Role Of Spinal P2X Receptor Positive Microglia In The Mechanism Of Electroacupuncture Relieving Neuropathic Pain

BackgroundAlthough electroacupuncture(EA)is effective in the relief of neuropathic pain,the underlying mechanisms remain unclear.Previous studies have reported excessive release of interferon-?(IFN-?)after nerve injury transformed quiescent spinal microglia into an activated state with more neuropathic pain,associated with purinergic receptor P2X4 expression.However,the inhibition of spinal P2X4 receptor positive(P2X4R~+)microglia only significantly relieved tactile allodynia induced by nerve injury,but not thermal hyperalgesia.This study aimed to examine whether EA treatment exerts its analgesic effect via attenuating IFN-?release and subsequent generation of P2X4 receptor positive(P2X4R~+)microglia or via down-regulating spinal P2X7 receptor positive(P2X7R~+)microglia-mediated over-expression of interleukin(IL)-1?and/or IL-18.MethodsMale Sprague-Dawley(SD)rats underwent chronic constriction injury(CCI),IFN-?intrathecal injection or 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate(BzATP)intrathecal injection.Von Frey and Hargreaves tests were performed to evaluate the effect of EA on pain hypersensitivity.The spinal P2X4R,P2X7R,IFN-?,IL-1?,and IL-18 expression levels were determined via real-time polymerase chain reaction(PCR),western blot analysis,immunofluorescence staining,and enzyme-linked immunosorbent assay(ELISA).The selective P2X7R antagonist A-438079 was used to examine P2X7R~+microglia dependent release of IL-1?and IL-18.In vitro primary cultures of microglia were used to examine IFN-?activation of P2X4R~+cells and to assess the P2X7R~+microglia-induced IL-1?and IL-18 release.ResultsIn CCI rats,EA treatment significantly increased paw withdrawal threshold relative to control.IFN-?facilitated P2X4R~+microglia activation both in vitro and in vivo.EA also down-regulated both P2X4R and IFN-?expression in the spinal cord after CCI.However,EA did not exert the same analgesic effect after intrathecal IFN-?injection.EA treatment significantly improved the pain thresholds and inhibited the spinal P2X7R~+microglia activation induced by CCI or BzATP administration,which was accompanied by the suppression of spinal IL-1?and IL-18 over-expression.Moreover,A-438079 also improved the pain thresholds and suppressed the over-expression of IL-1?in the CCI-and BzATP-injected rats.The analysis of cultured microglia further demonstrated that A-438079 markedly decreased the BzATP-induced IL-1?release.ConclusionsEA treatment relieves nerve injury-induced pain hypersensitivity via the inhibition of spinal IFN-?-mediated P2X4R~+microglia activation and spinal P2X7R~+microglia-mediated IL-1?over-expression.