Mechanical allodynia was assessed by measuring the paw withdrawal threshold with a set of Von Frey filaments (0.04–2 g; North Coast medical). Mice were placed on an elevated metal grid. The filament was applied to the plantar surface in vertical angle for 1–2 s from the bottom. 50% withdrawal threshold values were determined using the up-down method.

Thermal hyperalgesia was assessed by measuring the paw withdrawal latency to radiant heat stimuli. Mice were placed in elevated chambers with Plexiglas floor and allowed to habituate for 20 min. The radiant heat source (IITC Inc life science) was applied to the center of the plantar surface in the hind paw for 4 times with at least 5 min intervals. The average withdrawal latency of the 4 trials was recorded as the response latency.

For the tail flick test, mice were restrained in a cylinder with tail hung out. The mice were then placed on the test panel with the tail covered a detection hole at a fixed distance to the tail root. The same radiant heat was focused on the tail at the detection hole position. Tail flick will expose the hole to the light and trigger the sensor to record the latency time. The tests were done for 4 times with at least 5 min intervals. The average withdrawal latency of the 4 trials was recorded as the flick response latency.

The rotarod tests were performed using a four-lane Rotarod apparatus (Med Associates Inc). The rota-rod speed started from 4 Round Per Minute (RPM) and uniformly accelerated to 40 RPM in 5 min. Each mouse was tested for 3 times with 5 min intervals for the first day and one time for the next day.

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