Expt. 10 effect of spasmogens and spasmolytics using rabbit jejunum

Experiment No. 10
Effect of spasmogens and spasmolytics
using rabbit jejunum
Mr. Vishal Balakrushna Jadhav
Assistant Professor (Pharmacology)
GES’s Sir Dr. M. S. Gosavi COPER, Nashik-5

Overview of Discussion
 Objective
 Principle
 Requirements
 Experimental specifications (conditions)
 Drugs and solutions used in rabbit intestine experiment
 Preparation of Tyrode solution (PSS)
 Procedure
 Kymographrecording of contractions
 Observation table
 Result and interpretation
2

Objective
To study the effects of ACh, Physostigmine, Barium Chloride (BaCl2),
Atropine, Adrenaline and Propranolol on isolated rabbit jejunum.
3

Principle
 Rabbit intestine is a smooth muscle which shows regular pendular
movement (i.e. continuous contraction and relaxation). Therefore,
to study the effect of drugs on the intestinal movement rabbit
intestine is an ideal preparation. Rabbit intestine is supplied by
autonomic nervous system. It consists of muscarinic (M3) and
adrenergic (α1, β1 and β2) receptors. Muscarinic receptor agonists
like ACh produces contraction of rabbit intestine and
physostigmine increases the spasm and pendular movements.
These muscarinic actions and effects are blocked by muscarinic
blockers like atropine.
 Barium chloride (BaCl2) acts by increasing tone of rabbit intestine
and thereby increasing spasm and pendular movements of
intestine.
 Adrenaline acts on α and β adrenoceptors and exhibits an
inhibitory influence of pendular movements of intestine. These
actions of adrenaline are blocked by α- and β-blockers.
4

Requirements
Animal: Medium sized rabbit (12 hour fasted)
Physiological solution: Tyrode solution.
Drug- Epinephrine (Adrenaline) (10 μg/ml), Propranolol (200
μg/ml), Acetylcholine (10 μg/ml), Physostigmine (10
μg/ml), Atropine sulphate (100 μg/ml), Barium chloride
(10 mg/ml)
Chemical- Fixing solution.
Instruments: Sherrington recording drum, Student organ
bath, Aerator, Insulin or tuberculin syringe to inject drugs
in small fractions, Dissecting board and various dissecting
instruments. Simple straw lever or frontal writing lever
and stand, Pipette, Stop watch etc.
Miscellaneous: Kymograph paper, plasticin, clips, and
thread.
5

Experimentalspecifications(conditions)
 Isolated tissue- Isolated rabbit jejunum preparation
 Drug- Epinephrine (Adrenaline) (10 μg/ml), Propranolol (200
μg/ml), Acetylcholine (10 μg/ml), Physostigmine (10 μg/ml),
Atropine sulphate (100 μg/ml), Barium chloride (10 mg/ml)
 Physiological salt solution (PSS)- Tyrode solution.
 Time cycle- Total- 5 minutes, Base line- 30 seconds, Contact
time- 30 seconds, Washing period- 3 minutes
 Applied load/ tension- 0.5 g
 Bath capacity- 40Acetylcholine ml
 Bath temperature- 37°C
 Speed of rotation of drum- 0.25 mm/ second
 Magnification value (Mf) = d (F-W)/ d (F-T)
 Aeration- Normal air (1- 2 bubbles/ second)
6

Drugs and solutions used in rabbit intestine experiment
7
Sr.
No.
Drug Dose
(μg)
Concentration
(μg/ml)
1 Epinephrine(Adrenaline) 2 10
2 Propranolol 200 1 mg/ml
3 Acetylcholine 2 10
4 Physostigmine 2 10
5 Atropine sulphate 20 100
6 Bariumchloride 2000 10 mg/ml

