicate growth cones. Scale bar, 20 μm. Modified from ref. (7).

driven by V++ imaging software (Digital Optics Ltd., Auckland,

New Zealand) or MetaMorph™ software (Molecular Devices,
Chicago, IL) to acquire images and control an electronic shutter
(Vincent Associates, Rochester, NY) in the excitation pathway.
Neurons are identified based on their morphological features
using differential interference contrast and fluorescence micros-
copy (see Notes 23 and 24). For neurons that extend processes
beyond the field of view, acquire multiple images and merge
together using Adobe Photoshop CS2 (Adobe Systems Inc.,
San Jose, CA) (see Note 25). The images of cultured hip-
pocampal neurons grown on PEI substrates and exposed to
water-soluble CNTs (SWNT-PEG and SWNT-PABS) are
shown in Fig. 2.
3. We analyze cell morphology using the OLYMPUS MicroSuite
™ Basic software to quantify the neuronal morphological char-
acteristics. We examine six parameters: neurite number, total
neurite length, mean neurite length, number of growth cones,
number of branches, and number of branches per neurite (see
Fig. 3). For neurite length measurements, manually trace
neurites on a computer monitor using a computer mouse.
After selecting the “polygon length” mode, manually trace the
neurite by clicking the mouse at various loci of the neurite to
assign its segments, as illustrated in Fig. 4. Sum the lengths of
all individual segments for a particular neurite to calculate the
full length of that neurite (see Note 26).
 23 Dissociated Cell Culture for Testing Effects of Carbon Nanotubes… 271

Neurite length (µm) Number of growth cones

6 3
Number of neurites
** **
per neuron

per neuron
4 2

* **
2 1

0 0
400 100
Total neurite length

** **
per neuron (µm)

300 75

200 50

100 25

0 0
60 15
Number of branches

Number of branches
*
per neuron

per neurite
40 10

20 5

0 0
control SWNT-PABS SWNT-PEG SWNT-PABS SWNT-PEG
0.1µg/mL 0.1µg/mL 1µg/mL 1µg/mL
(76) (60) (56) (44) (43)

Fig. 3. Parameters of neuronal growth and morphology. Neurons grown on PEI-coated glass coverslips (control) were
treated with SWNT-PABS or SWNT-PEG each at two different concentrations (0.1 and 1 μg/mL). Bars represent
means ± standard errors of means. Numbers in parentheses indicate the number of neurons studied in each condition.
Asterisks indicate a significant difference in measurements (*p < 0.05, **p < 0.01). For a description of the effects that
water-soluble SWNTs exert of neuronal morphology, consult the original work (7). Reproduced from ref. (7).

4. Notes

1. Unless stated otherwise, the water used in all of our proce-

dures is purified by the Milli-Q® Synthesis system (Millipore
Corp.; http://www.millipore.com/pressroom/cp3/5khpn7).
This ultra-pure water has 18.2 MΩ cm resistivity, less than 5
parts per billion of organics content, and pyrogen content less
than 0.001 EU/mL. It can be sterilized by autoclaving (121°C,
20 psi, 45 min).
2. This PEI stock solution can be stored for 4–6 weeks at 4°C.
Alternatively, aliquot in appropriate amounts (100 μL) and
store at −20°C for up to 1 year.
272 W. Lee and V. Parpura

Fig. 4. Neurite tracing approach. Shown here are examples of neurite length measurements, where neurites were manually
traced by an investigator marking various loci of an individual neurite (ticks). Lengths of individual segments (between tick
marks) were summed to calculate the full length of a neurite (segmental lines).

3. The borosilicate glass coverslips used have been pretested for

mammalian neural cell culture. These glass coverslips (12 mm
in diameter, thickness #1, 0.13–0.16 mm; D-263 glass, Erie
Scientific Company) can be purchased via Fisher Scientific (cat.
no. 12-545-82-12CIR-1D). Prior to subsequent coverslip
processing procedures, clean the glass coverslips (one ounce
packet contains ~500 coverslips) by placing them in 2% v/v
RBS 35 (Pierce) detergent dispersion in Milli-Q water and
boiling for 15 min. Next, rinse the coverslips by running distilled
water for 30 min and soak inside a beaker containing Milli-Q
water overnight. To remove possible residual detergent film
retained on coverslips, wash each individual coverslip by using
a clean metal forceps to hold the coverslip and dipping it three
times in Milli-Q water in a beaker. Repeat this step for two
additional times in two separate beakers. Air-dry the washed
coverslips in a laminar flow hood by placing them at a slanted
angle onto a drying scaffold, which ensures that both sides of
the coverslips are air-dried properly. The drying scaffold can be
made by making accordion folds (~1 cm) of a 90-mm diameter
filter paper. Store the dried washed coverslips in Petri dishes with
sheets of filter paper between them to prevent scratching.
Sterilize the washed coverslips under UV light prior to PEI coating.
The time for washing one packet of coverslips takes ~3 h.
23 Dissociated Cell Culture for Testing Effects of Carbon Nanotubes… 273

4. UV sterilization is done using the GS Gene Linker™ UV

Chamber (Bio-Rad; Power set at Str, 2 × 90 s). Alternatively,
one can use UV lamps in the laminar flow hood, but the dura-
tion of exposure needs to be adjusted according to the manu-
facturer’s recommendation.
5. Rat pups can be obtained from a breeding colony or from
timed-pregnant female Sprague-Dawley rats that are commer-
cially available (e.g., Charles River Laboratories).
6. A blunt glass probe can be made using a Pasteur pipette with
the tip sealed and curved (like a hockey stick) by heating it on
a Bunsen burner.
7. We use appropriate plastic pipette tips to cover the tips of all
tools in order to protect them from mechanical damage before
placing tools into the glass dish.
8. This calculation is needed because various batches of papain
may have different enzyme activity leading to variable total
content of international units (IU) in 100 mg of papain. It
takes about ~1 h at room temperature to dissolve papain, a
process which can be assessed visually as the turbid mixture
becomes a clear solution.
9. The amount of bicarbonate added to the medium is adjusted
so that medium reaches the appropriate pH (7.35) when main-
tained in a 95% air/5% CO2 atmosphere incubator. The pH of
medium needs to be checked (measured) and the level of bicar-
bonate readjusted if needed. The addition of glutamine to the
mito-MEM is recommended even though MEM already con-
tains glutamine. However, glutamine has a limited storage life
in the refrigerator and it breaks down more quickly at higher
temperatures.
10. We use Thermo Scientific* HyClone USDA Tested Fetal
Bovine Serum (cat. no. SV30014) sterile filtered by the
manufacturer. We test each serum batch for various functional
properties of neurons (including neurite outgrowth) and astro-
cytes. Based on our previously used batches, the company
selects new serum with matching properties (various parame-
ters used) and puts a desired amount of serum on reserve for
4–6 weeks until we thoroughly test it in our experimental para-
digms. Presently, we use the batch FTM33793, while in the
past 5 years, we used three other batches (in reverse chrono-
logical order): FRE26364, FPC20883, and FNS18021. We
provide these batch numbers for guidance only.
11. We use a custom-made open diamond bath imaging chamber
with a circular recess at its bottom to accommodate mounting
of a coverslip (12 mm in diameter, thickness #1). Similar imag-
ing chambers are commercially available (e.g., cat. no. RC-25
or RC-25, Warner Instruments).
274 W. Lee and V. Parpura

12. We repackage the vacuum grease into a 3-mL syringe to which

we attach a 1-cm-long 18-gauge blunt needle. This handheld
syringe/needle is used to apply the vacuum grease at the circu-
lar recess to the bottom of the chamber in order to attach the
coverslip. Note that the grease displays fluorescent properties
and should be used parsimoniously without smudging the bot-
tom of the coverslip with it, which would occur if too much
grease is applied.
13. To prepare dry DMSO, open an ampoule containing 5 mL of
sterile filtered DMSO (Sigma-Aldrich) and pour it into a
15-mL conical centrifuge tube filled to the 2 mL level with
molecular sieve beads (sodium aluminosilicate molecular
sieves, 8–12 mesh beads, cat. no. M-2635, Sigma-Aldrich) to
absorb water. Keep the tube tightly capped and wrapped in
aluminum foil, as DMSO is hygroscopic and light sensitive.
Store at room temperature. The beads are reusable. However,
note that beads contain indicator beads that change color
from blue to pink when saturated with water. At that juncture,
they need to be replaced.
14. If dishes containing PEI-coated coverslips are not used imme-
diately, their lids can be sealed with Parafilm© and the dishes
stored in a sterile container at room temperature for up to 1
week of PEI coating application. Do not expose these PEI-
coated coverslips to UV light as this polymer is sensitive to
UV light.
15. It is cost-effective to have all segments within a Petri dish
populated with PEI-coated coverslips as this allows reduced
usage of culture medium. However, the cell culture is also suc-
cessful when number of coverslips is reduced from a maximal
of 4 to 2–3 coverslips per dish.
16. For best results, it is advisable to dissect no more than one pup
while learning the procedure. However, even for a proficient
user, we do not recommend dissecting more than two pups at
any one sitting. As the number of rats increases, the time
needed for dissection increases. During the preparation of neu-
ronal cell cultures, speed is of the essence. Once the rat pup
dies, the brain tissue will begin to deteriorate. We have observed
a decrease in cell viability which coincides with increases in the
number of pups dissected.
17. To prepare calcein-AM loading solution, add 1 μL of pluronic
acid stock solution to 998 μL of external solution. Vortex and
then add 1 μL of calcein-AM stock solution. Vortex and transfer
to a 35-mm Petri dish. This solution can be used for loading
cells attached to multiple coverslips, up to several hours. We
normally prepare this solution twice a day.
18. Minimize the transfer time to avoid fluctuations of the medium
pH, which can affect the health of the cell culture.
23 Dissociated Cell Culture for Testing Effects of Carbon Nanotubes… 275

19. When pressing the coverslip attached to the imaging chamber

against the Kimwipe™, make sure that you place the wipe on a
clean flat surface and that you do not apply too much pressure,
otherwise the coverslip can crack. If you crack the coverslip,
replace it after cleaning the chamber and reapplying the grease.
20. By initially placing the chamber-coverslip assembly on the
Kimwipe™, you will notice some wetting of the wipe because
some of the culturing medium will be retained at the bottom
surface of the coverslip. Subsequently, lift up and place the whole
assembly onto a different spot of the wipe. Leaks are easily rec-
ognized, as the chamber will quickly empty to wet the wipe.
21. Regardless of whether the chamber leaks or not, after pressing
the assembly against the wipe, you may also notice a circular
grease imprint on the wipe. If any, this should be minimal. If in
excess, next time, mount the coverslip with somewhat less
grease, but not an insufficient amount necessary for sealing.
Do not slide the assembly across the wipe at any time as you
will cause smudging of the grease onto the coverslip. This will
affect the quality of images since (1) the grease does not mix
with the oil used on the objective and hence it would generate
the distortion of images, and (2) the grease fluoresces and thus
increases the background.
22. We place our microscopes on antivibration isolation tables.
We find this approach necessary in order to prevent possible
movement artifacts.
23. We confirm morphological identification of neurons by labeling
with the neuron-specific markers: anti-tubulin III (also referred
to as Tuj1), anti-neuron-specific enolase, and the FITC-
conjugated C-fragment of tetanus toxin (6, 7).
24. When acquiring images of calcein-loaded cells, one should be
aware of possible photobleaching of the fluorescent dye which
can be minimized by the use of neutral density filters inserted
in the excitation pathway and by adjusting the camera integra-
tion time. Obtained images must have fluorescence intensity
values within the dynamic range of the camera (0-4095 for a
12-bit camera) and prominently display the full extent of neu-
rites and their branches.
25. It is sometimes necessary to take several images to capture the
entire morphology of the cell when the cell’s processes extend
beyond the field of view. If multiple image acquisition is
required, make sure that the focus of the cell remains unchanged
during the entire image acquisition process as even a minute
change in focus might affect the proper merging of images,
especially when smaller cellular structures such as fine cell pro-
cesses are in question. Merging images can be done by using
the automated photomerge function in the File tab of
Photoshop CS2. If Photoshop CS2 software is not available,
276 W. Lee and V. Parpura

an alternative free image merging software that can be used is

ImageJ (NIH, USA) with the stitching plug-in installed.
26. Alternatively, analysis of cell morphology can be automated by
using the Neurite application module of MetaMorph™ soft-
ware ver. 6.1 (Molecular Devices) to quantify the neuronal cell
morphological characteristics after neurons were grown on
CNT scaffolds/substrates (9). Using this approach, we examine
eight parameters: neurite number, mean neurite length, maxi-
mum neurite length, neurite straightness, total neurite out-
growth, number of growth cones, number of branches, and
cell body area (see Fig. 4 of ref (9)).

Acknowledgments

We would like to thank Yingchun Ni and Hui Hu for providing

Figure 4, and Randy F. Stout, Jr. for comments on a previous
version of the manuscript. This work was supported by the National
Science Foundation (CBET 0943343).

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 Chapter 24

High-Resolution Imaging and Evaluation of Spines

in Organotypic Hippocampal Slice Cultures
Frederik Sündermann, Nataliya Golovyashkina, Christian Tackenberg,
Roland Brandt, and Lidia Bakota

Abstract
Dendritic spines act as sites of excitatory neuronal input in many types of neurons. Spine shape correlates
with the strength and maturity of synaptic contacts. Thus, evaluation of spine morphology is relevant for
studies on neuronal development, for determination of morphological correlates of learning and memory,
and for analysis of mechanisms of neurodegeneration. Here, we describe a method to determine spine
morphology in an ex vivo model of organotypic hippocampal slice cultures prepared from transgenic or
non-transgenic mice. Spines are imaged using confocal high-resolution imaging and evaluated by algorithm-
based analysis. The approach permits semiautomated determination of spine density and classification of
different spine types in dendritic segments from hippocampal subregions to evaluate intrahippocampal
connectivity.

Key words: Dendritic spine, Hippocampus, Organotypic culture, Laser scanning microscopy,
Automated morphology analysis

1. Introduction

Dendritic spines are small protrusions from dendrites and consti-

tute the primary site of excitatory synaptic input in many principal
neurons of the brain including pyramidal cells in the neocortex and
hippocampus, and Purkinje neurons in the cerebellar cortex (1).
Spine volumes range between 0.01 and 0.8 μm3 and are thought
to function as microcompartments for segregating postsynaptic
responses. Evidence indicates that spine alterations are related to
learning, memory, and neurodegeneration and that spines show
high structural dynamics (2, 3). However, it is still unclear which
mechanisms are involved and what is the functional or pathological
role of spine changes during development and degeneration.

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_24, © Springer Science+Business Media, LLC 2012

277
278 F. Sündermann et al.

Organotypic hippocampal slice cultures combine the accessibility

and maintenance of in vitro culture systems with the preservation of
intact hippocampal synaptic circuitry and anatomy (4). The latter is
especially important for spine analysis, since spine changes can occur
in a subset of neurons in a region-specific manner. Preparation of
slice cultures from mice provides the further advantage that the
advances in transgenic mouse technology, which resulted in the gen-
eration of a variety of different mouse models, can be used to deter-
mine the contribution of single gene products on spine morphology.
An example presents the use of mouse models that are transgenic for
mutated amyloid precursor protein (APP), which is known to be
involved in the development of Alzheimer’s disease (AD). Using this
model, we have demonstrated that the density and shape of spines
are altered compared to cultures from non-transgenic animals (5).
Algorithm-based analysis of spine density and morphology
requires fluorescence labeling of individual neurons in a slice prep-
aration. One way to label neurons is the use of a Sindbis virus
expression system, which permits effective infection of neurons in
organotypic slices with this neurotropic alphavirus (6). By using
fusion constructs of green fluorescent protein (GFP) with coding
sequences of interest, the effect of the synthesized protein on spines
can be studied in infected neurons in an authentic environment,
which also can be exploited to perform live-cell imaging of degen-
erating neurons (7). This approach has, for example, been taken in
our lab with AD-like modified cytoskeletal proteins (5, 8). The
production of Sindbis virus vectors for gene transfer has been
described in many excellent papers and manuals and will not be
covered in this article (see e.g., (9)). As an alternative to virus infec-
tion, algorithm-based analysis is also possible with slices that have
been prepared from genetically modified mice that express fluores-
cent proteins in selected neurons (e.g., (10)). The following chap-
ter describes a method to determine spine morphology in an ex
vivo model of mouse organotypic hippocampal slice cultures using
confocal high-resolution imaging and algorithm-based analysis.

2. Materials

2.1. Components for 1. Animals: 6–8 days old mouse pups (e.g., mouse lines with
Slice Preparation, C57BL/6 background; see Note 1).
Culture, Infection, and 2. MEM: Minimum Essential Medium Eagle, HEPES
Fixation Modification without L-glutamine (Sigma-Adrich).
3. BME: Basal Medium Eagle with Earle’s, without L-glutamine
(Gibco BRL Life Technologies).
4. NB: Neurobasal Medium without L-glutamine (Gibco BRL
Life Technologies).
 24 High-Resolution Imaging and Evaluation of Spines… 279

5. Horse serum.
6. Glutamine, 200 mM.
7. Pen-Strep: Penicillin/Streptomycin, 100× (PAA Laboratories).
8. Fungizone Gibco Fungizone® Antimycotic, liqid (Invitrogen).
9. N1-supplement: N1 medium supplement, 100×, for neural
cell cultures (Sigma-Adrich).
10. Virus: Virus constructs encoding fluorescent protein (e.g.,
SinRep5-eGFP) that can be optionally tagged with other pro-
tein of interest.
11. Confocal matrix : Micro-Tech-Lab (Graz, Austria).

2.2. Media, Prepare all media under sterile conditions using sterile filter such as
Supplements, 50 mL Steriflip or 500 mL Steritop-GP 0.22 μm ExpressTM
and Other Solutions Membrane (Millipore), depending on the volume of medium
needed.
1. Heat-inactivate horse serum at 56°C for 45 min. The aliquots
can be stored at −20°C for at least 1 year. (see Note 2).
2. Dissolve glucose powder in double distilled H2O (dd H2O) to
20% w/v and sterile-filter using a 0.22 μm filter. Store at 4°C.
3. Dissection medium: 1% glutamine and 1% Pen-Strep in MEM.
To 98 mL MEM, add 1 mL glutamine and 1 mL Pen-Strep.
The medium can be stored at 4°C for 24 h. (see Note 3).
4. Culture medium: 25% horse serum, 25% BME, 3% glucose, 1%
glutamine, 0.5% Pen-Strep, and 0.5% fungizone in MEM. In a
50 mL sterile conical tube add 12.5 mL horse serum, 12.5 mL
BME, 1.5 mL glucose, 0.5 mL glutamine, 0.25 mL Pen-Strep,
and 0.25 mL fungizone; bring volume to 50 mL with MEM
and filter sterilize. Preheat only the amount of medium that is
needed for a medium change on the same day (count 2–4 mL
additionally for pH adjustment). Adjust the pH value to 7.2
with HCl/NaOH. If not preheated, culture medium can be
stored at 4°C for at least 1 month. (see Note 4).
5. NB-N1 medium: 3% glucose, 1% glutamine, 1% N1-supplement,
0.5% horse serum, 0.5% Pen-Strep, and 0.5% fungizone in MEM.
In 50 mL sterile conical tube, add 1.5 mL glucose, 0.5 mL glu-
tamine, 0.5 mL N1-supplement, 0.25 mL horse serum, 0.25 mL
Pen-Strep, and 0.25 mL fungizone; bring volume to 50 mL with
NB and filter sterilize. Preheat only the amount of medium that
is needed for a medium change on the same day (count 2–4 mL
additionally for pH adjustment). Adjust the pH value to 7.2 with
HCl/NaOH. If not preheated, NB-N1 medium can be stored at
4°C for at least 1 month. (see Note 4).
6. Fixation solution: preheat 25 mL of phosphate buffed saline
(PBS) to 70°C. Add 1 g paraformaldehyde, mix and let it cool
down. Add 1 g sucrose. Store at −20°C.
280 F. Sündermann et al.

2.3. Culture and 1. Cell culture inserts. Millicell cell culture inserts, 30 mm, orga-
Dissection Dishes notypic hydrophilic PTFE, 0.4 μm. #PICM0RG50, Millipore.
(see Note 5).
2. Multidish 6-well plates (Nunclon™). Alternatively, single 3.5-cm
culture dishes can be used.
3. Bacterial dish (10 cm) as a dissection dish.

2.4. Dissection Tools 1. Large surgical scissors. Type: standard. Alloy: stainless steel.
(see Note 6; Fig. 1a) Tip shape: sharp/blunt. Tip angle: straight. Length: 13 cm.
Effective cutting edge: 42 mm. (#14001-13).
2. Small scissors. Type: standard. Alloy: stainless steel. Feature:
toughcut (this feature is not crucial). Tip shape: sharp/sharp.
(#14058-11).
3. Graefe forceps. Length: 10 cm. Alloy: stainless steel. Tip shape:
curved. Tips: serrated. Tip dimensions: 0.8 mm × 0.7 mm.
(#11052-10).
4. Dumont #5 Forceps. Length: 11 cm. Alloy: titanium (or inox).
Tip shape: straight. Tip dimensions: 0.05 mm × 0.02 mm.
(#11252-40).
5. Hippocampus tool (spatula) (×2). Length: 16 cm. Alloy: stain-
less steel. Tip shape: straight. Tip diameter: 8.5 mm × 0.3 mm.
(see Note 7).
6. Forceps. Length: 10.5 cm. Alloy: remanit 4301. Tip shape:
angled. Tips: serrated. (#2854.1).
7. Spatula (×2). Length: 13 cm. Alloy: stainless steel. End shape:
rounded. (see Note 7).

2.5. Large Equipment 1. Laminar flow CL2 safety cabinet.

for Preparation and 2. Sterile dissection safety cabinet.
Culturing Organotypic
3. McIlwain tissue chopper. Standard razor blades can be used
Slices
with the tissue chopper.
4. Dissection microscope (e.g., Leica, but any 5–10× magnifying
dissection microscope is suitable).
5. Cell culture incubator at 37°C and 5% CO2.

2.6. Equipment for Confocal Laser Scanning Microscope (e.g., Nikon Eclipse TE2000-U
Image Acquisition or Zeiss LSM 510) equipped with 10× and 20× air, 40× and 60×/63×
oil immersion objectives (suitable for fluorescence imaging), lasers
and filter sets corresponding to the fluorescent proteins (e.g., 488 nm
argon laser for GFP), and image acquisition software (e.g., EZ-C1
software, Nikon or LSM 5 software, Carl Zeiss).

2.7. Software 1. Autodeblur 9.3 software.

and Hardware 2. 3DMA Version 0204 software.
Requirements
 24 High-Resolution Imaging and Evaluation of Spines… 281

Fig. 1. Preparation of organotypic hippocampal slices from mouse brain. (a) Preparation tools. (b) Dissection setup.
(c) Decapitation of a 1 week old mouse. (d) Incision of the skin along the midline of the head. (e) Removal of the skin to the
sides with the scissors. (f) Removal of the skull with the curved forceps. (g) Removal of the brain with the spatula.
(h) Cutting off the cerebellum. (i) Separation of two brain hemispheres with the spatula. (j) Dislodgement of the midbrain
with the spatula. (k) Hippocampus, prepared for cutting into slices. (l) Cutting of the hippocampus with the tissue chopper.
(m) Cut and separated slices. (n) Placing a slice onto a membrane insert within 6-well plate. (o) Three slices placed on the
membrane insert.

3. PC with “Ubuntu Linux 6.06 LTS” as operating system and

the following additional packages: Jgraph (http://www.cs.utk.
edu/~plank/plank/jgraph/jgraph.html), xv (http://www.trilon.
com/xv/).
4. PC with “Microsoft Windows XP” as operating system.
282 F. Sündermann et al.

3. Methods

3.1. Preparation, Organotypic slice cultures from hippocampus are cultivated according
Culturing, Infection, to the membrane interface technique (4) (see Note 8).
and Fixation of
Hippocampal Slices

3.1.1. Dissection The procedure takes place under the sterile culture bench.
Preparation
1. Add 1 mL of culture medium to each well of a 6-well plate.
2. Place a membrane insert into each well with sterile forceps (see
Note 9).
3. Place the plates and dissection medium on ice.
4. Prepare two 50-mL centrifuge tubes with sterile PBS or ddH2O
(see Note 10).
5. Prepare sterile eppendorf cups on ice if tissue collection for
genotyping is required.
6. Cut rounds from Whatman paper, place one into each of the
dissection dishes and sterilize under UV (see Note 11).

3.1.2. Dissection The procedure takes place under the sterile preparation bench. The
of the Hippocampi tools are sterilized with 70% ethanol or by autoclaving. Solid cool-
ing pads can ensure low temperature during dissection procedure
(see Fig. 1b).
1. Decapitate a 6–8 day old mouse and place the head into the lid
of the sterile dissection dish (see Note 12; Fig. 1c).
2. Spray the head with 70% ethanol to avoid contamination of the
samples originating from the surface of the skin.
3. Fix the head at the eyes with the angled forceps, and using
small scissors, make an incision in the skin along the midline of
the head (see Fig. 1d).
4. Remove the skin on both sides with the scissors (see Note 13;
Fig. 1e).
5. Cut in the middle of the skull with the curved forceps
pulling it neatly below the skull from rostral to caudal end
(see Note 14).
6. Move away the sectioned skull with forceps (see Fig. 1f).
7. Remove the brain with spatula by gently reaching below (in
between the brain and skull (see Note 15)) and lifting it up
(see Fig. 1g).
8. Place the brain into the prechilled dissection medium.
9. Gently fix the brain at the position of the cerebral cortices with
one of the spatulas and remove the cerebellum with the other
(see Fig. 1h).
 24 High-Resolution Imaging and Evaluation of Spines… 283

10. Further using the spatula, make a sagittal cut between the two
hemispheres to separate them (see Fig. 1i).
11. Flip one of the hemispheres onto the convex side and remove
the thalamus and the basal ganglia situated on top of the hip-
pocampus under a dissection microscope (see Fig. 1j).
12. Cut out the hippocampus from the underlying cortex (see
Fig. 1k).
13. With fine forceps clean away the vessels around the hippocam-
pus (see Note 16).
14. Repeat the procedure with the other hemisphere.
15. Place the two hippocampi in a small petri dish with prechilled
dissection medium on ice.

3.1.3. Preparation The procedure takes place under the sterile preparation bench.
of Hippocampal Slices
1. Using the spatulas, place two hippocampi on a teflon stage of
with Tissue Chopper
the tissue chopper.
2. Aspirate the excess medium with help of a sterile pipette (see
Note 17).
3. Cut rapidly 400 μm thick slices with the tissue chopper (see
Note 18; Fig. 1l).
4. Transfer the slices back into the small petri dish and place them
on ice (see Note 19).
5. Identify intact individual slices under the dissection microscope
and transfer them with the small spatulas onto the membrane
inserts (see Note 20; Fig. 1n).
6. Change the culture medium below the inserts to fresh, cooled
medium (see Note 21).
7. Place the 6-well plate with slices into the incubator at 37°C
with 5% CO2.

3.1.4. Maintenance and The procedure takes place under the sterile culture bench.
Infection of the Slices
1. Exchange the culture medium in the culture dishes every 2–3
days (see Note 22).
2. On day 11 post-preparation, change the culture medium to
NB medium containing N1 supplement (see Note 23).
3. On day 12 post-preparation, apply the virus with the droplet
method (see Note 24).

3.1.5. Fixation of Cultures are fixed at day 3 postinfection to ensure the highest
Hippocampal Slices expression of the fluorescent protein and best signal-to-noise ratio
for appropriate image processing.
1. Let the slices remain attached to the culture plate membrane
to preserve hippocampal structure and rinse with PBS within a
6-well plate (see Note 25).
284 F. Sündermann et al.

2. Apply ~2 mL/well cold fixing solution for 2 h at 4°C.

3. Wash with PBS at least three times for 15 min each.
4. Cut out a piece of membrane with the attached slices using a
scalpel and transfer the membrane with forceps onto a glass
slide (see Note 26).
5. Mount the cultures with confocal matrix and cover with cover-
slip (see Note 27).

3.2. Microscopy of 1. Locate the effectively infected slices using 10× objective
Hippocampal Slices through eyepiece.
2. With 20× objective identify the regions of the hippocampus
and the cells that will be imaged.
3. With 40× objective identify the dendritic branches of individ-
ual pyramidal neurons on the apical or basal side, respectively.
4. Image CA1 and CA3 pyramidal neurons with voxel size of
0.08 × 0.08 × 0.25 μm in the x–y–z directions with 60×/63×
objective. Adjust the image size according to the length and
shape of the imaged dendritic fragment (see Note 28).

3.3. Semiautomated 1. Open the file in the Autodeblur program.

Analyses of Dendritic 2. Open the 3D Deconvolution menu (see Fig. 2) at Deconvolution
Spines → 3D Deconvolution.
3.3.1. Image Processing 3. Optionally you can insert a cutting step (see Note 29; see Fig. 3).
with Autodeblur 4. Choose “Adaptive PSF (Blind)” as deconvolution method.
5. Click on <change> in the “Optics Settings” to adjust the image
and PSF settings. These options depend on the confocal setup
(numerical aperture, magnification, refractive index) and the
emission wavelength of the used fluorescent markers.
6. The “Deconvolution Settings” depend on the level of noise of
the images. For confocal images an iteration number from 10 to
15 and a level of noise between low and medium is sufficient.
7. Adjust the “Output Settings” to your needs. For 3DMA analysis,
a 16-bit tiff image format is recommended.
8. Click on <Start> to start the process or on <Batch> to add
another file for deconvolution. The batch process can be started
from “Batch processing” in the “Deconvolution” menu.

3.3.2. Analysis with 3DMA Autodeblur is run on a PC with “Windows XP” as an operating
system. The original “3DMA” software is created for operating
under “Unix/Linux” systems. To perform 3DMA based analysis, a
“Windows XP” PC for deconvolution and a “Unix/Linux” PC
(Ubuntu 6.06 LTS) for 3DMA analysis is required. For data trans-
fer, a “Share” folder, which can be accessed by both operating sys-
tems, is recommended (see Note 30).
 24 High-Resolution Imaging and Evaluation of Spines… 285

Fig. 2. Deconvolution settings dialog window. In this dialog window deconvolution settings
can be adjusted as described in the protocol (refer to Subheading 3.3.1, steps 4–7). The
shown preselections for spine analysis are recommended by the authors.

1. Within the “Linux” operating system, open a terminal (xterm)

and switch in superuser mode (sudo su).
2. After deconvolution it is necessary to create a new folder for
your analysis. Change to this folder and type “create_wdir”
(see Fig. 4 line: 01) in order to automatically create the required
subfolders.
3. Copy the image data (*.tif and *.aqh files) into the “raw”
folder. Change to the “raw” folder (see Fig. 4 line: 02).
4. To make the files readable for “3DMA” convert them into a
“rsz”-format (see Fig. 4 line: 03-05).
5. Switch to the “Recompiled Fixed Source” mode by typing
“3dmaswitch” in the terminal window.
6. Convert the file by typing “aqh2mtif <filename>” (without
suffix, e.g., filename.tif will be filename).
286 F. Sündermann et al.

Fig. 3. Results of the deconvolution. (a) The deconvolution algorithms enhance edges
within images and can produce artefacts like black borders around bright image com-
pounds. (b) Other artefacts are shadows of brighter structures imprinted in dark planes of
their neighborhood. The intensity of these imprints is slightly higher than the background.
(c) Projection of an appropriately deconvolved image stack. Scale bar, 2 μm.

01:> create_wdir
02:> cd raw
03:> 3dmaswitch
04:> aqh2mtif <file_without .tif>
05:> 3dmaswitch
06:> cd ../cases
07:> 3dmaGUI_lnx

Fig. 4. List of command lines for 3DMA file processing.

7. When all files are converted, switch back to “Original Source”

mode by typing “3dmaswitch” again.
8. Change to the “cases” folder and start 3DMA by “3dmaGUI_
lnx” (see Fig. 4 line: 06-07).
9. After the welcome screen you are asked to choose an applica-
tion. Choose <Dendritic Spine>.
10. In the “Application Routine Menu” (see Fig. 5a) you can
choose between different routines. Start with “Spine Detection
Routines.”
11. Choose the options as shown in (see Fig. 5b) and click on
<continue>.
12. In the “Data and Parameter Settings Section” switch to <resized>
and limit the listed files to files with “rsz” extension by typing in
the “Data Filename in raw directory” field “../raw/*.rsz.” After
clicking <Load Data> your files will appear in the file section.
Choose one file for processing by clicking on it. Remove the
other files from the list by pressing “space” key repeatedly.
 24 High-Resolution Imaging and Evaluation of Spines… 287

Fig. 5. Screenshot of 3DMA menus. (a) Applications essential for spine analysis are listed: “Spine Detection Routines,” “Edit
Detected Spines,” and “Static Spine Analysis”. (b) After choosing “Spine Detection Routines” default options are recommended.

Fig. 6. Data and parameter selection for spine detection.

13. Select the options as shown in (see Fig. 6): uncheck <Use fiducial
polygon>, select <Simple Thresholding>. Enter a threshold
level in <Enter lower threshold>. As an alternative, choose
<Indicator Kriging> and enter a lower and an upper threshold
level (see Note 31; Fig. 7).
14. Click <Save Simple Settings> to save the entered settings. After
confirmation start your analysis by clicking <Run>.
15. After finishing the processing of the file start a <New Run>,
choose <Dendritic Spine> as application, and select the <Edit
Detected Spines> routine for manual correction.
16. In the menu which appears select the file to edit by double
clicking. Deselect falsely detected spines and dendrites by right
mouse click (see Fig. 8). Click <Save> to finish the editing
process (see Note 32).
288 F. Sündermann et al.

Fig. 7. Effects of thresholding. (a,c,e) Comparison of different threshold levels for spine detection to visualize the effects of
thresholding. (b,d,f) Projections of thresholded images with its detected spines. (b) Image with threshold set too low shows
a bias toward stubby spines. (d) Optimally thresholded image with no bias toward any spine type. (f) Images with threshold
set too high shows segmented dendritic backbone and a bias toward thin and mushroom spine types.

Fig. 8. Cropped dialog window for manual editing of detected spines. On the left side in the full dialog view, an overview
projection of the dendritic segment with colored spines is shown.
 24 High-Resolution Imaging and Evaluation of Spines… 289

17. To complete your analysis and obtain the statistical data, start
again a <New Run>, select the <Dendritic Spine> application,
and choose the <Static Spine Analysis> routine.
18. In this menu load your data by clicking <Load Data> and set
the output filename. Alternatively, change the output folder as
well. The default options are “../spine/detect/analysis.txt.”
19. The analysis file can be read with every text or word editor (see
Note 33).

4. Notes

1. Different transgenic mouse lines can be used with C57BL/6

background, e.g., APP transgenic line, as an Alzheimer’s
disease model.
2. Use aliquots from the same batch of serum, since horse serum
can differ from batch to batch.
3. About 25 mL are needed for the dissection dish and about
3 mL for each small dish with hippocampi. Hence, 50 mL of
dissection medium are enough for ~5 pups.
4. Antibiotics can have additional effects on slices in culture. e.g.,
penicillin can reduce GABAergic transmission (11). If one is
able to successfully culture slices without using antibiotics, they
should be omitted from the medium. However, once started it
is important to use antibiotics throughout the entire experi-
mental procedure to avoid differential influence on the results.
5. Different inserts are commercially available. However, only
Millipore provides PTFE membranes. The material is crucial
for appropriate slice development (certain degree of flattening
during the culture time) and subsequent confocal microscopy
(the PTFE membrane is optically clear in contrast to mem-
branes from other materials).
6. One can use dissection tools with the same basic features (e.g.,
size and material) from other providers.
7. Above-mentioned tools are purchased from Fine Science
Tools GmbH, Heidelberg and Carl Roth GmbH & Co. KG,
Karlsruhe.
8. Organotypic cultures are often made also according to roller-
tube technique (12, 13). However, during cultivation with
the roller-tube method the slices thin to a practically one-cell
layer. The membrane interface culture method maintains a
thickness of slices at ~100–150 μm during the cultivation
period, which makes this method more suitable for the study
of 3D spine analyses.
290 F. Sündermann et al.

