The Role Of Glia Cells Of Spinal Dorsal Horn In Peripheral Nerve Damage Induced Neuropathic Pain

Background：Over the past few years, glial cells has been considered as apromising target against neuropathic pain. Indeed, changes in glial phenotypeshave been reported throughout the entire nociceptive pathway, from peripheralnerves to higher integrative brain regions and pharmacological inhibition ofsuch glial reactions reduces the manifestation of pain in animal models. Thiscomplex interplay between glia and neurons relies on various mechanismsdepending both on glial cell types considered (astrocytes, microglia, satellitecells or Schwann cells), the anatomical location of the regulatory process(peripheral nerve, spinal cord or brain) and the nature of the chronic painparadigm. Intracellularly, recent advances have pointed out to the activation ofspecific cascades, such as mitogen associated protein kinases (MAPK) and Wnt-β-catenin pathway in the underlying processes behind glial activation.Reports suggest that microglia play a key role in spinal nerve ligation(SNL)-induced neuropathic pain, and toll-like receptor3(TLR3) has asubstantial role in the activation of spinal microglia and the development oftactile allodynia after nerve injury. In addition, ketamine application couldsuppress microglial activation in vitro, and ketamine could inhibitproinflammatory gene expression possibly by suppressing TLR-mediated signaltransduction. But there is no related reports about whether ketamine can treatneuropathic pain by inhibiting microglia TLR signaling pathways.Astrocyte also played a key role in SNL-induced neuropathic pain. Theactivation of astrocyte was later and longer than microglia which was accordingto the chronicity of neuropathic pain. It was considered synaptic plasticity play akey role in the chronicity of neuropathic pain. Wnt pathway was proved playinga key role in regeneration of hippocampus neuron. But whether the Wnt signalpathways are involved in the peripheral nerve injury inducted synaptic plasticityof spinal dorsal horn in rat, there is still no related report.Aim: Based on the present research situation and task group preliminary studiesaccumulation, the present study was designed to disclose whether intrathecalketamine could suppress SNL-induced spinal microglial activation and exertsome antiallodynic effects on neuropathic pain by suppressing TLR3activation.Moreover, we explored whether Wnt signal pathways are involved in thechronicity of peripheral nerve damage-induced neuropathic pain as well as thecorresponding control mechanism.Methods: Behavior pharmacology method, immunofluorescence, RT-PCR and Western blot were used to observe the activation of microglia and astrocyte andthe change of TLR3and Wnt-β-catenin pathway in spinal cord.Results: The experiment one: Behavior pharmacology results show thatintrathecal ketamine (30,100and300μg/kg) exerted an effective and reliableeffect on SNL-induced mechanical allodynia in a dose-dependent manner.Immunohistochemical fluorescence results show that SNL induced microgliaactivation in spinal cord3d after surgery, at the same time, ketamine (30100300μ g/kg) can restrain SNL induced microglia activation, and there exists dosedependent relationship. SNL induced significant upregulation of p-p38in theipsilateral spinal dorsal microglia, almost all p-p38-positive cells wereOX42-positive microglia. The results from the western blot showed that SNLinduced an upregulation of p-p38in the spinal dorsal horn in the SNL-salinegroup vs sham-saline group. On the other hand, Poly I:C induced significantmechanical allodynia, and microglia activation was detected. But ketaminereversed Poly I:C injection-induced mechanical allodynia and spinal dorsalmicroglia activation.The experiment two: Immunohistochemical fluorescence results are shownSNL can induce astrocytes activation from3d to21d after SNL. Almost allWnt-3a-positive cells are GFAP-positive astrocytes. Frizzled-4and β-cateninpositive staining are localized in the neuron. The real-time PCR results showthat SNL can induce high expression of Wnt-3a gene3d after SNL, and theexpression is related to the progress of pain,35d to the peak,42d fell down.Frizzled-4show a high expression from7d after SNL, continuing to28d,35dfell down. β-catenin also express higher from7d after SNL,21d to the peak,continuing to35d, felling42d, but still in high level. Western blot results show that Wnt-3a highly express from3d after SNL, maintaining in high level from14d to35d. The expression of Frizzled-4slightly rise from3d to35d afterSNL. Î'-catenin expression rise from14d after SNL, and maintain in a highlevel to35d. Behavior pharmacology results show that either Wnt-3a antibodyor inhibitor of Frizzled-4failed to attenuate neuropathic pain by disturbing Wntpathway. Inhibitor of astrocytes also cannot affect the mechanical allodyniainduced by SNL. However, disturbing Wnt pathway or inhibitor of astrocytesactivation on3d or7d after SNL could reverse mechanical allodynia.Conclusion: Both microglia or astrocytes play an important role in peripheralnerve injury-induced neuropathic pain. Intrathecal injection of ketamine couldrelieve SNL-induced mechanical allodynia, probably by suppressingTLR3-induced spinal microglia activation during the initiation of SNL-inducedneuropathic pain. Obstructing Wnt pathway or suppressing the activation ofastrocyte can significantly reduce SNL-induced neuropathic pain three daysafter surgery, this may be the underlying mechanism of chronicity ofneuropathic pain. Therefore, spinal dorsal horn glia cells could be new target ofneuropathic pain, which will provide a new idea for neuropathic pain treatmentin clinic.