Mechanical and Thermal Pain Assessment in Rats

To measure the paw withdrawal mechanical threshold (PWMT), rats were placed in a plastic cage with a metal mesh floor, and Von Frey filaments (North Coast Medical, San Jose, CA, United States) ranging from .4 to 26 g were applied vertically to the plantar surface of the left hind paw until the filament was slightly bent, as described in our previous studies (Shen et al., 2020 (link); Zhang et al., 2022 (link)). The paw withdrawal threshold was determined using the up-and-down method. The PWMT was recorded as the minimum stimulus force required to induce withdrawal responses (lifting or licking).
We used a thermal pain test instrument (Tes7370, Ugo Basile, Comerio, Italy) to test the paw withdrawal thermal latency (PWTL) (Shen et al., 2020 (link); Zhang et al., 2022 (link)). Briefly, rats were placed in the same chamber, which was placed on a 2-mm-thick heat-conducting glass plate. The rats were irradiated with a radiant heat source stimulator (cut-off time: 30 s) on the plantar surface of the left hind paw, and the thermal withdrawal latency was recorded. We repeated the measurement three times at 5-min intervals, and the mean latency was calculated as the PWTL.
The PWMT and PWTL of rats were measured 1 day before and 1–7 days after surgery, respectively. All behavioral tests were performed after the rats were acclimated to a specific individual chamber for at least 30 min.