Acetic acid-induced writhing test

Writhing was induced in mice by intraperitoneal (i.p.) injection of a 0.6% acetic acid aqueous solution²⁶. Drugs or control (vehicle) were s.c. administered, and after 25 min (5 min prior to testing), each animal received i.p. acetic acid solution. Each mouse was placed in individual transparent Plexiglas chambers, and the number of writhes was counted during a 10 min observation period. Antinociceptive activity, as percentage decrease in the number of writhes compared to the control group, was calculated according to the following formula: % inhibition of writhing = 100 × [(C—T)/C], where C is the mean number of writhes in control animals, and T is the number of writhes in drug-treated mice. For the antagonism study, naltrexone (1 mg/kg) was s.c. administered 10 min before the opioid agonist, and writhing as assessed as described above. Each experimental group included five to six animals.