Behavior test

Changes in rat behavior were detected from three aspects, namely, mechanical withdrawal threshold (MWT), paw withdrawal thermal latency (PWTL) and number of withdrawal upon cold stimulation, reflecting the resistance against mechanical, thermal and cold stimulations, respectively. The behavior tests were performed at night on 0, 5, 10, 15 and 20 d post CCI surgery.

MWT was detected by a monitor Electronic von Frey Plenthysmometer (IITC Life Science, Woodland Hills, CA, USA) based on the method proposed by Vivancos G et al. (Vivancos et al., 2004). The rat was placed in a transparent glass box to adapt to the environment. The back heels of both sides were stimulated by a metal probe vertically, with gradually increasing stimulus intensity. The rat withdrew or touched its feet quickly, and the minimal stimulus intensity that could lead to withdrawals was recorded. Detection on the same rat was repeated for 3 times, with 1 min of interval between two detections. MWT was calculated as the mean of 3 repeated results.

PWTL was measured by a monitor Plantar Test Apparatus (IITC Life Science) (Hargreaves et al., 1988). The rat back feet were exposed to the light of the equipment. When the rat withdrew or touched its feet, the light exposure duration was recorded. Detection on the same rat was repeated for 3 times, with 3 min of interval between two detections. PWTL was calculated as the mean of 3 repeated results.

The number of withdrawal upon cold stimulation was measured based on the method proposed by Datta et al. (2010). Briefly, 0.1 mL of acetone was dropped on the rat back foot, and the rat withdrew or touched its foot upon the acetone-induced cold stimulation, which was considered to be positive reaction. Detection on the same rat was repeated for 3 times, with 3 min of interval between two detections. The number of withdrawal per minute was recorded.