9. Take care that air bubbles do not remain under the membrane.
The membranes will become completely transparent when
wetted.
10. One of the tubes with sterile PBS/ddH2O may serve to clean
most of the blood and tissue from the tools before placing
them into 70% ethanol, the other to rinse the tools from etha-
nol before continuing to use them for further dissection.
11. Preparing the whole package of dishes at once, and packing
them back into the original sterile bag after sterilization together
with Whatman paper ensures a ready dissection dish for many
preparations. The Whatman paper provides a better contrast
and a non-slippery surface during the dissection procedure.
12. Cut off an ~3 mm portion of the tail if genotyping is required.
The tail should be placed immediately into a sterile eppendorf
cup kept on ice. Pay attention that the dish in which the decap-
itation is taking place is cleaned throughout with 70% ethanol
after each mouse to avoid false-positive PCR results due to
cross contamination of the genetic material.
13. The upper and lateral skull should be completely clean and
accessible for further preparation.
14. Care must be taken to avoid injury during this procedure.
Pulling the forceps slightly upward, while sliding it might help
avoid injury to the brain.
15. During this move the cranial nerves are cut so that the brain
can be flipped out.
16. The remaining connective tissue might block the separation of
the chopped slices.
17. Avoid injuring the slices but gently correct their position
with the pipette tip to align them perpendicular to the axes
of the blade.
18. Set the blade arm of the chopper in the position closest to the
hippocampi to minimize the cutting time. Add a drop of cold
dissection medium onto the slices immediately after chopping.
19. During this procedure gently pick up the slices with help of the
big spatulas and try to separate the slices without injuring them
by fine, round movements of the spatulas close to one another
(see Fig. 1m). Keep the slices on ice for 30 min before continu-
ing the procedure.
20. Up to 3 or 4 slices can be cultured on one insert (see Fig. 1o).
21. This procedure takes place under the sterile culture bench. Suck
all the medium from the dish with a sterile glass pasteur
pipette during each of the medium changes otherwise the new
medium will increase in volume due to the residual medium
resulting in leveling above the membrane and eventually to
 24 High-Resolution Imaging and Evaluation of Spines… 291

suffocation of the cells within slices. Carefully place the fresh

medium below the membrane and not on the slices by pulling
it out from the pipette tip between the insert and the wall of
the culture dish.
22. To avoid temperature changes within the slice during medium
exchange keep the 6-well plate on a warm pad while it is out of
the incubator. The culture medium during slice cultivation
should always be applied pre-warmed.
23. Changing to a different medium prior to infection is required
to drastically lower the serum content that might counteract
the virus infection but in parallel it complements some of the
serum proteins to maintain adequate culture conditions.
24. The use of Sindbis virus was chosen for this approach because
it infects neurons with high efficiency and provides a fast syn-
thesis of the fluorescence protein on levels that ensure good
image quality. Preparation of Sindbis virus is carried out under
safety level 2 conditions according to the Sindbis Expression
System Manual (Catalog no. K750-01). A small aliquot of the
virus is thawed on ice and 1 μL of the viral solution is gently
applied with a pipette onto each slice without touching the
surface of the slice. Silicone-coated tips have reduced surface
tension that helps in dispensing the infectious particles.
25. For an extensive rinse use ~2 mL/well of PBS (one below and
one above the membrane).
26. It is easier to cut the membrane if the insert is transferred onto
a piece of cardboard box. Suck off the medium prior to the
procedure and soak up on paper the excessive liquid.
27. Due to the thickness of the slices, place two small, round cov-
erslips on the glass slide, flanking the membrane as elevators.
Cover the sample together with the small coverslips with one
bigger coverslip. Avoid air bubbles under the coverslip, as that
would disturb imaging process.
28. The length of the imaged dendritic segments should be
between 20 and 30 μm. Choose a dendritic segment that is not
crossed by other dendrites.
29. Deconvolution is also an edge-sharpening procedure. Therefore,
it can introduce some artefacts, like a darker border around
brighter image components and an imprint of the brighter areas
in darker planes (see Fig. 3). To reduce these artefacts cut the
empty planes around the area of interest during a preprocessing
step and adjust the options in the “Deconvolution Settings”,
like iteration number and level of noise.
30. 3DMA-Neuron as a software package for automated neuronal
morphology analysis is no longer available from the homepage
of the authors (W.B. Lindquist and C.M. Weaver). It has been
292 F. Sündermann et al.

commercialized by Nihon Visual Science, Inc. In case of

interest to purchase the software, contact Nihon Visual Science,
Inc. (E-mail: [email protected]). Other programs such as
NeuronStudio, a noncommercial program created at Mt. Sinai
School of Medicine by the Computational Neurobiology and
Imaging Center (14), may also be applicable for spine analysis
but has not been tested by us. We are also currently developing
a robust and easy-to-use software tool, which we will provide
free of charge upon request.
31. Choosing an appropriate threshold level is the most critical
step during spine analysis. If the threshold is too low, the vol-
ume of the dendritic segment and the spines appear larger,
which results in a bias toward stubby spine type. If the thresh-
old is too high, the volume of the dendritic segment and the
spines appear smaller than they are which results in a bias
toward thin and mushroom spine types. A threshold chosen
much too high will segment the dendritic backbone and make
further analysis impossible (see Fig. 7).
32. Open the file with “rsz” extension, to look at the original image
in parallel. This will help to determine falsely detected spines.
33. In the analysis file, the following parameters are listed: total
number of spines, number of mushroom, stubby and thin
spines, spine density, dendrite radius, and length and volume
of each spine.

Acknowledgements

This work has been supported by the Deutsche

Forschungsgemeinschaft (DFG BR1192/11-2) and a Lichtenberg-
Fellowship within the graduate college “Membranes and cellular
communication” (supported by the state of Lower Saxony; FS).
We appreciate the valuable work of Neelam Shahani and Tobias
Wolf during the establishment of this technique.

References

1. Harris KM, and Kater SB (1994) Dendritic dynamics of dendritic spines in memory and
spines: cellular specializations imparting both cognition. Trends Neurosci 33, 121–129
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Annu Rev Neurosci 17, 341–371 simple method for organotypic cultures of ner-
2. Tackenberg C, Ghori A, and Brandt R (2009) vous tissue. J Neurosci Methods 37, 173–182
Thin, stubby or mushroom: spine pathology in 5. Tackenberg C, and Brandt R (2009) Divergent
Alzheimer’s disease. Curr Alzheimer Res 6, pathways mediate spine alterations and cell
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3. Kasai H, Fukuda M, Watanabe S, Hayashi- tau, and R406W tau. J Neurosci 29,
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6. Ehrengruber MU, Lundstrom K, Schweitzer 10. Feng G, Mellor RH, Bernstein M, Keller-Peck C,
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C, and Brandt R (2006) Tau aggregation and 13. Gähwiler BH, Capogna M, Debanne D,
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phology after targeted expression of Alzheimer’s come of age. Trends Neurosci 20, 471–477
disease-relevant tau constructs in organotypic 14. Rodriguez A, Ehlenberger DB, Dickstein DL,
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9. Ehrengruber MU, and Lundstrom K (2007 ) Three-Dimensional Detection and Shape Classifi-
Alphaviruses: Semliki Forest virus and Sindbis cation of Dendritic Spines from Fluorescence
virus vectors for gene transfer into neurons. Microscopy Images. PLoS ONE 3, e1997
Curr Protoc Neurosci Chapter 4:Unit 4.22
 Chapter 25

Imaging Amyloid Precursor Protein In Vivo:

An Axonal Transport Assay
Tomás L. Falzone and Gorazd B. Stokin

Abstract
Fusion of fluorescent probes to axonally transported proteins represents an established approach that
enables live imaging of axonal transport. In this approach, in vivo examination of fluorescent particle
dynamics provides information about the length, directionality, and the velocity by which axonally trans-
ported proteins travel along axons. Analysis of these parameters provides information about the distribu-
tion of axonal proteins and their dynamics in and between different subcellular compartments. Establishing
the movement behavior of amyloid precursor protein within axons indicated that live imaging approaches
offer the opportunity to significantly enhance our understanding of the biology as well as pathology of
axonal transport. This chapter provides a fluorescence-based procedure for measuring axonal transport of
APP in cultured newborn mouse hippocampal neurons.

Key words: Amyloid precursor protein, Axonal transport, Fluorescent probes, In vivo fluorescent
imaging, Primary hippocampal cell culture

1. Introduction

Axonal transport is a complex system that guarantees the delivery

of proteins and organelles to synapses, axonal compartments, and
cell bodies in a timely manner. This transport system, which
supplies high synaptic protein turnover with the appropriate cell-
derived commands and delivers environmental signals from differ-
ent presynaptic environments to cell bodies, relies on molecular
motors moving cargoes along microtubule tracks at the expense of
ATP hydrolysis. The force-generating molecular motors transport
cargoes either toward the synapses or toward cell bodies by means
of anterograde or retrograde axonal transport, respectively (1).
Amyloid precursor protein (APP) is a type II integral mem-
brane protein (2). In neurons, APP-containing vesicles undergo

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_25, © Springer Science+Business Media, LLC 2012

295
296 T.L. Falzone and G.B. Stokin

constitutive bidirectional axonal transport. Several observations

link APP to the pathogenesis of Alzheimer’s disease (AD). In brief,
aberrant App genotypes segregate with AD phenotype (3), and
aberrant accumulation of proteolytic fragments of APP in the form
of amyloid peptides and plaques are considered a neuropathological
hallmark of AD brains (4). Intriguingly, aberrant accumulation of
APP and its proteolytic fragments has been long described within
axons of AD brains (5) as well as in several other diseases ranging
from traumatic brain injury to multiple sclerosis. The early patho-
logical presence of APP in the axons points to a role of APP beyond
AD, suggesting that APP may represent a bona fide surrogate
marker of axonal injury (6, 7).
Early work on axonal APP focused largely on immunochemical
studies. These approaches indisputably localized APP within axons
and uncovered its aberrant accumulation in disease (8). Axonal
transport of APP was first suggested in a sciatic nerve ligation study,
which showed APP synthesized in the dorsal root ganglia accumu-
lating proximally to the ligation site within axons (9). It was after
the development of fluorescent approaches, however, when bidirec-
tional movement of APP was first visualized, together with the
assessment of its dynamic behavior within axons (10, 11). Although
the function of axonal APP movement behavior and the mechanisms
regulating axonal transport of APP remain largely unknown, these
approaches indisputably revolutionized our understanding of APP
biology and allowed the identification of APP axonal transport
impairments in animal models of AD, which added a new twist to
the current pathogenesis of AD (12–15). In conclusion, novel flu-
orescent approaches to studying axonal transport can be applied to
any axonally transported transmembrane or soluble protein. The
tracking and surveillance of these proteins have only started pro-
ducing a wealth of new data that will significantly further our
understanding of the axonal milieu (16, 17). This chapter describes
a detailed protocol in which the above approach is applied to the
axonal tracking of APP in cultured mouse hippocampal neurons.

2. Materials

2.1. Primary 1. Cold Hank’s balanced salt solution (HBSS, Gibco). Store at
Hippocampal Neuron 4°C (see Notes 1– 4).
Cultures 2. Hank’s buffer: to 500 mL HBSS, add 0.4 g of D-glucose
(Sigma-Aldrich), 0.834 g of 4-(2-hydroxyethyl)-1-pipera-
zineethanesulfonic acid (HEPES, Sigma-Aldrich), 5 mL of
100× penicillin-streptomycin (Invitrogen) and filter-sterilize
using 0.22-μm-diameter filter cartridge. Store at 4°C.
 25 Imaging Amyloid Precursor Protein In Vivo: An Axonal Transport Assay 297

3. Disaggregation buffer: add to phosphate buffered saline (PBS,

Gibco) 1.5 mM DL-cysteine HCL (Sigma-Aldrich), 0.025%
bovine serum albumin (BSA, Sigma-Aldrich), and 35 mM
D-glucose (Sigma-Aldrich). Store at −20°C.

4. Papain solution: add 45 U of lyophilized papain (Worthington)

and 0.05% DNase I (Boehringer Mannheim) to disaggregation
buffer. Once papain is dissolved, filter-sterilize by passing
through a 0.22-μm-diameter filter cartridge.
5. Complete Dulbecco’s Modified Eagle’s Medium (DMEM,
Gibco): supplement DMEM with 10% fetal bovine serum
(FBS, Gibco) and 500 μM L-glutamine (Sigma-Aldrich). Store
at 4°C.
6. Complete Neurobasal medium: enrich Neurobasal medium
(Gibco) with 1:50 (v/v) of 50× serum-free supplement B27
(Invitrogen) and 500 μM L-glutamine (Sigma-Aldrich). Store
at 4°C.
7. Boric acid buffer: to 400 mL of water, add 1.24 g of boric acid
(Sigma-Aldrich) and 1.9 g of borax (Sigma-Aldrich), then fil-
ter-sterilize the solution. Store at 4°C.
8. Poly-D-lysine-coated coverslips: wash round coverslips (Fisher
Scientific) three times using 100% acetone with gentle agita-
tion, then three times with 100% ethanol, and finally three
times with sterile water. When dried, coverslips are incubated
with 100 μg of poly-D-lysine (MW 30,000–70,000) per mL of
boric acid buffer for several hours at room temperature, then
washed thoroughly with sterile water, and placed in a 24-well
dish (Falcon, BD biosciences) for cell plating.
9. Stereo dissecting scope (SMZ660, Nikon).
10. Fine forceps and scissors (Roboz).

2.2. Transfection 1. Pcdna3 CMV-APP-YFP vector expressing a protein fusion

between APP695 (APP) and yellow fluorescent protein (YFP)
at the C-terminus of APP (see Note 5) (10).
2. Lipofectamine 2000 (Invitrogen). Store at 4°C.
3. 10-day-old primary hippocampal cell culture.

2.3. Axonal 1. Inverted epifluorescence microscope (TE-2000U, Nikon)

Transport Imaging 2. Heat- and CO2-controlled chamber (Harvard Apparatus,
Holliston, MA)
3. Photometrics CoolSNAP HQ-cooled CCD camera (Roper
Scientific, Ottobrunn, Germany)
4. Glass bottom plates (MatTek, Ashland, MA)
5. MetaMorph 6.0 (Universal Imaging Corporation, Marlow, UK)
298 T.L. Falzone and G.B. Stokin

3. Methods

3.1. Harvesting Cells 1. Primary hippocampal cell cultures are harvested from postnatal
from Hippocampi day 1 mice (see Note 6). Using fine forceps under a dissecting
scope, cut the skin above calvarium by a midline anteroposte-
rior incision. Separate from the underlying bones of the calva-
rium both skin flaps generated by the incision. Incise the cranial
bones in the top midline portion and chip away to expose the
brain.
2. Detach the brain from the cranial nerves, remove from the
skull, and cut in half. Use a small spoon to remove the hip-
pocampi from the brain and meninges, and place into cold
HBSS (see Note 7).
3. Under a tissue culture hood, wash the excised hippocampi with
10 mL of HBSS at 4°C and then digest in 0.22-μ-filtered
papain disaggregation buffer for 20 min with moderate
shaking at 37°C.
4. After digestion, wash hippocampi twice with 10 mL of
complete DMEM (see Note 8), triturate 12 times with a 1-mL
micropipette in 2 mL of complete DMEM, and plate on top of
poly-D-lysine-coated coverslips at 60,000 cells per well in a
24-well dish (see Note 9).
5. After allowing 2 h for neurons to attach, replace the complete
DMEM with 500 μL of complete Neurobasal medium.
Maintain cell cultures at 37°C in 5% CO2 incubator.

3.2. Transfection Ten- to fourteen-day-old primary hippocampal cell cultures are

transfected with pcdna3 CMV-APP-YFP expression vector using
Lipofectamine 2000.
1. For each well, dilute 0.8 μg of endotoxin-free purified DNA in
50 μL of plain Neurobasal medium and gently mix. Similarly,
gently mix and dilute 2 μL of Lipofectamine 2000 in 50 μL of
plain Neurobasal medium and then incubate for 5 min.
2. After incubation, combine the diluted DNA with diluted
Lipofectamine 2000 to a total volume of 100 μL, mix gently,
and incubate for 20 min.
3. Remove 300 μL of complete Neurobasal medium from each
well and add 100 μL of DNA-Lipofectamine complexes to
each well containing primary hippocampal cells (see Note 10).
Mix gently by rocking the plate back and forth.
4. Incubate cells at 37°C in a 5% CO2 incubator and replace
medium to complete Neurobasal medium 2–4 h later (see
Note 11).
 25 Imaging Amyloid Precursor Protein In Vivo: An Axonal Transport Assay 299

3.3. In Vivo Imaging 1. In vivo transport of APP-YFP vesicles is imaged 16 h after

transfection using a heated stage at 37°C and in a 5% CO2
chamber using an inverted epifluorescence microscope con-
nected to a Photometrics CoolSNAP HQ-cooled CCD camera
and driven by MetaMorph 6.0. (see Note 12).
2. Remove transfected cells from multiwell plates by lifting the
coverslip with forceps and flipping it over a glass bottom plate
containing complete Neurobasal medium equilibrated in a 5%
CO2 incubator (see Note 13).
3. Identify fluorescent (transfected) cell bodies using an oil immer-
sion super apochromatic 100× lens. Follow axonal projections
at least two fields away from the cell body view. Anterograde
and retrograde axonal transport is determined depending on
the orientation of cell bodies and projection tips.
4. Register axonal dynamics of fluorescent APP-YFP vesicles in
15-s stacks format (tiff) and capture at a speed of ten frames
per second at ×100 magnification and 2 × 2 binning for
continuous registration (see Note 14, Fig. 1). Kymographs are
generated from stacks and analyzed using MetaMorph or
converted to QuickTime or AVI movie format for presenta-
tions. Calibration of movie pixel magnification and number of
frames per second should be established prior movie registra-
tion and used across experiments.

Fig. 1. Axonal transport movie registered in primary hippocampal neurons transfected with
APP-YFP using an inverted epifluorescence microscope. 100-ms frame image montage of
a movie registered in a transfected axon showing dynamics of APP-YFP vesicles. White
arrows indicate stationary (1, 4, 7), anterograde (2, 3) or retrograde (5, 6) moving vesicles,
respectively. Time corresponds to frames extracted from a 30-s movie. Bar equals 10 μm.
300 T.L. Falzone and G.B. Stokin

a
Anterograde Retrograde

30 sec.
10 um

b 1 2 3 4 5 6 7

10 um

Fig. 2. Kymograph generated from movie showing APP-YFP axonal transport dynamics.
(a) Kymograph generated from movie in Fig. 1 showing the distance displacement of
anterograde, stationary, and retrograde APP-YFP vesicles along 30 s. (b) Schematic
representation of the above kymograph in which all APP-YFP vesicles were tracked and
plotted. Straight, dashed, and dotted black lines correspond to anterograde (2, 3), retrograde
(5, 6), and stationary (1, 4, 7) particles, respectively. Bar equals 10 μm.

3.4. Kymograph 1. Open movie stack in a working station computer using

Generation MetaMorph (see Note 15). Draw a line of five-pixel width
and Analysis from cell body to axon tip to define the precise location of the
measurements and to track the length of the axon in cap-
tured movies.
2. Using the kymograph function in MetaMorph, generate a
montage image from the plotted line in which distance and
time are represented by the X and Y axes, respectively (Fig. 2a).
3. Using MetaMorph, in each kymograph plot lines following all
visible straight and descending fluorescent particles generated
by APP vesicle dynamics along time (Fig. 2b). Transfer measure-
ments extracted from lines such as angle, X and Y axes, and
start and end points obtained from pixel identification to an
Excel file and convert to microns by time for analysis (Fig. 3a).
APP-YFP vesicle movement is approximated from kymograph
as a percentage of clearly defined, stationary, anterogradely or
retrogradely moving APP-YFP out of defined particles, which
started and ended movement within the time and distance
captured in the kymograph. Average speed, distance, and
directionality of axonal transport are calculated in Excel files
for further statistical analysis (Fig. 3b).
4. In line with scientific rigor, all kymographs are coded before
the beginning of each experiment and scored blind to the
identity of the sample. Only upon completion of particle
plotting and of all data collection, the samples are uncoded
to allow analysis.
 25 Imaging Amyloid Precursor Protein In Vivo: An Axonal Transport Assay 301

b
50
Particle Proportion (%)

40

30

20

10

1.4 40

1.2 35
Run length (µm)

30
Speed (µm/sec)

1
25
0.8
20
0.6
15
0.4
10
0.2 5
0 0

Fig. 3. Axonal transport measurements obtained from kymograph analysis. (a) Excel file showing calculations extracted
from lines plotted on kymograph from Fig. 2b. Each line was separated by decreasing angle and corresponds to antero-
grade, stationary, and retrograde particles. Distance, time, and speed are calculated from the kymograph. (b) Results of
average particle proportion (%), speed (μm/s), and run length (μm) calculated from Excel file. Black, gray, and white bars
correspond to anterograde, stationary, and retrograde transport, respectively.
302 T.L. Falzone and G.B. Stokin

4. Notes

1. Prepare all solutions using ultrapure water by purifying

deionized water to attain a sensitivity of 18 MΩ cm at 25°C.
2. Prepare, store, and manipulate all reagents at room temperature,
unless indicated otherwise.
3. Follow regulations strictly for disposal of waste materials.
4. Hank’s buffered salt solution is fairly complicated to make,
which is why most people buy it.
5. Any APP cDNA, or other axonally transported protein cDNA,
fused to a fluorescent probe can be used for the in vivo imaging
of protein movement within axons. Alternatively, or simultane-
ously, fluorescent dyes may be used for labeling proteins or
organelles. In example, mitochondria can be dynamically
visualized using Mitotracker (Invitrogen).
6. Unless otherwise stated, all steps of the described method are
performed at room temperature and pressure.
7. Hippocampal spoon (Fine Science Tools) can be used to
facilitate detachment of hippocampi from cornu Ammonis.
8. Complete DMEM used for washing allows FBS to inactivate
papain activity.
9. Pipette trituration is a key step. Do it too much and you’ll kill
the cells, do it too little and you’ll have many clumps.
10. Little drops can be delivered at different points of the round
coverslips to facilitate distribution. Make sure the medium cov-
ers the entire well surface.
11. The same medium removed before starting transfection can be
used to replace Lipofectamine transfection medium. This proce-
dure helps cells recover after transfection due to factors and
proteins released by cells during their development.
12. Continued registration can be done with different camera
software devices in TIFF format and then opened in a different
computer station for analysis.
13. High magnifications (×100) require a short working distance
from specimen to lens. The sandwich made by flipping the cov-
erslips inside the glass bottom plate (1 mm) allows the cells to be
in the lens working distance for imaging and movie registration.
14. Cooled CCD camera allows fast continuous registration at low
sensitivity reaching the speed of ten frames per second at a time
resolution of 100 ms per frames. 2 × 2 binning was set up to favor
high-speed acquisition by reducing the recorded information.
15. Kymograph can also be generated in ImageJ software using
“multiple kymograph” plug-in.
 25 Imaging Amyloid Precursor Protein In Vivo: An Axonal Transport Assay 303

Acknowledgements

This work was supported by PICT-2008-0293 (ANPCyT)(T.L.F.),

Alzheimer Association NIRG-10-172840 Grant (T.L.F.) and
P3-0338 grant from the Slovenian Research Agency (G.B.S.).

References
1. Hirokawa N, and Noda Y (2008) Intracellular study in cultured hippocampal neurons. Mol
transport and kinesin superfamily proteins, Biol Cell 11, 1213–1224
KIFs: structure, function, and dynamics. 11. Goldsbury C, Thies E, Konzack S, and
Physiol Rev 88, 1089–1118 Mandelkow EM (2007) Quantification of amy-
2. Kang J, Lemaire HG, Unterbeck A, Salbaum loid precursor protein and tau for the study
JM, Masters CL, Grzeschik KH, et al (1987) of axonal traffic pathways. J Neurosci 27,
The precursor of Alzheimer’s disease amyloid 3357–3363
A4 protein resembles a cell-surface receptor. 12. Stokin GB, Lillo C, Falzone TL, Brusch RG,
Nature 325, 733–736 Rockenstein E, Mount SL, et al (2005)
3. Bertram L, Lill CM, and Tanzi RE (2010) The Axonopathy and transport deficits early in the
genetics of Alzheimer disease: back to the pathogenesis of Alzheimer’s disease. Science
future. Neuron 68, 270–281 307, 1282–1288
4. Goedert M, and Spillantini MG (2006) A cen- 13. Stokin GB, Almenar-Queralt A, Gunawardena
tury of Alzheimer’s disease. Science 314, S, Rodrigues EM, Falzone T, Kim J, et al
777–781 (2008) Amyloid precursor protein-induced
5. Cras P, Kawai M, Lowery D, Gonzalez-DeWhitt axonopathies are independent of amyloid-beta
P, Greenberg B, and Perry G (1991) Senile peptides. Hum Mol Genet 17, 3474–3486
plaque neurites in Alzheimer disease accumu- 14. Falzone TL, Stokin GB, Lillo C, Rodrigues
late amyloid precursor protein. Proc Natl Acad EM, Westerman EL, Williams DS, et al (2009)
Sci USA 88, 7552–7556 Axonal stress kinase activation and tau misbe-
6. Stokin GB, and Goldstein LS (2006) Axonal havior induced by kinesin-1 transport defects.
transport and Alzheimer’s disease. Annu Rev J Neurosci 29, 5758–5767
Biochem 75, 607–627 15. Araki Y, Kawano T, Taru H, Saito Y, Wada S,
7. Stokin GB, and Goldstein LS (2006) Linking Miyamoto K, et al (2007) The novel cargo
molecular motors to Alzheimer’s disease. J Alcadein induces vesicle association of kine-
Physiol Paris 99, 193–200 sin-1 motor components and activates axonal
8. Joachim CL, Duffy LK, Morris JH, and Selkoe transport. EMBO J 26, 1475–1486
DJ (1988) Protein chemical and immunocy- 16. Eva R, Dassie E, Caswell PT, Dick G, ffrench-
tochemical studies of meningovascular beta- Constant C, Norman JC, et al (2010) Rab11
amyloid protein in Alzheimer’s disease and and its effector Rab coupling protein contrib-
normal aging. Brain Res 474, 100–111 ute to the trafficking of beta 1 integrins during
9. Koo EH, Sisodia SS, Archer DR, Martin LJ, axon growth in adult dorsal root ganglion neu-
Weidemann A, Beyreuther K, et al (1990) rons and PC12 cells. J Neurosci 30,
Precursor of amyloid protein in Alzheimer dis- 11654–11669
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Proc Natl Acad Sci USA 87, 1561–1565 Greensmith L, and Schiavo G (2010) Deficits
10. Kaether C, Skehel P, and Dotti CG (2000) in axonal transport precede ALS symptoms
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distinct carriers: a two-color video microscopy 20523–20528
 Chapter 26

The Use of Specific AAV Serotypes to Stably

Transduce Primary CNS Neuron Cultures
Saafan Z. Malik, Margaret A. Maronski, Marc A. Dichter,
and Deborah J. Watson

Abstract
Although primary neuronal cell cultures are a valuable source of in vitro insight for many neurobiologists,
all current gene expression technologies for these cells have significant drawbacks. Some of these limitations
of current gene expression protocols include toxicity, transient expression, a requirement for postnatal
neurons, and/or low efficiency. To date, many types of experiments were not possible because of these
limitations. Here, we outline a methodology by which primary cultured neurons can be transduced at any
age, after plating, with virtually no toxicity and continued gene expression for the lifetime of the culture.
This method involves the use of adeno-associated viral vectors, which have the potential to be highly useful
for either upregulation or downregulation of single or multiple genes, including neurotrophins, other
neuroprotective genes, and neurotoxins.

Key words: Viral vector, Gene delivery, Cultured neurons, Neurotrophins, Immunocytochemistry,
Neuroprotection assays, Glutamate toxicity

1. Introduction

Reliable testing of candidate genes in primary cultured neurons is

a critical technique required to identify trophic and protective
factors for a variety of neurodegenerative diseases. Neuroscientists
would benefit from the ability to design reliable neurotoxicity
assays and test potentially neuroprotective agents on primary cultured
neurons. However, common techniques such as recombinant
Sindbis, SV40, or Semliki forest viral vectors as well as plasmid
transfection using calcium phosphate, commercially available lipids,
or electroporation suffer from many drawbacks, including toxicity,
transient expression, the requirement for immature neurons, and/or
low efficiency any of which could prevent an essential experiment.

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_26, © Springer Science+Business Media, LLC 2012

305
306 S.Z. Malik et al.

We and other groups have shown that a few well-known

adeno-associated viral (AAV) vectors can mediate efficient, stable,
and nontoxic transduction in vivo and in vitro. (e.g. refs 1–3).
Recently, we published a detailed comparative study of AAV vec-
tors packaged with each of seven naturally occurring serotypes in
addition to four engineered capsid proteins (3). One of the impor-
tant findings of that study was the particular usefulness of AAV1
capsids to mediate gene expression in a very high percentage of
cultured neurons. Of note, there is another potential benefit of
AAV vectors: the ability to use the same vector directly in an
in vivo experiment with proper planning.
Here, we detail the planning and methodology for designing
and producing such a vector, testing it in vitro, using it for neuro-
protection assays in vitro, and issues of note when moving into
in vivo studies.

2. Materials

There is a “canonical” book in this area to which readers are

referred for general methodology. (see “Rat Hippocampal Neurons
in Low-Density Culture” in (4)). We have summarized the overall
technique with specific modifications necessary for the assays in
this chapter (see below and also (5)).

2.1. Poly-L-Lysine 1. Many cultured cells, especially neurons, will not adhere to plastic
Coating and or glass without prior coating of the surface. Poly-L-lysine
Preincubation (Peptides International) has a positive charge which attracts
of Tissue Culture the negative surface charge of the neurons to the plate or well.
Plates with or Without Poly-L-lysine is made up in borate buffer.
Glass Coverslips 2. Borate buffer: add a stir bar to 500 mL of endotoxin-free water
(BioWhittaker/Lonza) and set on a stir plate in the biosafety
hood to stir. Add 2.38 g boric acid and 1.27 g borax to the
water and continue stirring for approximately 15 min or until
all the powder is dissolved. Resterilize the solution through a
0.2-mm filter and store in a labeled, sterile bottle at room
temperature for up to 6 months.
3. Poly-L-lysine solution: dispense 100 mL borate buffer into a
sterile 500-mL screw-cap bottle under the hood. Add one
bottle (100 mg) poly-L-lysine (molecular weight 70–150 kDa,
Peptides International, Louisville, KY) to the borate buffer and
swirl until all the poly-L-lysine has dissolved in the buffer, about
2 min.
4. Preincubation medium: combine 500 mL of Neurobasal
medium and 5 mL (one bottle) of B27 (with antioxidants)
supplement.
 26 The Use of Specific AAV Serotypes to Stably Transduce Primary… 307

5. Plates and coverslips are required according to the specific

experimental conditions. Coverslips must be 12-mm round
German glass coverslips (Fisher Scientific). Autoclave before use.

2.2. Primary 1. One liter bottle of Dulbecco’s modified Eagle’s medium

Hippocampal Cell (DMEM) containing 25 mM HEPES buffer, 4.5 g/L glucose,
Culture no glutamine (e.g., BioWhittaker/Lonza BW12-709 F).
2. Fetal calf serum (Hyclone). Store in 50 mL aliquots at −20°C.
3. Trypsin, 2.5% (Invitrogen).
4. HEPES-buffered saline solution (HBSS) and water
(BioWhittaker/Lonza).
5. Trypan blue, 0.4% in saline.
6. A 37°C water bath with a shelf above the water.
7. A timer.
8. Glass Pasteur pipettes and a rubber bulb.
9. Bunsen burner.
10. 35-, 60-, and 100-mm-diameter tissue culture plates.
11. Poly-L-lysine-treated culture plates.

2.3. Construction 1. A plasmid containing the AAV inverted terminal repeats (ITRs)
of AAV Genomic and polyA sequence that has been propagated in recA- compe-
Plasmid tent cells and sequenced.
2. Additional plasmids containing the expression elements of
interest.
3. Standard molecular cloning reagents, including restriction
enzymes and buffers, ampicillin and/or kanamycin, LB broth
and plates, agarose gels, 37°C shaking incubator.
4. Competent bacterial cell lines such as SURE2 or STABL2 that
are recA-. These must be kept at −80°C and thawed on ice a
few minutes before transformation.
5. Primers that allow sequencing of the final plasmid, especially
the ITRs.

2.4. Packaging of AAV 1. Contact information for vector preparation experts (see
Subheading 3.5 for recommendations).

2.5. Concentration 1. An AAV-green fluorescent protein (GFP) vector with a titer of

Response of at least 5× (10) GC/mL.
Neurotoxic Agent 2. Poly-L-lysine plates or coverslips of your choice.
3. L-glutamate.
4. Phosphate-buffered saline.
5. Microcentrifuge tubes for making dilutions.
308 S.Z. Malik et al.

6. 10 mL sterile cell culture reservoir.

7. 10% neutral buffered formalin and appropriate waste
containers.
8. VECTASHIELD with DAPI (Vector Laboratories).
9. Parafilm.
10. Microscope slides and nail polish.
11. A fluorescent microscope with a digital imaging camera, attached
computer and software. For example, a Nikon 80i microscope,
a CoolSnap cf2 monochrome camera, and ImagePro software.

2.6. Quantification 1. Permeabilization buffer: Tris-buffered saline (TBS) containing

of Neuroprotection 0.3% Triton (TBST).
2. Blocking reagent: 5% goat or donkey serum in TBS.
3. Appropriate combinations of primary and secondary antibod-
ies and detection reagents. For identification of neurons, rab-
bit polyclonal antimicrotubule-associated protein 2 (1:500),
Biotin-SP-conjugated goat anti-rabbit IgG (Biotin-SP is
Jackson ImmunoResearch’s trade name for biotin with a
6-atom spacer positioned between biotin and the protein to
which it is conjugated), and Alexa594-conjugated streptavidin
(1:500 each). For astrocytes, monoclonal anti-glial fibrillary
acidic protein (GFAP) antibody (Sigma, 1:500), Biotin-SP-
conjugated donkey anti-mouse IgG, and Alexa594-conjugated
streptavidin (Molecular Probes, 1:500 each). Other primary
antibodies such as anti-NeuN or anti-GFP will also be useful.

3. Methods

3.1. Preincubation 1. Observing sterile technique, use sterile forceps to add one
of Plates and/or coverslip per well of a 24-well plate or five coverslips to a
Coverslips 35-mm-diameter tissue culture plate (Nunc). Alternatively, use
a 24-well plate with no coverslips. The specific setup depends
on the type of experiment you wish to perform.
2. Set the plates in a single layer on an incubator tray and keep
inside the hood at room temperature.
3. For a 35-mm plate, add 1.5 mL of the poly-L-lysine solution
and make sure the coverslips, if present, are submerged and
arranged in a single layer on the bottom of the dish. Store at
room temperature for 24 h.
4. The next day, aspirate the poly-L-lysine solution and add
1.5 mL of BioWhittaker water to each dish, again being sure to
submerge the coverslips, and arrange in a single layer on the
 26 The Use of Specific AAV Serotypes to Stably Transduce Primary… 309

bottom of the dish. It is important that the coverslips remain

in the same orientation as previously (i.e., do not flip over and do
not move or overlap). Store for a further 24 h inside the hood.
5. The next day, aspirate the water and add 1.0 mL of the prein-
cubation medium for approximately 72 h. The use of German
glass and this preincubation step promotes greater viability in
older cultures. The reduction or lack of serum, in the culture,
reduces or eliminates glia.
6. Gridded coverslips can be used if necessary (see Note 1).

3.2. Serum-Free 1. Immediately before use, add 0.5 mL of thawed trypsin to

Primary Hippocampal 4.5 mL of sterile HBSS with HEPES at room temperature in a
Neuron Cultures (see 15-mL centrifuge tube, mix and add to 35-mm dishes containing
Notes 2 and 3) the dissected material. Place each of the 35-mm dishes into
the bottom of a 100-mm dish and carry to 37°C water bath.
The 35-mm dishes should have their lids on and remain sterile
at all times. Place the 100-mm dishes on a shelf above the
water level and cover the bath. Set timer for 15 min.
2. After 15 min, return trypsinized hippocampi to the hood.
Carefully pour the mixture into sterile 60-mm plates and add ~10 mL
of sterile HBSS with HEPES to dilute the trypsin. Return to
the water bath. After 5 additional minutes, retrieve the 60-mm
dish and remove as much HBSS with HEPES as possible without
suctioning up any hippocampi. Then add another sterile 10 mL
HBSS with HEPES to the dish, place it back into the 100-mm
bottom dish, and place in water bath for another 5-min wash.
Afterwards, dispense the hippocampi into a 15-mL centrifuge
tube until all hippocampal material is in tube (~5 mL). Discard
the rest of the isolation medium containing trypsin and HBSS
with HEPES.
3. Constrict the opening of the sterile 9-in. Pasteur pipette with a
Bunsen burner until resistance forms in the suction bulb, indi-
cating the opening is sufficiently small. With the constricted
Pasteur pipette, triturate hippocampi gently 14 times until all
tissue is broken up into individual cells and cannot be seen
individually any more with the solution appearing cloudy with-
out any material floating in it. Avoid bubbles by keeping the
tip of the pipette in the solution while triturating. Add 5 mL of
Dulbecco’s modified Eagle’s medium supplemented with 10%
fetal bovine serum.
4. Count the cells using 10 mL of cell suspension drawn from
the center of the tube and 90 mL of trypan blue. Calculate the
number of cells/mL and the percent viability.
5. For this protocol, use 100,000 cells/mL. Add additional
Neurobasal medium with B27 to bring up the volume to the
required concentration for plating.
310 S.Z. Malik et al.

6. For 24-well plates, use 0.5 mL or 150,000 cells/well. For

96-well plates, use 500 mL per well. For 35-mm plates containing
coverslips, add 1.5 mL into each plate at 100,000 viable cells/mL
equaling a total of 150,000 cells total per plate.
7. Store the cultures in a 37°C, 5% CO2 humidified incubator.
For reduced number of glia in cultures, use Neurobasal plus
B27. Most glial cells are inhibited by lack of serum in the
medium, but a small percent will survive in the Neurobasal/
B27. If your experiments dictate that absolutely no glia should
be present, you can add a mitotic inhibitor 24 h after plating
the neurons.
8. Neuronal cell cultures are generally used from 7 days up to
5 weeks. Cultures should be fed by adding a few drops of new
Neurobasal/B27 medium to each plate. There is a greater risk
of contamination at the time of feeding than of the cultures
metabolizing the medium in the plates, so the cells can be fed
about once per week.
9. The same protocol can be used for cortical cultures.

