The detailed experimental design was illustrated in Fig 1. Chemicals other indicated were obtained from Beyotime and Boster (Shanghai, China). Four times injections of fentanyl (60 μg/kg per injection, s.c.) at 15 min intervals, resulting in a cumulative dose of 240 μg/kg [10 (link), 11 (link)] were conducted to induce OIH in this study. The control animals received an equal volume (1.2 ml/kg) of physiological saline. KN93(N-[2-[[[3-(4’-Chlorophenyl)-2-propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4′-methoxybenzenesulfonamide phosphate salt) and KN92 (2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, monohydrochloride)were purchased from Cayman (Ann Arbor, MI) and were dissolved in 50% dimethyl sulfoxide (DMSO). 50% DMSO was used as a vehicle control. Intrathecal (i.t.) injections were performed manually between the L5 and L6 inter-vertebral space in conscious rats as previously described [12 (link)]. The injection was performed using a glass microsyringe (RWD Life Science, Shenzhen, China). Each rat was injected with a volume of 0.45 μl. Success of the i.t. injection was verified by a lateral tail-flick.
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