2.6.2 Hargreaves test
Paw withdrawal latency to radiant heat was evaluated by the Hargreaves test (Hargreaves, Dubner, Brown, Flores & Joris, 1988). Animals were placed individually in Plexiglas cages positioned on a heated glass floor (30°C) and allowed to habituate for at least 30 min before testing. A radiant heat source was positioned underneath the animal and aimed at the plantar surface of each hind paw. The time interval before the animal withdrew its paw from the heat stimulus was measured by an automatic plantar stimulator meter (IITC model 390, Woodland Hills, CA) and recorded as the paw withdrawal latency (PWL). This test was repeated three times at 5-min intervals. If no behavioral response was elicited after 30 s (cutoff time), the stimulus was discontinued to avoid sensitization and prevent tissue damage. The average PWL across the three trials was used for data analysis.