EVALUATION OF OSTEOARTHRITIC ACTIVITY OF TEST COMPOUND IN EXPERIMENTAL RATS

                                                              STUDY PROTOCOL

EVALUATION OF OSTEOARTHRITIC ACTIVITY OF TEST COMPOUND IN EXPERIMENTAL RATS

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 : Rattus norvegicus

Strain : Sprague-Dawley

Age : 4-5 Weeks

Body Wight : 180-200g

Sex : Male

No. of animals : 8 /Group

Total No. of animals : 50


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

10 animals will be taken as extra for disease induction mortality.

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 rats will be anaesthetized with an intramuscular injection of a mixture of ketamine (40–100 mg/kg) plus xylazine (5–13 mg/kg).

· Knees of rat 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 1.5 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.

· Make sure 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.

· All animals will be treated with Na CMC or reference drug or test compound at day 0 and continue till day 28.

· Pain threshold will be measure using hot plate analgesiometer at day 0, 7, 14, 21, 28.

· Knee joint thickness will be measure daily using venire caliper.

· 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

· Knee joint thickness (daily basis).

· Hot plate test (Day 0, 7, 14, 21, 28).

· 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).

· Serum CRP protein.

6. REFERENCE(S):

6.1 Shetty YC , Singh VK , Manjesh PS, Vetrivel Babu Nagarajan, Patil P, Chawda M, Rege NN. Evaluation of the effect of an ayurvedic formulation Myostaal Forte tablets on chondroprotective biomarkers in an experimental model of osteoarthritis in rats. Phytomedicine Plus. Phytomedicine Plus 1 (2021) 100082.

6.2 De Sousa Valente J. The Pharmacology of Pain Associated With the Monoiodoacetate Model of Osteoarthritis. Front. Pharmacol. 10:974. doi: 10.3389/fphar.2019.00974.

6.3 Shunsuke Jimbo, Yoshinori Terashima, Atsushi Teramoto, Tsuneo Takebayashi, Izaya Ogon, Kota Watanabe, Tatsuya Sato, Nobutoshi Ichise, Noritsugu Tohse, Toshihiko Yamashita. Antinociceptive effects of hyaluronic acid on monoiodoacetate-induced ankle osteoarthritis in rats. Journal of Pain Research 2019:12

6.2 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.

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You may like to read theses links:   

1. List of All SOPs and Documents for In-vivo Laboratory

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