1. OBJECTIVE
Evaluation of the efficacy of TWO TEST SAMPLES in a mouse model of MOG (MOG35-55) induced experimental autoimmune encephalomyelitis (EAE).
2.1.1 Mouse
|
Species
|
: |
Mouse
(Mus musculus) |
|
Strain
|
: |
C57BL/6 (Preferred) |
|
Source |
: |
|
|
Sex |
: |
Female |
|
Age
|
: |
6-8
weeks |
|
No. of animals/group |
: |
10 |
|
Randomization |
: |
Animals
will be randomized initially based on body weight |
|
Acclimatization |
: |
Animals
will be acclimatized for one week in the animal holding area |
|
Identification of animals |
: |
Cage
labeling for group identification and tail markings with permanent markers for
identification of animals within a group. |
2.2
Allocation
of Animals
|
Group |
Treatment |
Dose |
No. of Animals |
|
G1 |
Normal Control |
0.5% MC; p.o.; q.d. |
10 |
|
G2 |
Disease Control |
0.5% MC; p.o.; q.d. |
10 |
|
G3 |
Reference
Control – Prednisolone |
6 mg/kg, p.o., q.d. |
10 |
|
G4 |
Test Sample A |
300 mg/kg; p.o., b.i.d. |
10 |
|
G5 |
Test Sample A |
900 mg/kg; p.o., b.i.d. |
10 |
|
G6 |
Test Sample A |
2700 mg/kg; p.o.,
b.i.d. |
10 |
|
G7 |
Test Sample B |
200 mg/kg; p.o., b.i.d. |
10 |
|
G8 |
Test Sample B |
600 mg/kg; p.o., b.i.d. |
10 |
|
G9 |
Test Sample B |
1800 mg/kg; p.o., b.i.d. |
10 |
2.3
Animal
welfare
The laboratory
animal facility is registered for experiments on animals with the Committee for
the Control and Supervision of Experiments on Animals (CCSEA),
Department of Animal Husbandry and Dairying, Ministry of Fisheries, Animal
Husbandry and Dairying, Govt. of India bearing registration no. The experimental protocol- ------- is
approved by the Institutional Animal Ethics Committee (IAEC) and the husbandry
conditions will be maintained as per the CCSEA recommendations.
2.4
ANIMAL
HUSBANDRY conditions
|
Target Room Temperature |
: |
21
± 3 °C |
|
Target Relative humidity |
: |
30
to 70% |
|
Light/dark cycle |
: |
12-hourly |
|
Feed |
: |
Standard pellet diet (gamma-irradiated) purchased
from a commercial supplier- ----- would be provided ad libitum
to the animals. |
|
Water |
: |
RO
drinking water ad libitum. |
2.5
Experimental
Procedure(S)
· Healthy animals will be selected after the completion of the quarantine period. Subsequently, they will be randomly allocated into 11 different groups consisting of 8 animals each based on their body weight.
2.5.1 Development
of the EAE model
· Animals of Group G2-G9 will be anaesthetized by Ketamine and xylazine. Then 100 μl of antigen/CFA emulsion was injected subcutaneously into two different sites on each hind flank.
· 200 µL of pertussis toxin (PTX) will be injected intraperitoneally. A second dose of PTX will be injected after 48 hours of immunization.
·
Body weight of the animals and clinical
score (0 to 5) will be evaluated daily from day 0.
|
Scoring |
Indication/Observation |
|
0 |
Indicates no disease |
|
1 |
Indicates limp tail |
|
2 |
Impaired righting
reflex |
|
3 |
Indicates partial hind
limb paralysis |
|
4 |
Indicates complete hind
limb and partial front limb paralysis |
|
5 |
indicating moribund
condition/death |
2.5.2 Test
and reference substance administration
·
Animals will receive vehicle/test
substance for 10 days before induction by oral route which continues after
induction from day 0 up to day 30 of the main study, in a dose volume of 20 mL/kg
·
Animals will receive reference substance
orally from day 9 of the study (after the onset of symptoms) up to day 30 in a dose
volume of 20 mL/kg
·
Group G1 will serve as normal control and
will be administered 0.5% MC, q.d.
·
Group G2 will serve as disease control and
will be treated with 0.5% MC, q.d.
·
Animals of group G3 will be treated with
reference substance- Prednisolone at the dose of 6 mg/kg, q.d.
·
Animals of groups G4, G5, and G6 will
be treated with Test Sample A dose
levels of 300, 900, and 2700 mg/kg, orally, b.i.d.
·
Animals of groups G7, G8, and G9 will
be treated with Test Sample B at
the dose levels of 200, 600, and 1800 mg/kg, orally, b.i.d.
2.5.3. Study termination –Blood collection and harvesting of organs
· On day 30 of the main study, the animals will be euthanized and just before the animal dies, blood will be collected by cardiac puncture and serum will be separated for the estimation of Cytokines e.g. IFN-γ, TNF-α, IL-2, IL-10, IL-17A, IL-6, IL-1β, IL-1α, and prostaglandin E2 (PGE2) will be assayed.
2.5.5 Histopathological
analysis
· Brain and Spinal cord will be collected after perfusion and fixed in 10% formaldehyde for paraffin embedding. Luxol Fast Blue (LFB) and hematoxylin/eosin (H&E) for myelin assessment and quantification of cell infiltration respectively. Immunohistochemical (IHC) analysis will be done to observe the protein expression of NG2 and MBP in mice brains.
2.5.6 Parameters
to be evaluated
·
Body weight: Daily
·
Clinical Score: Daily
· End point parameters: Serum cytokines level, brain weight, body weight, clinical sign evaluation, histopathological and immunohistochemical evaluation.
2.6 Statistical
analysis
The data will be expressed as mean ± standard error of the mean (SEM) for each group. Statistical analysis will be done using GraphPad Prism version 7.04 software using oneway and/or two ANOVA analyses of Variance followed by Dunnett’s multiple comparison post-hoc test or Tukey’s multiple comparison test. A p-value <0.05 will be considered statistically significant.
14.0 REFERENCES
1. McFarland, H. F., & Martin, R. Multiple sclerosis: a complicated picture of autoimmunity. Nat. Immunol. 8, 913-919, doi:10.1038/ni1507 (2007).
2. Wiendl, H. Neuroinflammation: the world is not enough. Curr. Opin. Neurol. 25, 302-305, doi:10.1097/WCO.0b013e3283534abf (2012).
3. Van Der Star, B. J. et al. In vitro and in vivo models of multiple sclerosis. CNS Neurol. Disord. Drug Targets. 11, 570-588 (2012).
4. Pachner, A. R. Experimental models of multiple sclerosis. Curr. Opin. Neurol. 24, 291-299, doi:10.1097/WCO.0b013e328346c226 (2011).
5. Bittner, S., Afzali, A.M., Wiendl, H., Meuth, S.G. Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice. J. Vis. Exp. (86), e51275, doi:10.3791/51275 (2014).
6. Tao Yang, Qi Zheng, Su Wang, Ling Fang, Lei Liu, Hui Zhao, Lei Wang and Yongping Fan Yang et al. Effect of catalpol on remyelination through experimental autoimmune encephalomyelitis acting to promote Olig1 and Olig2 expressions in mice BMC Complementary and Alternative Medicine (2017) 17:240
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