3.3. Generation of AAV 1. AAV is a small (20 nm), single-stranded, icosahedral, nonrepli-
Vectors for Neuronal cating DNA virus composed of a ~4.7-kb genome comprised
Cell Transduction: of one ITR on each end of the genome and two open reading
Design Considerations frames which generate the multiple genes necessary for the
AAV life cycle and capsid production. Recombinant AAV vec-
tors are generated by packaging an engineered AAV genome
into an AAV capsid sequence that may or may not be derived
from the same AAV serotype as the genome. To generate
recombinant vectors, the rep/cap sequences can be delivered
in trans, allowing for more flexibility in vector design. Currently,
genome plasmids are generally based on the AAV2 ITRs
because they can be easily cross-packaged into capsids from
AAV1, 5, 6, 9, etc. Each capside sequence probably binds to a
separate receptor, though not all of them are known. These vec-
tors are called AAV2/5 or AAV2/9, etc., as specified by the
user. To generate the genome of the vector, the plasmid must
contain all the expression components in one continuous
sequence. (The total size of the entire plasmid is not critical
but must contain the antibiotic resistance gene, etc.) The usual
arrangement of AAV elements is 5¢ITR (usually derived from
AAV2) followed by a promoter, a gene of interest, a polyA
sequence, and the 3¢ITR. The entire sequence between the
5¢ITR and the 3¢ITR should not be much larger than the size
of the wild-type genome (~4.7 kb), otherwise the recombinant
genome will not be packaged efficiently. Unrelated “stuffer”
sequence is sometimes included if needed to bring the sequence
up to 4.7 kb. Maximal packaging size is about 5.2–5.3 kb (6–8).
To overcome some aspects of the size limitations, trans-splicing
vectors have been developed (for more details, see (9)).
26 The Use of Specific AAV Serotypes to Stably Transduce Primary… 311

2. The length of the ITRs is fixed at ~145 bp each, so scientists

have worked hard to discover, generate, or optimize short
promoters that are cell-type specific, regulated in some way, or
active in all cells. Short promoters would of course allow more
space for longer genes, multiple genes, other regulatory
elements, and/or reporter genes. Promoters that are in use
currently in neuroscience research are (among many others)
hCMV immediate early promoter and enhancer (620 bp),
GUSB promoter (378 bp), CBA promoter (1.7 kb), and EF1a
(1,194 bp). More cell-type-specific expression can be achieved
by using the neuron-specific synapsin-1 promoter (470 bp)
and neuron-specific enolase promoter (1.8 kb) or the glial-
specific GFAP promoter (2.2 kb) (2, 10, 11). Other promoters,
including various drug-regulated promoters and combination
promoters, are constantly being identified, optimized, and
tested. Finally, a novel avenue of approach is to use a promoter
which is active only during certain physiologic states important
for neuronal or glial cell biology, for example, apoptosis, excito-
toxicity, or neurotrophin response. We have noted that the
hCMV promoter takes a few days to generate detectable
expression in cultured neurons (3). Cell-type-specific promot-
ers may speed up the process, or faster expression of the trans-
gene may be achieved by the use of self-complementary
genomes which bypass the rate-limiting step of second-strand
synthesis (12).
3. It is frequently useful to include a reporter gene if the size of
the construct allows it. The internal ribosomal entry site
(IRES) initiates cap-independent translation of a second cis-
tron, with the caveat that expression of the second cistron is
always weaker than the first cistron. The benefit is that, if you
can detect expression of the reporter, it suggests that the
cDNA in the first cistron is being expressed well. Multiple
IRES elements from viruses and mammalian cell have been
identified; Our recent experience suggests that the IRES
from the foot-and-mouth disease virus produces reasonable
cap-independent translation of GFP in 293T cells (S. Malik
and D. Watson, manuscript in preparation.) The IRES-GFP
component in our constructs is 1,164 bp. An alternative
strategy is to include a second promoter to drive expression
of the second cDNA; however, they may compete for tran-
scription factors.
4. Finally, most AAV constructs contain the woodchuck hepatitis
virus posttranscriptional regulatory element before the polyA
sequence to increase transgene expression (13).
5. If you plan to extend your in vitro findings to in vivo experiments,
it is essential to determine whether your serotype transduces
cells in the brain region and/or in the cell type of interest. For
this, a literature search or pilot experiment is necessary.
312 S.Z. Malik et al.

6. For this, a literature search or pilot experiment is necessary. (14, 15).

7. For a more detailed review of these issues, see (16) and refer-
ences therein.

3.4. Construction 1. To construct your genomic plasmid, begin with a plasmid con-
of AAV Genomic taining the ITRs and as many other components as desired.
Plasmid Many useful plasmids are available from academic investigators
and from an extensive AAV plasmid catalog available at the
National Gene Vector Biorepository (https://www.ngvbcc.
org/ReagentRepository.action), as well as from commercial
sources. Be sure a map is supplied and a sequence if possible. It
is highly advisable to sequence the AAV parts of the plasmid
before continuing.
2. It is ESSENTIAL to use competent bacteria that lack recA so
that the ITRs stay intact. For this, a literature search or pilot
experiment is necessary. If the provider of your source plasmid
has not done this, you can be sure that the ITRs are not com-
plete and you should not continue to use that plasmid.
Unfortunately, this is not an uncommon event. Again, this is
extremely important. Appropriate bacterial strains include
SURE2 and STABL2 cell lines which are recA-. Clone in your
promoter and cDNA of interest, as well as other elements. The
AAV genomic plasmids usually contain the polyA element
already; this should be confirmed. Again, you MUST use
competent cells lacking recA to propagate the intermediates.
Sequence the final product, especially the ITRs.
3. Confirm that the cassette is active by transfecting it into 293T
cells. Your gene of interest should be detectable by Western
blot, immunostaining, or other means.

3.5. Packaging of AAV 1. Methods for the packaging and purification of recombinant
AAV vectors are continually improving. For experiments using
neurons in vitro, clinical grade vectors are not required;
research grade vectors are sufficient. If financial resources are
available, we strongly recommend working with an experi-
enced core facility, such as the one at the University of
Pennsylvania (http://www.med.upenn.edu/gtp/vectorcore/),
the University of Michigan (http://www.med.umich.edu/
vcore/), or many other academic vector cores that package
AAV vectors for outside investigators. Alternatively, you can
contact a commercial AAV production company, although it
should be noted that packaging with certain serotypes is only
available at some companies. We would suggest that commer-
cial kits for packaging and purifying AAV vectors in your own
laboratory should be a last resort. We have observed significant
toxicity of purification by-products on neuronal cell cultures.
 26 The Use of Specific AAV Serotypes to Stably Transduce Primary… 313

Also, please note that as of this writing, the National Gene

Vector Laboratory no longer produces vectors.
2. Titering of AAV vectors is also critical to match titers if you are
testing multiple serotypes for your application. Neuronal cell
cultures do not respond well to volumes above ~50 mL in a
35-mm plate. Therefore, the more concentrated vector you
can use, the better. For example, in previous studies, we have
used vectors with titers in the 5 × 10 (12) or 5 × 10 (13) genome
copies; more concentrated vectors will be available as purification
methods become more sophisticated. Controls should include
(1) cultures receiving the same dose in microliter of the vehicle
only (in our case, PBS plus 5% glycerol) or (2) cultures with no
treatment at all. If possible, it is also helpful to test more than
one independent batch of vector to rule out any problems that
might have occurred during production.
3. The issue of serotype may require some testing for your particular
application. In our experience, using reporter genes, AAV1, 8,
and 9 were particularly efficient and nontoxic to neuronal cell
cultures, whereas AAV2 was relatively ineffective (3). AAV5
and AAV6 caused toxicity at the same high doses, which was
measured on a scale of 0–3 (see below). At lower doses, these
two vectors were nontoxic but less efficient. It should be noted
that another group ((1) found AAV6 to be sufficient for neuronal
cell transduction). AAVRh10 shows very promising expression
in vivo (17, 18).

3.6. Concentration Before performing any neuroprotection assays, the dose-response

Response of curve must be determined. This should be performed with a GFP
Neurotoxic Agent reporter vector of the same serotype as the experimental vectors.
Vectors are stored at −80°C in small aliquots and thawed on ice
before use.

3.6.1. Transduction For 96-well plates:

of the Cells for
1. Plate dissociated primary hippocampal neurons (100,000
Neurotoxicity Experiments
cells/mL) in poly-L-lysine-coated 96-well plates.
2. One week after plating, transduce each well with the GFP
reporter vector, at an approximate multiplicity of infection
(MOI) 1.5 × 105. The volume of vector added should not
exceed about 1/10 of the media volume. At 21 days in vitro
(DIV), the cells should be approximately 90% GFP positive.
For 24-well plates:
1. Add a single dose of the GFP reporter vector to cultured rat
hippocampal cells on DIV 7. Expression of GFP should be detect-
able in about 90% of the cells by 1-week posttransduction.
314 S.Z. Malik et al.

Check for any changes in morphology (axonal retraction or

beading) indicating cell toxicity.
For coverslips in a 35-mm plate:
1. Add a single dose of 2.0–2.5 × 1011 genome copies of the GFP
reporter vector to cultured rat hippocampal cells on DIV 7.
The volume of vector added should not exceed about 50 mL in
a 35-mm plate. Expression of GFP should be detectable in
about 90% of the cells by 1-week posttransduction or earlier.

3.6.2. Addition 1. Two weeks after plating, add L-glutamate (0–400 mM) to the
of the Neurotoxic Agent wells, which should still contain the regular medium as well.
There should be a minimum of three replicates per experimen-
tal condition. As a control, transduce some cells but do not
treat with glutamate. These will be processed the same as the
other wells and will serve as the denominator for the calcula-
tion of percent toxicity (see below).
2. Twenty-four hours after addition of glutamate, aspirate the
medium and fix all cells with 10% neutral buffered formalin for
10 min at room temperature in a fume hood. Remove the for-
malin and dispose of it in chemical waste containers. Gently
wash the cells twice in PBS before proceeding. Add a small
drop of VECTASHIELD containing DAPI to each well. At
this stage, the plate can be sealed with parafilm and kept at 4°C
for up to a few days before imaging. Alternatively, the cover-
slips should be mounted on glass slides with the same mount-
ing medium and sealed with nail polish.

3.6.3. Quantification 1. An overall sense of the toxicity can be gained by a quick visual
of Neurotoxicity by Intrinsic inspection of the wells. We use a scale of 0–3, where 0 = healthy
GFP Fluorescence cultures with uniform GFP in the processes; 1 = beaded GFP
expression in the processes but little or no cell loss; 2 = substantial
cell loss and/or fragmented GFP fluorescence in the processes
and cell body; and 3 = majority of cells dead.
2. After that, live cells are quantified. Carefully seal the plate with
parafilm to prevent leakage of any fluid or mounting medium.
In an initial experiment, we obtained digital images of three non-
overlapping fields at 10× with the plate inverted on the micro-
scope stage, which was set to its lowest setting to accommodate
the plate (or with slides mounted normally on the microscope).
3. Manually count cells on each image, three nonoverlapping
fluorescent 10× fields per well and three wells per condition.
To insure reproducibility, it helps to have multiple lab mem-
bers count the cells. Calculate cell survival as percentage of the
untreated wells.
4. Immunostaining is also possible (see Subheading 3.7).
 26 The Use of Specific AAV Serotypes to Stably Transduce Primary… 315

3.6.4. Quantification 1. Once an initial manual cell count is complete, an automated

of Neurotoxicity acquisition protocol can be established. Again, this should be
by an Automated established with a healthy culture first.
Acquisition Protocol
2. First, determine image acquisition settings with your software
that clearly visualize all the DAPI-stained nuclei in a selected
field of a healthy culture (not treated with glutamate) and save
that setting. The nuclei should appear oval to round and bright
enough to be seen clearly without overexposure. Use an objective
of high enough power to see the fluorescence easily, but not so
high that the field of interest contains less than 50 cells. We
generally use a 10× objective.
3. Next, while keeping the microscope on the same field, increase
the image acquisition time so that all of the GFP-positive cells
in that field are clearly visualized. Save that setting as well.
4. Perform this protocol on at least five nonoverlapping fields in
each channel and save the images separately, with filenames
labeled by glutamate dose, fluorescent agent, and field. It is
helpful to save the images by field as you acquire them so it is
easy to assign fields to images as you go along.
5. Overlay the images for each field to confirm that the GFP-
positive cells each contain a DAPI-positive nucleus.
6. Returning to the individual images, use the captured images to
perform an automated count of the cells. It is usually easier to
do this with the nuclei, which are clearly delineated. The settings
for counting can be adjusted to include cells of a particular
range of pixels. These settings should also be saved and used
for all the wells. In our experience, it is sometimes difficult to
determine whether a glutamate-treated cell should be counted
or not, because of its altered morphology. Establishing imaging
and counting protocols on healthy cultures and using the same
parameters for glutamate-exposed cultures will help to keep all
the quantification assays uniform.
7. To validate the automated imaging and counting protocols, con-
firm that the automated values match the manual cell counts.
8. Finally, calculate the percent of live cells for each concentration
of glutamate, using the number of cells in the healthy cultures
as the denominator.

3.6.5. Determine 1. Plot the dose of glutamate against the percentage of live cells.
the Dose-Response Curve Choose the concentration of glutamate to use in all the follow-
ing neuroprotection assays. We prefer a concentration of gluta-
mate that represents about 30% cell survival so that we can
(1) determine whether a neuroprotective response is significant;
(2) determine whether there may be an unexpected result of
neurotoxicity by a supposedly neuroprotective construct; and
(3) have the potential to see significant intermediate degrees of
316 S.Z. Malik et al.

protection since it is unlikely that any given agent will protect

all the cells. In other words, we try to avoid a ceiling or a floor
effect, while retaining the ability to detect degrees of protec-
tion below a complete rescue.
2. If there is any indication that the GFP itself is toxic to the
cultures, substitute a vector of the same serotype containing
LacZ. Alternatively, if you suspect the serotype is toxic, try one
or more alternative serotypes containing GFP.

3.7. Quantification 1. This should be performed with a vector of the same serotype as
of Neuroprotection the reporter vector, but containing the gene of interest. This
technique does not require a GFP-containing vector (although
it does not preclude the use of one). Surviving cells are detected
by immunostaining for the protein of interest.
2. Fix the cells as described above but do not use mounting
medium.
3. A general immunostaining procedure is followed. After fixation,
permeabilize with TBST and block in 5% goat or donkey serum
in TBS. Expose to the primary antibody overnight at 4°C,
wash and incubate with the corresponding secondary antibody,
wash and incubate with the fluorescent streptavidin (1 h at
room temperature each), and then wash again and invert onto
glass slides with VECTASHIELD mounting medium contain-
ing DAPI to identify nuclei. If two antibodies are to be used at
the same time, note that one should be directly conjugated and
the other should be biotinylated. Similarly, the species of the
two antibodies should be different so that the secondary anti-
bodies do not bind to both primary antibodies. As to the fluo-
rescent tags, they should not be in the green range (488 nm) if
a GFP-expressing vector is being used and not in the blue
range (350 nm) to avoid conflict with the blue signal from the
DAPI. In general, we use a red fluorescent tag for the neurons
and, if necessary, a fluor in the 633-nm range for the second
antibody. After the last wash, apply VECTASHIELD mounting
medium with DAPI, about one drop per well. This mounting
medium does not set well, so it is essential to seal the plate well
or to invert the coverslips (if present) on a glass slide, dry, and
seal with nail polish.
4. For this protocol, neurons are identified by immunostaining
with rabbit polyclonal anti-microtubule-associated protein 2
(1:500) and detected with Biotin-SP-conjugated goat anti-
rabbit IgG and Alexa594-conjugated streptavidin (1:500 each).
Astrocytes are identified on separate coverslips with monoclo-
nal anti-GFAP antibody (1:500), Biotin-SP-conjugated donkey
anti-mouse IgG, and Alexa594-conjugated streptavidin (1:500
each). Confirm the specificity of staining by omitting the pri-
mary antibody in each case. Immunofluorescence is colocalized
 26 The Use of Specific AAV Serotypes to Stably Transduce Primary… 317

with GFP intrinsic fluorescence or immunofluorescence for

confirmation.
5. After immunostaining, keep the plates and/or coverslips in the
dark. To image, invert the plate on the lowered microscope
stage as described earlier or mount the glass slide onto the
stage. Keep the room dark to the extent possible.
6. The imaging protocol should be optimized as described above,
except that the fluorescent signal from the antibody should be
optimized on healthy cultures before moving on to the treated
cultures.
7. Confirm the dose response of glutamate by counting the DAPI
cells alone and plotting that number against the concentration
of glutamate. To calculate the percent transduction of live cells,
express the number of GFP-positive cells as (GFP+/DAPI+).
8. Plot the concentration of glutamate vs. the percentage of sur-
viving cells, using the untreated cultures (untreated with vector
and untreated with glutamate) as controls. A second control
should be included, which is the vector alone but no glutamate.
This will confirm that the gene of interest has no independent
effect on neuronal cell survival. We prefer a bar graph with the
healthy cultures at 100% as the first bar.

4. Notes

1. Gridded plastic coverslips may be used as an alternative to the

glass coverslips for some neurotoxicity protocols. Sterilize the
coverslips in ethanol in a covered container overnight at room
temperature. Clean coverslips by adding and aspirating ethanol
six times to multiwell plates that fit the coverslips and check
the orientation under the microscope. The letters should be
right side up. If not, flip the coverslip with sterile forceps but
avoid scratching them. Allow the ethanol to evaporate 30 min
then add 1 mL of poly-L-lysine solution to each well, making
sure each coverslip is submerged, and incubate overnight at
room temperature. Aspirate poly-L-lysine solution from wells,
add 1 mL (BioWhittaker/Lonza) tissue culture water to each
well, making sure coverslips are submerged, and store at room
temperature overnight. Aspirate water and allow it to evapo-
rate 24 h at room temperature. Recheck coverslip orientation
and store in a drawer until ready for preincubation.
2. We find it convenient to preincubate the plates from Friday to
Monday.
3. It is important to note that we do not include the protocol for
Cesarean section of the rat at stage E18 or E19 or the procedure
318 S.Z. Malik et al.

for sterile isolation of embryonic hippocampi in this chapter.

This is a complicated procedure that should be learned from an
experienced professional, should be approved by Animal Care
and Use Committee and your institution, and should conform
to federal guidelines (19). Our cultures are generally derived
from E19 Sprague Dawley rats.

Acknowledgments

The work in our laboratory is supported, in part, by grants from

the National Institutes of Health RO1 NS040978 (DJW), RO1
NS041811 (MAD), a pilot grant from the University of Pennsylvania
Institute for Medicine and Engineering (DJW), and by funds from
the University of Pennsylvania Department of Neurosurgery.
As always, we thank Dr. Sean Grady for mentorship and support.

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 Chapter 27

Preparation and Characterization of Biocompatible Chitosan

Nanoparticles for Targeted Brain Delivery of Peptides
Secil Caban, Yılmaz Capan, Patrick Couvreur, and Turgay Dalkara

Abstract
Here, we describe a nanocarrier system that can transfer chitosan nanoparticles loaded with either small
peptides such as the caspase inhibitor Z-DEVD-FMK or a large peptide like basic fibroblast growth factor
across the blood–brain barrier. The nanoparticles are selectively directed to the brain and are not measurably
taken up by liver and spleen. Intravital fluorescent microscopy provides an opportunity to study the penetration
kinetics of nanoparticles loaded with fluorescent agents such as Nile red, and has demonstrated that this
nanomedicine formulation is rapidly transported across the blood–brain barrier.

Key words: Nanoparticles, Brain drug delivery, Blood–brain barrier, Neurotrophic factors, Caspase
inhibitors, Neuroprotection

1. Introduction

The blood–brain barrier (BBB) is a diffusion barrier essential for

normal function of the central nervous system (1). Only lipophilic
or nonionic water soluble molecules with low molecular weight
can cross the BBB. Essential compounds such as amino acids,
hexoses, and peptides need specific carriers or transporters to
permeate the brain. Accordingly, the majority of available pharma-
ceutical agents cannot efficiently reach the brain parenchyma,
which is one of the bottlenecks in the treatment neurological and
psychiatric disorders (2). One of the strategies developed to over-
come this limitation is to conjugate the drug molecule to an anti-
body directed against one of the carrier proteins expressed on the
luminal surface of brain capillary endothelial cells (e.g. transferrin
receptor) (3–5). Although this approach has been shown to be
functional for the brain delivery of several BBB-impermeable
molecules, its efficiency is generally low because the ratio of drug
to carrier protein is not above 1:1 (6).

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_27, © Springer Science+Business Media, LLC 2012

321
322 S. Caban et al.

Instead of creating chimeric drug–antibody complexes,

conjugating the antibody to the nanoparticles loaded with sub-
stantial amounts of the drug molecule has recently emerged as an
alternative method to improve the efficiency of receptor-mediated
brain drug delivery (7, 8). In rodents, the transferrin receptor
appears to be a promising target for a number of reasons. First,
it is highly expressed on the luminal surface of brain capillary
endothelia (5); second, less than a quarter of the receptors are
saturated by the circulating transferrin (9), leaving plenty of
unoccupied receptors for the antibodies to bind; and finally, it is
rapidly shuttled between the luminal and abluminal membranes
(turnover half time is 2–3 min) to provide high throughput
transferrin delivery to the brain (10). Furthermore, using specific
antibodies that target the transferrin receptor subtype-1 located
mainly on the brain endothelium, nanoparticles can selectively be
targeted to the brain without affinity to the transferrin receptors in
other tissues predominantly expressing the subtype-2 (10–12).
Nanoparticles are generally made up of biocompatible poly-
mers (13). They are coated with hydrophilic polymers such as
poly(ethylene glycol) (PEG) to reduce rapid clearance from the
circulation by the reticuloendothelial system (14) (see Fig. 1).
Chitosan is a polymer obtained by deacetylation of chitin, a natu-
rally occurring polysaccharide in shells of crustaceans. Chitosan is
highly biocompatible; it does not lead to allergic reactions or rejec-
tion, and is degraded to nontoxic amino-sugars in tissues (15).
Another advantage of chitosan is that it can be prepared under
exceptionally mild conditions owing to its hydrophilic nature,
making it particularly attractive for loading delicate compounds
such as peptides and functional macromolecules (16–18).
Experimental studies suggest that neurotrophic factors provide
protection against acute as well as chronic neurodegeneration (19).
Similarly, the small peptide inhibitors of proteases that play
important roles in cell death (such as caspases) are also neuropro-
tective (20, 21). One important advantage of both classes of agents

Fig. 1. Schematic outline of nanoparticle preparation. Peptides (Z-DEVD-FMK or bFGF) or Nile red are loaded into CS–PEG–BIO
polymers and then nanosperes are formed by ionic gelation with tripolyphosphate solution (TPP). The nanoparticles are
conjugated with anti-mouse transferrin monoclonal antibodies (TFRMAb) by means of biotin–streptavidin bonds (reprinted
and modified from ref. (7) with permission).
 27 Preparation and Characterization of Biocompatible Chitosan… 323

is that they have a relatively long therapeutic time window in

acute disorders such as cerebral ischemia (21–23), increasing their
chance to be successfully translated to stroke treatment in the
clinic. Unfortunately, however, both groups of these peptidergic
agents cannot cross the BBB.
We have recently demonstrated that PEGylated chitosan
nanoparticles conjugated via avidin–biotin linkage with the anti-
transferrin antibody could efficiently deliver these promising agents
to the central nervous system, and provide neuroprotection when
given systemically (7, 24). This nanomedicine rapidly penetrates
the brain, possibly by combining the affinity of the antibody for the
transferrin receptor to trigger receptor-mediated transcytosis across
the BBB with the ability of cationic chitosan to interact with the
negative charges on the surface of the brain capillary endothelium
(25). The combined systemic administration of the nanoparticles
loaded with basic fibroblast growth factor (bFGF) and a caspase
inhibitor (Z-DEVD-FMK) substantially reduced infarct volume as
well as neurological deficit after experimental focal ischemia in the
mouse (26), similar to the results obtained with their intracere-
broventricular administration (27). Thus, chitosan nanoparticles
open new and exciting opportunities for brain delivery of biologically
active peptides including neurotrophic factors and hence hold
promise for the treatment of central nervous system disorders.

2. Materials

Prepare all solutions using ultrapure water prepared by purifying

deionized water to attain a sensitivity of 18 MΩ cm at 25°C. Animal
housing, care, and application of experimental procedures were all
done in accordance with institutional guidelines. All animal experi-
ments described in this and the previous section were approved by
Hacettepe University Ethics Committee. Modified chitosan poly-
mers were provided by Eduardo Fernandez-Megia, Ramon Novoa-
Carballal, and Ricardo Riguera from Universidad de Santiago de
Compostela, Spain.

2.1. Preparation of 1. Dissolve chitosan (CS) in water to obtain a concentration of

Chitosan and Surface- 1.75 mg/mL.
Modified Chitosan 2. Dissolve chitosan–polyethylene glycol (CS–PEG) in water to
Nanoparticles obtain a concentration of 1 mg/mL.
3. Dissolve chitosan–polyethylene glycol–biotin (CS–PEG–BIO)
in water to obtain a concentration of 1 mg/mL.
4. Dissolve sodium tripolyphosphate (TPP) in water at two
different concentrations: 0.4 mg/mL and 0.84 mg/mL (see
Notes 1 and 4).
324 S. Caban et al.

5. N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-
fluoromethyl ketone (Z-DEVD-FMK) (BACHEM, USA):
Centrifuge a vial containing 1 mg of the peptide and then
dissolve the contents in 75 μL dimethyl sulfoxide. Divide the
solution into aliquots. Next, dilute each aliquot with water and
further divide into smaller working aliquots. Store at −20°C
until use (see Note 5).
6. Centrifuge a vial containing 50 μg of bFGF (Invitrogen, USA)
and then dissolve the contents in 500 μL water to obtain a
solution of 100 μg/mL. Divide this solution into smaller
working aliquots and store at −20°C (see Note 5).
7. Nile red: prepare a 1 mg/mL solution in dimethyl sulfoxide.

2.2. Conjugation 1. Dissolve 380 mg of tetrasodium ethylenediaminetetraacetic

of Streptavidin and acid in 10 mL water to obtain a 0.1 M solution.
Monoclonal Antibody 2. Dissolve 7 mg of streptavidin (Sigma) in 250 μL water.
to CS–PEG–BIO
3. Dissolve 40 mg of Traut’s reagent (2-immunothiolane,
Nanoparticles Thermo Fisher Scientific Inc, Pierce, USA) in 10 mL water to
yield a 4 mg/mL solution.
4. Prepare a solution of 34 mg NaOH, 310 mg boric acid, and
373 mg potassium chloride dissolved in 100 mL water.
5. Functional grade anti-mouse monoclonal antibody to the
transferrin receptor (TfRMab) (functional grade purified anti-
mouse CD71, eBioscience, USA, clone R17217): prepare a
1 mg/mL solution.
6. Phosphate buffered saline (PBS, pH 7.4) is used as the release
medium in release studies.

3. Methods

3.1. Preparation 1. Dissolve CS or the surface-modified CS (CS–PEG or CS–PEG–

of Nanoparticles BIO) polymers in water by magnetic stirring at 700 rpm. CS is
dissolved in half an hour whereas dissolving CS–PEG and
CS–PEG–BIO requires 45–50 min (see Fig. 2) (28).
2. Dissolve TPP in water by magnetic stirring at 700 rpm.
3. Add Z-DEVD-FMK or bFGF or Nile red solution into the
polymer solution after the polymer is dissolved. The final con-
centration of polymer must be kept the same with the begin-
ning concentration mentioned in Materials (see Note 2).
4. Add TPP solution drop-wise onto the polymer solution while
stirring. Volumetric ratio of the polymer to TPP, which is a
cross-linker used to form nanoparticles, depends on nanopar-
ticle type. For CS nanoparticles the ratio is 1/1 mL, whereas it
 27 Preparation and Characterization of Biocompatible Chitosan… 325

Fig. 2. Chemical structures of chitosan (Chi) and sodium tripolyphosphate, and schematic illustration of chitosan nanoparticle
preparation procedure with ionotropic gelation method. (TPP: tripolyphosphate solution). (Modified from ref. (28) with
permission).

is 5/2 mL for CS–PEG and CS–PEG–BIO nanoparticles (see

Note 3).
5. Continue stirring the nanoparticle suspension for 15 min.
6. Centrifuge the nanoparticle suspension at 10,000 rpm
(9,277 g) at +4°C for 1 h.
7. Discard the supernatant and resuspend in water for in vivo
administration or resuspend in PBS for release studies (see
Tables 1 and 2, Fig. 3).

3.2. Conjugation 1. Add 1,900 μL of borate buffer onto 100 μL of Traut’s reagent
of TfRMAb to and stir for 5 min on a magnetic stirrer.
Nanoparticles 2. Take equal volumes of the above and of the streptavidin
solution and mix together on a magnetic stirrer for 90 min to
obtain thiolated streptavidin.
3. In parallel, stir 100 μL of TfRMAb solution (1 mg/mL) with
5 μL of m-maleimidobenzoyl-N-hydroxysuccinimide solution
(5 mg/mL in dimethylformamide) for 30 min at room tem-
perature. This will transform some of the amino groups into
maleimide groups.
326 S. Caban et al.

Table 1
Examples of nanoparticle size and zeta potential

Polymer type Particle size (nm) Zeta potential (mV)

CS (blank) 301 22
CS (Z-DEVD-FMK loaded) 650 20
CS (bFGF loaded) 424 23

Table 2
Examples of drug association efficiency and loading
capacity of nanoparticles

Nanoparticles % AEa % LCb

CS (Z-DEVD-FMK loaded) 23 0,019 × 10−3

CS (bFGF loaded) 21 2,739 × 10−3
a
% Association Efficiency (% AE) = 100 × (total peptide amount − free peptide
amount)/total peptide amount
b
% Loading Capacity (% LC) =100 × (total peptide amount − free peptide
amount)/nanoparticle weight

Fig. 3. Scanning Electron Microscopy (SEM) images of loaded and PEGylated chitosan
nanoparticles.
 27 Preparation and Characterization of Biocompatible Chitosan… 327

Fig. 4. A typical in vitro release profile of a peptide from chitosan nanoparticles.

4. After preparing CS–PEG–BIO nanoparticles, add 12.5 μL of

streptavidin solution and 12.5 μL of antibody solution to the
nanoparticle suspension and incubate for 30 min at room
temperature.

3.3. Release Studies 1. Add 1 mL of PBS in each release compartment after discarding
from Nanoparticles supernatant.
2. Place each eppendorf tube in a water bath at 37°C on a horizontal
shaker.
3. Centrifuge nanoparticles at 10,000 rpm (9,277 g) for 10 min
and then filter at predetermined time intervals (see Note 6).
4. Determine the concentration at each time point and calculate
the cumulative values (see Fig. 4).

3.4. In Vivo Monitoring 1. Anesthetize mice with isoflurane during surgery and with
of Nanoparticle urethane (750 mg/kg, intraperitoneal, followed by 500 mg/kg
Penetration 30 min later) during the experiment.
into the Brain 2. Monitor body temperature with a rectal probe and maintain at
37.0 ± 0.2°C using a homeothermic blanket control unit
(Harvard Apparatus). Monitor systolic blood pressure nonin-
vasively by using a cuff and tail probe (NIBP controller;
ADInstruments). Monitor pulse rate and oxygen saturation by
an oxymeter using amini Y clip on the left lower extremity
(V3304 Tabletop Pulse Oxymeter; Surgivet) (see Note 7).
3. Open a cranial window of 5 × 5 mm over the parietotemporal
cortex and leave the dura intact to maintain physiological con-
ditions. Seal the window with dental acryl and then fill it with
artificial cerebrospinal fluid (124 mM NaCl, 5 mM KCl, 1.25 mM
NaH2PO4, 1.3 mM MgSO4, 2.4 mM CaCl2, 25 mM NaHCO3,
and 10 mM glucose; pH 7.4) at 37°C. (see Notes 8 and 9).
328 S. Caban et al.

Fig. 5. Nanoparticles are rapidly transported to brain parenchyma after systemic administration. (a) The graph illustrates
the change in fluorescence recorded from the brain over the course of 3 h after injection of Nile red loaded to TfRMAb-
conjugated or unconjugated (TfRMAb-free) nanoparticles. The difference between the two lines (triangles) reflects the fluo-
rescence coming from the nanoparticles within the parenchyma and illustrates the time course of nanoparticle penetrance
to the brain. (b) Nile red concentration in brain postvascular tissue increased only when TfRMAb-conjugated nanoparticles
were administered. The graph illustrates spectrophotometric measurements at 549 nm from brain homogenates obtained
1 h after injection of TfRMAb-conjugated or unconjugated nanoparticles or from sham-operated mice. Only values above
the horizontal thick line, below which values correspond to the tissue background readings, were taken into consideration.
*p < 0.05 compared with the other groups. (c) Confirmation of penetration of the nanoparticles to the parenchyma by fluo-
rescence microscopy on brain sections obtained 1 h after injection. FITC-conjugated anti-rat IgG antibody (green) labeled
the nanoparticles bearing TfRMAb, clearly demonstrating that the nanoparticles were dispersed within the extracellular
space (arrowheads) outside the vessel lumens (longitudinal structures). Some FITC-conjugated nanoparticles exhibited
green as well as red fluorescence because they had not released of all the Nile red loaded within an hour (images on the
right ). Scale bars: (c), left, 15 μm; (c), right, 5 μm. (Reprinted from ref. (7) with permission).
 27 Preparation and Characterization of Biocompatible Chitosan… 329

4. Capture fluorescent images under a microscope at 100× magni-

fication in a dark room by using appropriate camera and
imaging software (see Note 10; Fig. 5).
5. Save images in TIFF format, and calculate the mean fluoro-
scence intensity of the area imaged with the software.
6. Record sequential images before starting the experiment to
obtain baseline. After the systemic (intravenous) injection of
Nile red-loaded nanoparticles, record pictures at predeter-
mined time intervals (e.g. 1, 5, 10, 20, and 30 min, and then
every 15 min for 3 h) using the same exposure time and gain
settings.
7. Determine the changes in fluorescence (red) intensity from
baseline after injection of the antibody-conjugated or the
unconjugated nanoparticles. Since unconjugated nanoparticles
cannot penetrate the brain, they reflect the Nile red fluores-
cence coming from the nanoparticles within the circulation.
Subtract this signal from the signal obtained with antibody-
conjugated nanoparticles to obtain the net signal coming from
the nanoparticles penetrating the brain.
8. At the end of the experiment, perfuse mice transcardially with
saline to flush intravascular content including the nanoparticles
and then extract the brain, liver, and spleen. Tissues are imme-
diately frozen and kept at −80°C until the determination of
Nile red by UV spectrophotometry, which gives an estimate of
the nanoparticles translocated to the parenchyma of the tissues
examined (see Fig. 5).

3.5. Histological 1. Obtain fresh frozen, 20 μm-thick coronal brain sections.

Detection of 2. Fix the sections with 96% alcohol for 10 min, wash with PBS,
Nanoparticles and then immunostain with FITC-conjugated goat anti-rat
in the Brain IgG antibody (Sigma, at 1:100 and 1:200 dilutions, in PBS) at
room temperature for 60 min to detect the nanoparticles
conjugated with TfRMAb, which is a rat IgG2a (see Note 11).
3. Block the sections with 10% normal goat serum for 10 min at
room temperature and immunostain with anti-FGF-2/bFGF
polyclonal antibody (Millipore, 1:100 in PBS) at +4°C over-
night. After washing with PBS for 5 min two times, incubate
with cy3-conjugated AffiniPure goat anti-rabbit IgG (Jackson
Immunoresearch, 1:200 in PBS) at room temperature for
90 min to immunostain human bFGF-loaded nanoparticles.
4. Finally, coverslip with mounting medium containing Hoechst
33258 to counterstain the nuclei in order to obtain tissue ori-
entation under the microscope.
5. Check co-localization of the IgG immunoreactive spots (green)
under a fluorescent microscope with Nile red fluorescence or
with immunoreactivity for human bFGF, depending on
330 S. Caban et al.

whether the nanoparticles have been loaded with Nile red or

bFGF (see Fig. 5).
6. We routinely carry out negative controls by omitting the primary
antibody.

