Chronic Constriction Injury Model

The rat CCI model of neuropathic pain was produced according to the method described by Bennett and Xie (Bennett and Xie, 1988). Adult male SD rats were anesthetized by isoflurane delivered by using the anesthetic machine. The right common sciatic nerve was isolated at the mid-thigh level and loosely ligated using four chromic gut (4–0) ties separated by intervals of 1 mm. For sham surgeries, the right sciatic nerve was exposed using the same procedure, but the nerve was not ligated. On the 9th day after surgery, Ro5-4864 or vehicle (5% DMSO) was administered i. p. 50 min before the measurement of mechanical allodynia, and koumine was administered s. c. 60 min before the measurement of mechanical allodynia. To determine whether koumine potentiated the analgesic effects of Ro5-4864, koumine (0.28 mg/kg, s. c.) or vehicle was injected 10 min before Ro5-4864 (0.0625–1.0 mg/kg, i. p.) injection on day 9 after CCI surgery, and the MWT was determined 50 min after the last injection. Animals were randomized for treatment and behavioral test were performed in a blinded manner. Mechanical allodynia was measured using the procedures described below. Only rats with mechanical withdrawal threshold scores (calculated by CCI ipsilateral paw mechanical withdrawal threshold/contralateral paw mechanical withdrawal threshold) on day 8 after CCI surgery >0.75 were used in the study. Moreover, animals demonstrating motor deficit were excluded (Xu et al., 2012).