2.6. Hot Plate Test

Mice were tested to assess central antinociceptive activity according to the method described by Sahley and Berntson [24] and adapted by Matheus et al. [25]. Initially, all mice were evaluated for the determination of their individual baselines. Mice were placed over the stainless-steel heating plate that was at a temperature of 55 ± 0.1°C, and the time that it took for each mouse to remove one hind paw from the surface of the plate was timed. These verifications were taken 60 and 30 minutes before mice underwent inhalation. The baseline of each animal was calculated as the mean of the timed trials. The dwell limit time for each mouse was set at three times the baseline value. Baseline determinations were performed 2 days before the pharmacological test. After 2 days, mice were subjected to inhalation. Five and 30 minutes after the end of the inhalation test, mice were placed on the hot plate under same experimental conditions described above. The time that each mouse took to lift a hind paw was timed and scored for final analysis.