The Role Of HCN Channels At Spinal Dorsal Horn In Neuropathic Pain

Neuropathy pain after nerve injury is difficult to cure. The mechanism of neuropathy pain is a current research focuses in the field of pain. Previous studies showed that hyperpolarization-activated cyclic nucleotide-gated cation（HCN） channels play a critical role in neuropathic pain. HCN channels are expressed in dorsal spinal horn. However, the role of HCN channels at spinal dorsal horn in neuropathic pain is not clear. P2 X receptors play a critical role in neuropathic pain signaling. Several P2 X receptor subtypes are expressed in spinal dorsal horn.Whether HCN channels modulate the function of P2 X receptor is not clear. In this proposal, some methods are used to investigate : 1)the role of HCN channels in neuropathic pain by detecting the behavioral responses and the expression of HCN channels and P2 X receptors in dorsal horn; 2) The effect of HCN channels on the function of P2 X receptors in cultured spinal dorsal horn neurons. The research works will clarity the role and relative mechanism of HCN channels in neuropathic pain, and provide some evidence for developing effective analgesics bases on HCN channels as new therapeutic targets.Objective: 1. To investigate the role of HCN channels at spinal dorsal horn in neuropathic pain.2. To Observe the role of HCN channels in regulation the action of P2 X receptors in dorsal horn neurons.Methods: 1.In vivo:Forty-eight healthy male SD rats（7⁸ weeks old） were divided into 6 groups:（1）sham group（n=8）,（2）CCI group（intrathecal injection of saline,n=8）,（3）⁶CCI+ ZD7288 group（intrathecal injection of 1,10,30,50μg ZD7288, respectively, n=8）. In the sham-operated rats, the sciatic nerve was visualized and left intact. SD rats in other 2 groups were intrathecally cathetered at 5 d before CCI operation. the thermal withdrawal latency（TWL）were measured.The expression of HCN1,3,4,P2X2,6 and phospho-protein kinase A（P-PKA）was detected by western blot.2. In vitro:Spinal dorsal horn neurons of newborn SD rat（﹤3d）were cultured and divided into 7 groups: 1control; 2ATP（100μmol/L）; 3Ca2+-free solution（for 20min）+ATP;4TNP-ATP（10μmol/L for 10 min）+ATP; 5ZD7288（1,10,100,1000umol/L for 10 min）+ ATP; 68-Br-c AMP（c AMP analog,100μmol/L for 10 min）+ATP; 7ZD7288（100μmol/L for 10 min）+8-Br-c AMP（100μmol/L for 10 min）+ ATP.Changes of [Ca2+]i in neurons were detected by laser scanning confocal microscopy.Results: 1. TWL in CCI group was significantly lower than that in the sham group after operation（P<0. 05）. Intrathecal administration of ZD7288（HCN channels antagonist,30μg）markedly suppressed the decrease of TWL after CCI operation（P<0.05）. The Western blot assay showed that the expression of HCN1,3,4,P2X2,6 and P-PKA was markedly enhanced in the ipsilateral dorsal horn on days 7（P<0.05）and 14（P<0.05）after nerve injury. Intrathecal administration of ZD7288 markedly suppressed the increased expression of HCN1,3,4,P2X2,6 and P-PKA after CCI（P<0.05）.2. ATP（100μmol/L）caused the increase of [Ca2+]i transients in spinal dorsal horn neurons via activation of P2 X receptor（P<0.05）;HCN channels antagonist ZD7288（10-1000umol/L, incubation for 10 min）inhibited ATP（100μmol/L）-induced [Ca2+]i increase in spinal dorsal horn neurons with a dose-dependent manner.8-Br-c AMP（100μmol/L）enhanced ATP（100μmol/L）-induced [Ca2+]i increase in spinal dorsal horn neurons.The effect of the 8-Br-c AMP can be reduced by ZD7288（incubation for 10 min）（P<0.05）.Conclusion: 1. Activation of HCN channels in spinal dorsal horn can promote the occurrence of neuropathic pain induced by CCI. HCN channel can promote the expression of purine P2 X receptors in spinal dorsal horn at neuropathic pain state.2. HCN channel antagonists ZD7288 significantly inhibited P2 X receptor-induced [Ca2+]i increase in dorsal spinal horn neurons, which indicates that HCN channels can enhance the action of P2 X receptors.