Tizanidine hydrochloride

The drugs administered during the experiments were amiloride hydrochloride hydrate (molecular weight [MW] 266.09; Sigma-Aldrich, USA), tizanidine hydrochloride (MW 290.17; Sigma-Aldrich, USA) and yohimbine hydrochloride (MW 390.90; Sigma-Aldrich, USA). All drugs were dissolved in normal saline. Amiloride and tizanidine were each intrathecally administered 10 min before the formalin test. To assess the dose dependency and time courses of the antinociceptive action of intrathecal amiloride, tizanidine and coadministered amiloride-tizanidine, the animals were randomly assigned to different groups receiving intrathecal injections of different doses of amiloride (12.5 μg, 25 μg, 50 μg, 100 μg), tizanidine (0.5 μg, 1 μg, 2.5 μg, 5 μg) or amiloride plus tizanidine 10 min before formalin injection. In the control group, intrathecal saline was given. Yohimbine was intrathecally administered 10 min before 5 μg tizanidine, 100 μg amiloride or the dose that resulted in 50% of the saline response (ED₅₀)/2 of amiloride + tizanidine ED₅₀/2 mixture. The agents were delivered using a microsyringe in a total volume of 10 mL, followed by 10 mL of saline to flush the catheter.