Behavioral tests

Rats were habituated to the testing environment for 3 days before baseline testing. The behaviors of all rats were tested blindly. Paw withdrawal thresholds (PWT) was considered as mechanical allodynia. PWT was measured by stimulating the mid-plantar surface of the hind paw with a Dynamic Plantar Aesthesiometer (Ugo Basile, Comerio, Italy). The rats were placed in a test cage with a wire mesh floor, and a thin rod with a diameter of 0.5 mm was applied to the skin of the midplantar area of the hind paw. The filament exerted an increasing force ranging from 0 to 50 g over a period of 20 s at a rate of 2.5 g/s. When the animal withdrew its hind paw, the steel rod stimulus was halted, and the force at which the animal withdrew its hind paw was recorded. The mean of five consecutive trials with an interval of at least 5 min was used for analysis.

Paw withdrawal latency (PWL) was considered as thermal hyperalgesia. PWL was tested using a Hargreaves radiant heat apparatus (IITC Life Science, Woodland Hills, CA, USA) with the basal paw withdrawal latency adjusted to 10 to 14 s and a cutoff of 20 s to prevent tissue damage. The stimulation was applied five times with an interval of at least 10 min.

A rotarod system (IITC Life Science) was used to assess motor function. Rats were tested in three trials separated by 15-min intervals. The rotation started at a speed of 5 rpm and accelerated constantly up to a maximum speed of 20  rpm within 300 s. The fall latency was recorded and averaged.