Behavioral assessment of allodynia Baseline (BL) responses to light mechanical touch were assessed using the von Frey test after animals were habituated to the testing environment, as previously described (Chaplan et al. 1994; Milligan et al. 2000). Briefly, rats were placed atop 2-mm bars with 8-mm spacing between parallel bars for approximately 45 min for five days. All behavioral testing was performed

during the first half of the light cycle in a sound-, light-, and temperature-controlled room. The von Frey test utilizes a series of calibrated monofilaments (3.61–5.18 Inhibitors,research,lifescience,medical log stimulus intensity; North Coast Medical, Morgan Hills, CA), applied randomly to the left and right plantar surface of the hindpaw for 8 sec. Lifting, licking, or shaking the paw was considered a response. Following CCI or sham surgery, animals were behaviorally tested on Day 3 and 10. On Day 10 post-surgery after behavioral assessment, all animals received an i.t. AM1241 or vehicle injection followed by behavioral reassessment Inhibitors,research,lifescience,medical at 30 min intervals for 5 h and again at 24 h. Testing was performed in a blinded fashion. CCI surgery Following BL behavioral assessment, the surgical procedure for chronic constriction of the sciatic nerve was completed as previously described (Bennett and Xie 1988). Briefly, isoflurane (induction 5% volume followed Inhibitors,research,lifescience,medical by 2.5% in oxygen),

anesthetized rats had their mid-to-lower back and the dorsal left thigh shaved and cleaned with diluted Bacti-Stat AE, (EcoLab HealthCare Division, Mississauga, Ontario, Canada). Using aseptic procedures, the sciatic nerve

was carefully isolated and loosely ligated with 4 segments of chromic gut sutures (Ethicon, Somerville, NJ) with each suture approximately 1 mm apart. Sham surgery was identical to CCI surgery but without the Inhibitors,research,lifescience,medical nerve ligation. The overlying muscle was sutured closed with two 3–0 sterile silk sutures (Ethicon, Somerville, NJ), and animals recovered from anesthesia within approximately 5 min. Intrathecal (i.t.) injection AM1241 was administered via acute i.t. catheter. Injections were performed as previously described (Milligan et al. 2005b). Briefly, rats Inhibitors,research,lifescience,medical were anesthetized with isoflurane and an 18-gauge sterile, hypodermic needle, with the plastic hub removed was inserted between lumbar vertebrae L5 and L6. The PE-10 injection catheter was marked between 7.7 and 7.8 cm from an open end, with the other end inserted into a 30-gauge needle. A sterile Hamilton whatever syringe was fitted with the 30-gauge check details needle and the attached PE-10 catheter, and collectively referred to as an injection catheter. Either 10 μl drug or equivolume vehicle was withdrawn from respective vials via the open end of the PE-10 injection catheter, which was gently inserted into the placed 18-gauge needle and threaded rostrally to the 7.7 cm marking on the injection catheter. The resulting position of the inserted tip of the PE-10 catheter occurs at the i.t. lumbosacral enlargement (~L4–L5).