circadian rhythm in Spinal cord injuries

To evaluate the level of oxidative and antioxidative
parameters and its relationship with pain symptoms
in patients with Spinal Cord Injuries
PROF. GHIZAL FATIMA
Department of RHEUMATOLOGY,
King George’s Medical University,
Lucknow, India.

Introduction: Why we did this study
• Spinal cord injuries (SCI) cause myelopathy or damage to nerve
roots or myelinated fiber tracts that carry signals to and from the
brain.
• The prevalence of spinal cord injury is not known in India and
many other countries.
• According to known data, about 450,000 people in the United
States live with spinal cord injury (one in 670), and there are
about 11,000 new spinal cord injuries every year (one in 30,000).

• The damage begins at the moment of injury when displaced
bone fragments, disc material, or ligaments bruise or tear into
spinal cord tissue.
• The pathophysiological disturbances of SCI are not well
known. Redox imbalance are associated with symptoms
suggestive of SCI.
• It is possible that oxidative stress, antioxidative enzymes is
involved in SCI.
Introduction: cont.

• Furthermore, oxidative and antioxidative parameters in India
has been investigated only since the last few years in SCI
patients and very few association studies have been performed
in these patients when compared to the global reports,
establishing oxidative, antioxidative association studies in
North Indian population is essential for understanding the
significance of inflammation in the development of SCI.
Introduction: cont.

• Oxidative stress means an alteration in the delicate balance
between free radicals and the scavenging capacity of
antioxidant enzymes in the body.
• There are several clinical conditions associated with increased
oxidative stress, but novel data suggest a relationship between
oxidative stress and SCI. Furthermore, there is little
information available in scientific literature about oxidative
stress in SCI patients.
• In the present study we examined the involvement of oxidative
and antioxidative parameters in patients with Spinal Cord
Injuries (SCI) and also evaluated its correlation with the
severity of its pain symptoms.
Introduction: cont.

Objectives
• Assessment of the Oxidative stress parameter like Lipid Peroxides (LPO)
and Protein carbonyl group in 14 SCI patients and 14 healthy controls
without SCI.
• Assessment of the Antioxidative stress parameters like catalase ,
Glutathione peroxidase (GPx) and Glutathione Reductase (GR) ) in 14 SCI
patients and 14 healthy controls without SCI.
• Assessment of Pain symptoms of SCI by Visual Analog Scale (VAS).
• To evaluate its relationship with pain symptoms and oxidative stress
parameters and Antioxidative stress parameters in SCI patients and healthy
controls without SCI.
• .

Materials and Method
• Oxidative stress was determined by measuring the levels of Lipid Peroxides (LPO)
(Ohkawa et al 1979) and Protein carbonyl group ((Levine and Williams, 1994) in plasma
in 14 SCI patients and 14 healthy controls without SCI.
• Antioxidative stress parameters was determined by measuring the levels of catalase ,
Glutathione peroxidase (GPx) and Glutathione Reductase (GR) ) in lysate in 14 SCI
patients and 14 healthy controls without SCI.
• Assessment of Pain symptoms of SCI was done by Visual Analog Scale (VAS).
• Clinical assessment was done by following questionnaires
1- General Assessment Questionnaire (self designed)
2- Pain symptoms of SCI was done by Visual Analog Scale (VAS).

Inclusion and exclusion criteria
Inclusion Exclusion
Patients who fulfilled the criteria
developed by the American Spinal
Injury Association (ASIA) were
included in the study.
Smokers and those using any oral
contraceptives were excluded from the
study as these factors can influence the
oxidative stress and antioxidative
parameters.
Informed consent for inclusion in
the study were taken from all the
subjects.
Moreover, subjects with known co-morbid
conditions like diabetes mellitus and other
endocrinal disorder (thyroid and Pituitary
dysfunction), psychiatric patients will be
excluded from the study. Subjects suffering
from Multiple Myeloma will also be
excluded from the study.

Diagnosis of patients: (SCI)
Traumatic spinal cord injury is classified into five categories on the ASIA
Impairment Scale. The criteria is-
• A indicates a "complete" spinal cord injury where no motor or sensory function is preserved in the
sacral segments S4-S5.
• B indicates an "incomplete" spinal cord injury where sensory but not motor function is preserved
below the neurological level and includes the sacral segments S4-S5. This is typically a transient
phase and if the person recovers any motor function below the neurological level, that person
essentially becomes a motor incomplete, i.e. ASIA C or D.
• C indicates an "incomplete" spinal cord injury where motor function is preserved below the
neurological level and more than half of key muscles below the neurological level have a muscle
grade of less than 3, which indicates active movement with full range of motion against gravity.
• D indicates an "incomplete" spinal cord injury where motor function is preserved below the
neurological level and at least half of the key muscles below the neurological level have a muscle
grade of 3 or more.
• E indicates "normal" where motor and sensory scores are normal. Note that it is possible to have
spinal cord injury and neurological deficits with completely normal motor and sensory scores.