4. Notes

1. The concentrations of the polymer and cross-linker solutions

are very critical; concentration changes may result in undesirable
aggregation or precipitation of nanoparticles or may change
the particle size. Use the same concentration and timing to
obtain uniform nanoparticle batches.
2. If you want to load a new peptide or another hydrophilic
substance into the nanoparticles, you may either prepare a solu-
tion of substance and add it into the polymer/cross-linker
solution or directly dissolve it in the polymer/cross-linker solu-
tion. Attention must be paid to the final concentration of
the solution if you choose to add the drug solution into the
polymer/cross-linker.
3. Also, use a fixed TPP dropping rate into polymer solution to
obtain uniform batches. Using a syringe without plunger may
provide reproducible drop rates for every trial.
4. Use a vial with a cap to prevent contamination of solutions
from environmental pollutants during magnetic stirring.
5. While working with peptides like bFGF or Z-DEVD-FMK, it
is a good idea to separate them into small aliquots and store
them at −20°C until use. Keep the volume as low as possible
because every aliquot can be used only once.
6. During release studies, filter the supernatant through a 13 mm
diameter 0.22-μm cellulose acetate syringe filter before deter-
mining the concentration.
7. For intravital microscopy experiments, it is essential to maintain
the physiological conditions (e.g. normal body temperature,
blood pressure, and oxygenation) so as not to cause cerebro-
vascular dysfunction.
8. Wash the cranium frequently with saline to avoid heating while
drilling the cranial window.
9. Drill the whole window area until the bone is thinned to a film.
Gently lift and peel the bony film with a pair of forceps without
damaging the underlying dura mater and cortex.
10. Make sure that the cranium is well fixed and not moving with
animal’s respiration, and that the artificial cerebrospinal fluid
 27 Preparation and Characterization of Biocompatible Chitosan… 331

level over the window and the lighting conditions are constant
while recording sequential fluorescent images.
11. For histological detection of nanoparticles in the brain paren-
chyma, it is preferable to use two markers fluorescing at different
wavelengths (e.g. anti-IgG (green) and Nile red) and co-localize
both signals to ensure that the observed spots are not micro-
scopic staining artifacts.

Acknowledgements

We wish to thank all of our colleagues including our collaborators

in Spain (Eduardo Fernandez-Megia, Ramon Novoa-Carballal,
and Ricardo Riguera from Universidad de Santiago de Compostela)
for their important contributions to the development of the nano-
particles used in studies cited in this chapter. Dr. Turgay Dalkara’s
work is supported by the Turkish Academy of Sciences.

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 Chapter 28

[3H]Serotonin Release Assay Using Antigen-Stimulated

Rat Peritoneal Mast Cells
Stephen D. Skaper and Laura Facci

Abstract
The concentration of nerve growth factor (NGF) is elevated in a number of inflammatory and autoimmune
states in conjunction with increased accumulation of mast cells. Mast cells, which are of hematopoietic
lineage, and NGF appear to be involved in neuroimmune interactions and tissue inflammation. Mast cells
themselves are capable of producing and responding to NGF. Here we describe a protocol for the isolation
and culture of peritoneal-derived rat mast cells, together with a [3H]serotonin release assay which is useful
in assessing the effects of antigens and neurotrophic factors on mast-cell activation.

Key words: Mast cells, Antigen, Degranulation, Serotonin, Nerve growth factor, Nerve–immune
interaction

1. Introduction

Neurotrophic proteins promote neurite outgrowth, neuronal cell

differentiation, and survival in vivo and in vitro. Nerve growth fac-
tor (NGF) represents the first and best characterized member of
the neurotrophin family (1). We now know that NGF displays bio-
logical activities in a broad spectrum of cell types outside the ner-
vous system and is produced by a wide range of cell populations
not normally considered targets for innervation by NGF-dependent
neurons, including cells of the immune–hematopoietic lineage.
Mast cells are a heterogeneous immune-effector cell type found in
connective tissues throughout the body, occur adjacent to blood
and lymphatic vessels, and are concentrated beneath mucosal sur-
faces (2). Mast cells were the first cells of the immune lineage to be
recognized as a target for NGF, both in vitro (3) and in vivo (4).
In addition, NGF stimulates the proliferation of both B and T
lymphocytes (5, 6), promotes human hematopoietic cell growth

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_28, © Springer Science+Business Media, LLC 2012

333
334 S.D. Skaper and L. Facci

and differentiation (7), and acts as an autocrine survival factor for

memory B lymphocytes (8). Moreover, human monocytes (9),
activated CD4+ T-cell clones (10, 11), and lymphocytes (12)
express the NGF receptor TrkA.
Mast cells occur in many peripheral tissues, in perivascular
regions in close apposition to innervating sensory or autonomic
nerve fibers, and also within the peripheral and central nervous
systems. Mast cells secrete and respond to mature NGF (13–15)
and are capable of synthesizing higher molecular weight forms of
biologically active NGF and other neurotrophins (16), suggesting
the possibility for autocrine/paracrine effects of these neurotrophic
factors on mast cells. Given the interconnecting links between
inflammation, pain, NGF, and mast cells (17), procedures for their
isolation and analysis of NGF effects represent an important ele-
ment in the study of neurotrophic factors. The following chapter
presents a protocol for the isolation and purification of rat perito-
neal mast cells, and their application to the measurement of [3H]
serotonin release.

2. Materials

2.1. Equipment 1. Laminar flow biological safety cabinet (CL2)

and Labware 2. Humidified, water-jacketed culture incubator at 37°C and 5%
CO2/95% air
3. Water bath set at 37°C
4. Dissecting tools (Fine Science Tools (InterFocus Ltd) are very
high quality for the price)
5. Bench centrifuge to accommodate 15- and 50-mL tubes
6. 15- and 50-mL polypropylene plastic centrifuge tubes (sterile)
7. 0.45-mm filters (Millipore)
8. 50-mL syringes
9. Syringe needles (1.5-in.)
10. 0.5-mL Eppendorf ® LoBind microcentrifuge tubes
11. Vacuum-driven filter units (0.45-mm pore size, 250- and
500-mL capacity) from Millipore (Stericup®)

2.2. Cell Culture Media, 1. Bovine serum albumin (BSA), Cohen fraction V (Sigma)
Supplements, and [ 3H] 2. RPMI-1640 medium (Invitrogen)
Serotonin Release
3. L-Glutamine (200 mM, sterile, for cell culture) (Invitrogen)
Assay Reagents
4. Penicillin/streptomycin (10,000 U/mL penicillin and
10,000 mg/mL streptomycin) (Invitrogen)
5. Trypan blue stain 0.4% (Invitrogen)
 28 [3H]Serotonin Release Assay Using Antigen-Stimulated Rat Peritoneal Mast Cells 335

6. Glutaraldehyde (25% in water) (Sigma)

7. Safranin O (Sigma)
8. Mouse monoclonal anti-DNP IgE, clone SPE-7 (Sigma)
9. Dinitrophenylated human serum albumin (DNP-HSA)
(Sigma)
10. [3H]serotonin (Perkin Elmer)
11. b-NGF (recombinant human) (Peprotech)
12. Brain-derived neurotrophic factor (BDNF, recombinant
human) (Peprotech)
13. Neurotrophin-3 (NT-3, recombinant human) (Peprotech)
14. Neurotrophin-4 (NT-4, recombinant human) (Peprotech)
15. Substance P (Sigma)
16. Phosphate-buffered saline (PBS) (Invitrogen)

2.3. Culture Medium 1. PBS + 0.6 % (w/v) glucose: Dissolve 0.6 g of D-glucose in
and Solutions 100 mL of PBS and filter-sterilize.
2. 40 % (w/v) BSA in PBS/0.6 % glucose: Dissolve 40 g of BSA in
100 mL of PBS and filter-sterilize (see Note 1).
3. 30 % (w/v) BSA: Dilute the 40% glucose solution (75 mL) in
PBS/0.6% glucose (25 mL).
4. Culture medium: To 100 mL of RPMI-1640 medium, add
1 mL of 200 mM L-glutamine stock solution (2 mM final) and
0.5 mL penicillin/streptomycin 100× stock solution (50 U/
mL penicillin and 50 mg/mL streptomycin, final).
5. PIPES buffer pH 7.10: 25 mM PIPES (7.56 g/L), 100 mM
NaCl (5.84 g/L), 5 mM KCl (0.372 g/L), 1 mM CaCl2·2H2O
(0.147 g/L), MgCl2×6H2O (0.81 g/L), and 5.6 mM D-glucose
(1.00 g/L).

3. Methods

3.1. Isolation 1. Adult male CD Sprague Dawley rats (200–250 g) are used. The
of Mast Cells recommended procedure for euthanasia is CO2 asphyxiation
and cervical dislocation. This should be carried out in strict
compliance with approved institutional and national guidelines.
2. Using a 50-mL syringe (1.5-in. needle), inject into the perito-
neal cavity 40 mL of sterile PBS/0.6% (w/v) glucose.
3. Gently massage the abdomen for about 90 s.
4. Carefully open the peritoneal cavity and collect the fluid contain-
ing peritoneal cells. Take care to avoid entering internal organs,
which can contaminate the wash solution (see Notes 2 and 3).
336 S.D. Skaper and L. Facci

5. Transfer the peritoneal collection fluid into 50-mL polypropyl-

ene tissue culture centrifuge tubes. Do not place on ice.
6. Centrifuge at 200 × g for 5 min at room temperature.
7. Remove the supernatant with a 10-mL pipette, leaving 5 mL
of liquid in the bottom of the tube.
8. To a 50-mL polypropylene tissue culture centrifuge tube, add
5 mL of the 40% BSA solution, then carefully layer 5 mL of the
30% BSA solution over this (see Note 4).
9. Layer over this BSA gradient up to 20 mL of cell suspension
carried over from the initial centrifugation in step 5. Using
volumes greater than 20 mL will decrease the efficiency of the
separation process—use multiple gradients for large samples.
10. Centrifuge the BSA gradient tube(s) at 200 × g for 5 min at
room temperature.
11. Remove the BSA phases with a 10-mL pipette, leaving the bot-
tom 10 mL in each tube.
12. Using a long (9-in.) Pasteur pipette, continue removing liquid
from the tubes in step 9, leaving only the last 2 mL in the bot-
tom of each tube.
13. To each tube, now add 10 mL of PBS/0.6% glucose.
14. Centrifuge at 200 × g for 5 min, room temperature.
15. Remove the supernatant(s) and resuspend each cell pellet in
2 mL of RPMI-1640 + glutamine + penicillin/streptomycin.
16. To 90 mL of this cell suspension, add 10 mL of 0.4% trypan
blue.
17. Count cells (see Chap. 3). Expect a yield of 300,000–400,000
cells per rat.
18. Expect a final purity of mast cells >90%. If you want to check
this for yourself, you can perform Alcian blue and Safranin
staining (optional) (18). See Subheading 3.2 below.
19. Plate cells according to experimental design, using the above
RPMI-1640 culture medium.

3.2. Safranin Mast cells can be divided into two types: connective tissue mast
O Staining cells and mucosal mast cells. Peritoneal mast cells belong to the
of Mast Cells first type. Mast-cell phenotypes can be classified in terms of their
profiles of proteinases and proteoglycans. In this regard, mucosal
mast-cell granules stain with Alcian blue rather than with Safranin,
while connective tissue mast cells stain with Safranin but not with
Alcian blue:
1. This procedure makes use of the Shandon Cytospin three
centrifuge (Block Scientific, Bohemia, NY, USA, www.
blockscientificstore.com).
 28 [3H]Serotonin Release Assay Using Antigen-Stimulated Rat Peritoneal Mast Cells 337

2. A 0.1-mL cytofunnel disposable sample chamber is adequate

for this purpose. Pipette into the chamber 0.1 mL of mast-cell
suspension, which has been diluted to ~400,000 cells per milliliter
in culture medium.
3. Cytospin the mast cells onto a glass slide at 700 rpm for 5 min.
The cytofunnel chamber is attached to the glass slide. The
manufacturer-supplied directions provide illustrative examples
of the technique and should be followed in this regard.
4. Fix the cells with 0.1% glutaraldehyde (diluted 1:250 from a
25% stock solution in water) in PBS for 20 min at room
temperature.
5. Rinse three times with PBS.
6. Rinse quickly with 1% acetic acid (1:100 (v/v) of glacial acetic
acid, in double-distilled H2O) for 10 s.
7. Stain in 0.1% Safranin O solution (0.1 g in 100 mL of 0.125 N
HCl) for 5 min.
8. Rinse twice with PBS.
9. Keep the slide(s) in double-distilled H2O or PBS.
10. Observe under a light microscope without phase contrast
optics—Safranin-stained mast-cell granules will appear red.

3.3. Measuring [ 3H] Mast cells have high-affinity IgE receptors (FceRI) (19) and have
Serotonin Release been used extensively to study the signaling pathways leading to
from Antigen- exocytotic release of inflammatory mediators during antigen
Stimulated Mast Cells stimulation of mast cells (20, 21). In this assay, a mouse monoclo-
nal IgE which is specific for dinitrophenol (DNP) haptens (clone
SPE-7) is used. DNP-HSA is employed as the triggering agent in
this assay. Cells are preloaded with [3H]serotonin (5-[1,2-3H(N)]
hydroxytryptamine binoxalate) which is released upon FceRI
cross-linking.
1. Isolate rat peritoneal mast cells, as described in Subheading 3.1
above.
2. Plate mast cells in a 96-well tissue culture plate (brand is not
important) at a density of 50,000 cells per well in RPMI-1640
medium with 2 mM L-glutamine and 50 U/mL penicil-
lin + 50 mg/mL streptomycin + 0.1 mCi of [3H]serotonin. Final
volume: 100 mL per well.
3. Incubate cells for 18 h in a 5% CO2 incubator (37°C).
4. Replace this medium with 100 mL of PIPES buffer containing
anti-DNP IgE (0.3 mg/mL) to sensitize the cells.
5. Incubate for 1 h at 37°C.
6. Replace the IgE solution with 100 mL per well of prewarmed
PIPES buffer containing 0.1–1.0 mg/mL of DNP-HSA.
338 S.D. Skaper and L. Facci

The level of conjunction averages 30–40 mol of DNP per mol

of albumin.
7. Incubate for 15 min at 37°C.
8. Collect the incubation medium and add 100 mL per well of 1%
Triton X-100 to solubilize the cells (see Note 5).
9. To one vial for liquid scintillation counting of radioactivity,
transfer 70 mL of incubation medium, and to a second vial,
transfer 70 mL of the Triton extract. Add to each vial an appro-
priate volume of liquid scintillation counting fluid and place in
a liquid scintillation analyzer.
10. Calculate the percentage of [3H]serotonin release as [released
dpm/(released dpm + cell-associated dpm)] × 100.
11. Always subtract the background (spontaneous release) from
the stimulated release value (“net” release).

3.4. Assay Application NGF is known to induce mediator release from mast cells (22).
to Study of NGF Effects of NGF on stimulus-evoked mediator release from mast
Regulation of Mast- cells can be assessed using the [3H]serotonin release assay described
Cell Function here. A representative experiment is now described, in which rat
peritoneal mast cells are incubated with NGF, together with spe-
cific antigen (15) or substance P (23) to induce degranulation. The
steps below are taken from Subheading 3.3 above:
1. Seed mast cells into a 96-well tissue culture plate, 50,000 cells
per milliliter.
2. Load with [3H]serotonin for 18 h.
3. Remove this solution and prime cells with anti-DNP IgE for
1 h at 37°C.
4. Replace the IgE solution with 100 mL of prewarmed PIPES
buffer containing DNP-HSA (0.1 mg/mL) or substance P
(30 mM).
5. In replicate wells containing DNP-HSA or substance P, add
the following neurotrophins to a final concentration of 100 ng/
mL: NGF, BDNF, NT-3, or NT-4. The neurotrophin is added
to the DNP-HSA or substance P–containing solution from a
25-mg/mL stock solution (i.e., 1:250) (see Note 6).
6. Continue incubation for a further 15 min.
7. Determine net release of [3H]serotonin.
8. Note that a significant increase in secretion of preloaded [3H]
serotonin occurred in mast cells treated with specific antigen or
substance P, in combination with NGF, but not BDNF, NT-3,
or NT-4 (see Table 1).
 28 [3H]Serotonin Release Assay Using Antigen-Stimulated Rat Peritoneal Mast Cells 339

Table 1
Exogenous NGF regulates mast-cell function

Net [3H]serotonin release (%)

Stimulus None NGF BDNF NT-3 NT-4

Antigen 9.3 ± 2.1 20.3 ± 4.2a 7.3 ± 3.1 6.1 ± 3.7 5.5 ± 1.8
Substance P 6.7 ± 2.9 18.9 ± 5.9a 5.9 ± 4.0 7.9 ± 3.5 –
3
Rat peritoneal mast cells were preloaded with [ H]serotonin and then chal-
lenged with either a specific antigen (IgE) or substance P (30 mM), without or
with the indicated recombinant neurotrophin (100 ng/mL). Net release of
[3H]serotonin was determined, as described in Subheading 3.3. Values are
means ± SD (three experiments). ap < 0.05. NGF alone did not affect basal
[3H]serotonin release. Reprinted with permission from Elsevier (17)

4. Notes

1. Dissolving this amount of BSA can be difficult. We find it easi-

est to add the required amount of PBS to a beaker with stir bar,
place on the magnetic stir plate, and add the entire amount of
BSA. Turn on the stir plate (low speed) and allow the BSA to
slowly dissolve from the interface between powder and liquid.
A 100-mL solution at 40% BSA will take several hours to fully
dissolve. Do not stir vigorously (avoid foaming) and do not
heat. Vacuum-driven filter units (0.45-mm pore size) from
Millipore (Stericup® or Steritop®) work well for sterilization.
2. This is a critical step: The peritoneal lavage should be straw-
colored and will be lightly opaque. The major contaminants
are blood cells and debris from compromised internal organs,
intestine in particular. Difficulty may be encountered when
withdrawing the lavage using the syringe used for injection of
the lavage solution. An alternative method collection is to
make a small incision on the side of the injection and enter the
peritoneal cavity with a 25-mL pipette. Withdraw the contents
of the cavity toward the periphery, again avoiding internal
organs (we actually prefer this route for collection).
3. Once proficiency is developed, it is possible to process up to
25 rats in a period of 90 min. However, the technique is not
foolproof, and even an experienced researcher should not be
surprised if he/she is unable to achieve a success rate better
than 80%.
4. Layering of the BSA solutions is best done using a 5-mL pipette
and automatic pipettor—the important thing is not to allow
340 S.D. Skaper and L. Facci

the two gradient solutions to mix, i.e., a steady hand is needed.

Doing this under a tissue culture flow cabinet, we usually do
this at eye level (which means getting down on your knees) so
that you can physically observe the 30% BSA solution flowing
on top of (but not into) the 40% solution beneath.
5. Care must be exercised when treating mast cells with com-
pounds where dimethyl sulfoxide or ethanol is used as solvent,
as these will decrease mast-cell release of serotonin. Dimethyl
sulfoxide or ethanol, if used, should not exceed 0.2% by
volume.
6. The neurotrophins are supplied as solids. When reconstitut-
ing, it is important to use a solution with carrier protein, for
example, PBS with 0.1% BSA. Neurotrophins (NGF, BDNF,
NT-3, NT-4) have a very high isoelectric point (which means
they are quite basic) and will stick to glass and plastic
(especially polystyrene) surfaces. We recommend reconstitu-
tion in the container provided by the supplier to a concentra-
tion ³25 mg/mL. Aliquot this stock into 0.5-mL
low-protein-binding Eppendorf tubes (10–20 mL/tube) and
store at −20°C for up to 6 months. Once thawed, an aliquot
can be safely kept in the refrigerator for up to 4 weeks. Do
not subject BDNF to repeated freeze–thaw cycles.

Acknowledgments

L. Facci was supported by Fondazione CARIPARO Progetto

Dottorati di Ricerca Anno 2009.

References
1. Levi-Montalcini R (1987) The nerve growth 6. Otten U, Ehrhard P, and Peck R (1989) Nerve
factor 35 years later. Science 237, 1154–1162 growth factor induces growth and differentia-
2. Galli SJ, Nakae S, and Tsai M (2005) Mast cells tion of human B lymphocytes. Proc Natl Acad
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3. Böhm A, Aloe L, and Levi-Montalcini R (1986) Denburg JA (1988) Nerve growth factor pro-
Nerve growth factor enhances precocious dif- motes human hemopoietic colony growth and
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10. Ehrhard PB, Erb B, Graumann U, and Otten U active high molecular weight neurotrophins.
(1993) Expression of nerve growth factor and Mol Brain Res 97, 177–185
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Proc Natl Acad Sci USA 90, 10984–10988 functions. Mol Neurobiol 24, 183–199
11. Lambiase A, Bracci-Laudiero L, Bonini S, 18. Mayrhofer G (1980) Fixation and staining of
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12. Santambrogio L, Benedetti M, Chao MV, 19. Metzger H (1992) The receptor with high
Muzaffar R, Kulig K, Gabellini N, et al (1994) affinity for IgE. Immunol Rev 125, 37–48
Nerve growth factor production by lympho- 20. Benhamou M, Stephan V, Robbins KC, and
cytes. J Immunol 153, 4488–4495 Siraganian RP (1992) High-affinity IgE recep-
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Johnson EM Jr (1993) Mediator release from kemia (RBL-2H3) cells induces early and late
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 Chapter 29

Rat Hippocampal Slice Culture Models

for the Evaluation of Neuroprotective Agents
Elisabetta Gerace, Elisa Landucci, Tania Scartabelli,
Flavio Moroni, and Domenico E. Pellegrini-Giampietro

Abstract
Organotypic slices cultured for weeks in vitro represent an extremely valuable strategy for the investigation
of the long-term properties of neuronal circuits under physiological and pathological conditions. Here, we
describe how to prepare rat organotypic hippocampal slice cultures and how to expose them for appropri-
ate periods of time to excitotoxic agents or to oxygen and glucose deprivation conditions, in order to
mimic the pattern of pyramidal cell damage which is observed in vivo and in other in vitro models. This
preparation is very useful not only to study synaptic plasticity or the pathways and mechanisms of neuro-
degeneration but also to evaluate the effects of neuroprotective agents.

Key words: Organotypic hippocampal slices, Neuroprotection, Oxygen-glucose deprivation,

N-methyl-D-aspartate, (S)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, Kainate, Glutamate
receptors

1. Introduction

Organotypic slices cultured for weeks in vitro represent an extremely

valuable strategy for the investigation of the long-term properties
of neuronal circuits under physiological and pathological condi-
tions (1). In particular, organotypic slice cultures from the hip-
pocampus not only retain a tissue organization and a distribution
of glutamate receptors that is very similar to that observed in situ
(2) but also exhibit synaptic plasticity mechanisms and a respon-
siveness to pathological insults (e.g., excitotoxicity, hypoxic, or
ischemic conditions) that are comparable to what is obtained
in vivo and in other in vitro models (such as acute slices or primary
neuronal cell cultures). For example, ischemic-like insults produce
a selective damage in the CA1 pyramidal cell layer, whereas kainic
acid damages predominantly the CA3 region.

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_29, © Springer Science+Business Media, LLC 2012

343
344 E. Gerace et al.

In the past few years, we have shown that organotypic

hippocampal slices exposed to oxygen-glucose deprivation (OGD)
for periods ranging from 15 to 75 min exhibit 24 h later a time-
dependent and gradual increase in CA1 injury that builds up following
selective apoptotic degeneration of pyramidal cells (3, 4). In our
experience, a period of 30 min OGD has proven optimal to detect
the effects of drugs that attenuate CA1 injury (4–7) as well as those
that produce an aggravation of OGD toxicity (4, 8). However, in
some cases, a “sublethal” period of OGD (20 min) appears to be
more appropriate to reveal both the toxic properties of pharmaco-
logical agents and the concentration dependency of the protective
effects of some antagonists (9).
The concentrations of drugs used in organotypic hippocampal
slice experiments are somewhat higher than those expected from
their Kd values and those used in cell cultures. Higher concentra-
tions of drugs are required to saturate receptors in brain slices, due
to the fact that they diffuse slowly through the thickness of brain
tissue in vitro. Moreover, in the case of organotypic slices statically
cultured on semiporous membrane inserts at the interface between
culture medium and gas atmosphere, the concentrations of drugs
need to be further increased because only the bottom of the slice is
exposed to the bathing medium to which the drug is added and
only a fraction of the drug diffuses across the membrane and
reaches the tissue (4, 7). The following chapter presents a protocol
for the preparation of rat organotypic hippocampal slice cultures
and their exposure to excitotoxic agents or to OGD conditions, in
order to mimic the pattern of pyramidal cell damage observed
in vivo and in other in vitro models.

2. Materials

All solutions are prepared using ultrapure water (prepared by puri-

fying deionized water to attain a sensitivity of 18.2 MΩ-cm at room
temperature) and analytical grade reagents. Deionized-purified
water, nonsterile glassware and plasticware, and surgical tools and
equipment are sterilized by autoclave before use. All procedures
are carried out using standard tissue culture sterile techniques.

2.1. Dissecting This is the medium used for hippocampal dissection and slice prep-
Medium aration. The solution is prepared under a sterile laminar flow
hood:

1. Prepare 100 mL of 45% glucose solution by combining 45 g of

D-(+)glucose and 80 mL of sterile water in a glass flask. Stir and
 29 Rat Hippocampal Slice Culture Models… 345

heat gently to dissolve. Add water to 100 mL, filter (0.2 μm),
and collect into a sterile recipient. Aliquot in Eppendorf sterile
tubes and store at −20°C for 1–2 months.
2. Prepare approximately 100 mL of Dissecting Medium in two
50-mL Falcon sterile tubes placed in a test-tube rack under the
hood at room temperature: Add 50 mL of Hanks’ Balanced
Salt Solution (HBSS, Sigma H9269) (see Note 1) to each of
the Falcon tubes using a sterile pipette. Then add 0.5 mL of a
45% glucose solution to each of the tubes and 0.75 mL of
amphotericin B (see Note 2).
3. Vortex to mix all ingredients and store at 4°C for maximum
1–2 weeks.

2.2. Normal Medium This is the medium used to grow and maintain the hippocampal
slice cultures. Prepare approximately 100 mL of Normal Medium
in two 50-mL Falcon sterile tubes placed in a test-tube rack under
the hood at room temperature, as follows:
1. Add 25 mL of Eagle’s Minimal Essential Medium (MEM,
Sigma M2279) (see Note 3) to each of the Falcon tubes using
a sterile pipette. Then add 12.5 mL of HBSS to each of the
tubes, 12.5 mL of Horse Serum (see Note 4), 0.5 mL of a 45%
glucose solution, and 0.75 mL of amphotericin B.
2. Finally, add 0.25 mL of 200 mM glutamine (see Note 5) to
each of the tubes.
3. Vortex to mix all ingredients and store at 4°C for maximum
1–2 weeks.
4. Warm up to 37°C in a water bath before use.

2.3. Serum-Free This is the medium used for excitotoxicity experiments and for
Medium the 24-h recovery period following OGD and excitotoxicity exper-
iments. Prepare approximately 100 mL of Serum-Free Medium in
two 50-mL Falcon sterile tubes placed in a test-tube rack under the
hood at room temperature, as follows:
1. Add 37.5 mL of Eagle’s MEM to each of the Falcon tubes
using a sterile pipette. Then add 12.5 mL of HBSS to each of
the tubes, 0.5 mL of a 45% glucose solution, and 0.75 mL of
amphotericin B.
2. Finally, add 0.25 mL of 200 mM glutamine to each of the
tubes.
3. Vortex to mix all ingredients and store at 4°C for maximum
1–2 weeks.
4. Warm up to 37°C in a water bath before use.
346 E. Gerace et al.

2.4. Glucose- and This is the medium used for OGD:

Serum-Free Medium
1. We prepare 100 mL of Glucose- and Serum-Free Medium in
two 50-mL Falcon sterile tubes placed in a test-tube rack under
the hood at room temperature.
2. Add 37.5 mL of Eagle’s MEM to each of the Falcon tubes
using a sterile pipette. Then add 12.5 mL of HBSS and 0.75 mL
of amphotericin B to each of the tubes.
3. Finally, add 0.5 mL of 200 mM glutamine to each of the
tubes.
4. Vortex to mix all ingredients and store at 4°C for maximum
1–2 weeks.
5. Warm up to 37°C in a water bath and saturate by bubbling
with 95% N2/5% O2 for 15 min before use.

2.5. Experimental 1. Propidium iodide (PI, Sigma P4170, see Note 6): Prepare
Reagents approximately 10 mL of a 1-mg/mL solution in sterile water,
filter (0.2 μm), aliquot in foil-wrapped Eppendorf tubes, and
store in a closed box at 4°C. PI is a highly polar compound
which is normally excluded from cells with an intact plasma
membrane. When the membrane is damaged, PI enters the
cells and upon binding to exposed DNA becomes highly fluo-
rescent (the absorption maximum is 535 nm, and the fluores-
cence emission maximum is 617 nm).
2. Glutamate (Sigma G8415): Prepare a 100-mM stock solution
in water. Add a few drops of 0.1 N NaOH to dissolve. Filter
(0.2 μm), aliquot in Eppendorf tubes, and store at −20°C.
3. N-methyl-D-aspartate (NMDA, Sigma M3262): Prepare a
100-mM stock solution in water. Filter (0.2 μm), aliquot in
Eppendorf tubes, and store at −20°C.
4. (S)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA, Ascent Scientific 005): Prepare a 10-mM stock solu-
tion in water. Filter (0.2 μm), aliquot in Eppendorf tubes, and
store at −20°C.
5. Kainic acid (Tocris Bioscience 0222): Prepare a 20-mM stock
solution in water. Filter (0.2 μm), aliquot in Eppendorf tubes,
and store at −20°C.

2.6. Special Equipment 1. Laminar flow hood. Both a vertical or horizontal laminar flow
hood can be used.
2. Cell culture CO2 incubator. The conditions are 37°C and 100%
humidity in 95% air/5% CO2.
3. McIlwain tissue chopper (Mickle Laboratory Engineering Co.
Ltd., UK). Use a new double-edge blade (Gillette Platinum or
from the chopper manufacturer) for every slice preparation
session.
 29 Rat Hippocampal Slice Culture Models… 347

4. Millicell-®CM (0.4 μm, 30-mm diameter) semiporous cell

culture inserts (Millipore PICM03050).
5. Dissecting stereo microscope. We use an Olympus SZ51
Stereozoom microscope.
6. Fiber optic cold light source. We use a KL200 Schott illumina-
tor for stereo microscopy.
7. Inverted fluorescence microscope. We use an Olympus IX-50
microscope equipped with a Xenon-arc lamp, a low power
objective (4×), and a rhodamine filter set (such as Omega
Optical filter sets O-5725 and O-5729).
8. Paddle pastettes (Alpha Laboratories LW4295).
9. Modular incubator hypoxia chamber (Billups-Rothenberg
MIC-101). Airtight sealed chamber with inlet and outlet valves
for fast gas exchange, which allows for rapid and economic
creation of a nonfluctuating hypoxic environment.
10. Diazocarb (95% N2/5% O2) cylinder (50 L) with flow meters.
11. Low light level CCD camera (we use a Basler SCA640 camera)
controlled by software (we use InCyt Im1™, Intracellular
Imaging Inc.) running on a personal computer.

3. Methods

3.1. Preparation 1. Under a sterile laminar flow hood, fill each well of 3 or more
of Hippocampal Slice 6-well sterile Corning cell culture plates (see Note 7) with
Cultures 1.2 mL of prewarmed (37°C) sterile Normal Medium. Place
one Millicell-®CM insert into each well and keep the plates in
an incubator at 37°C and 100% humidity in 95% air/5% CO2
until use.
2. Place the following items on an aluminum foil sheet under the
hood: one Whatman (Grade No. 1) filter paper disc (diameter:
9–10 cm) for each pup, one polyethylene disc (diameter: 2 cm)
for each pup (see Note 8), a dissecting scalpel with a new blade,
two pairs of scissors (one large and one small), two pairs of
small tweezers (one straight and one curved), 3–4 paddle
pastettes, and one plastic teaspoon. Turn on the UV light for
15 min and then turn it off before starting.
3. For each pup, fill a 100 × 15-mm sterile Petri dish with 5 mL of
ice-cold Dissecting Medium and place a polyethylene disc on
top of the cutting table of the chopper (see Note 9). Insert the
Whatman filter paper disc inside the inverted lid of the Petri
dish and place the two scissors, the curved tweezers, and the
teaspoon into a 100-mL beaker (filled to one-third with 70%
ethanol). Take the beaker and the Petri dish lid with the filter
paper to the animal house.
348 E. Gerace et al.

Fig. 1. How to remove the brain from a rat pup. After removing the skin (a), cut the skull bone with small scissors along the
sagittal middle line from back to front (b), ending with two diagonal cuts toward each eye (c). Remove the skull bone with
curved tweezers and, using a teaspoon, transfer the whole pup brain (d) to a filter paper placed on an inverted Petri dish lid.

4. Cut the head of a rat pup (see Note 10) with the large pair of
scissors. Use the small scissors to delicately remove the skin,
cut the skull bone along the sagittal middle line from back to
front, ending with two diagonal cuts toward each eye (see
Fig. 1). Remove the skull bone with the curved tweezers, and
using the teaspoon, transfer the whole pup brain to the filter
paper on the Petri dish lid.
5. Under the sterile hood, place the Petri dish lid with the brain
onto a piece of black cardboard and illuminate with the Schott
cold light source. Divide the two hemispheres with the scalpel
and, with the use of the paddle pastette, remove the cerebel-
lum, brainstem, and midbrain. Cut out the portion of the cor-
tex which is rostral to the hippocampus with the scalpel. Insert
the paddle pastette between the remaining cortex and the hip-
pocampus, raise and remove the hippocampus, and place it on
the polyethylene disc on top of the cutting table of the chop-
per (see Note 11).
6. Arrange the two hippocampi on the polyethylene disc, side by
side. Start the chopper (set at 420 μm) and cut the whole
length of both hippocampi. Remove the polyethylene disc with
the straight tweezers, turn it over, and dip it gently into the
Petri dish containing the 5 mL of ice-cold Dissecting Medium,
thus releasing the slices into the medium.
7. Allow gentle separation of the slices using a paddle pastette.
Take out the 6-well plates with the Millicell-®CM insert from
the incubator. Select the best slices (see Note 12) using the
stereomicroscope and place four of them very gently onto each
humidified insert using a paddle pastette. Place the plates in an
incubator at 37°C and 100% humidity in 95% air/5% CO2.
 29 Rat Hippocampal Slice Culture Models… 349

8. Change the Normal Medium every 2–3 days (1 mL per well).

After 24 h, the slices will start to adhere to the insert mem-
branes. The slices will be mature and ready for the experiments
after 12–14 days in vitro (DIV).

3.2. Excitotoxicity Hippocampal slices cultured for 14 DIV retain an organotypic orga-
in Organotypic nization in which the pyramidal and granule cell layers can be clearly
Hippocampal Damage defined when observed under phase-contrast microscopy or follow-
ing toluidine blue staining (4, 7). Control slices incubated with PI
display very low fluorescence levels, but when cultures are exposed
to glutamate receptor agonists, PI staining increases dramatically.
Maximal damage is achieved in this system by exposing the slices to
10 mM glutamate for 24 h: Virtually all neuronal populations are
destroyed, and PI fluorescence in CA1 and CA3 increases several-
fold above background levels (see Fig. 2). In order to obtain the
layer-selective injury shown in Fig. 2, we use relatively low concen-
trations of NMDA (10 μM), AMPA (10 μM), or kainate (5 μM) for
24 h. It is also possible to expose the slices to higher concentrations
of NMDA (300 μM), AMPA (100 μM), or kainate (100 μM) for a
brief period of time (60 min), but in this case, the damage will be
more diffuse and less selective (see examples in 8). All slices used in
one experimental session are prepared from the same litter, i.e.,
from neonatal rat pups born on the same day:
1. Before the experiment, perform a “PI viability test” (see Note
13) by adding 6 μL of the 1-mg/mL stock solution of PI into
the wells containing the cultures slices (final concentration:
5 μg/mL). Rinse gently and observe under the fluorescence
microscope 30 min later. Cultures displaying distinct PI fluo-
rescence in the pyramidal cell layers are excluded. A mild PI
uptake is sometimes observed in the dorsal blade of the den-
tate gyrus, even in healthy slices.
2. Briefly rinse the slices and then incubate with 1.2-mL Serum-
Free Medium that has previously been prewarmed to 37°C.
3. Under the hood, add to each well the appropriate microliters
from the stock solutions (use intermediate dilutions if needed)
in order to reach the desired final concentration of glutamate
receptor agonists: 10 mM glutamate, 10 μM NMDA, 10 μM
AMPA, and 5 μM kainate. Treat control slices with Serum-
Free Medium.
4. Incubate for 24 h by placing the 6-well plates in the incubator
at 37°C and 100% humidity in 95% air/5% CO2 for recovery.
Neuronal injury is evaluated 24 h later.

3.3. OGD in To mimic the conditions that occur following cerebral ischemia
Organotypic in vivo, 14 DIV organotypic hippocampal slices are exposed to
Hippocampal Slices OGD in a hypoxia chamber for selected periods of time. Exposure
350 E. Gerace et al.

