Experimental Protocol and Carrageenan induced paw oedema

Both CB2 agonist (GW 405833) and CB2 antagonist (AM630) were given in doses of 3 and 1 mg/kg, respectively, by an intravenous route from rat tail after dissolving in 2% DMSO. The doses of CB2 agonist and antagonist compounds were chosen according to literature (16,17). After non-selective cyclooxygenase (COX) inhibitor drug, diclofenac, and carrageenan were dissolved in 1% sterile saline, diclofenac was administered at a dose of 10 mg/kg. The rats were randomly assigned 7 groups, with 6 animals in each group (n=6): 1: Saline control, 2: CAR (carrageenan), 3: CAR+DIC (carrageenan plus diclofenac), 4: CAR+AGO (carrageenan plus CB2 receptor agonist), 5: CAR+ANTA (carrageenan plus CB2 receptor antagonist), 6: CAR+AGO+ANTA (carrageenan plus CB2 receptor agonist plus CB2 receptor antagonist), 7: CAR+Vehicle (carrageenan plus DMSO). Antagonist was administered at 5 min. prior to agonist. Diclofenac was applied intraperitoneally, carrageenan was applied as intraplantar injection.

Ten minutes after the last drug administration, into the plantar surface of the right hind paw of all rats, except saline control group, were given a 0.1 ml volume of the previously prepared 1% carrageenan solution, resulting in the include of the paw oedema. All local injections were made at same volume. The oedema formation was assessed as an increase in paw thickness at the dorsal-planter axis at the metatarsal level using a calliper. Basal thickness was determined before animals were given the drug. The changes in the paw thickness were measured at 0, 1, 2, 3, and 4 h. Relation between paw thickness and basal values was found and noted. To determine the anti-inflammatory potency, the relation between all drug-given groups and 1% carrageenan group was expressed as %.

Animals were anesthetized with ketamine (50 mg/kg, IP) and xylazine (5 mg/kg, IP). They were monitored for loss of the tail reflex, which was defined as loss of the twitching or movement of the tail pinched using the fingers.

Blood was collected by cardiac puncture and centrifuged at 3000 rpm for 10 min to obtain serum. Serum samples were stored at −80°C up to analysis. Animals were killed by neck dislocation and soon paw tissue and skin samples were taken for histopathological evaluation and biochemical analyses.