Spinal Microglia Modulate LTP Of Nociceptive Synaptic Transmission: Study On A Role Of P2X7 Receptor

Tetanic stimulation of the sciatic nerve induced long-term potentiation (LTP) of spinal cord neurons and pain behavioral hypersensitivity. The present work was to investigate the role of microglia in the modulation of central sensitization of spinal pain pathway induced by tetanic stimulation of the sciatic nerve (TSS) in rats.LTP, considered as the substrate of central sensitization in spinal pain pathway, can be induced by TSS, natural noxious stimulation of skin or nerve injury. Furthermore, the tetanic stimulation with the identical parameters used in electrophysiological recording induced hypersensitized pain behavior, including allodynia and hyperalgesia. However, the exact mechanisms of the development of spinal LTP remain obscure.Numerous works have proved that glia in the spinal cord, especially microglia, play a vital role in the development of pathological pain. Microglia can be activated in both inflammatory and neuropathic pain. Pre-application of glial metabolic inhibitor prevents the induction of spinal LTP and alleviates hypersensitized pain behavior, indicating the involvement of microglia in the induction of LTP of spinal nociceptive responses.P2X7 receptor (P2X7R) is excessively expressed in glia and considered as a pivotal mediator in the crosstalk between neuron and glia. The activation of P2X7R by exocytic ATP promoted significant release of glutamate, which is necessary for the induction of hippocampal LTP, and proinflammatory cytokines, such as interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α). It has been reported that spontaneous firing of spinal cord neurons and pain hypersensitivity are significantly suppressed by selective P2X7R antagonists. Moreover, P2X7 knockout mice showed no chronic inflammatory and neuropathic pain. Therefore, P2X7R may be a vital molecule in mediating the involvement of microglia in the induction of spinal LTP.By means of electrophysiological recording, mechanical allodynia test, immunohistochemistry and Western blot, the present study was to investigate the role of P2X7R in microglia in the induction of spinal LTP and long-term pain hypersensitivity induced by TSS. The main findings were as follows:Spinal LTP of C-fiber-evoked field potentials was induced by TSS and was blocked by intrathecal administration of microglial metabolic inhibitor minocycline 1 h before tetanic stimulation; the mechanical allodynia induced by TSS was partially alleviated by consecutively intrathecal administration of minocycline 1 h before and 7 days once daily after tetanic stimulation. These findings indicate that spinal microglia are involved in the induction of spinal LTP and pain hypersensitivity induced by TSS.Pre-injection of P2X7R antagonist oxATP or BBG 0.5 h before tetanic stimulation and P2X7-siRNA 7 days before TSS blocked spinal LTP; preincubation of spinal slices with BBG 1 h before tetanic stimulation blocked the spinal LTP of field excitory postsynaptic potentials (fEPSPs) induced by high frequency stimulation of Lissauer's tract without affecting the baseline responses, suggesting a pivotal role of P2X7R in the induction of spinal LTP.Pre-injection of P2X7R antagonist oxATP or BBG 0.5 h before tetanic stimulation and P2X7-siRNA 7 days before TSS partially alleviated bilateral allodynia on day 3,5 and 7 after tetanic stimulation; pre-injection of BBG 0.5 h before tetanic stimulation suppressed the upregulation of Fos expression 2 h after TSS, indicating the significant inhibition of excessive excitation of spinal pain sensitization neurons by the antagonism of P2X7R. Immunohistochemistry results showed that P2X7R was predominantly colocalized with microglia marker OX-42 but not with astrocyte marker GFAP and neuron marker NeuN; the activation of microglia and the upregulation of P2X7R induced by TSS was suppressed by preadministration of P2X7 antagonist BBG 0.5 h before tetanic stimulation, indicating a specific role of P2X7R in the induction of spinal LTP.The upregulation of phospharylated p38 mitogen-activated protein kinase (MAPK),IL-1βand GluR1 induced by tetanic stimulation was reversed by pre-injection of BBG 0.5 h before tetanus; the IL-1βantibody IL-1ra blocked the induction of spinal LTP and inhibited the increased expression of GluR1, indicating that IL-1βsignaling pathway was involved in the induction of spinal LTP after the activation of P2X7R.Imunohistochemistry results demonstrated that microglia and astrocytes were activated on day 3 and day 7 after TSS, respectively; Western blot results showed that the increased expression of interleukin-18 (IL-18), downstream molecule of P2X7R, as well as interleukin-18 receptor (IL-18R) was significantly inhibited by consecutive application of microglial metabolic inhibitor minocycline 1 h before and 7 days after TSS; IL-18 was predominantly colocalized with microglia marker Iba-1 and slightly with astrocyte marker GFAP but no colocalization with neuron marker NeuN; IL-18R was almost completely colocalized with astrocyte marker GFAP. These results suggest that the IL-18 signaling pathway contributes to the exacerbation of pathological pain by mediating the interaction of microglia and astrocytes.In conclusion, the present work demonstrated that P2X7R in microlia played a vital role in the induction of spinal LTP and persistent pain hypersensitization and IL-1βsignaling pathway was involved in the process. The interaction of microglia and astrocytes, mediated by a downstream molecule of P2X7R IL-18, contributed greatly to the maintenance of pathological pain.