Fig. 2. Neuronal death induced by glutamate receptor agonists in rat organotypic hip-
pocampal slices. Cultured slices are exposed to drugs, incubated with PI (5 μg/mL), and
observed under fluorescence optics. Hippocampal slices under normoxic and drug-free
(control) conditions display background PI fluorescence. Hippocampal slices observed fol-
lowing 24 h exposure to 10 mM glutamate display a maximal degree of neuronal death in
all hippocampal layers. Hippocampal slices observed following 24 h exposure to 10 μM
NMDA display more intense fluorescence in the CA1 pyramidal cell layer, whereas follow-
ing exposure to 10 μM AMPA PI labeling is abundant in all layers. Following 24 h exposure
to 5 μM kainate fluorescence is more intense in the CA3 pyramidal cell layer.
 29 Rat Hippocampal Slice Culture Models… 351

100

% of glutamate-induced PI fluorescence
**
80
**

60

20 min OGD

40

*
20

0 10 20 30
OGD duration (min)
30 min OGD

Fig. 3. Neuronal death induced by OGD in rat organotypic hippocampal slices. Cultured
slices are exposed to OGD for the indicated period and 24 h later incubated with PI for
fluorescence detection of its optical density in the CA1 region. OGD causes increasing
levels of CA1 injury when applied for 20–40 min. Data are expressed as percentage of
maximal damage produced by 24 h exposure to 10 mM glutamate. Hippocampal slices on
the left, photographed under fluorescence optics, display intense PI labeling in the CA1
subregion when exposed to increasing periods of OGD. Values represent the mean ± SEM
of at least five experiments performed in triplicate. *P < 0.05 and **P < 0.01 vs. basal PI
fluorescence (ANOVA + Tukey’s w test).

to OGD for periods ranging from 20 to 40 min leads to significant

increases in the levels of PI fluorescence in the CA1 region, as
measured 24 h after the insult (4, 7) (see Fig. 3).
1. Perform the “PI viability test” as indicated in Subheading 3.2,
step 1.
2. Briefly rinse the slices and then incubate with 1.2-mL Glucose-
and Serum-Free Medium that has previously been prewarmed
to 37°C and saturated with 95% N2/5% O2.
3. Place one or two multiwell plates (without the closing lid)
under the hood in the Billups-Rothenberg hypoxia chamber.
Seal the chamber, attach the gas tube to the inlet valve, and
flush with 95% N2/5% O2 at 2–3 psi for 10 min keeping the
outlet valve open. Clamp the outlet valve and flush for another
minute, clamp the inlet valve and then place the airtight cham-
ber in the incubator at 37°C for the desired period of time (see
Note 14). Treat control slices with glucose-containing and
normoxic Serum-Free Medium for the same period of time.
352 E. Gerace et al.

4. At the end of the OGD exposure, remove the Glucose- and

Serum-Free Medium and replace with glucose-containing and
normoxic prewarmed (37°C) Serum-Free Medium.
5. Return the multiwells to the incubator at 37°C and 100%
humidity in 95% air/5% CO2 for recovery. Neuronal injury is
evaluated 24 h later.

3.4. Assessment Cell injury is assessed in organotypic hippocampal slices by measur-

of Neuronal Damage ing the intensity of PI fluorescence (4, 7). Preliminary experiments
in our laboratory, as well as a number of previous studies using
organotypic hippocampal slices exposed to ischemia-related insults
(10, 11), have shown that there is a linear correlation between rela-
tive PI fluorescence and the number of injured cells as detected by
morphological criteria:
1. At the end of the 24-h recovery period, add 6 μL of the 1-mg/
mL stock solution of PI into the wells containing the cultures
slices (final concentration: 5 μg/mL). Rinse gently and observe
under the fluorescence microscope 30 min later.
2. Digitize the image using a CCD camera controlled by software
and store the images in the hard disc of the personal
computer.
3. To quantify cell death, the CA1, CA3, and dentate gyrus hip-
pocampal subfields are identified and encompassed in a frame
using the drawing function of an image software (ImageJ;
NIH, Bethesda, USA). A PI fluorescence intensity measure-
ment for any given subfield consists of the mean of the fluores-
cence intensity values of each pixel in the area defined by the
frame.
4. Percentage of cell death in each experiment is expressed as %
death = 100 (F24 − FB)/(Fmax − FB) (see Subheading 4), where F24
is subfield fluorescence 24 h after exposure to excitotoxins or
OGD, FB is background PI fluorescence determined in control
slices, and Fmax is maximal fluorescence after complete neuronal
death induced by 24 h exposure to 10 mM glutamate.

4. Notes

1. HBSS is shipped at room temperature. Store at 4°C after

opening.
2. Amphotericin B (Sigma A2942) is shipped as a 250-μg/mL
solution in a 100-mL bottle. Aliquot upon arrival in 1.5-mL
sterile Eppendorf tubes and store at −20°C for 2–4 months.
 29 Rat Hippocampal Slice Culture Models… 353

3. Eagle’s MEM must always be stored at 4°C.

4. Horse Serum (Gibco 26050) is shipped in dry ice in 500-mL
bottles. Thaw at 37°C in a water bath, aliquot in sterile 50-mL
Falcon tubes, and store at −20°C until use.
5. Glutamine (Sigma) is shipped as a 200-mM solution in a 100-
mL bottle. Aliquot upon arrival in sterile 1.5-mL Eppendorf
tubes and store at −20°C for 2–4 months. This ingredient
should be added last in all solutions to avoid precipitation.
Vortex the Eppendorf tube before use.
6. PI is extremely volatile as a powder and light-sensitive; there-
fore, extreme care should be exercised when handling. Moreover,
wear gloves and a mask because PI is a known mutagen.
7. We usually prepare three 6-well Corning plates for each rat lit-
ter (approximately 10 pups).
8. We cut the 2-mm polyethylene discs from the bottom of the
wrappers of the Millicell-®CM inserts.
9. It is very important to secure the polyethylene disc under the
clips of the cutting table to prevent displacement of the hip-
pocampi while cutting the sections.
10. We use Wistar Hannover rats at postnatal day 8–9 (P8-P9),
weighing approximately 15 g.
11. In order to spread the hippocampi straight on the polyethylene
disc, we use two paddle pastettes soaked with Dissecting
Medium. This is also helpful to avoid damaging the
hippocampus.
12. Select slices in which the CA3 and CA1 pyramidal cell layers
and the dentate gyrus granule cell layer are both compact and
clearly visible. Exclude those with any visible damage and the
slices from the dorsal hippocampus (which are smaller and less
resistant).
13. If PI is thought to interfere with the experiment, slice cultures
can also be observed under phase-contrast optics. Damaged
cells will appear brownish, and if they are numerous, the slice
should be definitely discarded.
14. To achieve a reliable, intense, and selective damage of the CA1
pyramidal cell layer (see Fig. 3) that can be attenuated by neu-
roprotective drugs (4, 7), we perform an OGD of 30 min,
which is calculated by adding a 10-min flush with the open
outlet valve +20 min in which the airtight sealed hypoxia cham-
ber is kept at 37°C in the incubator. To obtain a milder degree
of CA1 damage, which is particularly useful to detect exacerba-
tion of OGD induced by pharmacological agents, we expose
the slices to 20 (10 + 10) min OGD (9).
354 E. Gerace et al.

Acknowledgements

This work was supported by grants from the University of Florence,

the Italian Ministry of University and Research (MIUR, PRIN
2008 project), and the Compagnia di San Paolo (Turin, Italy).

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come of age. Trends Neurosci 20: 471–477 7. Pellegrini-Giampietro DE, Peruginelli F, Meli
2. Bahr BA (1995) Long-term hippocampal slices: E, Cozzi A, Albani-Torregrossa S, Pellicciari R,
a model system for investigating synaptic et al (1999) Protection with metabotropic glu-
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Cozzi A, Picca R, et al (2001) Poly(ADP- 1607–1619
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mental models of cerebral ischemia. Cell Death Differential role of mGlu1 and mGlu5 recep-
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5. Cozzi A, Meli E, Carlà V, Pellicciari R, Moroni Seeley PJ, Sundstrom LE (1997)
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temically active metabotropic glutamate 1 3538–3553
 Chapter 30

A 6-Hydroxydopamine In Vivo Model of Parkinson’s Disease

Giulia Mercanti, Gianfranco Bazzu, and Pietro Giusti

Abstract
Animal models of Parkinson’s disease are essential to explore pathophysiological hypotheses and to test
new treatment options, including neurotrophic factors. Catecholaminergic neurotoxins used to generate
such models are 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. These neurotoxins
predominantly kill dopaminergic neurons through oxidative damage and mitochondrial failure, although
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine fails to induce a significant dopaminergic neurodegeneration
in rats. The present chapter describes a protocol for the 6-hydroxydopamine rat model based on stereotaxic
injection performed only unilaterally, which mimics an early-to-mid stage of the disease.

Key words: Parkinson’s disease, Animal models, Neurotoxins, Dopaminergic neurons,

6-Hydroxydopamine, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine

1. Introduction

Parkinson’s disease (PD) is a slowly progressing neurodegenerative

disease where the midbrain dopaminergic neurons are lost. The
symptoms become evident when about 60–80% of the dopamine
(DA) content is lost, which corresponds to about 50–60% loss of
DA neurons in the substantia nigra pars compacta (SNpc) (1). PD
is characterized by cardinal motor symptoms including rigidity,
postural imbalance, bradykinesia, akinesia, and resting tremor (2).
Experimental animal models represent a critical component of any
cascade directed to the identification and development of new
therapeutics for PD, including neurotrophic factors (3–7). The
ideal PD model would mimic the human etiopathological condi-
tion in both the distribution of damage and its temporal profile.
The etiopathology of PD is currently unknown, and different
models may therefore mimic only particular aspects of the neuro-
degenerative process, such as mitochondrial dysfunction, oxidative
stress, and misfolded protein aggregation. The most common

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_30, © Springer Science+Business Media, LLC 2012

355
356 G. Mercanti et al.

model reproduces the DA deficiency syndrome rather than the

process of progressive dopaminergic degeneration and involves
stereotaxic injection of specific catecholaminergic neurotoxins into
different brain regions involved in voluntary motor coordination.
Catecholaminergic neurotoxins used to generate such models are
6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) (8) and predominantly kill dopamin-
ergic neurons through oxidative damage and mitochondrial failure
through inhibition of respiratory chain complexes (9). While they
elicit motor deficits in rodents and nonhuman primates, MPTP
fails to induce a significant dopaminergic neurodegeneration
in rats (10).
Severe lesioning of the nigrostriatal DA system is required for
studies of restorative or symptomatic treatments. 6-OHDA is a
toxin that enters DA neurons through the high affinity DA trans-
porter and accumulates in the cytosol, where it inhibits mitochon-
drial respiratory chain complexes I and IV (9). At the same time,
free radicals form which finally results in DA degeneration from
oxidative stress (11). The toxin does not cross the brain–blood bar-
rier and has to be injected directly into areas containing DA fibers.
The protoxin MPTP is highly lipophilic and, when systemically
administered, crosses the brain–blood barrier to enter the brain
parenchyma where it is oxidized to 1-methyl-4-phenyl-2,3-dihy-
dropyridinium in cells containing monoamine oxidase B. 1-Methyl-
4-phenyl-2,3-dihydropyridinium then undergoes spontaneous
oxidation to the active toxic molecule 1-methyl-4-phenylpyridin-
ium (MPP+) and is released into the extracellular space. MPP+
enters DA neurons via the DA transporter and is concentrated
within the mitochondria, where it impairs oxidative phosphoryla-
tion by inhibiting complex I activity (1). Alterations in energy
metabolism and generation of free radicals lead to neurodegenera-
tion of DA neurons. MPTP neurotoxicity in nonhuman primates
was the first effective primate model of PD. Surprisingly, MPTP,
for reasons unknown, is unable to destroy the dopaminergic
innervation in other species, except for certain strains of mice (C57
black and Swiss Webster) (12).
The MPTP-treated mouse, although widely used, is not repro-
ducible and robust. Stereotaxic injection of MPP+ in rats has been
used but offers no advantages over the 6-OHDA rat model, and it
is not routinely employed (12). For these reasons, this chapter will
focus on the 6-OHDA rat model based on stereotaxic injection.
The stereotaxic lesion is most commonly performed only unilaterally
because animals subject to bilateral destruction of the DA system
develop aphagia and adipsia and require extensive monitoring and
care to survive (13). The complete lesion model mimics a late stage
of the human disease. The unilateral model, on the other hand,
mimics an early-to-mid stage of the disease and has the advantage
of exhibiting a behavioral impairment on the side contralateral to
the lesion, leaving the intact side as an internal control. This model
 30 A 6-Hydroxydopamine In Vivo Model of Parkinson’s Disease 357

can be well standardized; the lesion obtained is quite stable for 2

weeks following neurotoxin injection and is cost-effective. Different
levels of lesion can be produced by injecting the neurotoxin at
different levels of the nigrostriatal pathway: a partial lesion, by
injecting toxin directly into the striatum (which is taken up by axon
terminals and causes retrograde degeneration in the SNpc); a very
severe state of DA depletion, corresponding to an advanced stage
of PD (14) if the injection site is the medial forebrain bundle
(MFB); or a more specific but moderate DA depletion, when the
lesion is performed in the SNpc.

2. Materials

2.1. Surgical 1. Kopf 900 stereotaxic instruments supplemented with ear bars
Apparatus and Tools (Kopf instruments).
2. 10-μL Hamilton syringe (Hamilton Bonaduz AG, Bonaduz,
GR, Switzerland).
3. Microinjection needles (27–30 gauge) connected to the
Hamilton syringe with a polyethylene tubing (PE 20, Becton
Dickinson) of about 25-cm length.
4. Surgical tools: scissors, sharp forceps, scalpels, sterile cotton
pads, syringes and needles, metal clips, suture clips and apply-
ing forceps, and ear punch (Fine Science Tools).
5. Electric shaver.
6. Dental cement.
7. Dremel 300 Series drill with engraving cutter 109 (Dremel
Italia).
8. Harvard compact infusion syringe pump (American Laboratory
Trading, Inc. East Lyme, CT, USA).
9. Heating pad for rodents and small animals (two Biological
Instruments, Besozzo, Italy).

2.2. List of Materials 1. Fentanyl citrate salt, 50 μg/mL (Sigma-Aldrich.), + medeto-

midine hydrochloride (DormitorVet®, Pfizer Animal Health)
50 μg/mL, 20:1 mixture.
2. Atipamezole hydrochloride (Antisedan®, Pfizer Animal
Health).
3. Ketamine 80 mg/kg + xylazine 12 mg/kg (ketamine hydro-
chloride/xylazine hydrochloride solution, Sigma-Aldrich).
4. Chloral hydrate: dissolve 4 mg in 100 mL of sterile 0.9% (w/v)
NaCl (Sigma-Aldrich).
5. 6-OHDA-HCl (Sigma-Aldrich). Dissolve in oxygen-free water
containing 0.02% ascorbic acid. The solution should be
358 G. Mercanti et al.

prepared fresh every 90 min, kept on ice, and protected from

exposure to light. Oxidized solutions turn red.
6. Sterile saline solution: 0.9% (w/v) NaCl with 0.02% ascorbic
acid (Sigma-Aldrich).
7. Sterile deionized water.
8. Ethanol 70%.

3. Methods

3.1. Surgical All the animal procedures are carried out following guidelines
Procedures governing animal experimentation (European Union decree of
and Administration 24/11/1986 (86/609/IIC)) for the techniques described.
of 6-OHDA Before starting with the lesion, prepare all reagents and materials
that will be used. Once the animal is under anesthesia, everything
must be ready and near the stereotaxic instrument to avoid any
distraction from the surgical procedure.
1. Prepare a vial containing the solution of sterile 0.9% NaCl with
0.02% (w/v) ascorbic acid and put on ice.
2. Aliquot small amounts of approximately 1–2 mg of 6-OHDA-
HCl into Eppendorf® tubes and cover with aluminum foil to
avoid exposure to light. Store the aliquots at −20°C and avoid
repeated thawing/freezing. Be careful because 6-OHDA rap-
idly oxidizes when exposed to light and room temperature and
takes on a pinkish color.
3. Set up the surgical work station with all the surgical tools listed
above; disinfect with 70% ethanol.
4. Connect the needle with the PE tube to the Hamilton syringe.
Test by filling the syringe with the saline–ascorbic acid solution
and press it through the syringe and needle. This procedure
needs some practice and may seem time-consuming but is criti-
cal that there is no leakage from the PE tube or any occlusion
as this could prevent the toxin from reaching the target struc-
tures at the correct concentration (see Notes 1–3).
5. Place the needle on the stereotaxic frame and make sure that its
orientation is perfectly vertical and straight to avoid injection
on the wrong site.
6. Turn on the heating pad in such way that it is warm enough
when the animal will be placed in the stereotaxic frame.
7. Once everything is ready, weigh and anesthetize the animal.
Surgery can be performed using one of the different injectable
anesthesias proposed below:
● Fentanyl citrate salt 50 μg/mL + medetomidine HCl 50 μg/
mL, 20:1 mixture, 6.3 mL/kg intraperitoneal. To wake
 30 A 6-Hydroxydopamine In Vivo Model of Parkinson’s Disease 359

up the animals faster, use atipamezole hydrochloride

(Antisedan®) 1 mg/kg as an antagonist.
● Ketamine 80 mg/kg + xylazine 12 mg/kg intramuscular
(ketamine hydrochloride/xylazine hydrochloride solution).
Weigh the animal and give 1 μL solution per gram body
weight.
● Chloral hydrate, 400 mg/kg intraperitoneal (weigh 4 g of
chloral hydrate and dissolve in 100 mL of sterile 0.9% NaCl).
8. Afterwards, leave the animal in a cage with bedding until
unconscious (see Note 4).
9. Wipe the fur on the top of the skull with a cotton pad soaked
in 70% ethanol and shave the fur with an electric shaver. This
facilitates suturing of the skin and decreases the chance of
infection at the incision site.
10. Place the animal in the stereotaxic frame on a flat-skull position;
enter the ears with the ear bars and fix the skull to avoid left-
right movements of the head (see Fig. 1a). Next, fix the teeth
of the animal on the tooth bar so as to prevent the head moving
up and down (see Fig. 1b). This procedure is delicate and takes
time, but it is essential that the head is positioned correctly so
that the bregma and lambda are at the same horizontal level.

Fig. 1. Stereotaxic surgery. Flat-skull positioning of the animal with ear bars (a) and tooth bar (b) on the stereotaxic frame
before starting surgery. (c) Shows the operatory field with skull bone exposure.
360 G. Mercanti et al.

Only in this way it is possible to correctly calculate the

coordinates to find the right injection site (see Note 5).
11. Perform a midline incision on the skin of about 2 cm in length
starting between the eyes and remove the connective tissue
above the bone so that the bregma can be easily located. With
the two metal clips, keep the skin open (see Fig. 1c).
12. Injection coordinates are expressed in millimeters relative to
bregma and the dural surface (15):
To target the sensorimotor part of the striatum: AP = +0.5;
L = −2.5; DV = −7.0 (4 μL).
To target the SNpc: AP = −5.7; L = −2.0; DV = −8.7 (4 μL).
To target the MFB: first injection AP = −4.4; L = −1.2;
DV = −7.8, tooth bar = −2.3 (2 μL); second injection AP = −4.0;
L = −0.8; DV = −8, tooth bar = +3.4 (2 μL).
13. Find the bregma and point the needle exactly on it, read the
coordinates anteroposterior (AP) and lateral (L) on the manip-
ulator (x, y, z), then retract the needle and calculate the
location of injection subtracting the coordinates for different
lesion sites (see Fig. 2). Move the manipulator on the injection
site and mark it with a pencil (see Note 6).

Fig. 2. Stereotaxic coordinates. Dorsal and lateral views of rat skull. Antero/posterior (A/P) and lateral (L) coordinates are
referred to bregma point; dorsal/ventral (D/V) coordinates are calculated from the dural surface.
30 A 6-Hydroxydopamine In Vivo Model of Parkinson’s Disease 361

14. Retract the needle and drill a small hole in the skull bone above
the injection coordinate. Be careful not to rupture the
meninges when drilling (see Note 7).
15. Calculate the dorsal-ventral (DV) coordinate of the injection
having the dural surface as a reference plane, then make a small
hole in the meninges with either sharp forceps or an injection
needle. This will facilitate the injection and avoid damage to
the needle tip.
16. Dissolve 4 mg of 6-OHDA-HCl in 1 mL of sterile 0.9% NaCl
with 0.02% ascorbic acid. The concentration of 6-OHDA-HCl
in this solution should be 3.6 mg/mL (corresponding to
3.0 mg/mL free base 6-OHDA). Always keep the aliquots in
the freezer until use and check the color of the solution. Once
dissolved, keep on ice and in the dark because 6-OHDA is
light and temperature sensitive and rapidly oxidizes. Prepare a
fresh solution every 90 min, approximately.
17. Rinse the syringe with saline containing 0.02% ascorbic acid.
18. Fill the syringe with the 6-OHDA solution.
19. Place the Hamilton syringe on the pump, connect the needle
with the PE tube to the Hamilton syringe, fill the circuit with
the toxin solution, and set the infusion flow rate to 0.5 μL/min.
20. Slowly lower the injection needle down to the desired depth.
21. Start the infusion pump and pressure-eject the desired volume
of 6-OHDA solution at a maximum rate of 0.5 μL/min.
22. Leave the needle in place for 5 min before slowly retracting it
(see Fig. 3).
23. Empty the syringe and clean with two washes of ethanol and
one of saline containing 0.02% ascorbic acid.
24. Rehydrate the wound area with sterile water and close by
suturing.
25. Apply analgesic postoperatively and mark the tail or the ear
with a progressive identification number.
26. Inject 2 mL of saline subcutaneously to prevent dehydration
during recovery from anesthesia. For ethical reasons, analgesic
treatment should be administered to the animals before they
regain consciousness following anesthesia. The postoperative
analgesia consists of:
● Glucose solution (glucose 5%): 3 mL subcutaneously
● Steroidal anti-inflammatory (i.e., betamethasone disodium
phosphate 1.5 mg/mL, Bentelan®, Glaxo Wellcome):
100 μL intramuscular
● Nonsteroidal anti-inflammatory (i.e., indomethacin,
Liometacen®, Chiesi): 100 μL intramuscular
● Antibiotic (i.e., ceftriaxone, Rocefin® 1, Roche): 100 μL
subcutaneously
362 G. Mercanti et al.

Fig. 3. Schematic representation of lesion procedure. The toxin 6-OHDA-HCl is injected into the selected area by means of
a 27/30-G needle connected to a polyethylene tube and a Hamilton syringe. A precision infusion pump has a constant rate
of 0.5 μL/min. At the end of toxin infusion, the needle is left in place for additional 5 min, retracting it slowly.

27. Place the animal in a clean cage until it regains consciousness

and recovers.
28. Disinfect the surgical tools and the table.

3.2. Postoperative 1. To prevent dehydration, administer sterile physiological saline

Care or glucose–saline solution subcutaneously (5 mL) immediately
after surgery.
2. During surgery, the body temperature of the rat decreases. To
offset this temperature loss, place a wool pad underneath the
animal’s body. When surgery is finished, place the rat into its
cage and cover the body with a cloth and the eyes with paper.
The latter is especially important if the rat is put under a heat-
ing lamp—this is to prevent the eyes from drying.
3. On the days following surgery, check that the animals resume
their normal activity, that the wound heals, and that they do not
lose weight. If some animals have problems eating, give them a
special diet with food pellets completely soaked in 15% sugar/
water solution, which should be put directly into the cage.

3.3. Comments: Choice 6-OHDA does not cross the blood–brain barrier and therefore is
of Lesion Site classically injected into the SNpc and/or the MFB and produces a
massive, virtually complete lesion of nigral dopaminergic cell
bodies; SNpc neurons begin to die within the first 12 h of injection,
 30 A 6-Hydroxydopamine In Vivo Model of Parkinson’s Disease 363

and marked lesion of striatal dopaminergic terminals, paralleled by

dopamine depletion, is established within 2–3 days (16). The injec-
tion is commonly carried out unilaterally, with the controlateral
hemisphere serving as control. Bilateral injections are generally
avoided, due to the high mortality rate associated with this proce-
dure, which induces marked aphagia and adipsia in the operated
animals (13) that requires intense nursing care. In the mid-1990s,
a variant of the original procedure was proposed, in which 6-OHDA
is injected into the striatum, where the terminals of SNpc neurons
are located. The intrastriatal injection of 6-OHDA induces prompt
damage of striatal terminals, followed by delayed, progressive cell
loss of SNpc neurons, which are secondarily affected through a
“dying back” mechanism. The degree of SNpc damage obtained
with this procedure is less marked, compared with the intra-SNpc
injection, remaining confined to 50–70% of the nucleus (17).
However, the target site for administration of a neurotoxin in the
brain for producing lesions that mimic parkinsonism in animals
remains ambiguous (18).

4. Notes

1. An alternative to using a Hamilton microinjection needle is a

glass capillary with an outer diameter of approximately 50 μm
at the tip (the diameter can be checked on a microscope). The
purpose of the glass capillary is to provide an injection cannula
of the smallest possible size in order to reduce nonspecific
damage to the tissue. The inner diameter of the capillary at the
end opposite to the tip should not be smaller than 0.8 mm to
fit on the Hamilton syringe needle. Place a small amount of
parafilm around the needle of the Hamilton syringe and insert
the needle into the glass capillary. Make sure the needle is
sealed and does not leak. Test this by filling the syringe with
saline containing 0.02% ascorbic acid and pass the solution
through the capillary. This procedure needs some practice and
may seem time-consuming on the first trial. However, it is crit-
ical that there is no leakage from the borders of the capillary,
since this could prevent the toxin from reaching the target
structure in the brain.
2. Mount the syringe with or without the capillary on the stereo-
taxic frame and make sure that the needle/capillary assembly is
vertically straight in its orientation.
3. Pay particular attention to the cleanliness of the needle to prevent
infection and the formation of blood clots which can cause
mechanical damage to brain tissue during subsequent injections.
4. Test reflexes by pinching the tail or paws to ensure that the
animal is anesthetized.
364 G. Mercanti et al.

5. While the animal is fixed to the stereotaxic frame, pay particular

attention to keep the head up to avoid that the animal may
suffocate.
6. Note the time when the anesthesia is done, the coordinates of
the injection site for each animal, and the time when 6-OHDA
is dissolved.
7. When drilling the hole in the skull bone, hold the drill in a
vertical position and avoid applying too great a pressure on the
bone which may cause the drill bit to penetrate the dura. Make
small circular and concentric movements with the drill bit and
make an opening not too large.

References
1. Dauer W, and Przedborski S (2003) Parkinson’s 10. Przedborski S, Jackson-Lewis V, Naini A,
disease: mechanisms and models. Neuron Jakowec M, Petzinger G, Miller R, Akram M
39:889–909 (2001) The parkinsonian toxin 1-methyl-4-
2. Recchia A, Rota D, Debetto P, Peroni D, phenyl-1,2,3,6-tetrahydropyridine (MPTP): a
Guidolin D, Negro A, Skaper SD and Giusti P technical review of its utility and safety. J Neuro-
(2008) Generation of alpha-synuclein-based chem 76:1265–1274
rat model of Parkinson’s disease. Neurobiol Dis 11. Olanow CW (1993) A radical hypothesis
30:8–18 for neurodegeneration. Trends Neurosci
3. Kordower JH, Emborg ME, Bloch J, Ma SY, 16:439–444
Chu Y, Leventhal L et al (2000) Neurode- 12. Jenner P (2008) Functional models of Parkinson’s
generation prevented by lentiviral vector delivery disease: a valuable tool in the development
of GDNF in primate models of Parkinson’s of novel therapies. Ann Neurol 64(Suppl
disease. Science 290:767–73 2):S16–S29
4. Gash DM, Zhang Z, Ovadia A, Cass WA, Yi A, 13. Ungerstedt U (1971) Adipsia and aphagia after
Simmerman L et al (1996) Functional recovery 6-hydroxydopamine induced degeneration of
in parkinsonian monkeys treated with GDNF. the nigro-striatal dopaminergic system. Acta
Nature 380:252–255 Physiol Scand Suppl 367:95–122
5. Biju K, Zhou Q, Li G, Imam SZ, Roberts JL, 14. Ungerstedt U (1968) 6-Hydroxy-dopamine
Morgan WW et al (2010) Macrophage- induced degeneration of central monoamine
mediated GDNF delivery protects against dop- neurons. Eur J Pharmacol 5:107–110
aminergic neurodegeneration: a therapeutic 15. Paxinos G, Watson C (1986) Rat Brain in
strategy for Parkinson’s disease. Mol Ther; Stereotaxic Coordinates San Diego, Academic
doi:10.1038/mt.2010.107 Press
6. Yasuda T and Mochizuki H (2010) Use of 16. Deumens R, Björklund A and Prickaerts J (2002)
growth factors for the treatment of Parkinson’s Modeling Parkinson’s disease in rats: an evalua-
disease. Expert Rev Neurother 10:915–924 tion of 6-OHDA lesions of the nigrostriatal
7. Laganiere J, Kells AP, Lai JT, Guschin D, pathway. Exp Neurol 175:303–317
Paschon DE, Meng X et al (2010) An engi- 17. Lee CS, Sauer H and Bjorklund A (1996)
neered zinc finger protein activator of the Dopaminergic neuronal degeneration and motor
endogenous glial cell line-derived neurotrophic impairments following axon terminal lesion
factor gene provides functional neuroprotection by intrastriatal 6-hydroxydopamine in the rat,
in a rat model of Parkinson’s disease. J Neurosci Neuroscience 72:641–653
30:16469–16474 18. Sindhu KM, Banerjee R, Senthilkumar KS,
8. Lambert CE, and Bondy SC (1989) Effects of Saravanan KS, Raju BC, Rao JM et al (2006)
MPTP, MPP+ and paraquat on mitochondrial Rats with unilateral median forebrain bundle,
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44:1277–1284 neurotoxins MPP + or rotenone display
9. Glinka Y, Gassen M, and Youdim MB (1997) differential sensitivity to amphetamine and
Mechanism of 6-hydroxydopamine neurotox- apomorphine. Pharmacol Biochem Behav
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 Chapter 31

Brain Microdialysis in Freely Moving Animals

Gianfranco Bazzu, Alice Biosa, Donatella Farina, Ylenia Spissu,
Giammario Calia, Sonia Dedola, Gaia Rocchitta, Rossana Migheli,
Pier Andrea Serra, and Maria Speranza Desole

Abstract
Brain microdialysis is an analytical technique used for the dynamic monitoring of brain neurochemistry in
awake, freely moving animals. This technique requires the insertion of a small dialysis catheter, called a microdi-
alysis probe, into a specific brain region, and its perfusion with an artificial extracellular fluid. The
microdialysate samples, obtained from the probe outlet, can be analysed using high-performance liquid
chromatography with electrochemical detection for the quantification of oxidizable molecules recovered
from the extracellular space. In this chapter, we describe a protocol for performing a microdialysis setup and
experiment in freely moving rats and mice. Furthermore, the high-performance liquid chromatographic
determination of ascorbic acid, uric acid, catecholamines, indolamines and derivatives is described in detail.

Key words: Ascorbic acid, Amperometric detection, Dopamine, 3,4-Dihydroxyphenylacetic acid,

L-3,4-Dihydroxyphenylalanine, High-performance liquid chromatography, Homovanillic acid,
5-Hydroxyindoleacetic acid, In vivo microdialysis, 3-Methoxytyramine, Noradrenaline, Serotonin,
Stereotaxic surgery, Uric acid

1. Introduction

Microdialysis is an analytical technique used for continuous mea-

surement of the concentrations of low molecular weight dialysates in
the extracellular fluid of virtually any tissue. The microdialysis prin-
ciple was first employed in the 1960s when rudimentary dialysis
systems were implanted into animal tissues, mainly into rat brain, to
study the neurochemistry in a dynamic manner (1). The modern
brain microdialysis technique was used for the first time in 1972 (2),
and in 1974, it was employed for monitoring dopamine release (3).
Today, studied compounds include endogenous molecules such as
neurotransmitters (to determine their variations) or exogenously
administered drugs (to determine their pharmacokinetic).

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_31, © Springer Science+Business Media, LLC 2012

365
366 G. Bazzu et al.

The brain microdialysis technique requires the insertion of a

small microdialysis probe into a discrete brain region to mimic a
small blood capillary having a diameter around 200 μm. The microdi-
alysis probe encloses a cylindrical semipermeable membrane (hollow
fibre) perfused with an isosmolar liquid similar, in composition, to
the extracellular fluid. The membrane is connected to a perfusion
circuit by means of a piece of polyethylene tubing (inlet), while a
second piece of tubing (outlet) allows the recovery of the microdi-
alysate sample: the perfusion fluid enriched with the unbound
dialysates present in the extracellular space passively diffused through
their concentration gradient. In a similar manner, drugs can be
added to the perfusion fluid and locally administered (intracerebral
retrodialysis). The low perfusion flow rate, ranging from 0.1 to
5 μL/min (1), allows for maximum efficiency of recovery. The
microdialysis technique has some unique advantages including the
continuous monitoring of the extracellular space of (virtually) any
tissue, high spatial resolution, limited local traumatisation and lack
of tissue proteins (including enzymes) in the microdialysate. The
principal disadvantage is the poor temporal resolution (minutes)
compared to other techniques (4). Microdialysis is mainly used for
studying the changes in extracellular concentrations of analytes. It is
difficult to calculate the real analyte concentrations in the extracel-
lular space due to the irregular geometry of this compartment;
moreover, a total equilibrium cannot be established because of the
constant perfusion of the probe with fresh perfusate (1). Microdialysis
probes can be calibrated by either measuring the loss of a compound
using an analyte-containing perfusion fluid or the gain of compound
using an analyte-containing calibration solution. The most fre-
quently used calibration methods are no-net-flux methods (5, 6)
and the retrodialysis method (7).
Today, brain microdialysis is used not only in awake, freely mov-
ing animals (mice, rats, primates etc.) but also in humans (8, 9). In
this chapter, we describe the materials and the methods necessary
for performing a microdialysis setup and experiment in freely mov-
ing rats and mice. We have selected the striatum as the most tar-
geted region for microdialysis probe implantation and the analysis
of ascorbic acid, uric acid, catecholamines, indolamines and deriva-
tives by high-performance liquid chromatography (HPLC) as one
of the most used techniques for analysing microdialysate samples.