• Statistical analysis was done using SPSS statistical software (16.0 versions).
• Quantitative variables of SCI patients and controls were presented as the
mean ± standard deviation, and are compared by independent t-test.
• Pearson correlation was done to find the pattern of associations between the
groups.
• A value of p<0.05 was considered statistically significant and p<0.01 is
considered highly significant.
• .
Statistical analysis

Clinical and Biochemical Characteristics among Study and Control groups
Parameters SCI=14
[mean ± SD]
Controls=14
[mean ± SD]
P-value
Age (years) 34.7±9.8 32.8±10.5 N.S
ESR 27.2±9.7 24.9±8.2 N.S
ALT 39.8±14.1 37.6±14.4 N.S
VAS 87.2±11.4 35.0±8.3 <0.05

Oxidative Parameters
Study (n=14)
(Mean ± SD)
Control (n=14)
(Mean ± SD)
P-Value
Lipid Peroxides (LPO)
3.31±0.57 2.24±0.35
p<0.01
Protein carbonyl group
1.90±0.45 1.32±0.26
p<0.01
Oxidative stress parameters among Study and Control groups

Comparison of Lipid Peroxide level in SCIpatients and Control Group
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Patients (n=14) Control (n=14)
Lipid Peroxide
Lipid Peroxide

Comparison of Protein Carbonyl Group level in SCIpatients and Control
Group
0
0.5
1
1.5
2
2.5
Protein Carbonyl Group
Protein Carbonyl
Group

Antioxidative parameters among Study and Control groups
Antioxidative Parameters
Study (n=14)
(Mean ± SD)
Control (n=14)
(Mean ± SD)
P-Value
Catalase
41.0±5.1 57.3±8.6 <0.01
Glutathione peroxidase
(GPx)
28.2±3.7 38.7±5.1 <0.01
Glutathione Reductase
(GR)
24.4±4.2 28.1±6.2 <0.01

Comparison of Catalase in SCI patients and Control Group
0
10
20
30
40
50
60
70
Catalase
Catalase

Comparison of Glutathione peroxidase in SCI patients and Control Group
0
5
10
15
20
25
30
35
40
45
50
GPx
GPx

Comparison of Glutathione reductase in SCI patients and Control Group
0
5
10
15
20
25
30
35
40
GR
GR

Pearson correlation analysis in Lipid Peroxides (LPO), Protein carbonyl
group with age and VAS
Parameters Groups Age VAS
r r
Lipid Peroxides (LPO)
SCI patients 0.084 0.533*
Control 0.043 0.129
Protein carbonyl group
SCI patients 0.038
0.434*
Control -0.064
0.115

Pearson correlation analysis in Catalase, Glutathione peroxidase and
Glutathione reductase with age and VAS
Parameters Groups Age VAS
r r
Catalase
SCI patients -0.093 -0.182
Control .114 0.072
Glutathione
peroxidase (GPx)
SCI patients -0.138 0.081
Control -.133 0.125
Glutathione
Reductase (GR)
SCI patients 0.058 0.084
Control -.101 0.043

• In conclusion, the present study indicate that SCI patients are exposed to
oxidative stress and this increased oxidative stress may play a role in the
etiopathogenesis of SCI. Moreover, our results also show that increased
oxidative stress parameters are more strongly associated with Pain symptoms
in SCI patients. The limitations of the study is the sample size we need good
sample size to draw much better conclusion.
• Therefore, an intervention study is recommended to address the question of
whether improved oxidative stress parameters in SCI patients can lead to
decrease in its symptomatology.
Conclusion

Relevance of the Study
These findings resulted in better understanding of oxidative
stress parameters in SCI patients which is found elevated in
patients group as compared to the control group. Therefore,
increased oxidative stress levels may be responsible for the
worsening of SCI symptoms.

