Experimental protocol and drug delivery

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, 11] 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]. 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.

Experiment 1: To investigate the effect of CeLC CaMKIIα antagonism on the level of CaMKIIα activity in CeLC, PAG, RVM and spinal cord regions and the behavioral hyperalgesia induced by fentanyl, rats were first implanted with CeLC cannulas before the induction of OIH by fentanyl. Then the Control group (n = 6) and OIH group (n = 6) received 50%DMSO (vehicle) 0.3 μl. OIH+KN92 10 nmol group (n = 6) and OIH+KN93 10 nmol group (n = 6) received equal volume (0.3 μl) of KN92 10 nmol and KN93 10 nmol respectively 6.5 h after the last injection of fentanyl. Experiment 2: To test whether spinal cord has a reverse effect on amygdala CaMKIIα signal, animals were randomly divided into four groups (n = 6). The Control group and OIH group received 50% DMSO (vehicle) 0.45 μl. OIH+KN92 45 nmol group and OIH+KN93 45 nmol group received equal volume (0.45 μl) of KN92 45 nmol and KN93 45 nmol respectively 6.5 h after the last injection of fentanyl. Experiment 3: To confirm whether p-CaMKIIα in vlPAG is involved in fentanyl-induced hyperalgesia and whether there is connection between PAG and other regions including CeLC, RVM and spinal cord in OIH, animals were randomly divided into four groups (n = 6). Control group and OIH group received 50% DMSO (vehicle) 0.3 μl. OIH + KN92 10 nmol group and OIH + KN93 10 nmol group received KN92 10 nmol and KN93 10 nmol respectively 6.5 h after the last injection of fentanyl. Experiment 4: To determine if there were differences between the Control rats and OIH rats in the synaptic transmission, and whether CaMKIIα modulates synaptic transmission in PAG neurons in OIH rats, the PAG slices of control rats (n = 6) and OIH rats (n = 6) were obtained 12 h after the last injection of saline or fentanyl. KN93 (10 μM) were added in the recording well 10 min after the baseline recording. Experiment 5: To further confirm whether inhibition of CeLC CaMKIIα activity has a direct effect on the enhanced synaptic transmission of vlPAG neurons in OIH rats, the Control+KN92 (n = 11) group and OIH+KN92 (n = 8) received CeLC injection of KN92 (10 nmol) and Control+KN93 group (n = 8) and OIH+KN93 (n = 8) group received CeLC injection of KN93 (10 nmol) respectively 6.5 h after the last injection of saline or fentanyl.