2. Materials

2.1. Chemicals 1. Artificial extracellular fluid (aECF): dissolve 13.32 mg of CaCl2

and Solutions in 100 mL of double-distilled water and stir the solution for
10 min until completely dissolved. Add 20.14 mg of KCl,
858.48 mg of NaCl and 8.1 mg of MgCl2 and stir briefly.
 31 Brain Microdialysis in Freely Moving Animals 367

The final solution will have the following concentration:

147 mM NaCl, 1.2 mM CaCl2, 2.7 mM KCl and 0.85 mM
MgCl2. Filter the solution using 0.22-μm (pore diameter)
syringe filters (Millex-HP, Millipore, Billerica, USA). Divide
the solution in aliquots of 5 mL each and store at −20°C. The
solution is stable for several weeks.
2. Standard solution for microdialysis probe calibration:
(a) Ascorbic acid (AA): dissolve 1.76 mg in 1 mL of mH3PO4
1%.
(b) Uric acid (UA): dissolve 1.68 mg in 1 mL of mH3PO4 1%.
(c) Norepinephrine (NE): dissolve 1.62 mg in 1 mL of mH3PO4
1%, then dilute by adding 10 μL of this solution to 1 mL
of mH3PO4.
(d) L-Dihydroxyphenylalanine (L-DOPA): dissolve 1.97 mg in
1 mL of mH3PO4 1%, then dilute by adding 10 μL of this
solution to 1 mL of mH3PO4.
(e) Dihydroxyphenylacetic acid (DOPAC): dissolve 1.68 mg
in 2 mL of mH3PO4 1%.
(f) Dopamine (DA): dissolve 1.90 mg in 1 mL of mH3PO4
1%, then dilute by adding 10 μL of this solution to 1 mL
of mH3PO4.
(g) 5-Hydroxyindolacetic acid (5HIAA): dissolve 1.91 mg in
2 mL of mH3PO4 1%.
(h) Homovanillic acid (HVA): dissolve 1.82 mg in 2 mL of
mH3PO4 1%.
(i) 3-Methoxytyramine (3MT): dissolve 2.04 mg in 1 mL of
mH3PO4 1%, then dilute by adding 10 μL of this solution
to 1 mL of mH3PO4.
(j) 5-Hydroxytryptamine (5HT): dissolve 4.05 mg in 1 mL of
mH3PO4 1%, then dilute by adding 10 μL of this solution
to 1 mL of mH3PO4.
The final solution is prepared by adding different ali-
quots of solutions a–j above to 2,796 μL of aECF: 30 μL
of AA, 15 μL of UA, 30 μL of NE, 30 μL of L-DOPA,
3 μL of DOPAC, 30 μL of DA, 3 μL of 5HIAA, 3 μL of
HVA, 30 μL of 3MT and 30 μL of 5HT. The final concen-
trations will be AA 100 μM, UA 50 μM, NE 1 μM, L-DOPA
1 μM, DA 1 μM, DOPAC 5 μM, 5HIIA 5 μM, HVA
5 μM, 3MT 1 μM and 5HT 1 μM. Filter the solution using
0.22-μm (pore diameter) syringe filters (Millex-HP,
Millipore). Divide the solution in aliquots of 500 μL each
(using 1.5-mL Eppendorf vials) and store at −80°C. The
solution will be stable for few weeks.stock solutions of
neurochemicals are prepared in 1% mH3PO4, obtained by
368 G. Bazzu et al.

dissolving 1 g of mH3PO4 in 100 mL of distilled water,

corresponding to a concentration of 102.04 mM.
3. Anaesthetic and euthanasia solutions: prepare a 4% chloral
hydrate solution for anaesthesia by dissolving 4 g of solid in
100 mL of sterile saline (0.9% NaCl) and store at 4°C. Prepare
an 8% solution for euthanasia dissolving 8 g of chloral hydrate in
100 mL of sterile saline and store at 4°C. Before use, filter the
solutions using 0.22-μm syringe filters (Millex-HP, Millipore).
These solutions can be used for a week after preparation.
4. D-Amphetamine solutions for pharmacological treatments (10):
for subcutaneous (s.c.) administration, dissolve 1.2 mg of
D-amphetamine hydrochloride in 2.0 mL of sterile saline. For
retrodialysis administration, prepare a 200 μM solution by dis-
solving 1.35 mg of powder in 1 mL of sterile water, then dilute
40 μL of the former solution into 1,960 μL of aECF solution.
These solutions must be prepared immediately before use and
filtered using 0.22-μm syringe filters (Millex-HP, Millipore).
5. HPLC mobile phase: prepare 1 L of mobile phase by dissolving
84.5 g of KH2PO4 and 2.4 g of K2HPO4 in 850 mL of Milli-Q
water. Add 292.24 mg of EDTA and stir for few minutes until
completely dissolved. Add 150 mL of HPLC grade methanol
and at the end, add 80 mg of 1-octanesulfonic acid sodium
salt. Adjust to pH = 2.7 with concentrated H3PO4. Degas the
mobile phase solution using an ultrasonic bath for at least
30 min.
6. Standard solution for HPLC calibration:
stock solutions of neurochemicals are prepared in 1%
mH3PO4 as described in Subheading 2.1, step 1.
(a) AA: dissolve 1.76 mg of powder in 1 mL of mH3PO4 1%.
(b) UA: dissolve 1.68 mg of powder in 1 mL of mH3PO4 1%
and dilute by adding 10 μL of this solution to 1 mL of
mH3PO4.
(c) NE: dissolve 1.62 mg of powder in 1 mL of mH3PO4 1%
and dilute by adding 1 μL of this solution to 1 mL of
mH3PO4.
(d) L-DOPA: dissolve 1.97 mg in 1 mL of mH3PO4 1% and
dilute by adding 1 μL of this solution to 2 mL of
mH3PO4.
(e) DOPAC: dissolve 1.68 mg in 1 mL of mH3PO4 1% and dilute
by adding 10 μL of this solution to 1 mL of mH3PO4.
(f) DA: dissolve 1.90 mg in 1 mL of mH3PO4 1% and dilute
by adding 1 μL of this solution to 2 mL of mH3PO4.
(g) 5HIAA: dissolve 1.91 mg in 1 mL of mH3PO4 1% and dilute
by adding 10 μL of this solution to 1 mL of mH3PO4.
 31 Brain Microdialysis in Freely Moving Animals 369

(h) HVA: dissolve 1.82 mg in 1 mL of mH3PO4 1% and dilute

by adding 10 μL of this solution to 1 mL of mH3PO4.
(i) 3MT: dissolve 2.04 mg in 1 mL of mH3PO4 1% and dilute
by adding 1 μL of this solution to 2 mL of mH3PO4.
(j) 5HT: dissolve 4.05 mg in 1 mL of mH3PO4 1% and dilute
by adding 1 μL of this solution to 2 mL of mH3PO4.
The final solution is prepared by adding different ali-
quots of solutions a–j above to 2,851 μL of aECF: 2.3 μL
of AA, 30 μL of UA, 9 μL of NE, 7.6 μL of L-DOPA,
18 μL of DOPAC, 4.2 μL of DA, 15 μL of 5HIAA, 15 μL
of HVA, 30 μL of 3MT and 18 μL of 5HT. The final con-
centrations will be AA 7.5 μM, UA 1.0 μM, NE 15 nM,
L-DOPA 12.7 nM, DA 7.0 nM, DOPAC 0.6 μM, 5HIIA
0.5 μM, HVA 0.5 μM, 3MT 50 nM and 5HT 30 nM.
Filter the solution using 0.22-μm (pore diameter) syringe
filters (Millex-HP, Millipore). Divide the solution in ali-
quots of 50 μL each (using 0.5-mL Eppendorf vials) and
store at −80°C. The solution is stable for several weeks.

2.2. Animals and 1. Animals: male Wistar rats weighing 300 g or 10-week-old
Stereotaxic Surgery C57BL/6 mice (weighing 30 g) from Charles River (Ballina,
Italy) can be used for in vivo microdialysis experiments. The ani-
mals must be maintained under standard animal care conditions
(12:12-h light/dark cycle, lights coming on at 7 a.m., room
temperature 21°C), with food and water ad libitum. Prior to the
start of any experiment, the health of the rodents has to be
assessed according to published guidelines (11). All procedures
need to be specifically licenced under the European Community
directive 86/609 included in national legislatures (i.e. Decreto
No. 116/1992 of the Ministry of Public Health in Italy).
2. Brain atlas for stereotaxic coordinates: the George Paxinos’ vol-
umes entitled “The Rat Brain in Stereotaxic Coordinates” (12)
and “The Mouse Brain in Stereotaxic Coordinates” (13) are
the reference books for setting up a microdialysis experiment
in rats and mice, respectively.
3. Stereotaxic apparatus: stereotaxic frame for small animals
(Model 900, David Kopf Instruments, UK) for rats equipped
with a mouse adaptor for mice (Kopf Mouse Adaptor from
2Biol, Varese, Italy). We recommend placing the stereotaxic
frame on a vibration-free marble table to increase precision
during probe implantation and to reduce brain damage.
4. Neurosurgery materials: alcohol-based permanent marker
(OHPen 96 fine, Stabilo, Germany), Betadine disinfectant
(Meda Pharma, Italy), sterile cotton wool, anatomical and sur-
gical tweezers, scalpel and scalpel blades, 27-G needles, 1- and
2.5-mL disposable syringes (for intraperitoneal (i.p.) injection
370 G. Bazzu et al.

of the anaesthetic solution), scissors, crocodile clips, Dremel

drill and tips (small tip for screw hole and trephine tip), small
screws and dental cement (Paladur, Hereus Kulzer GmbH,
Hanau, Germany), 5/0 suture needles and needle holder.

2.3. Microdialysis 1. Microdialysis probe construction: razor blades, scalpel, scalpel

Probe and Circuits blades, alcohol-based permanent marker, 100 mm of microdi-
alysis hollow fibre (AN69 Hospal Industrie, France) obtained
from a blood dialysis cartridge, epoxy resin (Pattex, Henkel
Italia, Milano, Italy), low-magnification (2–20×) stereo
microscope, 100 mm of PE50 tubing (0.58 I.D. polyethylene
tubing, Intramedic BD Becton Dickinson Italia S.p.A.,
Milano, Italy), 75 mm of silica capillary (Polymicro
Technologies, Phoenix, AZ, USA), 23-Gauge (23 G) and
27-G needles (hypodermic needle, Artsana S.p.A.,Casnate,
como Italy), diamond wheel (Dremel® 545 Diamond Wheel,
Dremel, Milano, Italy), drill (Dremel® 200 Series, Dremel)
and needle holder
2. Microdialysis probe in vitro calibration: 300 mm of PE50 tub-
ing, 1-mL Hamilton syringe, 250-μL Eppendorf vials, 27-G
needles and microinfusion pump (KDS-101-CE, KD Scientific,
Halliston, MA)
3. In vivo microdialysis: 800 mm of PE50 tubing, 1-mL Hamilton
syringe, 250-μL Eppendorf vials, 27-G needles, swivel (375/22
Single channel swivel, 22 gauge, Instech Laboratories, Inc.,
Plymouth Meeting, PA USA) and microinfusion pump (KDS-
101-CE)

2.4. HPLC 1. Alltech 426 HPLC pump (Alltech Srl, Milan, Italy)
2. Rheodyne injection valves (Alltech Srl) equipped with a 25-μL
loop
3. 50-μL Hamilton syringes (Alltech Srl)
4. Alltech Adsorbosphere™ C18 Column (100 × 4.6 mm;
Alltech Srl)
5. Antec EC controlled equipped with VT03 cell having a glassy
carbon working electrode, a Ag/AgCl reference electrode and
a stainless steel counter electrode (Alfatech Srl, Milan, Italy)
6. HPLC polyetheretherketone (PEEK) tubing and accessories
for 25-μL loop and connections (Alfatech Srl)
7. Varian data acquisition software (Varian Star v. 5, Varian Inc.,
USA) and hardware (Varian 800 MIB, Varian)
8. Personal computer (1 GB RAM and 250 GB hard disk)
equipped with an Ethernet board (operating system: Windows
XP/Vista/Windows 7)
 31 Brain Microdialysis in Freely Moving Animals 371

2.5. Statistical Software for data storage, scientific graphing and statistical analysis:
Analysis Microsoft Excel 2007 (Microsoft Inc., USA), GraphPad Prism 5.0
(GraphPad Software, La Jolla, CA, USA), KaleidaGraph 4.0 (Synergy
software, Reading, PA, USA)

3. Methods

3.1. Microdialysis 1. Prepare three short microdialysis hollow fibres (25 mm in

Probe Construction length) cutting a straight microdialysis fibre with a sharp razor
blade (see Fig. 1a).

Fig. 1. Microdialysis probe construction procedure. See text for a detailed explanation of
the steps necessary for obtaining a working probe implantable in rat or mice striatum.
372 G. Bazzu et al.

2. Prepare the epoxy resin (see Note 1) on a small piece of

polycarbonate (usually a piece of transparent film measuring
5 × 5 cm) and seal one tip of the hollow fibre immersing it for
1 mm in the epoxy resin. The glue will rise in the hollow fibre
by capillary action, forming a cylinder with a meniscus in the
upper part (see Fig. 1b, arrow). Perform this step under a
microscope. Repeat this for each of the three fibres and leave
them to dry for at least 30 min at room temperature.
3. Cut one piece (30 mm in length) of PE50 tubing using the
razor blade and mark it with the permanent marker at 7 mm
from one end (see Fig. 1c, arrow).
4. Cut one silica capillary (25 mm in length) using the scalpel
blade (see Fig. 1d).
5. Cut a 23-G needle (10 mm in length) using the drill mounting
a diamond wheel. Gently curve the 23-G needle tip to obtain
an L-shape using a needle holder (see Fig. 1e).
6. Insert a 27-G needle in the PE50 and perforate the tube wall
in the direction of the permanent mark (see Fig. 1f); leave the
needle in this position.
7. Insert the silica capillary inside the 27-G needle up to two-
thirds of its length (see Fig. 1g) and gently remove the 27-G
needle while maintaining the position of the silica capillary. At
the end of this operation, the silica capillary will be inside the
PE50 tubing as illustrated in Fig. 1h.
8. The silica capillary has to be repositioned until it comes out
10 mm from the PE50 outlet (see Fig. 1h, arrows).
9. Insert the tip of the silica capillary (at the opposite side of the
outlet) inside the 23-G needle (prepared in the step 5) as illus-
trated in Fig. 1i.
10. Prepare the epoxy resin and glue the silica capillary, the PE50
tubing and the 23-G needle at the level of the permanent mark
(see Fig. 1i). At this point, the probe skeleton is made, and it
needs to dry for 30 min at room temperature.
11. Cut the previously prepared hollow fibres (14 mm in length
starting from the epoxy meniscus) with a sharp razor blade.
12. Under microscope, insert the silica capillary inside the straight
hollow fibre up to 0.5 mm from the epoxy meniscus (see
Fig. 1l).
13. Prepare the epoxy resin and glue the hollow fibre to the PE50
tubing (see Fig. 1l; Note 2). Leave the microdialysis probe to
dry for at least 30 min at room temperature.
14. Prepare the epoxy resin and ‘paint’ the external surface of the
hollow fibre using a 27-G needle (previously immersed in the
glue) for 6 mm (rats) or 8 mm (mice) starting from the PE50
 31 Brain Microdialysis in Freely Moving Animals 373

tubing (see Fig. 1l). Gently remove the excess epoxy using a
clean 27-G needle. During this step, the membrane will be
shielded which allows the dialysis process only in the probe’s
active area (around 4 mm for rats and 2 mm for mice). Leave
the microdialysis probe to dry for at least 30 min at room
temperature.
15. Store the microdialysis probe in a fresh and dark environment,
far from dust and moisture, and keep it in place for at least
24 h before use to allow complete polymerization of the epoxy
resin.

3.2. Microdialysis (a) Cut one piece (200 mm in length) of PE50 tubing using a
Probe In Vitro razor blade.
Calibration (b) Cut a second piece of PE50 tubing (50 mm in length).
3.2.1. Circuit for In Vitro (c) Cut a 23-G needle (10 mm in length) using the drill mounting
Probe Calibration (see a diamond wheel.
Fig. 2) (d) Insert the 23-G needle (for 4 mm) inside the probe outlet.
(e) Fill the Hamilton syringe (1 mL) with the aECF (see Note 3).
(f) Connect one end of the 200 mm PE50 tubing to the Hamilton
syringe needle and the other end to the probe inlet.
(g) Connect one end of the 50 mm PE50 tubing to the 23-G
needle previously inserted in the probe outlet.
(h) Place the Hamilton syringe on the infusion pump and fill the
circuit using the maximum flow rate (see Note 4).

3.2.2. In Vitro Probe (a) Place the microdialysis probe on the stereotaxic frame holder
Calibration (see Fig. 2) and set the infusion pump flow rate to 1.5 μL/min.
(b) Unfreeze one Eppendorf vial, containing the calibration solu-
tion, to room temperature.
(c) Insert the probe membrane (hollow fibre) in the calibration
solution and wait 10 min to allow for stabilisation of the
microdialysis gradient.
(d) Insert the free end of the 50 mm PE50 tubing in a 250-μL
Eppendorf vial and wait 20 min.
(e) Remove the Eppendorf vial (containing 30 μL of dialysate)
and store at −80°C until HPLC analysis (see Subheading 3.5).
At the same time, take a 30 μL sample of the calibration solu-
tion (in which the probe is immersed) and store at −80°C.
Repeat this step two more times.
(f) After HPLC analysis, calculate the percent recovery of the
probe for every neurochemical component in the calibration
solution, using the following formula:
Recovery (%) = [microdialysate] / [calibration solution] × 100 (1)

and determine the mean ± standard deviation (see Note 5).

374 G. Bazzu et al.

3.3. Neurosurgery 1. Anaesthetize the animal using the previously prepared 4%

chloral hydrate solution (400 mg/kg intraperitoneal) and switch
on the heating mat (37°C).
2. Shave the animal’s head and disinfect the skin over the skull
using a polyvinylpyrrolidone-based disinfectant (Betadine,
Meda Pharma, Italy).
3. Fix the animal’s head on the stereotaxic frame (rat) or in the
mouse adapter (mouse) (see Fig. 3a) and be sure that the body
is in contact with the heating mat.
4. Cut the skin over the skull in the middle line using a sharp
scalpel blade, following the rostro-caudal direction, starting
from the frontal region up to the occipital bone (see Fig. 3b).
5. Fix the skin borders using small surgical crocodile clips, remove
the periosteum from the skull using the scalpel blade (see Note 6)
and mark the bregma with the permanent marker (see Fig. 3c).
6. Calculate the coordinates (rostro-caudal and middle lateral) of
the right striatum using the Paxinos atlas of rat (or mouse)
brain mounting a 27-G needle on the stereotaxic frame holder.
Mark the corresponding point on the skull using the perma-
nent marker (see Figs. 1 and 3d).
7. Make the screw hole on the right parietal bone (see Figs. 2
and 3d) using the Dremel drill mounting a small tip and cut a
small disk of bone centred on the marked point (see Figs. 1
and 3e) using the trephine tip (cylindrical blade) exposing the
dura mater. Insert the screw in the parietal hole (see Figs. 2
and 3e).
8. Place the microdialysis probe on the stereotaxic frame holder
and recalculate the rostro-caudal and middle-lateral coordi-
nates from the bregma. Rest the tip of the probe on the dura
mater and calculate the dorso-ventral coordinate. Cut the dura
mater with a 27-G needle (see Note 7) and gently insert the
probe in the brain (see Fig. 3f).
9. Fix the microdialysis probe on the skull using dental cement
and anchor it to the screw previously inserted in the right pari-
etal bone (see Fig. 3g). Leave the dental cement to dry for at
least 10 min.
10. Remove the probe from the stereotaxic holder and close the
skin using a small semicircular suture needle and non-absorb-
able material (5/0 fine silk filament).
11. Disinfect the skin and remove the animal from stereotaxic
frame.
12. Place the animal in a warm and ventilated cage until complete
recovery from anaesthesia (see Note 8).
 31 Brain Microdialysis in Freely Moving Animals 375

Fig. 2. Circuit for the in vitro calibration of the microdialysis probe before in vivo use. The ratio between the concentrations
of neurochemicals, determined in the microdialysate and in the calibration solution, represents the probe recovery.

Fig. 3. Neurosurgery procedure used for implantation of the microdialysis probe in the striatum of anaesthetized rats or
mice. A stereotaxic frame for small animals is used for rats while it needs to be equipped with a mouse adaptor for mice.
See text for a detailed explanation of the steps illustrated in the figure.
376 G. Bazzu et al.

Fig. 4. Circuit for in vivo microdialysis in freely moving animals. The introduction of the liquid swivel prevents the formation
of sharp bends and folding of the polythene tubing during the animal’s rotations.

3.4. In Vivo (a) Cut two pieces (300 mm in length each) of PE50 tubing using
Microdialysis a razor blade.
3.4.1. Circuit for In Vivo (b) Cut a third piece of PE50 tubing (150 mm in length) and roll
Microdialysis Experiment up it to obtain three or four coils. Fix the coiled tubing with a
(see Fig. 4) small piece of adhesive tape as illustrated in Fig. 4.
(c) Cut a 23-G needle (10 mm in length) using the drill mounting
a diamond wheel.
(d) Insert the 23-G needle (for 4 mm) inside the probe outlet.
(e) Fill the Hamilton syringe (1 mL) with the aECF (see Note 3).
(f) Connect one end of the first 300 mm PE50 tubing to the
Hamilton syringe needle and the other end to the liquid swivel
inlet.
(g) Connect one end of the second 300 mm PE50 tubing to the
liquid swivel outlet and the other end to the probe inlet (the
probe is inserted in the brain).
(h) Place the Hamilton syringe on the infusion pump and fill the
circuit using the maximum flow rate (see Note 4).

3.4.2. In Vivo Experiment (a) Set the infusion pump flow rate to 1.5 μL/min and wait 60 min
with Freely Moving Animals to allow for stabilisation of the microdialysis gradient.
(see Fig. 4) (b) Connect one end of the 150 mm PE50 tubing to the 23-G
needle previously inserted in the probe outlet and wait 20 min.
 31 Brain Microdialysis in Freely Moving Animals 377

(c) Remove the 150 mm PE50 tubing from the animal head dis-
connecting it from the 23-G needle attached to the probe
outlet.
(d) Aspirate the microdialysate present inside the 150 mm PE50
tubing using a 50-μL Hamilton syringe, transfer it (around
30 μL) to a 250-μL Eppendorf vial and store at −80°C (see
Note 9) until HPLC analysis (Subheading 3.5). Repeat steps
b–d for every microdialysis sample recovered during the in vivo
experiment.
(e) Usually three consecutive microdialysis samples are sufficient
for defining the baseline of the examined neurochemicals (see
Note 10).
(f) After baseline sample recovery and if desired, a pharmacologi-
cal treatment can be performed via systemic or intracerebral
drug administration. D-Amphetamine prepared as previously
described (Subheading 2.1, step 4) can be injected subcutane-
ously (2 mg/kg corresponding to 1.0 mL in rats and 100 μL
in mice) or by intracerebral retrodialysis. In the second case,
the aECF containing 2 mM D-amphetamine needs to replace
the perfusion fluid present in the microdialysis circuit (see Note 11)
during the entire period of treatment (usually comprising
between 20 and 60 min, corresponding to 1–3 microdialysis
samples). Immediately after the systemic drug administration
or during its intracerebral retrodialysis, the microdialysis sam-
ples are recovered as illustrated previously (steps b–d).
(g) After the end of the intracerebral drug administration, the solu-
tion containing only aECF has to be restored (see Note 11).
(h) After the pharmacological treatments, microdialysates can be
recovered for a period varying from 120 to 240 min (corre-
sponding to 3–6 samples).
(i) At the end of the experiment, disconnect the animal from the
microdialysis circuit and sacrifice it using the euthanasia solu-
tion (described in Subheading 2.1.3) injected intraperitoneum
(2.0 mL in rats or 200 μL in mice). At this point, you can
verify the correct positioning of the microdialysis probe as
described elsewhere (10).

3.5. HPLC Setup 1. As illustrated in Fig. 5, the mobile phase is moved by an iso-
and Sample Analysis cratic pump, through PE tubes, within the Rheodyne injector
equipped with a 25-μL loop. Through the injector, 30 μL
sample is loaded and channelled into the C18 column where
the substances are separated, revealed by electrochemical cell
and quantified by the proper software (14, 15).
2. One hour prior to starting the experiment, check the electro-
chemical cell for applied potential (+780 mV vs. Ag/AgCl ref-
erence electrode), then increase gradually the mobile phase
378 G. Bazzu et al.

Fig. 5. HPLC setup for ascorbic acid, uric acid, catecholamine and indolamine quantification in microdialysate samples
from the striatum of freely moving animals. The electrochemical detection (EC) allows the amperometric oxidation of the
abovementioned neurochemicals using a +780 mV potential vs. Ag/AgCl reference electrode. The circuit is completely
metal-free for preventing sample degradation during the analysis. WE glassy carbon working electrode; RE Ag/AgCl refer-
ence electrode; CE stainless steel counter electrode.

flux from 0.3 mL/min (not-operating flux) to 1.2 mL/min

and let stabilise for 45 min.
3. After stabilisation, inject 30 μL of the same aECF solution
used for the microdialysis experiment (see Note 12).
4. Thaw and inject 30 μL of standard solution and with the proper
software, calculate each peak area. After each injection, care-
fully wash the syringe with ultrapure water.
5. Thaw (if necessary) and inject 30 μL of microdialysate sample.
6. By means of the provided software, calculate sample area peaks
and the concentration of each compound using the following
equation:
[sample] = (area of microdialysis sample / area of standard solution) (2)
× [standard solution].

7. Repeat steps 5 and 6 for each microdialysate sample.

8. Report microdialysis sample concentrations (for every neuro-
chemical component analysed) in a spreadsheet and perform
statistical analysis.
 31 Brain Microdialysis in Freely Moving Animals 379

3.6. Microdialysis Data The concentrations in the dialysate will be expressed in nM (DA,
Analysis L-DOPA, NE, 3HT and 3MT) or μM (AA, UA, DOPAC, HVA
and 5-HIAA) and given as mean ± SEM. D-Amphetamine effects
on neurochemicals can be evaluated as absolute or percent changes
from the preceding baseline (for this purpose data need to be
expressed as percentage of baseline). Statistical significance is
assessed using analysis of variance (ANOVA) for differences over
time. Differences within groups are determined by paired t tests,
with the Newman-Keuls multiple comparison adjustment. Pear-
son’s correlation coefficient between neurochemicals (i.e. DA vs.
3-MT, AA vs. DA or DA vs. DOPAC + HVA) could be calculated
in each animal after drug administration.

4. Notes

1. The preparation of the epoxy resin is a key point of the entire

process. It is necessary to mix the two epoxy components (resin
and hardener) in equal amounts for 2–3 min, and wait for
obtaining the desired consistency before use. Although the
epoxy resin becomes solid in few minutes, it needs at least 24 h
for complete polymerization.
2. The epoxy resin used in this step has to be less consistent: mix
70% of resin and 30% of hardener. Apply a small amount of
epoxy resin and pay attention to the capillary effect (suction
effect) inside the PE50 tubing.
3. Prevent the formation of air bubbles inside the syringe by gen-
tly aspiring the aECF.
4. Prevent the formation of air bubbles inside the entire circuit
(and in particular in the dialyzing part of the probe) filling the
tubing by gently pushing the syringe piston.
5. The percent recovery of the probe is an important parameter
for comparing microdialysis performance among probes made
by the same operator or different operators. Differences in the
construction process (mainly related to probe active surface
area) can influence this parameter. It is necessary to minimise
the standard deviation around the mean for each neurochemi-
cal to be sure that the calibration results are consistent.
6. Periosteum is rich in nociceptors while they are not present in
the brain; it is thus necessary to remove this membrane to pre-
vent pain in the postoperative period and during the microdi-
alysis experiment.
7. Dural venous sinuses (also called dural sinuses) are venous
channels found between layers of dura mater and are particu-
larly present under the cranial sutures. A lesion at this level can
induce copious bleeding.
380 G. Bazzu et al.

8. It could be useful to monitor body temperature, breathing and

animal reflexes at regular intervals. A tardive side effect of chlo-
ral hydrate is the production of pulmonary secretions treatable
with subcutaneous adrenaline.
9. Alternatively, you can directly analyse the microdialysis sample
by introducing it in the Rheodyne injector as illustrated in
Fig. 5.
10. Sometimes, baseline levels of neurochemicals (DA in particular)
are not so stable. In this condition, wait until the percent varia-
tion of three consecutive samples is lower than 10%.
11. For facilitating and speeding up the solution exchange proce-
dure, you can disconnect the fluidic circuit upstream of the
microdialysis probe and reconnect it filled with the new solu-
tion (see also Notes 3 and 4).
12. No chromatographic peaks are visible in normal conditions. If
oxidizable interferents are visible after aECF injection, a con-
taminant is present in the aECF solution. Replace the solution.

Acknowledgments

The authors would like to thank Mr. Gianni Esposito, HPLC

expert and brilliant laboratory technician, for his past work and
future suggestions. This work was supported by University of
Sassari (ex 60% fund), Fondazione Banco di Sardegna and Regione
Autonoma della Sardegna.

References

1. Chaurasia CS, Müller M, Bashaw ED, Benfeldt of intracellular water space in humans. Am
E, Bolinder J, Bullock R, et al (2007) AAPS- J Physiol 253, 228–231
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Principles, Application and Regulatory microdialysis under transient conditions. Anal
Perspectives Pharm Res 24, 1014–1025 Chem 65, 1017–1022
2. Delgado JMR, DeFeudis FV, Roth RH, Ryugo 7. Wang P, Wong S, and Sawchuk RJ (1993)
DK, and Mitruka BM (1972) Dialytrode for Microdialysis calibration using retrodialysis and
long-term intracerebral perfusion in awake mon- zero-net flux: application to a study of the dis-
keys. Arch Int Pharmacodyn Ther 198, 7–21 tribution of zidovudine to rabbit cerebrospinal
3. Ungerstedt U, and Pycock C (1974) Functional fluid and thalamus. Pharm Res 10, 1411–1419
correlates of dopamine neurotransmission. Bull 8. Charalambides C, Sgouros S, and Sakas D
Schweiz Akad Med Wiss 30, 44–55 (2010) Intracerebral microdialysis in children.
4. O’Neill RD, Rocchitta G, McMahon CP, Serra Childs Nerv Syst 26, 215–220
PA, and Lowry JP (2008) Designing sensitive 9. Nordström CH (2010) Cerebral energy metab-
and selective polymer/enzyme composite bio- olism and microdialysis in neurocritical care
sensors for brain monitoring in vivo. Trends Childs Nerv Syst 26, 465–472
Anal Chem 27, 78–88 10. Miele M, Mura MA, Enrico P, Esposito G,
5. Lönnroth P, Jansson PA, and Smith U (1987) A Serra PA, Migheli R, et al (2000) On the
microdialysis method allowing characterization mechanism of d-amphetamine-induced changes
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in glutamate, ascorbic acid and uric acid release 14. Rocchitta G, Migheli R, Esposito G, Marchetti
in the striatum of freely moving rats. Br J B, Desole MS, Miele E, et al (2006) Endogenous
Pharmacol 129, 582–588 melatonin protects L-DOPA from autoxida-
11. Morton DB, and Griffiths PHM (1985) tion in the striatal extracellular compartment of
Guidelines on the recognition of pain, distress the freely moving rat: potential implication for
and discomfort in experimental animals and a long-term L-DOPA therapy in Parkinson’s dis-
hypothesis for assessment. Vet Rec 116, 431–436 ease. J Pineal Res 40, 204–213
12. Paxinos G, and Watson C (1986) The Rat 15. Rocchitta G, Migheli R, Mura MP, Grella G,
Brain in Stereotaxic Coordinates. Academic Esposito G, Marchetti B, et al (2005) Signaling
Press, San Diego pathways in the nitric oxide and iron-induced
13. Paxinos G, and Franklin KBJ (2001) The dopamine release in the striatum of freely mov-
Mouse Brain in Stereotaxic Coordinates Second ing rats: role of extracellular Ca2+ and L-type
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 Chapter 32

Evaluating Motor Neuron Death in Neonatal Rats

Subjected to Sciatic Nerve Lesion
Andre Schwambach Vieira, Alexandre Cesar Santos de Rezende,
and Fabio Rogerio

Abstract
Neonatal sciatic nerve lesion is a useful experimental model for the study of neuronal cell death. Sciatic
nerve transection or crush is the most frequently used approach to evaluate motoneuron loss in the lumbar
enlargement of the spinal cord. Here we describe and illustrate the surgical procedures performed in our
laboratory to assess motoneuron cell death and the related cellular mechanisms.

Key words: Neonatal rat, Sciatic nerve, Transection, Crush, Motoneuron, Neuronal cell death

1. Introduction

During neurogenesis, programmed cell death is considered to play

a role in the establishment of neural pathways. Specifically, neurons
produced in excess would be excluded through cellular mechanisms
dependent on the availability of trophic factors (1, 2). Since the
original studies conducted by Rita Levi-Montalcini (3) on the nature
and characterization of neurotrophic factors, it has become clear that
distinct populations of neurons are sensitive to specific trophic
molecules. Innervated organs and cells of the central and peripheral
nervous systems are recognized as sources of such molecules. In
addition to the evidence obtained during embryogenesis, the
dependence of neuronal cells on trophic factors synthesized by
their targets can be experimentally highlighted after transectioning
or crushing the fibers of a peripheral nerve (1, 4–10).
A commonly used model to study the effects of the disruption
of peripheral trophic support on neurons is transectioning the sciatic
nerve of rodents within the first postnatal week. In this approach,
the nerve is completely sectioned and separated in two stumps,

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_32, © Springer Science+Business Media, LLC 2012

383
384 A.S. Vieira et al.

proximal and distal, which are kept distant one from the other.
Therefore, since physical contact between the stumps is not allowed,
axonal regeneration is impaired (11). When performed at the first
postnatal day (P0), such type of injury determines the loss of approx-
imately 100% of the lesioned motoneurons in the lumbar enlarge-
ment of the spinal cord. Such extensive neuronal cell loss is not
achieved when the lesion is performed in older animals, particularly
adults. Besides disruption of trophic support from the target, it is
hypothesized that immature nonneuronal cells present in the periph-
eral nerves, especially Schwann cells, would not be able to provide
the lesioned motoneurons with trophic factors. The opposite would
be true for the sciatic nerve of adult animals (5, 12–14).
Another experimental model used to evaluate motoneuron
damage is sciatic nerve crush. Conversely to the nerve fiber transection
model, the axons are not totally sectioned. In fact, as neural fibers
are disrupted by compression, the nerve stumps are thought to
remain connected, which would allow for neuronal regeneration.
Specifically, cellular recovery would be favored by a microenviron-
ment established at the injury site, in which growth factors and
chemokines released by cells and molecules of the extracellular
matrix would allow remodeling of the nerve stumps, axonal recovery,
and neuronal regeneration, at least in part, after the lesion. However,
it is important to emphasize that Schwann cells of neonatal rats
have reduced competence to support regenerating motoneurons
when compared to adult Schwann cells. One hypothesis that may
be put forward is that immature Schwann cells have reduced
production of trophic factors (1, 5, 15, 16).
Motoneuron lesion induced by sciatic nerve transection or
crush in neonatal rats is an experimental model that can be used in
many research fields of neuroscience, such as neuronal cell death
pathways, peripheral nerve regeneration, and functional recovery
and evaluation of trophic factors and neuroprotective agents. In
our laboratory, we have focused on the use of exogenous growth
factors and neuroprotective substances on motoneuron survival
after sciatic transection in neonatal rats. In particular, we have been
investigating neuronal cell survival and the possible related cellular
pathways through motoneuron counting and evaluating protein
and gene expression (9, 10, 17, 18). Here we describe the surgical
procedures currently performed in our laboratory to induce
motoneuron injury in the lumbar spinal cord of neonatal rats by
either transectioning or crushing the sciatic nerve.

2. Materials

2.1. Surgical 1. Microscissors

Procedure 2. Microforceps
3. Inverted microforceps
 32 Evaluating Motor Neuron Death in Neonatal Rats Subjected… 385

4. Microneedle holder
5. Scalpel #11
6. 8-0 suture silk
7. Glass Petri dish
8. Cotton swabs
9. 100-W incandescent lamp
10. Surgical microscope

2.2. Transcardiac 1. Ketamine (75 mg/kg)/xylazine (15 mg/kg)

Perfusion 2. 1-mL plastic syringe
3. Surgical table
4. Surgical scissors
5. Forceps
6. Hemostatic forceps
7. 28-G needle attached to flexible tube
8. Peristaltic pump
9. Microscissors
10. 0.9% NaCl solution containing 400 U/L of heparin
11. 4% Buffered formaldehyde solution

2.3. Spinal Cord 1. Surgical scissors

Dissection 2. Forceps
3. Scalpel #11
4. Microscissors
5. Microforceps
6. Surgical table
7. Surgical microscope

3. Methods

All the experimental procedures described herein were approved

by the Committee on Animal Care of the State University of
Campinas (protocol # 509-1).

3.1. Surgical 1. Separate rat pups from their mother in a small plastic box and
Procedures maintain them under a 100-W incandescent lamp until the
experiment is complete (see Note 1).
2. Immerse one rat in crushed ice for 4 min to induce anesthesia
by hypothermia (see Note 2).
386 A.S. Vieira et al.

Fig. 1. Anatomical location of the neonatal rat sciatic nerve as seen under the surgical microscope. (a) The approximate
location of the sciatic nerve (SN) is shown as a dotted line on the skin. The inset shows the region magnified in (a). (b, c)
The SN runs under the vastus lateralis (VL) and the adductor magnus (AM) muscles. (d, e) The VL and AM muscles were
removed to expose the SN. (f) Higher magnification of the SN. The arrow indicates the proximal region of the nerve. (c, d)
The nerve and the muscles are indicated by dotted lines.

3. Place the anesthetized pup on a Petri dish. From this moment

on, perform all procedures under the surgical microscope.
4. Make a 1-mm incision on the skin of the midthigh 2 mm poste-
rior to the upper half of the femoral bone (see Figs. 1 and 2).
5. Introduce the microforceps between the muscles vastus lateralis
and adductor magnus and separate them by repeatedly opening
and closing the forceps (see Figs. 1 and 2).
6. The sciatic nerve runs under the muscles mentioned above
(item 5). Using the microscissors and the microforceps, dissect
the nerve until the site where it crosses the incisura ischiadica.
7. For sciatic nerve transection, hold the nerve with the microfor-
ceps and cut it using the microscissors, 3 mm proximal and distal
to the region being held. Remove the nerve segment which
was isolated (see Note 3; Fig. 2).
8. For sciatic nerve crushing, press the nerve using the inverted
microforceps with fine tips. Keep such pressure for 30 s (see
Note 4).
9. Reapproximate the vastus lateralis and adductor magnus
muscles and suture the skin using the 8-0 silk.
 32 Evaluating Motor Neuron Death in Neonatal Rats Subjected… 387

Fig. 2. Anatomical location of the neonatal rat sciatic nerve as seen under the surgical microscope. (a) Incision on the
midthigh skin. (b) Exposed sciatic nerve after separation of the vastus lateralis and the adductor magnus muscles. (c) Higher
magnification of the region shown in (b). (d) The segment of nerve removed after transection is shown between the tips of
the microscissors. Bar: 3.0 mm (a, b, d), 1.2 mm (c).

10. Place the neonate rat under the 100-W incandescent lamp until
recovery is observed (see Note 5).
11. Return the pups to their mother (see Note 5).

3.2. Transcardiac 1. Separate rat pups from their mother in a small plastic box and
Perfusion maintain them under a 100-W incandescent lamp (see Note 1).
2. For anesthesia, administer the solution of ketamine (75 mg/kg)/
xylazine (15 mg/kg) intraperitoneally.
3. When the rat does not display any pedal reflex, fixate it on the
surgical table.
4. Make a skin incision through the midline, extending from the
sternal angle to the pubic region.
5. In the abdominal region, make a bilateral incision parallel to
the subcostal region and a midline linear incision. In this
procedure, the muscular and peritoneal membrane will be sec-
tioned, exposing the abdominal cavity.
388 A.S. Vieira et al.

6. Use small scissors to make an incision in the diaphragm in

order to access the thoracic cavity.
7. Make bilateral sections along each lateral part of the thoracic
cage. Lift the released ribs using the hemostatic forceps in
order to expose the heart.
8. Gently introduce the needle in the left ventricle and cut the
right atrium with microscissors (see Note 6).
9. Use a peristaltic pump to perfuse the rat with saline solution,
followed by a 4% buffered formaldehyde solution (see Note 7).

