The rats were anesthetized by sodium pentobarbital (40 mg/kg, i.p.) in order to implant intrathecal catheter for administration of drugs 3 days prior to each experiment. Briefly, one end of polyethylene-10 tubing was inserted intrathecally through an incision in the cisternal membrane and advanced 7-9 cm caudal until the tip of the catheter was positioned at the lumbar spinal level (L5 to L6). The other end of the intrathecal tubing was sutured to the musculature and skin at the incision site and externalized to the back of the rat. In each experiment, a Hamilton microsyringe (250 µl) was connected to the intrathecal tubing and used to deliver 100 μl of dimethyl sulfoxide (DMSO) as control, FSLLRY-NH2 (PAR2 antagonist, 10 μg), SB366791 (TRPV1 antagonist, 100 μM) and HC030031 (TRPA1 antagonist, 10 μg) (Kanai et al. 2006 ) (obtained from Sigma-Aldrich).
In a subset of studies, in order to examine the effects of PAR2 on expression of TRPV1 and TRPA1 and engagement of substance P and CGRP FSLLRY-NH2 (10 μg), SB366791 (100 μM) and HC030031 (10 μg) were intrathecally given using an infusion pump in control rats and rats 4 weeks following SCI, respectively. The pump was set to constantly deliver vehicle or the drugs over a period of 3 h. At the end of infusion, the superficial dorsal horn tissues were obtained under an anatomical microscope for Western Blot and ELISA experiments.
In a subset of studies, in order to examine the effects of PAR2 on expression of TRPV1 and TRPA1 and engagement of substance P and CGRP FSLLRY-NH2 (10 μg), SB366791 (100 μM) and HC030031 (10 μg) were intrathecally given using an infusion pump in control rats and rats 4 weeks following SCI, respectively. The pump was set to constantly deliver vehicle or the drugs over a period of 3 h. At the end of infusion, the superficial dorsal horn tissues were obtained under an anatomical microscope for Western Blot and ELISA experiments.