STUDY PROTOCOL
EVALUATION OF ANTI-ARTHRITIC POTENTIAL OF PLANT EXTRACT(S) USING COLLAGEN INDUCED ARTHRITIS (CIA) IN EXPERIMENTAL MICE
1.0 INTRODUCTION:
CIA has been the most widely studied model of rheumatoid arthritis (RA). It shares several pathological features with RA, and CII is a major protein in cartilage, the target tissue of RA. Additionally, of the antigen-defined models that are based on cartilage proteins, it has the shortest duration between immunization and disease manifestation. The CIA model has been used extensively to identify potential pathogenic mechanisms of autoimmunity, including the role of individual cell types in disease onset and progression, as well as to design and test new therapeutics. In recent years, the CIA model has been instrumental in the testing and development of the new biologically based therapeutics, such as those that target tumor necrosis factor-a, a cytokine produced by macrophages and T cells that is a dominant inflammatory mediator in the pathogenesis of RA. The development of these biologically based therapies has revolutionized the treatment of RA.
2.0 TEST SYSTEM DETAILS:
Species : Mus musculus (Mouse)
Strain : DBA/1
Age : 8-10 Weeks (at the time of immunization)
Body Wight : 25-30 g
Sex : Male or Female (Preferably Male)
No. of animals : 8 /Group
3.0 ALLOCATION OF GROUPS:
|
Groups |
Treatment |
Dose; ROA |
No. of Animals |
|
G1 |
Disease
Control |
Normal
saline or 0.25% Na-CMC |
8 |
|
G2 |
Reference
Drug- Dexamethasone |
0.5
mpk; p.o.* daily |
8 |
|
G3 |
Test
Sample-1 |
X
mpk; p.o. |
8 |
|
G4 |
Test
Sample -2 |
XX
mpk; p.o. |
8 |
|
G5 |
Test
Sample -3 |
XXX
mpk; p.o. |
8 |
4.0 METHODOLOGY:
· The study protocol (Form B) shall be approved from the IAEC before commencing the experiment.
· Animals shall be procured from the CPCSEA authorized vendor.
· Animals shall be quarantined for 1 week as per the in house SOP.
· Collagen Type II (CII) Emulsion Preparation:
· Collagen II in lyophilized form will be dissolved in dilute acetic acid (10 mM) at the conc. of 2-4mg/mL by gentle stirring for several hours to overnight.
· This shall be carried out at 4°C using reagents and glassware that will be pre-chilled to avoid denaturing C-II.
· Equal volumes of collagen solution will be emulsified with complete Freund’s adjuvant (CFA) on day 0 and incomplete Freund’s adjuvant (IFA) on day 21.
· Collagen Injection in Mice:
· On day 0, the mice will be immunized with 0.05 mL of collagen (emulsified in CFA) by intradermal (i.d.) injection site about 1.5 cm distal at the base of the tail.
· On day 21, mice will be given a booster dose of collagen (same dose emulsified in IFA) through the same route.
· Drug Treatment:
· Drug treatment will be initiated from day 0 (Immunization day) as follows:
o Group G1 animal will be treated as disease control and treated with normal saline or Na- CMC.
o Animals of group G2 will be treated orally with dexamethasone at the dose of 0.5mpk.
o Group G3, G4 and G5 animals will be treated with plant extract(s) at different dose levels.
· The clinical disease activity (CDA) will be scored every alternate day from day of immunization to last day of treatment.
· The arthritis scoring shall be done on a 4-point scale per paw: 0= normal, 1= erythema and mild swelling confined to the tarsals or ankle joint, 2= erythema and mild swelling extending from the ankle to the tarsals, 3= erythema and moderate swelling extending from the ankle to metatarsal joints, 4= erythema and severe swelling encompass the ankle, foot and digits, or ankylosis of the limb. Thus the maximum score of an animal will be 16.
· Clinical observation shall be done on every day.
· Body weight, CDA and Paw thickness will be assessed every alternate day.
· At the end of the experiment, blood will be removed via retro orbital plexus from all the animals and cytokine estimation will be performed.
· Animal will be humanely sacrificed; the fore limbs and hind limbs (paw, ankle and knee) will be removed surgically.
· Right hind will be stored in 10% buffered formalin for the histopathology whereas left hind limb and forelimbs will be processed for cytokine estimation.
5.0 END POINT PARAMETER(S):
· Incidence of arthritis
· Clinical Observation
· Body weight
· Clinical disease activity (CDA)
· Paw thickness
· Serum/paw tissue cytokines estimation- IL-6, IL-1β, TNF-α
· Histopathology of knee joint
6.0 REFERENCE(S):
6.1 David D Brand Kary A Latham & Edward F Rosloniec. Collagen-induced arthritis. Nature Protocols 1274; Vol.2 No.5; 2007.
6.2 K. M. Pietrosimone, M. Jin, B. Poston, and P. Liu. Collagen-Induced Arthritis: A model for Murine Autoimmune Arthritis. Bio Protoc. 2015 October 20; 5(20)
6.3 Aparanji Poosarla , B. Veerendra Kumar , T. Raghava Rao, D.N. Rao, and Rama Rao Athota. Alleviation of Collagen-induced Arthritis by Plumbago zeylanica in Mice. Pharmaceutical Biology 2007, Vol. 45, No. 1, pp. 54–59.
6.4 K. M. Pietrosimone, M. Jin, B. Poston and P. Liu. Collagen-Induced Arthritis: A model for Murine Autoimmune Arthritis. Bio Protoc. 2015 Oct 20; 5(20): e1626.
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