3.3. Spinal Cord 1. After perfusion, fixate the rat in the prone position (decubitus
Dissection ventralis) on a surgical table.
2. Make an extensive dorsal skin incision through the midline to
expose the paravertebral musculature. From this moment on,
perform all procedures under the surgical microscope.
3. Gently remove the paravertebral musculature using a scalpel
and the vertebrae with the microforceps. Then, section the
meningeal covering to expose the spinal cord (see Note 8).
4. For motoneuron investigation, isolate the lumbar enlargement
from the remaining spinal cord using a scalpel. Remove the
lumbar enlargement from the spinal canal, sectioning the nerve
roots and meningeal membrane using microscissors.
5. The isolated lumbar enlargement may be processed using stan-
dard histological protocols for paraffin embedding or frozen
processing (see Note 9).

4. Notes

1. We routinely use an incandescent lamp (100 W) under which

the pups are kept warm throughout the experiment, as they
are isolated from the mother and placed in a plastic cage.
A distance of approximately 20 cm between the lamp and the
rat is usually sufficient. For anesthetic recovery, the same
approach is performed.
2. Deep hypothermia in young rodents is an efficient technique
to induce anesthesia (19). Since the pups are not pharmaco-
logically treated, there is no risk of death associated with
overdose. After a short period of being unexposed to the
incandescent lamp, the rat must be totally immersed in crushed
ice until limb movements completely stop. A period of 4–5 min
of hypothermia (counting from the immersion) is sufficient to
elicit anesthesia for a surgical procedure of about 10 min.
3. It is crucial to keep the nerve stumps at a distance to avoid
regeneration. For this goal, an additional useful procedure is to
32 Evaluating Motor Neuron Death in Neonatal Rats Subjected… 389

retract the nerve endings in opposite directions using the

microforceps before approximating the muscles for wound
closure.
4. The inverted microforceps correspond to custom-made micro-
forceps with a 1-mm tip that opens when pressed and closes
when released, that is, the opposite to a standard microforceps.
In our experience, such inverted microforceps are extremely
helpful to ensure a constant and reproducible pressure applied
to the sciatic nerve in crushing experiments performed in
different occasions.
5. After the surgical procedure, dry the animals and place them
under the incandescent lamp until indirect signals suggestive of
anesthetic recovery are observed. We consider active respiratory
and upper limb movements as reliable observations that the
pup has recovered from anesthesia and is ready to be returned
to its mother. Before returning the pups to their mother, make
sure that the surgical wound is completely free of blood clots.
The presence of blood may interfere with the acceptance of
the mother to its pups and eventually with breastfeeding.
6. During transcardiac perfusion, take extreme care to avoid the
rupture of the cardiac septum. A useful procedure is to attach
a plastic catheter to the 28-G needle. The catheter should be
2 mm shorter than the needle in order to allow the introduc-
tion of only 2 mm of the needle inside the heart. The rupture
of the septum may be inferred by increase in lung volume and
elimination of liquids through the upper airways.
7. In our experience, the ideal perfusion rate is 3.0 mL/min. At
this rate, the perfusion with saline takes approximately 5 min,
and with formaldehyde, approximately 15 min. After the per-
fusion, we usually keep the whole rat immersed in a buffered
formaldehyde solution for 12–24 h. The goal of this step is to
avoid the occurrence of histological artifacts (see Note 8).
8. Extreme care should be taken when dissecting the spinal cord.
Excessive manipulation of inadequately perfused specimen or
after insufficient period of fixation may induce the occurrence
of the so-called dark neurons. In hematoxylin- and eosin-
stained sections, dark neurons are shrunken, irregular, and
densely basophilic cells. This particular histological artifact may
interfere with the morphological evaluation of motoneurons,
mainly being confused with dying neurons (20).
9. Morphological evaluation of spinal motoneuron loss induced by
sciatic lesion in neonatal rats may be performed through differ-
ent approaches. Since the sciatic nerve is constituted of motoneu-
rons whose bodies are in the ventrolateral region of the lumbar
enlargement (L4 and L5 segments mainly), a peripheral insult
allows one to carry out evaluation by counting the remaining
cellular bodies. A possible semiquantitative evaluation consists
390 A.S. Vieira et al.

Fig. 3. Transverse sections of the lumbar enlargement (L4) of rats 5 days after sciatic transection performed at an age of 2
days postnatally. (a) Reduction of the number of motor neurons is observed in the right ventrolateral region (shown in
(c)) as compared to the contralateral unlesioned side (shown in (b)). Paraffin-embedded sections stained with cresyl violet.
Bar: 500 μm (a), 100 μm (b, c).

of counting the nucleoli of motoneurons ipsilateral and

contralateral to the axonal injury. In general, we count nucleoli
in 20 serial paraffin sections (5 μm) per animal. Specifically, the
first section of every four is considered for counting. Then, a
ratio of the total number of nucleoli in the lesioned side to that
in the unlesioned side is calculated for each animal and used for
further statistical analyses (17, 18) (see Fig. 3). Alternatively, the
spinal cord may be subjected to stereological techniques for a
quantitative approach, such as the “optical fractionator” (21).
For this goal, we usually freeze the lumbar enlargement and
obtain 40-μm serial sections. Subsequently, a random systematic
uniform sampling is performed for the selection of sections used
for counting and for the placement of a grid of known size
in each sample. Then, an optical dissector, a three-dimensional
counting probe, is placed in each point of intersection of the
grid that falls in the region of interest. The total number of cells
is given by the formula C = SQ- . t/h . 1/asf . 1/ssf, where C is
the total number cells, SQ- is the sum of cells counted in the
optical dissector, t is the section thickness, h is the dissector
height, asf is the ratio between counting frame and grid areas,
and ssf is the ratio between sections used for counting and total
number of sections. For details on the fractionator and other
stereological tools, the reader is referred to an introductory
review by Gundersen et al. (22).

References
1. Kuno M (1990) Target dependence of the degeneration of motor neurons after
motoneural survival: the current status. J Neurosci axotomy. Nature 345, 440–441
Res 9, 155–172 5. Lowrie MB, and Vrbova G (1992) Dependence of
2. Oppenheim RW (1991) Cell death during postnatal motoneurones on their targets: review
development of the nervous system. Ann Rev and hypothesis. Trends Neurosci 15, 80–84
Neurosci 14, 453–501 6. Sendtner M, Holtmann B, Kolbeck R, Thoenen
3. Levi-Montalcini, R (1964) The nerve growth H, and Barde YA (1992) Brain-derived neu-
factor. Ann NY Acad Sci 118, 149–170 rotrophic factor prevents the death of motoneu-
4. Sendtner M, Kreutzberg GW, and Thoenen H rons in newborn rats after nerve section.
(1990) Ciliary neurotrophic factor prevents Nature 360, 757–759
 32 Evaluating Motor Neuron Death in Neonatal Rats Subjected… 391

7. Greensmith L, and Vrbova G (1996) neurotrophic factor in the sciatic nerve of the
Motoneurone survival: a functional approach. adult rat after lesion and during regeneration.
Trends Neurosci 19, 450–455 J Cell Biol 118, 139–148
8. Oliveira AL, Risling M, Negro A, Langone F, 16. Magill CK, Moore AM, Yan Y, Tong AY,
and Cullheim S (2002) Apoptosis of spinal MacEwan MR, Yee A, et al. (2010) The dif-
interneurons induced by sciatic nerve axotomy ferential effects of pathway- versus target-
in the neonatal rat is counteracted by nerve derived glial cell line-derived neurotrophic
growth factor and ciliary neurotrophic factor. factor on peripheral ner ve regeneration.
J Comp Neurol 447, 381–393 J Neurosurg 113, 102–109
9. Rezende ACS, Vieira AS, Rogerio F, Rezende 17. Rogerio F, De Souza Queiroz L, Teixeira SA,
LF, Boschero AC, Negro A, et al. (2008) Effects Oliveira AL, De Nucci G, and Langone F
of systemic administration of ciliary neurotrophic (2002) Neuroprotective action of melatonin
factor on Bax and Bcl-2 proteins in lumbar spinal on neonatal rat motoneurons after sciatic nerve
cord of neonatal rats after sciatic nerve transec- transection. Brain Res 926, 33–41
tion. Braz J Med Biol Res 41, 1024–1028 18. Rogerio F, Teixeira SA, Rezende ACS, Cofino
10. Rezende ACS, Peroni D, Vieira AS, Rogerio F, R, Queiroz LS, De Nucci G, et al. (2005)
Talaisys RL, Costa FTM, et al. (2009) Ciliary Superoxide dismutase isoforms 1 and 2 in lum-
neurotrophic factor fused to a protein trans- bar spinal cord of neonatal rats after sciatic
duction domain retains full neuroprotective nerve transection and melatonin treatment.
activity in the absence of cytokine-like side Dev Brain Res 154, 217–225
effects. J Neurochem 109, 1680–1690 19. Phifer CB, and Terry LM (1986) Use of hypo-
11. Vejsada R, Sagot Y, and Kato AC (1995) thermia for general anesthesia in preweanling
Quantitative comparison of the transient res- rodents. Physiol Behav 38, 887–890
cue effects of neurotrophic factors on axoto- 20. Cammermeyer J (1961) The importance of
mized motoneurons in vivo. Eur J Neurosci 7, avoiding “dark” neurons in experimental neu-
108–115 ropathology. Acta Neuropathol 1, 245–270
12. Schmalbruch H (1984) Motoneuron death 21. West MJ, Slomianka L, and Gundersen
after sciatic nerve section in newborn rats. J Comp HJG (1991) Unbiased stereological esti-
Neurol 224, 252–258 mation of the total number of neurons in
13. Schmalbruch H (1987) Loss of sensory neu- the subdivisions of the rat hippocampus
rons after sciatic nerve section in the rat. Anat using the optical fractionator. Anat Rec
Rec 219, 323–329 231, 482–497
14. Lowrie MB, Lavalette D, and Davies CE (1994) 22. Gundersen HJ, Bagger P, Bendtsen TF, Evans
Time course of motoneurone death after neo- SM, Korbo L, Marcussen N, et al. (1988) The
natal sciatic nerve crush in the rat. Dev Neurosci new stereological tools: dissector, fractionator,
16, 279–284 nucleator and point sampled intercepts and
15. Sendtner M, Stöckli KA, and Thoenen H their use in pathological research and diagnosis.
(1992) Synthesis and localization of ciliary APMIS 96, 857–881
 Chapter 33

Rodent Spinal Cord Injury Model and Application

of Neurotrophic Factors for Neuroprotection
Hari Shanker Sharma and Aruna Sharma

Abstract
Spinal cord injury (SCI) is a serious clinical problem that causes lifetime disabilities to victims and inflicting
huge social burden on our society. One of the main lacunae in developing potential therapeutic measures
in SCI is a lack of suitable animal models that could be comparable to clinical situations. Thus, develop-
ment of new animal models of SCI is highly needed to expand our knowledge on cell injury and repair
process in order to reduce cord pathology, and in translating advanced therapies in patients of SCI to
improve therapeutic strategies. Keeping these views in mind, a suitable animal model is developed in our
laboratory that can be used to explore new therapeutic tools in SCI. The details of our methods used to
induce SCI in rodents and neuroprotection achieved by use of selected neurotrophic factors are described
in this chapter.

Key words: Spinal cord injury, New model, Neurotrophic factors, Neuroprotection, Cell injury,
Neurorepair

1. Introduction

Spinal cord injury (SCI) is a devastating disease causing severe

disability, e.g., paralysis and paraplegia, in human populations
across the globe (1, 2). Thus, victims of SCI require lifetime reha-
bilitation and support causing serious burden on our society (3).
Unfortunately, the treatment strategies for SCI if delayed beyond
3 h may not achieve significant neuroprotection (4, 5). Thus,
efforts should be made to treat trauma patients as soon as possible
following primary insult to the central nervous system.
Available evidence suggests that most of the tissue destructive
changes in the spinal cord occur within the first hour after the
primary insult and progress with time (3, 6). The early conse-
quences of SCI include breakdown of the blood–spinal cord
barrier (BSCB), reduction in local blood flow, edema formation,

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7_33, © Springer Science+Business Media, LLC 2012

393
394 H.S. Sharma and A. Sharma

and neuronal, glial, and myelin damage (1, 3–5). These changes
will continue further with advancement of time beyond the injury
point across the spinal cord in rostrocaudal directions leading to
permanent disability (1, 4, 5, 7). Thus, a detailed knowledge on
early pathological events is necessary in exploring suitable
therapeutic strategies to treat SCI victims.
Recent studies show that treatment schedule initiated after 3–6 h
of primary insult is largely ineffective on the spinal cord pathology
(1, 4, 5). Thus, studies using early therapeutic interventions to
reduce spinal cord cell and tissue damage are needed. Obviously,
when spinal cord neurons along with nonneuronal cells, e.g., glial
cells, endothelial cells, and myelin, are protected after injury, then
only neurorecovery and/or neuroprotection could be achieved
(1, 8–12). Restoration of spinal cord cell and tissue function after
trauma is crucial to reduce sensory motor disturbances (1, 2, 13–17).
Accordingly, neuroregeneration and neurorepair mechanisms appear
to be the most important factors for any future spinal cord thera-
peutic strategies after injury.
Our studies have shown that topical application of neurotro-
phins, including nerve growth factor (NGF), brain derived neu-
rotrophic factor (BDNF), or other growth factors, e.g., glial-derived
neurotrophic factor (GDNF) and ciliary neurotrophic factor (CTNF),
reduces spinal cord pathology and functional disabilities in a rat
model of SCI when administered repeatedly within 10–30 min
after trauma (5, 6, 9–12). This suggests that neurotrophins could
be the potential and ideal candidates for effective therapeutic strat-
egies in SCI. However, further studies are needed to understand
the potential beneficial effects of various neurotrophins or other
compounds in combination to effectively reduce the pathological
consequences of SCI. For this purpose, an effective animal model
of SCI is needed to explore neuroprotective efficacy of various
compounds in the vicinity of the lesion site as well in the ipsilateral
or contralateral side of the cord (13, 14, 16, 17). However, to date,
there are no models of SCI in which this ipsi- or contralateral
aspects of spinal cord pathology can be examined in detail. This is
largely due to the fact that most models include compression,
weight drop, or transaction, leaving no scope to study the effects
of lesion or trauma around the injury site (1, 3–5, 15). Thus, effects
of drugs in the ipsilateral or contralateral cord are still lacking in
other models of SCI.
In this chapter, we describe a new model of SCI in rats and
mice in which both ipsilateral and contralateral cell changes can
be examined within the lesion site as well as in the rostrocaudal
directions of the cord after trauma. These features make this
model unique in expanding our understanding of the pathophysi-
ological consequences of trauma within the spinal cord in a very
precise manner.
 33 Rodent Spinal Cord Injury Model and Application of Neurotrophic… 395

2. Materials

2.1. Chemicals 1. Urethane.

and Other Reagents 2. Pentobarbital.
3. Ketamine.
4. Xylazine.
5. Equithesin (Swedish Pharmacy, Umeå, Sweden): chloral
hydrate 4.25 g, pentobarbital sodium 0.97 g, magnesium sul-
fate 2.1 g, propylene glycol 2.1 g, ethanol anhydrous 42.8 g,
and sterile water 9 g (100 mL solution). The anesthetic solu-
tion has a short expiry date of 1 month (see ref. (18, 19)).
6. BDNF, recombinant human (Sigma-Aldrich).
7. GDNF, recombinant human (Sigma-Aldrich).
8. Insulin-like growth factor-I, recombinant human (IGF-1)
(Sigma-Aldrich).
9. CNTF, recombinant rat (Sigma-Aldrich).
10. NGF-b from rat (Sigma-Aldrich).
11. Neurotrophin-3, recombinant human (NT-3) (Sigma-Aldrich).
12. Evans blue (Merck).
13. [131]-Iodine (Nordion, Belgium).
14. Paraformaldehyde.
15. Glutaraldehyde.
16. Lanthanum chloride.
17. Somogyi fixative (4% neutral paraformaldehyde, 0.05% glutar-
aldehyde, and 0.25% picric acid).
18. Osmium tetroxide.
19. Araldite.
20. Electron microscope grade Epon 812.
21. Toluidine blue.
22. Uranyl acetate.
23. Lead citrate.

2.2. Neurotrophin 1. Dissolve neurotrophic factors in phosphate-buffered saline to a

Stock Solutions concentration of 100 mg/mL and store at −20°C in a desiccator.
and Storage At the time of experiments, dilute the neurotrophic factors in
solution to a concentration of 1 mg/10 mL so that you could
adjust their dose by adjusting the volume (20, 21).
2. The diluted neurotrophic agent solutions can be safely stored
at 4°C in a desiccator for 3–7 days. If you need to use it longer
than a week, you could store it under desiccation at −20°C,
396 H.S. Sharma and A. Sharma

but avoid freeze–thaw cycles. CNTF, although stable at room

temperature for 3 weeks, is preferably stored at −20°C in a
desiccator.

2.3. Lab Apparatus 1. Liston bone cutter, straight: 14 cm/5.5 in. (Harvard Surgical
and Other Supplies Instruments)
2. Friedman Rongeur, curved: 14 cm/5.5 in. (Harvard Surgical
Instruments)
3. Dissecting stereomicroscope (Carl Zeiss, 20–30×)
4. Magnifying glass lamp (Magnifying Lamp 900-061).
5. Circular 22 W fluorescent light (Chicago electric)
6. Dual Lab standard stereotaxic frame for rats or mice, with 45°
ear bars (Harvard Apparatus)
7. EdgeAhead crescent knife, 1.75 mm edge length, 60°, bevel up
8. Gelco sponge (London)
9. PE 10 cannula
10. 21- and 36-gauge needles, butterfly needles
11. 50-mL-capacity Hamilton microliter syringes (HAMILTON
Bonaduz AG, Bonaduz, Switzerland)
12. Precision microinfusion pump (Razel Scientific Instrument
Inc, Stamford, CT, USA)
13. 3-in. Gamma counter (Packard)
14. Precision electronic balance (e.g., METTLER TOLEDO)
15. Oven capable of reaching 90°C (e.g., Class 100 Cleanroom
Ovens, Terra Universal)
16. Light microscope (Zeiss or similar type)
17. Ultramicrotome with diamond knife (LKB or comparable
supplier)
18. Phillips 400 transmission electron microscope (or comparable
model)

3. Methods

All the existing animal models have essentially some advantages

and drawbacks (1–5). The basic differences between the existing
animal models and human SCI are summarized in Table 1. Human
SCI results from four main vectors of forces, e.g., flexion, exten-
sion, rotation, and compression, that are working simultaneously
or in combination (1–3, 5, 6). Fracture of vertebral bodies or
luxation of vertebrae leads to concussion, contusion, or laceration
of the cord due to these multiple sheer forces. Thus, human SCI
 33 Rodent Spinal Cord Injury Model and Application of Neurotrophic… 397

Table 1
Animal vs. human SCI factors, variability, and differences

Clinical vs. experimental spinal cord injury (SCI)

Clinical Experimental

Multifactorial Simple
Flexion, extension, rotation, compression Weight drop, incision,a compression, transaction,
concussion, contusion, laceration hemisection, electrolytic lesion, contusion, clip
compression
Complex forces are active at the time of injury Only one injury factor is present
Closed vertebral system injury Open vertebral system injury
After laminectomy
Usually anterior compression Mainly posterior compression
Mainly occurs during state Produced under anesthesia conscious
Unlimited or uncontrolled time interval between Carefully controlled time schedule hospital
injury and first hand examination

Human vs. rat spinal cordb

Parameters Human Rat

Number of neurons 109 0.36

Length (cm) 43–45 8–10
Weight (g) 34 0.7
Proportion to brain (volume %) 2 35
Glial/neuron ratio 10–15 9–12c
Endothelial cell/neuron ratio 12–16c 10–14c
Weight-drop injury (g) 2,428 50b
971 20b
485 10b
Compiled from various sources (for details see ref. (1–8))
Data modified after Sharma (3–7) (for details see ref. (4, 5))
For details see: http://faculty.washington.edu/chudler/facts.html#spinal visited on Nov 25, 2010
a
Authors own investigation
b
Weight of rat spinal cord is about 50 times less than human spinal cord. Normally, 10–50 g weight is
dropped on the rat spinal cord for making injury (values represent static weight)
c
Rough estimate based on various sources on assumption based on 1 or 2 endothelial cells per capillary
(minimum)
398 H.S. Sharma and A. Sharma

includes various kinds of physical and mechanical damage that

finally determine the nature and extent of the cord pathology and
varies in each individual case (1). Furthermore, human SCI results
from the anterior cord compression or damage in conscious state
on the closed vertebral system, whereas, all animal models
represent posterior cord damage under anesthesia after laminec-
tomy (1–3, 8). In addition, the animal models of SCI involve one
force vector at one time.
To this end, we developed a new model of SCI in rats and mice
to study cell changes in the ipsilateral or contralateral cord in
rostrocaudal direction (see Fig. 1). The model consists of a longi-
tudinal incision to the right dorsal horn of the T10–11 segments.
The deepest part of the lesion is limited to the Rexed laminae VIII
(13–15). The model is quite reproducible, and the extent of lesion
varied only within a narrow range (3–7). The primary injury, i.e.,
the knife wound, is limited to the dorsal horn gray matter, leaving
white matter largely intact (16, 17), whereas in other injury models
using impact, compression, hemisection, or transection, direct
damage to white matter predominantly accounts for loss of sensory
motor functions in the ascending and descending spinal tracts
(see Table 2).

Fig. 1. Diagrammatic representation of spinal cord injury (SCI) in the rat and sampling of spinal cord tissues T9 or T12 for
morphological or biochemical analyses (a). (b) Depth of lesion (L) in the right dorsal horn and selection of tissue areas from
the ipsi- or contralateral cord in dorsal horn (1) or ventral horn (2) is clearly identified. Bar = 5 mm. Data modified after
Sharma (4, 5, 9, 20).
 33 Rodent Spinal Cord Injury Model and Application of Neurotrophic… 399

Table 2
Advantages and shortcomings of various experimental models of spinal cord injury

Type of injury Advantages Shortcomings

Weight-drop technique Since second century ad revised by Allen

(on exposed spinal (32)
cord) Widely used today in many species
Can be graded
Reproducible Results are variable
Closely simulates some of the Differs from clinical conditions
biomechanics of human SCI because of posterior
compression injury
Histopathology mimics human SCI, e.g., Variable results
hemorrhage, necrosis, cyst formation
Revised with electromechanical impact
device by Bresnahan et al. (33)
Better reproducibility Variable results
Balloon inflation in the
spinal extradural space
Photochemical No laminectomy required Differs from clinical conditions
infarction of vascular
endothelium
Incisiona Longitudinal by Sharma and Dey (34)
Modified by Sharma and Olsson (13)
Reproducible Differs from clinical conditions
Can be graded
Reproducible results
Transverse or longitudinal
White matter mainly intact (17) Does not mimic clinical injury
Minimal and focal injury
Changes can be seen ipsilateral/
contralateral
Spread of cell injury can be examined
in both rostrocaudal and ipsi-/
contralateral directions
A good model for studying secondary Involves simple “cut” factor
injury mechanisms Hemorrhage due to direct
blood vessel damage
Information compiled from various sources (for details see text). Data modified after Sharma and Westman
(3); Winkler et al. (7)
a
Authors’ own investigation

3.1. Animal Handling 1. Use rats or mice of specific age, sex, and strain according to
protocol. We used male Sprague–Dawley rats (180–250 g) or
C57 mice (30–35 g) from Alab, Stockholm, Sweden.
2. Keep the animals under controlled laboratory room temperature
(21 ± 1°C) with 12-h light and 12-h dark schedule, with food
pellets and tap water supplied ad libitum up to 6–7 days before
experiments.
400 H.S. Sharma and A. Sharma

3. Take individual rats/mice out from the cage by gently handling

the animal. Avoid lifting animals by the tail since this proce-
dure will induce severe stress, e.g., increase in heart rate and
respiration. Use good-quality sterile hand gloves (18, 19, 22).
4. Place the animals on a weighing pan large enough for easy
movement of animals. Do not use preweighed restraint boxes.
This will create unnecessary stress in animals. Record the body
weight, age, sex, and source of the animals. Date and time of
the experiments should also be recorded.
5. All experimental conditions for SCI and related techniques
should be performed following National Institute of Health
Guidelines for the care of laboratory animals and duly approved
by Local Institutional Ethics Committee.

3.2. Choice Several anesthetics can be used in SCI experiments depending on

of Anesthetics the local laboratory guidelines and permission from the respective
institutional authorities. A brief description of advantages and
disadvantages regarding use of particular anesthetics in SCI is
described below (see ref. (18, 19, 22)).

3.2.1. Urethane 1. Urethane (ethyl carbamate) is a colorless crystalline substance

readily soluble in water (»1 g in 0.5 mL water at room
temperature) with a neutral pH. This is a very safe anesthetic
as it induces only mild changes in the cardiovascular and respi-
ratory system without influencing the sympathetic nervous
system activity. For neurophysiological investigations, urethane
is a good choice for anesthesia because it largely acts on the
cortical level and does not depress the respiratory or cardiovas-
cular centers in the brain stem (19).
2. The long-lasting effects of urethane (>12 h) at the level of
surgical anesthesia can be achieved using a single dose ranging
between 1.5 and 1.8 g/kg, intraperitoneally, in rodents (22)
(see Note 1).

3.2.2. Pentobarbital 1. Surgical grade of anesthesia in rats can be achieved by sodium

pentobarbital in dose of 40–60 mg/kg, intraperitoneally.
However, this anesthesia induces profound bronchial secretions
that may be sometimes fatal.
2. To avoid bronchial secretions and poor respiration during the
experiment, pretreat with atropine (0.1 mg/kg, subcutane-
ously, 20–30 min before induction of anesthesia), if this drug
is not interfering with the experimental design and protocol.
3. The effects of anesthesia are short lasting (i.e., 60–80 min) and
require administration of maintenance doses at regular interval
of 45–60 min. The safety margin of pentobarbital anesthesia is
also quite narrow.
 33 Rodent Spinal Cord Injury Model and Application of Neurotrophic… 401

3.2.3. Combination 1. A combination of ketamine and a muscle relaxant like xylazine

Anesthesia is a good choice of anesthesia in various laboratories. However,
under this anesthesia, several reflexes, e.g., blinking of eyes,
swallowing, and movements of whiskers, or vibrissae, are present
although the animal does not respond to pain stimuli.
2. The duration of anesthesia is short lasting and depends on the
dose used. The safety margin is considerably higher than
sodium pentobarbital alone.

3.2.4. Mixture 1. In some laboratories, a mixture of various anesthetics is used

of Anesthesia to produce a safe anesthesia in animals of varying duration.
One such anesthetic that is widely used in small animals is
Equithesin.
2. Equithesin is a mixture of sodium pentobarbital, ketamine,
and muscle relaxant (see below). This anesthesia is very safe in
a dose of 3 mL/kg, intraperitoneal, for rats and mice. Respiratory
depression is seldom seen. However, the main drawback for
this anesthesia is its short duration, i.e., 30–40 min. Thus,
repeated maintenance doses are necessary to maintain a certain
level of anesthesia that is often difficult.

3.3. Administration 1 Hold rats/mice firmly from the back skin using a towel, and
of Anesthesia take care that the animal is not able to move its head otherwise
you may get bitten. While holding the animal, firmly turn it
around to expose its belly toward you.
2 Use an already prepared anesthetic in a syringe (for dose and
choice of anesthesia, see below). Gently administer the anesthetic
into the peritoneal space that can be reached from the lower
groin with a needle angle of 30° and pushing about 0.5–1 cm
inside using a 36-G stainless needle. Just before injection, pull
the plunger toward you to see that the needle is inside the
peritoneal cavity. In such case, a vacuum is created (18, 19)
(see Note 2).
3 If the syringe appears tight and pulling the plunger results in a
vacuum, then push the correct dose of anesthesia slowly within
20–30 s.
4 After completion of the injection, gently return the rat to its
cage and watch for the effects of anesthetics. The rat will show
signs of anesthesia and will gradually fall down on the cage floor
and go to sleep. Use a mild tail pinch or ear pinch to check
the stage of anesthesia. If the animal does not respond to these
stimuli, it is in the surgical grade of anesthesia (Grade IV) (18).

3.4. Exposing 1. After surgical grade anesthesia is obtained, shave hairs on the
the Spinal Column back skin gently and expose the skin area covering the spinal
column over the thoracic region. Wash the skin surface with
70% alcohol to sterilize the skin surface.
402 H.S. Sharma and A. Sharma

2. Make a longitudinal incision of the skin over the thoracic

vertebra pertaining to T8 to T12 (about 2.5–3 cm long). Wipe
out any blood coming from the underlying muscle. Identify
the thoracic T10 and 11 vertebras by its stout build just rostral
to the lumbar enlargement. Normally, this can be achieved by
counting vertebral spine from thoracic segments 1–12 (see
Note 3).
3. After identifying the T10–11 vertebra, make small incisions
around the vertebral muscle to clean the bone surface extending
between T9 and T12 vertebrae. Clean any oozing blood using
sterile cotton with some mild pressure over it (13, 14) (see
Note 4).
4. After exposing and cleaning the T10–11 vertebrae from blood,
apply cotton soaked with 0.9% saline over the exposed surface
and leave it for 5–10 min to stabilize the spinal column. This
will also alleviate stress reaction over the spinal cord due to any
pressure applied over the spinal column (13–15) (see Note 5).

3.5. Exposing 1. After exposing the T10–11 spines and articular processes,
the Spinal Cord insert one tip of a very-fine-pointed side-cutting bone cutter
(Laminectomy) carefully between T10 and T11 vertebral space.
2. Carefully remove the vertebral bone using space between the
two adjacent vertebrae. When you are able to perform on
the right side, use the same procedure to remove a part of the
vertebra on the left side as well. After that, use a precision
bone rongeur to widen the space by removing pieces of bones
slowly (see Note 6).
3. During the bone removal process, apply cold saline (4°C) over
the bone and spinal cord surface frequently to keep the tissue
wet all the time (16, 17).
4. Use a dissecting stereomicroscope to remove bone and to
avoid direct damage to the spinal cord, if necessary. However,
after some experience, a good magnifying glass lamp with a
circular 22 W fluorescent light will be enough to perform
laminectomy.

3.6. Making the Lesion 1. After laminectomy, stabilize the spinal column of the
rat or mice under a stereotaxic apparatus. Use constant
cold saline application over the exposed spinal cord.
2. Using one of the vertical axis manipulators of the stereotaxic
apparatus, fix the microknife with a straight bevel positioned
at 90° over one end of the exposed spinal cord (see Note 7).
3. Adjust the coordinates to fit the knifepoint over the right
dorsal horn (about 0.8 mm right to the dorsal spinal artery) of
the T10 segment. Carefully lower the tip of microknife to
 33 Rodent Spinal Cord Injury Model and Application of Neurotrophic… 403

touch the dura over the right dorsal horn without piercing it.
Any accidental piercing will result in leakage of spinal fluid.
4. After positioning the knife over the right dorsal horn, insert
the tip of the knife within the spinal cord up to 1.5 mm deep
to reach around the Rexed laminae VIII to IX ((13–15, 17);
see Fig. 1).
5. After reaching deeper into the desired area, gradually move the
knife using stereotaxic apparatus longitudinally backward up to
5 mm (13–17). After the lesion, remove the knife from the
spinal cord immediately and cover the wound with cotton soaked
in saline to prevent drying of the cord tissue. If there is any
bleeding, gently soak it with Gelco sponge (see Fig. 1; Note 8).

3.6.1. Alternative Methods 1. An experienced worker could also make a lesion using a #11
to Induce Spinal Cord sterile carbon steel surgical blade by hand under a magnifying
Injury fluorescent lamp in rats or in mice (13).
2. For making lesion by hand, the knife blade may be sealed with
araldite leaving only 1.5 mm tip open to avoid accidental
injury due to spinal reflex while making incision. The knife
preparation can be done one night before the experiment (13, 16)
(see Note 9).

3.7. Postoperative 1. After the lesion, remove the animals from the stereotaxic plat-
Care form and place them in a secure plastic cage on good surgical
bedding. Do not put spinal cord injured animals in the cage
with wooden scrap as small pieces of wood may choke these
anesthetized rats/mice causing to death.
2. Place each rat/mice in individual cages to avoid any possible
attack by other animal or laceration of the wound in case ani-
mals need to be revived for behavioral investigations. However,
for the study of spinal cord pathophysiology, it is not necessary
to awaken them. In such cases, maintain the anesthetic dose
for the entire survival period as needed by the experimental
protocol (19).

3.8. Application of To investigate the role of neurotrophins alone or in combination in

Neurotrophic Factors inducing neuroprotection after SCI in the above model, we use the
After Spinal Cord Injury following protocol for treatment (20, 21, 23–30).

3.8.1. Topical Application 1. Prepare the desired dilution of each neurotrophic factors in sep-
of Neurotrophins over arate 50-mL-capacity Hamilton microliter syringes. Connect the
the Spinal Cord syringe to a precision microinfusion pump for slow infusion.
Calibrate the speed of injection to deliver 10 mL/min to allow
0.25 mL over the spinal cord within 15 s (20, 21, 23, 24). Should
you choose for coapplication, replace with another neurotro-
phin-filled syringe after 15 s. Normally, a combination of 2–3
neurotrophic factors may be completed within less than a minute.
404 H.S. Sharma and A. Sharma

2. Alternatively, use microsyringes manually to administer

neurotrophic factors over the spinal cord by putting the tip
over the cord and rotate the plunger inward slowly to deliver
0.25 mL over 5–10 s. Adjust the concentration of neurotro-
phins from the stock solution or dilution to achieve the
desired doses of neurotrophins.
3. When using an infusion pump, attach a polythene cannula (PE
10) to the syringe and place the other end of the cannula over
the spinal cord either around the lesion site or over the normal
cord in the control group for spinal drug delivery.
4. In case of repeated topical application of neurotrophins alone
or in combination, use either manual or microinfusion pump
delivery according to the experimental protocol (see Fig. 1).
The same system can be adapted to mice model as well, if
needed (see Note 10).

3.8.2. Selection of Dosage 1. In one group of experiments, apply BDNF or IGF-1 in separate
and Combination of group of rats (0.1 mg/10 mL in phosphate-buffered saline)
Neurotrophic Factors in SCI repeatedly, starting from 30 min before injury followed by 10,
30, and 60 min after injury. This application may continue
then every hour until 240 min after injury (27–30).
2. In other group of animals, apply various combinations of
BDNF with GDNF, NGF, NT-3 or IGF-1, or GDNF starting
from 30 to 90 min after SCI (or according to your plan) and
allow the animals to survive the desired time after injury. You
can control the total dose of the neurotrophins adjusted
(0.5 mg each) in 10–30 mL solution (0.5 mg of BDNF + 0.5 mg
of GDNF, or IGF-1, NGF or NT-3) and apply topically over
the injured spinal cord within 10 s (20, 21, 23, 24). Control
group may receive 10–30 m L of 0.9% saline instead of
neurotrophins (25–28).

3.9. Parameters Spinal cord injury induces rapid leakage of proteins across the
for Assessing BSCB and will cause edema and cell injury. These cell and tissue
Neuroprotection injuries progress over time across the longitudinal and transverse
axis of the spinal cord, leading to functional disability. It is likely
that neurotrophic factors if applied soon after SCI will reduce
BSCB disturbances, edema formation, and cell injury (20, 29, 30).
The following subsections describe parameters, which may be used
to assess the neuroprotective effects of neurotrophins on SCI.

3.9.1. BSCB Permeability To measure BSCB breakdown in spinal cord injury, we use Evans
blue and radioiodine ([131]-Iodine) as tracers. These tracers when
introduced into the circulation bind to serum proteins. Thus, their
leakage within the cord represents tracer–protein complex indicating
vasogenic edema formation (18, 19).
 33 Rodent Spinal Cord Injury Model and Application of Neurotrophic… 405

1. Anesthetize the rats or mice with suitable anesthesia at the end

of the experiment and expose the right femoral vein by making
an incision on the skin of the hind leg.
2. Use a hypodermic 1-mL plastic syringe filled with these tracers
(Evans blue 2% solution, 3 mL/kg and radioiodine, 100 mCi/kg)
connected to a fine-tip 36-G needle. Using needle puncture,
with care slowly inject the tracers into the femoral vein (19).
3. Alternatively, insert a PE-10 cannula in the right femoral vein
after anesthesia to inject the tracer. However, in mice, right jugular
vein cannulation may be used to administer the tracer (6).
4. After 5 min of tracer injection, open the chest rapidly and
expose the heart. Insert a perfusion cannula (butterfly needle,
21 G) connected to either a perfusion apparatus or a peristaltic
pump attached to 0.9% sterile saline solution (250 mL) into
the left ventricle of the heart. After this procedure, cut the
right auricle and start saline perfusion at a pressure of 90 mmHg
for 45 s to wash out the remaining tracer within the blood
vessels (see Note 11).
5. After perfusion with saline (approximately 50 mL for rats and
20 mL for mice), dissect the spinal cord and identify several
regions around the rostral and caudal portion of the cord (see
Table 3). Analyze the leakage of Evans blue into the spinal
cord visually under a magnifying lens.
6. Leakage of Evans blue or radioiodine can be determined in the
blue areas by counting the radioactivity in a 3-in. Gamma
counter. The Evans blue could be measured using colorimetric
methods after detection of radioactivity biochemically as
described in details earlier (18, 19, 22). Leakage of Evans blue
or radioactivity is normally expressed as percentage increase
over the whole blood concentration (22).

