EVALUATION OF OSTEOARTHRITIC ACTIVITY OF PLANT EXTRACT(S)/ HERBAL FORMULATION IN NEW ZEALAND WHITE RABBITS

                                                                       STUDY PROTOCOL

EVALUATION OF OSTEOARTHRITIC ACTIVITY OF PLANT EXTRACT(S)/ HERBAL FORMULATION IN NEW ZEALAND WHITE RABBITS

1. INTRODUCTION:

Osteoarthritis (OA) is a degenerative disease of the joints followed by wear and tear of bones. Cartilage is the protective tissue that covers the ends of the bones. With OA, this cartilage breaks down, causing the bones within the joint to rub together. This can cause pain, stiffness, and other symptoms. OA can occur in any joint. However, the most commonly affected areas of the body include the hands, fingers, shoulder, spine (typically at the neck or lower back),hips joint, knees joints.

2. TEST SYSTEM DETAILS:

Species : Rabbit

Strain : New Zealand White

Age : 12-15 Weeks

Body Wight : 1.5-3.0 kg

Sex : Male

No. of animals : 8 /Group

Total No. of animals : 56

3. ALLOCATION OF GROUPS:

Groups	Treatment	Dose; ROA (p.o.)	No. of Animals
G1	Normal Control	0.25% Na-CMC	8
G2	Disease Control	0.25% Na-CMC	8
G3	Reference - Hylarunic acid	100 mpk; p.o.	8
G4	Plant Extract/Herbal Formulation-1	X mpk	8
G5	Plant Extract/Herbal Formulation-1	XX mpk	8
G6	Plant Extract/Herbal Formulation-2	X mpk	8
G7	Plant Extract/Herbal Formulation-2	XX mpk	8

4. METHODOLOGY:

· Induction of Osteoarthritis

· Healthy animals will be selected and placed in metallic cages that allow easy access to standard food and water ad libitum.

· Animas will be kept thermally regulated animal house (24˚C±1˚C) with relative humidity 50% ±20% or renewed air supply or 12 hour dark/light cycle.

· All rabbits will be anaesthetized with an intramuscular injection of a mixture of 5% Ketamine (20mg/kg) and 2% xylazine (0.1mg/kg) except normal control group.

· Both knees of rabbit will be shaved prior to injection over a sufficient large area and swab with betadine for disinfection.

· The left knee of animals will be injected by intra articular injection of 3 mg of MIA dissolved in 50 μL of sterile saline solution in a single dose while right knee injected with same dose volume of normal saline solution (without MIA) using a 29 G needle syringe.

· The needle will be inserted at an inclination of 45–55° above horizontal in order to ensure entry into the articular cavity and then inject slowly.

· No resistance will be felt when the needle is in articular space.

· Assessment of Osteoarthritis activity

· Animals are randomized based on the body weight and placed in their respective cages with cage identification tags.

· Group G1 animals will be served as normal control and treated with sodium CMC.

· Group G2 animals will be served as disease control and treated with sodium CMC.

· Group G3 will be served as reference drug and treated orally with reference drug hyaluronic acid at the dose of 100mg/kg b.wt.

· Group G4 and G5 treated orally with test compound at the dose of --------- respectively.

· Group G6 and G7 treated orally with test compound at the dose of ---------- respectively.

· All animals will be treated with Na CMC or reference drug or test compound at day 0 and continue till end of experiment (5 weeks).

· At the end of the experiment, blood will be collected and serum will be separated by centrifugation at 5000 rpm for 10 min.

· Serum sample processed for TNF-ɑ, IL-6 analysis.

· Animals will be humanely sacrificed; knee joint will be isolated and stored in 10% neutral buffer formalin or processed for histopathological analysis.

5. END POINT PARAMETER(S):

· Clinical observation.

· Food and water consumption.

· Body weight changes.

· Blood serum analysis for TNF-ɑ, IL-6 (w1, w2, w3, w4, w5).

· Knee joint thickness (daily basis).

· Histopathology of knee joints H & E staining, safranin-O, Alcian blue hematoxylin (ABH) staining as well as antibodies against rat type II collagen, type X collagen and matrix metalloproteinase (MMP-13).

· CRP protein.

6. REFERENCE(S):

6.1 Kim JE, Song D-h, Kim SH, Jung Y, Kim SJ (2018) Development and characterization of various osteoarthritis models for tissue engineering. PLOS ONE 13(3): e0194288. https://doi.org/10.1371/journal.pone.0194288.

6.2 M. Molinet, N. Alves, A. Vasconcelos, N.F. Deana. Comparative study of osteoarthritis induced by monoiodoacetate and papain in rabbit temporomandibular joints: macroscopic and microscopic analysis. Folia Morphol. Vol. 79, No. 3, pp. 516–527 DOI: 10.5603/FM.a2019.0104.

6.3 N. Guler, M. Kurkc¸u, G. Duygu, B. C¸ am: Sodium iodoacetate induced osteoarthrosis

model in rabbit temporomandibular joint: CT and histological study (Part I). Int. J. Oral Maxillofac. Surg. 2011; 40: 1289–1295.

6.4 Mohamed Ali Rebai, Nizar Sahnoun, Oussema Abdelhedi, Khaled Keskes, Slim Charfi, Fathia Slimi, Rim Frikha & Hassib Keskes (2020) Animal models of osteoarthritis: characterization of a model induced by Mono-Iodo-Acetate injected in rabbits, Libyan Journal of Medicine, 15:1, 1753943, DOI: 10.1080/19932820.2020.1753943.

6.5 Chiang E-R, Ma H-L, Wang J-P, Liu C-L, Chen T-H, Hung S-C (2016) Allogeneic Mesenchymal Stem Cells in Combination with Hyaluronic Acid for the Treatment of Osteoarthritis in Rabbits. PLoS ONE 11(2): e0149835. doi:10.1371/journal.pone.0149835.

6.6 M. Mihara Ph.D.*, S. Higo B.S., Y. Uchiyama M.S., K. Tanabe M.S. and K. Saito Ph.D. Different effects of high molecular weight sodium hyaluronate and NSAID on the progression of the cartilage degeneration in rabbit OA model. OsteoArthritis and Cartilage (2007) 15, 543e549.

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