Preparationof Tyrode solution (PSS)
 Prepare 1 litre of Tyrode solution by dissolving NaCl (8.0
g), KCl (0.2 g), MgCl2 (0.1 g), NaHCO3 (1.0 g), NaH2PO4
(0.05 g) and glucose (2.0 g) in distilled water.
 MgCl2 should be added at last.
 CaCl2 (0.2 g) should be dissolved separately in distilled
water to avoid chances of precipitation of salt.
 Mix CaCl2 solution to the higher volume of PSS.
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Procedure
 Fast a rabbit for 12 hour period.
 Set up the assembly for rabbit intestine experiment.
 Fill the outer jacket of student organ bath with water.
 Set the thermostat of the student organ bath at 37°C and
switch on the organ bath.
 Fill the reservoir with Tyrode solution and control the flow of
Tyrode solution to the inner organ tube through glass spiral
using haemostatic forceps.
 Once the temperature of Tyrode solution reaches 37°C, kill a
rabbit by giving a blow on its head and cutting the carotid
artery. Open the abdominal region and identify the intestine.
 Cut and remove a few centimeter long of the intestine portion
and immediately place it in the watch glass containing Tyrode
solution.
9

 Trim the mesentery and with gentle care clean the contents of the
intestine by pushing the Tyrode solution into the lumen. Utmost
care should be taken to avoid any damage to the gut muscle.
 Take a piece of intestine of 3-4 cm long and tie the thread to the
top and the bottom ends without closing the lumen, and mount
the tissue in the inner organ tube containing Tyrode solution
maintained at 37°C and bubbled with O2 or air or carbogen. A
tension of 0.5 g is applied and the tissue is allowed to equilibrate
for 30 minutes before adding the drugs to the inner organ tube.
 Record the normal pendular movement of rabbit intestine on the
kymograph fixed on Sherrington’s drum for 30 seconds. At the end
of 30 seconds of pendular base line, inject 0.1 ml of Epinephrine
(Adrenaline) (10 μg/ml) and record the response for 30 seconds. If
sufficient response is not there increase dose to 0.2 ml and so on.
 Follow the washing period 3 times after recording the response by
removing the Tyrode solution present in inner organ tube and
adding the fresh Tyrode solution for 60 seconds. Due to washing,
the writing point of frontal lever comes to the level of normal
pendular base line. 10

 Add 0.1 ml of Propranolol (200 μg/ml) into the inner organ tube,
allow it to act for 60 seconds and then record the response of
Epinephrine (Adrenaline) (10 μg/ml) in presence of propranolol.
Follow the washing period for recovery.
 Inject 0.1 ml of Acetylcholine (10 μg/ml) and record the response
for 30 seconds. If sufficient response is not there increase dose to
0.2 ml and so on.
 Add 0.1 ml of Physostigmine (10 μg/ml) into the inner organ tube,
allow it to act for 60 seconds and then record the response of
Acetylcholine (10 μg/ml) in presence of physostigmine. Follow the
washing period for recovery.
 Add 0.1 ml Atropine sulphate (100 μg/ml) into the inner organ
tube, allow it to act for 60 seconds and then record the response
of Acetylcholine (10 μg/ml) in presence of physostigmine. Follow
the washing period for recovery.
 Record the responses of different doses of Barium chloride (10
mg/ml) and follow the washing period for recovery.
11

 Observe the following parameters before and after drug
administration:
a. Force of contraction → amplitude (normal, increased or decreased)
b. Tone (normal, increased or decreased)
c. Frequency of contractions (per minute).
Parameters (a) and (b) are assessed by observing the recording. The
amplitude of contractions reflect the force. Shift in the mid point of
contractions indicate the change in tone. Rate is assessed by
counting movements of lever.
Precautions
 Give sufficient time for the intestine to recover between drug
administrations.
 Always note the parameter readings before and after giving drugs.
 Record frequency of contraction when there is maximum effect of
drug.
12

Kymographrecordingof contractions
Recordingof responses of Acetylcholine(ACh), Adrenaline
(Adr)and Bariumchloride (BaCl2)

Observationtable
14
Sr. No. Drug Frequency Amplitude Tone
1 Control(Tyrode solution) Normal Normal Normal
2 Epinephrine(Adrenaline) Decreased Decreased Decreased
3 Acetylcholine (ACh) Increased Increased Increased
4 Atropine sulphate Decreased Decreased Decreased
5 Bariumchloride (BaCl2) Increased Increased Increased

Result and interpretation
 Effect of various spasmogens and spasmolytics on
amplitude (force of contraction), tone and frequency of
contraction were analyzed using isolated rabbit intestine
(jejunum).
15

Expt. 10 effect of spasmogens and spasmolytics using rabbit jejunum

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Expt. 10 effect of spasmogens and spasmolytics using rabbit jejunum