3.9.2. Spinal Cord Edema Edema in the spinal cord could be determined by measuring water
Formation content of different spinal cord segments.
1. At the end of the experiment, remove the spinal cord and
divide it quickly into several segments such as T9, T10, T11,
and T12 and weigh them accurately using a precision
electronic balance (see Note 12).
2. Immediately after taking wet weight of the samples, place them
in an oven maintained at 90°C for 72 h. At this time, total
water may be evaporated and you could take the dry weight of
the sample (see Note 13).

3.9.3. Spinal Cord 1. Perfuse the rats or mice through the heart with either cold
Morphology 0.9% sterile saline (as mentioned above) or using cold phosphate-
buffered saline (0.1 M, pH 7.0) to washout blood inside the micro-
vessels (about 100–150 mL for rat and 60–80 mL for mice).
 Table 3
406

Postinjury treatment with neurotrophins either alone or in combination on spinal cord injury (SCI) induced blood–spinal
cord barrier (BSCB) permeability, spinal cord edema formation, and cell injury in the T9 segment in rats. The SCI was
performed by making a longitudinal incision into the right dorsal horn of the T10–11 segments, and the animals were
allowed to survive 5 h after trauma [for details see text]

Neurotrophins treatment alone (1 mg)a Neurotrophins combination (1 mg)a

BDNF GDNF NT-3 BDNF + GDNF BDNF + GDNF BDNF + NT-3 BDNF + NT-3
Parameters
measured Control 5 h SCI +30 min +30 min +30 min +60 min +90 min 60 min 90 min
H.S. Sharma and A. Sharma

BSCB permeability
Evans blue 0.24 ± 0.04 1.65 ± 0.12** 0.87 ± 0.32b 0.79 ± 0.22c 1.67 ± 0.13ns. 0.72 ± 0.18c 0.89 ± 0.23b 1.68 ± 0.34** 1.73 ± 0.14**
(mg %)
[131]
Iodine (%) 0.35 ± 0.06 1.96 ± 0.14** 0.94 ± 0.12c 0.89 ± 0.12c 1.88 ± 0.26ns 0.81 ± 0.16c 1.07 ± 0.24b 1.98 ± 0.44** 2.08 ± 0.51**
Edema formation
Water content 66.12 ± 0.18 69.34 ± 0.23** 67.67 ± 0.21b 67.23 ± 0.18b 68.86 ± 0.34ns 67.16 ± 0.12c 68.21 ± 0.12b 69.84 ± 0.13** 69.98 ± 0.43**
(%)
Cell injury
Neuronal Nil 4 2 ± 1d 2 ± 1d 4 ± 1ns 2 ± 2d 2 ± 2d 4±1 4±1
damage
Endothelium Nil 4 2 ± 1d 2 ± 2d 4±1 2 ± 1d 3 ± 1d 3 ± 1d 4±1
damage
(La3+)
Data modified after Sharma (5–8) (see text)
Values are mean ± SD of 5–6 rats in each group
*P < 0.05; **P < 0.01 (compared from control)
a
BDNF, GDNF, or NT-3 (Total amount, 1 mg in 10 mL) was applied topically in separate group of animals after SCI. In combination, BDNF, GDNF, or
NT-3 (0.5 mg each, total dose 1 mg) was used in identical manner
b
P < 0.05
c
P < 0.01 (compared from 5 h SCI), ANOVA followed by Dunnett’s test from one control
d
P < 0.05, Chi-square test from 5 h SCI group, ns not significant (from 5 h SCI group)
 33 Rodent Spinal Cord Injury Model and Application of Neurotrophic… 407

2. After the blood washout, perfuse with cold 0.4% buffered

paraformaldehyde or Simonyi fixative through the heart
(about 250–300 mL for rat and 150–200 mL for mice) (see
Note 14).
3. Cut small pieces of spinal cord tissues from T9, T10–11, or
T12 segments and embed them in paraffin using standard
procedures (14, 16).
4. Cut 3-mm thick paraffin sections from each block and stain the
sections using hematoxylin–eosin or Nissl staining to study
neuronal damage (15).
5. Examine these sections with a light microscope and save the
digital images at ×10 and ×20 magnifications using commercial
software for further analyses (see Note 15).

3.9.4. Ultrastructural 1. Perfuse with Somogyi fixative for better preservation of cell
Changes in the Spinal Cord membrane and nuclei (22).
2. To study BSCB breakdown at the ultrastructural level, add lan-
thanum chloride (2.5%, w/v) into the fixative before perfu-
sion. Lanthanum is an ion that can be seen at the ultrastructural
level across the microvessels without any special processing of
the tissue (25–28).
3. After perfusion, cut small tissue pieces from the desired spinal
cord segments and postfix them in osmium tetroxide and then
embed in araldite or electron microscope grade Epon 812.
4. Cut semithin sections (1 mm thick) from each block on a
microtome using a glass knife and stain them with toluidine
blue. Analyze these sections under a microscope and identify
special areas of the cord for ultrastructural observations (14,
15) (also see Fig. 2).
5. After identifying the desired areas of the spinal cord, trim the
block and cut ultrathin sections on an ultramicrotome using
diamond knife. Collect these ultrathin sections on a one whole
copper grid (or mesh grid) and counterstain them with uranyl
acetate and lead citrate before viewing them under a Phillips
400 transmission electron microscope.
6. Save the images using a digital camera using a basic magnification
of 4,000–6,000 of the grid for comparison (see ref. (4, 5)).
Lanthanum can be seen as crystallized dark black structures
around the microvessels and in the neuropil if BSCB break-
down occurred (4, 5) (see Fig. 3).

3.9.5. Pathological 1. Topical application of BDNF or IGF-1 in high quantity reduced

Findings After Neurotrophin the trauma-induced BSCB disruption to Evans blue albumin
Treatment in SCI and [131]-I (radioiodine) in the T9 and the T12 segments. In
these neurotrophin-treated animals, trauma-induced edema for-
mation and cell injuries are considerably attenuated (see Fig. 3).
408 H.S. Sharma and A. Sharma

Fig. 2. Epon-embedded toluidine blue–stained high-resolution (1 mm) thick sections from

spinal cord under light microscopy show profound expansion and edema in the T9 and
T12 segment of an untreated rat (a, b). Treatment with BDNF (c) or GDNF (d) alone remark-
ably attenuated spinal cord general expansion and edema after injury. Bar = 3 mm. Data
modified after Sharma (4, 5, 9).

2. Morphological examination revealed less distortion of nerve

cells, glial cells, and myelin vesiculation in the BDNF-treated
or IGF-1-treated injured rats (see Fig. 3). At the ultrastruc-
tural level, the neuropil is quite preserved, and vacuolation,
perivascular edema, and myelin damage were minimal in the
neurotrophin-treated animals (see Fig. 3). Infiltration of lan-
thanum across the spinal cord endothelial cell membrane and
in the basal lamina is also reduced in these neurotrophin-
treated rats (see Fig. 3).
3. These beneficial effects of neurotrophins were most pro-
nounced when the BDNF or IGF-1 was administered within
10 min of spinal trauma. Later application of neurotrophins
was less effective in reducing spinal cord pathology.

3.10. Neurotrophin 1. Application of BDNF in combination with NT-3 or NGF

Combination Is More (0.5 mg each) 30 min after SCI markedly improved spinal cord
Effective in Inducing morphology (see Table 3). This effect was markedly absent
Neuroprotection when neurotrophins were applied either 60 or 90 min after
SCI. Interestingly, even a triple combination of these neurotro-
phins (BDNF + NT-3 + NGF, 0.5 mg each) did not induce
 33 Rodent Spinal Cord Injury Model and Application of Neurotrophic… 409

Fig. 3. Repeated application of BDNF or IGF-1 over spinal cord following injury induces neuroprotection. (Aa) BDNF or IGF-1
was applied (1 mg in 10 mL) over the spinal cord starting from 30 min before injury followed by 5, 10, 15, and 30 min after
trauma, and thereafter, 60, 120, 180, and 240 min after injury. The animals were allowed to survive 5 h (a). BDNF or IGF-1
reduced BSCB breakdown to radiotracer in different segments (b) that coincided well with reduction in edema formation
(c). Morphological investigations showed preservation of myelin as seen using myelin basic protein (MBP) immunoreactiv-
ity (Bb) as compared to untreated spinal cord MBP (Ba). Ultrastructural studies showed exudation of lanthanum into the
spinal cord neuropil and across the microvessels (arrows Bc) that was significantly reduced by BDNF treatment (Bd).
Bar: B:a,b = 50 mm, B:c.d: 1 mm. Data modified after Sharma (4, 5, 9).
410 H.S. Sharma and A. Sharma

significant improvement in motor function beyond 60 min

SCI (results not shown). On the other hand, combining BDNF
with IGF-1 and GDNF (0.5 mg each) improved motor
function even after 90 min of SCI (see Table 3).
2. Profound reduction in the BSCB permeability to Evans blue
and radioiodine was also noted in traumatized animals that
received BDNF in combination with NT-3 or NGF 30 min after
SCI (see Table 3). No apparent reduction in BSCB perme-
ability was seen even when these neurotrophins were applied in
a triple combination (BDNF + NT-3 + NGF, 0.5 mg each)
beyond 60 min SCI (Sharma HS, unpublished observations).
However, a triple combination of BDNF with IGF-1 and
GDNF (0.5 mg each) significantly reduced the BSCB leakage
even after 90 min of SCI (see Table 3).
3. This combination of BDNF with NT-3 and/or NGF was also
effective in reducing edema formation if applied at 30 min but
not after 60 or 90 min SCI (see Table 3). On the other hand,
a combination of BDNF with IGF-1 and GDNF remarkably
reduced spinal cord edema formation when applied even
60–90 min after injury (see Table 3).
4. Cell changes in the spinal cord were markedly reduced by the
triple combination of BDNF, IGF-1, and GDNF treatment
that was effective when these neurotrophins were administered
60–90 min after SCI (see Table 3, Fig. 4). One the other hand,
a combination of BDNF with NT-3 and/or NGF did not
reduce cell changes in the spinal cord if applied 60 min after
SCI (see Table 3).

4. Notes

1. Urethane has some carcinogenic effects, and thus, care should

be taken for its use in the laboratory and exposure to humans.
Local safety regulations for the use and disposal of urethane
should be followed strictly (see ref. (18)).
2. If the needle pierces any other organ, pulling the plunger may
draw blood into the syringe. If this happens, then withdraw
anesthesia and try again after some time.
3. In rats, the cervical vertebra largely represents the similar area
of the spinal cord unlike humans where the vertebrae do not
normally represent the same level.
4. Sometimes, topical application of sterile saline (0.9%) over the
muscle will help reduce bleeding. Expose the vertebra slowly
and make it dry with often application of cold saline over the
vertebral column so that spinal cord is not damaged by direct
exposure to air.
33 Rodent Spinal Cord Injury Model and Application of Neurotrophic… 411

Fig. 4. A suitable combination of neurotrophic factors is more effective than their treatment
alone. Thus, treatment with BDNF, IGF-1, and GDNF in combination reduces motoneuron
damage after trauma markedly (c) as compared to untreated injured group (d). Whereas,
a combination of BDNF, NGF, and NT-3 was not that effective in reducing cell damage after
SCI either given 60 or 90 min after primary insult (a, b). Bar = 40 mm. Data modified after
Sharma (4, 5, 9, 20).

5. Watch carefully the respiration and also maintain animal body

temperature using a heating pad or any other device able to
control body temperature. Do not allow rectal temperature to
fall more than −0.5°C. Avoid direct heating of the spinal cord
or spinal column over the muscle or skin using infrared lamp.
This will increase the tissue temperature of the cord and spinal
column, leading to disturbances in respiration and other vital
organ functions.
412 H.S. Sharma and A. Sharma

6. Always use the bone rongeur moving outward to cut the bone
in order to avoid direct contact of metal parts with the spinal
cord. Try to make space over the dorsal spinal cord (about
5–6 mm), keeping the dorsal spinal artery in the middle. Avoid
any deep lateral opening over the spinal cord or spinal roots.
Also, make special care not to damage the dura matter while
removing the vertebral bone over the cord (6).
7. The Crescent microsurgery knife can easily penetrate tissue
without making injuries, and a precision lesion could be
produced without any laceration of the cord.
8. Take special care about the possible spinal reflex when any
metal touches the spinal cord. For this purpose, stabilization of
rat spinal cord is necessary by suitable clamps or using a firm
hold by hand over the back of the rat/mouse as and when
appropriate to avoid spinal reflex while making the injury (1, 4,
5). Normally, under surgical grade of anesthesia, spinal reflex
will be of minor degree during the lesion procedure. Any
unwanted movement of spinal cord due to spinal reflex will
damage spinal cord to different extents and result in variability
of data collection. If this happens, discard those animals from
data analyses.
9. Our laboratory compared results of spinal cord edema forma-
tion in rats and mice after microknife incision using stereotaxic
apparatus and free-hand lesion using scalpel blade (16). No
significant differences between handmade lesion and stereotaxic
apparatus-induced SCI could be noticed with regard to edema
formation or cell changes in the cord (16–19).
10. Repeated topical application of neurotrophins (0.1 mg–0.25 mg
/10 mL in phosphate-buffered saline) could gain rapid access
within the normal or injured cord tissue. Select the timing of
topical application starting from 10 to 30 min before injury
or to selected intervals after trauma ranging from 5 to 120 min
after the initial insult. An early manipulation of the spinal
cord after injury could result in thwarting spinal cord cell and
tissue injury and limit autodestructive changes in the cord.
11. Immediately before saline perfusion, take about 1 mL whole
blood sample after cardiac puncture and store it for analyses
of blood radioactivity or Evans blue concentration at the
time of killing.
12. The average wet weight of spinal cord segments could vary
from 60 to 90 mg in normal rats. Trauma to the cord may
increase the wet weight of the spinal cord samples from the
identical segments. Before weighing spinal cord injury samples,
remove large blood vessels or blood clots or hemorrhagic spots
over the injury site, as their presence will increase the wet
weight erroneously (19).
 33 Rodent Spinal Cord Injury Model and Application of Neurotrophic… 413

13. If in doubt, take dry weight at least three times after an interval
of 4–6 h. When the dry weight in the last three measurements
became stable, use this weight to calculate the water based on
the differences between wet and dry weight of these samples
(for details see ref. (14–16)).
14. For good fixation of spinal cord tissues, wrap the perfused
animals separately in aluminum foil and keep it at 4°C for
overnight. On the next day, remove the spinal cord and place
the samples in the same fixative at 4°C for 3–4 days.
15. If the images require processing using Photoshop or any
other image analysis program, always use identical filter or
color processing on the computer for control and experi-
mental samples (31).

Acknowledgments

Research work reported in this chapter is supported by grants

from Swedish Medical Research Council (2710 HSS), Göran
Gustafsson Foundation, Stockholm, Sweden; Alexander von
Humboldt Foundation, Bonn, Germany; and Ministry of Science
& Technology, Government of India, New Delhi. We very much
appreciate the technical assistance of Kärstin Flink (Uppsala) and
Franziska Drum (Berlin) in these investigations. We thank Tomas
Winkler (Uppsala), Conrad Johanson (Providence, RI, USA),
Rajendra D Badgaiyan (Boston, MA, USA), and Syed F Ali (NCTR,
US-FDA, Jefferson, AR, USA) for critically reading the manuscript
and providing important inputs for improvement.

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glutamate and GABA immunoreactivity in the I, p. 8–10
 INDEX

A 149–151, 153, 156, 161, 162, 165, 169, 172, 176, 177,
180–182, 184, 185, 191, 192, 194, 195, 216, 225, 227,
A2B5 monoclonal antibody ..............134, 139–142, 144, 245 230, 238, 239, 241, 243, 244, 250, 251, 297, 334–336,
Adeno-associated viral (AAV) vectors..................... 305–318 339, 340
Adrenal .................................................................... 223–234 Brain-derived neurotrophic factor (BDNF) ................... 3–6,
Adult....................... 14, 17, 19, 34, 39, 40, 67, 103–115, 117, 8, 24, 91–93, 96–98, 100, 104, 106, 108, 114, 117,
132, 138, 140–141, 143, 170, 171, 179–187, 120, 121, 125, 148, 150, 159, 161, 215, 216, 335,
189–199, 225, 230–232, 335, 384 338–340, 394, 395, 404, 406–411
Affinity purified antibodies ............................................. 139 BSA. See Bovine serum albumin (BSA)
Amperometric detection .................................................. 378 B27 supplement ............................. 25, 26, 41, 50, 51, 58, 59,
Amphotericin ................................... 191, 198, 345, 346, 352 62, 81, 82, 84–86, 92, 99, 104, 107, 108, 113, 119,
Amyloid β-peptide .................................8, 49, 57–64, 68, 85 121, 126, 133, 135, 143, 149, 151, 161, 163, 187, 297
Amyloid precursor protein (APP) ......57, 278, 289, 295–302
Anesthesia ....................................... 110, 358, 361, 364, 385, C
387–389, 398, 400, 401, 405, 410, 412 Campenot chambers ................................................ 214, 215
Antibody..................... 8, 17, 19, 45, 47, 93, 95, 97, 118, 122, Canine... ...........................................190–193, 195–196, 198
124, 127, 128, 134, 138–142, 149, 152–153, 192, Carbon nanotubes (CNTs) ...................................... 261–276
195, 197, 198, 235, 237–239, 241–246, 251, 253, Caspase .................................................................... 322, 323
254, 257, 308, 316, 317, 321–324, 327–330 Cell culture .............25, 26, 32–34, 40–41, 50, 53–55, 58–60,
Antigalactocerebroside antibody.............................. 134, 142 69, 72, 76, 80–85, 87, 92, 106, 117–128, 133, 134,
Apoptosis........................................ 23, 24, 31, 147, 213, 311 144, 149, 150, 161, 169, 181, 190, 202, 216, 218, 223,
APP. See Amyloid precursor protein (APP) 240, 248, 261–276, 279, 297, 307, 334, 343
Araldite............................................................ 395, 403, 407 Cell transduction ..................................................... 310–313
Ascorbic acid .....................357, 358, 361, 363, 366, 367, 378 Cerebellar granule neurons (CGNs) .................... 23–35, 160
Astrocytes ........... 44, 54, 67–76, 94, 117, 131, 132, 138, 139, Cerebellum ........ 23, 28, 29, 39–47, 52, 54, 71, 281, 282, 348
141–142, 144, 243–244, 266, 273, 308, 316 CGNs. See Cerebellar granule neurons (CGNs)
ATP................................................... 58, 59, 62–63, 210, 295 Chitosan .................................................................. 321–331
Automation ...............187, 249, 251, 254, 275, 276, 291, 315 Chloral hydrate ......................... 357, 359, 368, 374, 380, 395
Axonal transport .............................................. 214, 295–302 Chromaffin cells ...................................................... 223–234
Axons................................2, 13, 67, 117, 167–177, 179, 190, Ciliary neurotrophic factor (CNTF) .......................4, 8, 104,
202, 213, 247, 296, 357, 384 117, 120, 121, 134, 135, 141, 143, 148, 150, 152, 161,
163, 394–396
B
CNTs. See Carbon nanotubes (CNTs)
BBB. See Blood–brain barrier (BBB) Co-culture ................................................................... 79–88
BDNF. See Brain-derived neurotrophic factor (BDNF) Collagen ...........................114, 191, 198, 203, 204, 206, 209,
Biocompatible polymers .................................................. 322 214, 216–218, 220, 230, 232, 233, 251, 252, 258
Blood–brain barrier (BBB) ...................................... 321, 323 Collagenase ............................. 133, 135, 137–138, 141, 149,
Bone cutter ...................................................... 185, 396, 402 155, 161, 164, 171, 180, 181, 183, 185, 191, 194, 195,
Bovine......................... 26, 41, 44, 45, 61, 119, 133, 150, 161, 198, 225–227, 229, 230, 232, 233
191, 203, 224–230, 233, 250, 264, 273, 297, 309 Colorimetric assay ....................................................... 31, 58
Bovine serum albumin (BSA).................... 26, 27, 40, 42, 69, Compartmented chambers ...................................... 213–221
71, 97, 99, 100, 108, 114, 119, 120, 122, 123, Conditioned medium .......................104, 106–110, 114, 159
125–127, 133, 134, 136, 137, 139–142, 144, Cone photoreceptor cells ......................................... 147–157

Stephen D. Skaper (ed.), Neurotrophic Factors: Methods and Protocols, Methods in Molecular Biology, vol. 846,
DOI 10.1007/978-1-61779-536-7, © Springer Science+Business Media, LLC 2012

417
 NEUROTROPHIC FACTORS: METHODS AND PROTOCOLS
418 Index

Confocal ..............45, 209, 220, 237, 245, 278–280, 284, 289 Fibroblast growth factor (FGF).........................4, 15, 24, 40,
Cortex.................................40, 49, 51–53, 67–76, 82, 85, 87, 41, 43, 91, 114, 117, 120, 121, 148, 190, 191, 193,
230, 244, 267, 268, 277, 283, 327, 330, 348 322–324, 326, 329, 330
Counterstain ..............................................45, 192, 196, 197, Fluorescence ............................................ 45, 47, 95, 96, 106,
242, 329, 407 168, 174, 192, 197, 209, 235–238, 244, 255, 265,
Coverslips..... ..................29, 45, 92, 132, 149, 160, 172, 181, 270, 275, 278, 280, 291, 314, 315, 317, 328, 329,
203, 214, 224, 240, 262, 284, 297, 306–307, 329 346, 347, 349–352
Crush... .....................................................383–386, 388, 389 Fluoromount-G....................................................... 239, 242
Cryoprotectant.... ............................................................ 107 Forskolin.................................. 120, 121, 134, 135, 141, 143,
Cyclic AMP.. ..............................................24, 104, 108, 143 150–152, 161–163, 191, 193
Cytospin .................................................................. 336, 337 Freezing ........................................................... 203, 205, 358

D G
Degeneration ......................................... 8, 88, 147, 148, 190, GDNF. See Glial cell line derived neurotrophic factor
202, 277, 344, 356, 357 (GDNF)
Degranulation.................................................................. 338 GFAP. See Glial fibrillary acidic protein (GFAP)
Dendrites ............................ 19, 179, 248, 277, 287, 291, 292 GFP. See Green fluorescent protein (GFP)
Dendritic spine ................................................ 277, 284–289 Glia.....................................67, 68, 74, 75, 79–88, 94–95, 99,
Deoxyribonuclease ..................................................... 40, 225 112, 131, 132, 147, 183, 184, 186, 221, 309, 310
Differential attachment ..................................................... 75 Glial cell line derived neurotrophic factor
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (GDNF) .............................. 4, 8, 104, 106, 108, 114,
bromide (MTT) ...................... 16, 25–27, 31–33, 35, 120, 148, 159, 394, 395, 404, 406, 408, 410, 411
81, 86, 88, 96, 120, 125, 127 Glial fibrillary acidic protein (GFAP) ...................44, 73, 94,
Dinitrophenol (DNP) antibody....................... 335, 337, 338 239, 244, 245, 308, 311
Direct immunofluorescence ..................................... 237, 238 Glutamate...................................... 15, 16, 34, 67, 68, 93, 94,
Dispase .................................................................... 191, 194 126, 307, 314, 315, 317, 343, 346, 349–352
Dissection ................................ 25, 28–29, 32, 40–43, 45, 46, Glutaraldehyde ................................................ 335, 337, 395
50–54, 68, 70–71, 74, 92, 104, 118, 120–122, Glycogen synthase kinase-3 (GSK-3) ............................... 24
125, 132, 148, 160, 182–185, 187, 203, 206, Gradient ............. 53, 104, 105, 108, 111–113, 168, 170, 171,
207, 230, 262, 265–269, 274, 279–283, 289, 173–177, 219, 224, 229, 234, 336, 340, 366, 373, 376
290, 344, 385, 388 Green fluorescent protein (GFP) ....................209, 278–280,
Dissociation ....................27–29, 35, 52, 54, 74, 99, 111, 112, 307, 311, 313–317
120–122, 125, 126, 138, 139, 153–156, 164, 180, Growth cones .................................. 13, 14, 17, 19, 167–177,
181, 183–186, 190, 191, 193–194, 230, 231, 233 190, 217, 220, 248, 270, 271, 276
Dopamine ...............................91, 92, 98, 159, 201, 205, 210, GSK-3. See Glycogen synthase kinase-3 (GSK-3) ............ 24
355, 363, 365, 367
Dorsal root ganglion (DRG) ...........................168–172, 174, H
176, 177, 179–187, 190, 215, 216, 218–219, 296
Hamilton syringe .....................................357, 358, 361–363,
E 370, 373, 376, 377, 396
Hemocytometer....................25, 29–30, 35, 50, 82, 118, 132,
Embryonic ................................34, 40, 49, 51–55, 58, 60, 80, 149, 160, 169, 172, 184, 186, 187, 224, 226, 233, 252
87, 92, 103, 120, 125, 132, 138, 142, 176, 180, 318 High content analysis .............................................. 247–259
Enrichment ..............................................190, 192, 194–196 High performance liquid chromatography
Eppendorf tube ................................. 30, 100, 118, 132, 137, (HPLC) ........................ 366, 368, 370, 373, 377–378
172, 244, 327, 340, 346, 352, 353, 358 Hippocampus .................................. 39, 49, 51–55, 265, 268,
Evans blue ........................................395, 404–407, 410, 412 277, 280–284, 343, 348, 353
Excitotoxicity................................ 15, 68, 311, 343, 345, 349 Horse serum .................................... 107, 203, 205–207, 209,
215, 216, 220, 279, 289, 345, 353
F
Human ...................................3, 14, 39, 59, 67, 91, 119, 133,
FGI. See Fibroblast growth factor (FGF) 147, 161, 180, 189–194, 196–198, 202, 216, 248,
Fiber optic cold light source ............................................ 347 329, 333, 355, 366, 393
Fibroblast ........................................................141, 189, 190, 6-Hydroxydopamine (6-OHDA) ....................159, 202, 204,
192, 194–199, 234 208, 210, 355–364
 NEUROTROPHIC FACTORS: METHODS AND PROTOCOLS
Index
419

I Monoclonal antibody .................................19, 134, 246, 324

Morphology........................44, 139, 175, 196, 197, 262, 266,
Immunocytochemistry...............................46, 113, 184, 192, 269–271, 275, 276, 278, 291, 314, 315, 405, 407, 408
196–197, 233, 235, 251, 253–254 Morphometric analysis .................................................... 291
Immunofluorescence .................. 94, 209, 235–246, 316, 317 Motor neurons..................................103–115, 160, 383–390
Immunoglobulin (Ig) .....................................4, 5, 17, 18, 93, Mounting medium ............. 45, 239, 242, 244, 314, 316, 329
119, 120, 122–124, 127, 128, 133, 134, 139, 141, Mouse.....................................41, 49, 53–55, 58, 70, 87, 104,
144, 149, 153, 192, 195, 245, 308, 316, 328, 329, 119, 133, 148, 181, 192, 223, 244, 251, 270, 278,
331, 335, 337–339 296, 323, 335, 356, 369, 412
Immunopanning .............................................................. 144 Mowiol .................................................................... 239, 242
Immunostaining .............................. 41–42, 96, 97, 242–244, MPP+. See 1-Methyl-4-phenylpyridinium (MPP+)
246, 312, 314, 316, 317 MPTP. See 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine
Incubator for CO2 ..................................... 60, 70, 72, 73, 81, (MPTP)
83, 85, 109, 124, 140, 143, 172, 194, 195, 216, 240, Myelin....................................... 14, 15, 17–19, 67, 104, 105,
268, 298, 299, 337, 346 108, 111, 112, 168, 172, 190, 394, 408, 409
Indirect fluorescence antibody ......................................... 238
Insulin............................................ 4, 24, 119, 121, 133, 135, N
137, 140, 141, 143, 150–153, 155, 156, 161, 163,
Nanoparticles........................................................... 321–331
165, 169, 176, 203, 206, 395
Neonatal ....................51, 53, 67–76, 190, 244, 349, 383–390
Intraperitoneal ...................110, 327, 358, 359, 369, 374, 401
Nerve growth factor (NGF) ........................1, 3–8, 100, 117,
K 169–171, 176, 180–182, 184, 186, 187, 192,
201–208, 213, 216, 218, 219, 221, 248–250, 252,
Kainic acid ............................................................... 343, 346 255, 257, 258, 333–335, 338–340, 394, 395, 404,
Ketamine ...................107, 110, 357, 359, 385, 387, 395, 401 408, 410, 411
Kymograph .............................................................. 299–302 Nerve injury............................................................. 190, 248
Neurite .............................................. 1, 15, 87, 91, 106, 168,
L
180, 205, 214, 248, 270
Lactate dehydrogenase (LDH) ............................. 58, 60–62, Neurite outgrowth ..................... 1, 15–17, 19, 168, 171–173,
64, 81, 83, 87, 88 177, 180, 247–259, 261, 262, 273, 276, 333
Laminar flow hood ................................ 27, 32, 41, 142, 182, Neurobasal medium................................... 34, 41, 50, 51, 53,
221, 264–266, 272, 273, 344, 346, 347 58–60, 62, 80–82, 84–86, 92–94, 99, 108, 119–121,
Laminin ........................................... 114, 169, 170, 172, 174, 124, 187, 278, 297–299, 306, 309
177, 181, 182, 191, 193, 195, 196, 233 Neurons........................................... 1, 14, 23–35, 39, 49–55,
Laser scanning microscopy .............................................. 280 57–64, 67, 79–88, 91–100, 103–115, 117, 131, 147,
LDH. See Lactate dehydrogenase (LDH) 159, 168, 179–187, 189, 201–210, 213, 247, 266,
Lead citrate.............................................................. 395, 407 270, 271, 273–277, 295, 305–318, 333, 355,
Light microscope .............30, 32, 86, 123, 337, 396, 407, 408 383–390, 394
Lipofectamine ................................................. 297, 298, 302 Neuroprotection .............................. 8, 13–19, 25, 31, 33, 80,
83, 86, 147, 306, 308, 313, 315–317, 323, 393–413
M Neurotoxicity .............................................57–64, 83, 86, 96,
Mast cells................................................................. 333–340 305, 313–315, 317, 356
McIlwain tissue chopper ......................................... 280, 346 Neurotoxin ...........................................68, 91, 356, 357, 363
Merosin .............................119, 124, 149, 152, 155, 161, 163 Neurotrophin-3 ............................................... 114, 335, 395
Mesencephalon .................................................................. 94 Neurotrophin-4 ............................................................... 335
MetaMorph ...................................... 270, 276, 297, 299, 300 Neurotrophins ..................................... 1–9, 17, 58, 100, 104,
1-Methyl-4-phenylpyridinium (MPP+).................. 202, 204, 213–215, 311, 333, 334, 338–340, 394–396,
208, 210, 356 403–404, 406–408, 410, 412
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine NGF. See Nerve growth factor (NGF)
(MPTP)............................................................... 356 N-Methyl-D-aspartic acid ...................................... 160, 346
Metrizamide ......................149, 151, 156, 161, 162, 164, 165 N2 supplement ................68, 92, 99, 181, 182, 184, 215, 216
Microdialysis ........................................................... 365–380
O
Microglia .........67–76, 79–83, 85–88, 95, 117, 127, 243, 266
Microplate reader .................................................. 32, 58, 86 O2A progenitors ......................................131, 132, 137–144
Millicell® CM cell culture inserts .................... 347, 348, 353 OGD. See Oxygen-glucose deprivation (OGD)
 NEUROTROPHIC FACTORS: METHODS AND PROTOCOLS
420 Index

Oligodendrocytes .............................. 14, 15, 44, 67–76, 117, Rat........ ................................... 19, 23–35, 39–47, 49, 51–55,
131, 132, 135, 137, 139, 141, 142 57–64, 70–71, 80, 82, 85, 87, 91–100, 103,
Optic nerve ..........52, 117, 118, 121, 127, 131–144, 155, 164 118, 121, 124–125, 131, 147–157, 159–166,
Osmium tetroxide.................................................... 395, 407 168, 179–187, 189–199, 201–210, 216, 223,
Ovomucoid .................................27, 28, 52, 53, 71, 119, 120, 244, 262, 313, 329, 333–340, 343–353, 356,
122, 126, 133, 134, 136–138, 144, 149–151, 365, 383–390, 394
154–156, 161, 162, 164, 165 Release assay ................................................ 60–62, 333–340
Oxygen-glucose deprivation (OGD) ...................... 344–346, Retina.. ............................................ 118, 121–123, 125, 126,
349, 351–353 147, 148, 153–155, 159–166
Retinal ganglion cells (RGCs) ................................. 117–128
P RGCs. See Retinal ganglion cells (RGCs)
Paddle pastettes ............................................... 347, 348, 353
S
Papain.. .............................26–29, 50, 52–54, 69, 71, 74, 107,
108, 113, 118–120, 122, 125–127, 133, 135–139, SCG. See Superior cervical ganglia (SCG)
141, 142, 144, 149, 150, 153, 155, 161, 162, 164, Schwann cells ..................................................118, 189–199,
225, 232, 263, 264, 266, 268, 273, 297, 298, 302 221, 384
Paraformaldehyde .................................... 41, 44, 45, 99, 192, Sciatic nerve .................................................... 296, 383–390
197, 238, 244, 251, 253, 279, 395, 407 Selenite ....................................................119–121, 133, 135,
Parkinson’s disease models ....................... 201–210, 355–364 150, 151, 161–163, 169
PC12. See Pheochromocytoma cells (PC12) Sensory neurons ...................................1, 179–187, 213, 214
Peanut lectin .................................................................... 148 Serotonin ................................................................. 333–340
PEG. See Polyethylene glycol (PEG) Slice cultures ............................................180, 262, 277–292,
PEI. See Polyethyleneimine (PEI) 343–353
Peptide delivery ....................................................... 321–331 Spinal cord....................................... 17, 40, 67–76, 103–115,
Peristaltic pump ............................................... 385, 388, 405 160, 170, 179, 215, 267, 384, 393
Peritoneal fluid ................................................................ 336 Spinal cord injury .........................................8, 104, 393–413
Phase contrast optics ........................ 32, 86, 96, 97, 337, 353 Sprague–Dawley .......................................121, 262, 266, 273
Pheochromocytoma cells (PC12) ........................... 201–210, Stereo microscope....................................191, 198, 225, 232,
248–250, 258 347, 348, 370, 396, 402
Phosphatidylinositol 3-kinase (PI 3-kinase).........6, 7, 24, 25 Stereotaxic surgery........................................... 359, 369–370
Photoreceptor cells .................................................. 147–157 Sterilin plastic dishes ..................................68, 72, 73, 75, 80
PI. See Propidium iodide (PI) Streptavidin .............................................238, 246, 308, 316,
Pigment epithelium ......................................... 148, 159–166 322, 324, 325, 327
Platelet-derived growth factor (PDGF) ..................... 3, 134, Substance P ..................................................... 335, 338, 339
140, 143, 148, 159 Substratum .................................. 27, 70, 73, 81–82, 94, 169,
Polyethylene glycol (PEG) ..............................262, 263, 270, 182, 216–218, 220, 221
271, 322–327 Superior cervical ganglia (SCG) ............................. 201–210,
Polyethyleneimine (PEI) ........................................ 262, 265, 214, 216, 218–219
266, 268, 270–272, 274 Sympathetic neurons ............................8, 201–210, 214, 220
Poly-L-lysine ....................................... 26, 27, 31, 34, 69, 70,
72–74, 94–96, 99, 169, 181, 186, 191, 238, 240, 244, T
306–308, 313, 317 T3. See 3,3’,5-Triiodo-L-thyronine (T3) .................. 69, 119,
Poly-L-ornithine .................... 34, 95, 99, 181, 182, 191, 233 133, 150, 161
Progenitors ................... 39–47, 131, 132, 137–144, 148, 160 Targeted brain delivery ............................................ 321–331
Progesterone ............................................119–121, 133, 135, Thawing, cell lines ................................................... 204–205
150, 151, 161–163, 169 Thyroxine .......................................................... 69, 119, 133
Propidium iodide (PI) ..................................... 346, 349–353 Toluidine blue...........................................349, 395, 407, 408
Putrescine ................................................119, 120, 133, 135, Transection ....................................... 384, 386, 387, 390, 398
150, 151, 161, 162, 169 Transfection ..................................... 168, 169, 172, 174, 176,
177, 202, 209, 297–299, 302, 305
R
Transferrin ................................. 70, 119, 120, 133, 134, 150,
RAN-2 antibody .............................................120, 127, 134, 151, 161, 162, 169, 321–324
138, 139, 141, 144 3,3’,5-Triiodo-L-thyronine (T3) ........ 69, 119, 133, 150, 161
 NEUROTROPHIC FACTORS: METHODS AND PROTOCOLS
Index
421
Trypan blue ....................................... 26, 30, 35, 43, 44, 112, V
120, 124, 134, 191, 194–196, 218, 224, 226, 258,
307, 309, 334, 336 VectaShield ............................... 239, 242, 245, 308, 314, 316
Trypsin .......................................... 26, 40, 68, 107, 119, 133, Viral vector .................................................................. 8, 305
149, 161, 169, 180, 192, 203, 251, 264, 307
W
Tyrosine hydroxylase.................................................... 95, 97
Western blot .................................................................... 312
U
Y
Uranyl acetate .......................................................... 395, 407
Uric acid .......................................................... 366, 367, 378 Yellow fluorescent protein (YFP) ............................ 297–300