• Lipid peroxidation (LPO) (Ohkawa et al 1979)
• Mix 0.2 ml of blood plasma with 0.5 ml of acetic acid subsequently 0.5 ml of 8% SDS will be added and
shake the above mixture, after this, add 1.5 ml of 0.8% TBA solution. The reaction mixture was incubated in a
boiling water bath for one hour. After cooling at room temperature, add 3 ml n-butanol and this reaction
mixture will be centrifuged at 10,000 rpm for 15 minute. A clear supernatant obtained after centrifugation will
be used for measuring the absorbance at 532 mm against reagent blank. We will also run an appropriate
standard Melondialdehyde (MDA) 10 n mol of (=10l) simultaneously.
• Protein carbonyl group (Levine and Williams, 1994): Procedure: The experiment will be carried out in two
setups. In one set (experimental), 5 ml DNPH and 1.5 ml blood plasma will be taken and in the second set
(reference) 1.5 ml blood plasma will be added in the 5 ml of 2.5 M HCl instead of DNPH. Tubes will be left
for 1 hour at room temperature (in dark). The samples vortexes for 15 min. then 5ml of 20% TCA will be
added in both sets to a final concentration of TCA by 10% itself. Tubes then are kept to ice water for 30 min to
get protein precipitated and centrifuged. The protein precipitate collected, washed with 4 ml of 10% TCA and
recovered by centrifugations. Protein pellet washed 3 times with 4 ml mixture of ethanol: ethyl acetate (1:1
v/v) to remove unreacted DNPH and lipid components. Finally precipitate of experimental and reference
dissolved in 2ml of 6M guanidine HCl and left for 10 min at 37◦ C with general vortex mixing and insoluble
material will be removed by additional centrifugation. A clear supernatant obtained. The absorbance of this
will be measured at 365nm (UV) against guanidine HCl on spectrophotometer. The results expressed as
nmol/mg protein.

• Catalase (Aebi 1974): (Done in lysate)
• Procedure: 2ml of phosphate buffer and 1 ml. of diluted (0.2M) H2O2 will be taken in a cuvette, in
this we will add 0.02 ml enzyme source and mix thoroughly. The decrease in absorbance at 240nm
will be recorded after every 30 seconds for 3 minutes against reagent blank. One catalase unit is
defined as the amount of enzyme required to cause a decrease in optical density by 0.100 of
substrate (H2O2). We will also determine Protein content in enzyme source. The results will be
expressed as unit/mg protein.
• Glutathione Reductase (Hazelton and Lang; 1995) (Done in lysate)
• Procedure: The reaction mixture consist of 0.1 ml NADPH, 0.2 ml GSSG, 0.1 ml EDTA, 2.5 ml
buffer and 0.1 ml of enzyme source to a total volume of 3.0 ml. The reaction will be initiated by the
addition of enzyme source. Oxidation of NADPH followed at 340 nm. Blank reaction will be run
simultaneously. The decrease in absorbance at 340 nm is followed at 30 second intervals. We will
also determine Protein content in the enzyme source. Enzyme unit will be defined as nmole of
NADPH oxidized per minute per mg protein. The results will be expressed as unit/min/mg protein.
• Glutathione Peroxidase (Pagila and Valentine; 1967) (Done in lysate)
• Procedure: An incubation mixture containing 0.4 ml buffer, 0.2 ml of GSH, 0.2 ml EDTA, 0.2 ml
Sodium azide and 0.2 ml hydrogen peroxide will be pre-incubated at 37o C for 10 min. 0.1 ml of
enzyme source will be added and incubated at 37oC for 10 min. The reaction will then be
terminated by the addition of the 0.1 ml of 10% TCA. Supernatant taken and 3 ml of phosphate
buffer and 1 ml of DTNB will be added. The color developed read immediately at 412 nm in a
spectrophotometer. We will also estimate Protein in enzyme source. GPx enzyme activity will be
expressed as µg GSH oxidized per mg protein and the results will be expressed as unit/mg protein.

Centrifuge at 3000 rpm
for 15 min
Supernatant= Plasma
TBARS (LPO) analysis Pellet (RBCs)
Washed twice with 1 ml
normal saline (0.9% Nacl)
and Centrifuge at 3000 rpm
for 10 min, discarded the
supernatant (normal saline)
Pellet + Chilled Distilled water
(Equal volume to plasma)
Centrifuge at 5000
rpm for 20 min
Supernatant =
Lysate
(Enzymatic
analysis)
Preparation of Lysate
Blood Sample

• Using the Snedecor and Cochran(1989) sample size calculation formula
• Sample size (n) = 1 + 2C (SD/D)2
• (α) =0.05,
• (1-β) =0.9, then C=10.5
• n= 1+2*10.5(2/4)2
• n=30

circadian rhythm in Spinal cord injuries

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