2.5. Behavioral Test for Thermal Hyperalgesia

Thermal hyperalgesia was assessed employing a plantar analgesia meter (Model 390G, IITC Life Science, Woodland Hills, CA, USA) as described by Hargreaves et al. [42]. The rats were put into a clear Plexiglas chamber (18 cm × 29 cm × 12.5 cm) on a glass plate positioned above a lightbox. After a 15-min period of habituation to their environment, radiant heat aimed at the middle plantar skin surface of the right forepaw was produced by turning on the lightbox. When the rat withdrew its forepaw, the light beam was turned off immediately. The duration between the start of the noxious heating and the paw withdrawal reaction was recorded as paw withdrawal latency (PWL). A pain response was interpreted as positive if the rat exhibited abrupt paw withdrawal, shaking and licking. The machine would be cut off after 40 seconds to avoid paw tissue damage by heat. Three repetitive measurements were taken at an interval of five minutes. The average PWL of the three repetitive examinations was calculated for each rat on days 0 (pre-injury) and 1, 3, 5, and 7 post-injury. The data of day 0 (pre-injury) served as the baseline. All the thermal hyperalgesia tests were conducted with two investigators who were unwitting of the grouping and treatment.