| Objective To investigate the expression and distribution of G protein-coupled kinase6(GRK6) in the dorsal root ganglion (DRG) and spinal cord following chronic constriction injury (CCI) induced neuropathic pain in rats, and to explore the possible mechanism of GRK6in the regulation of neuropathic pain.Methods (1) CCI model were established in adult male rats by loosely ligating the sciatic nerve of the left hind leg, and the the nerves of Sham animals were identically exposed but not ligated. Mechanical allodynia and heat hyperalgesia were tested at different time points after CCI.(2) Western Blot was used to detect the protein expression of GRK6in the DRG and L4.5spinal segments after CCI. The celluar distribution of GRK6in DRG and spinal cord were detected by immunofluorescence double staining. Immunohistochemisty staining was applied to determine the staining and distribution alteration of GRK6in spinal dorsal horn after CCI.(3) The overexpression GRK6lenti virus was successfully constructed. A PE-10catheter was inserted into the L5spinal cord and lentivirus was injected3days before CCI surgery. Then the pain related behavior was tested to tesify the role of GRK6in this neuropathic pain model.(4) The protein expression of tumor necrosis factor-a (TNFa) after CCI was examined by Enzyme-linked immunosorbent assay (ELISA). Then the expression of GRK6in cultured spinal dorsal horn neurons were detected by Western Blot after being stimulated by TNF-a of different concentration. In order to testify the relation of GRK6and pro-inflammatory cytokines,1mg TNF-a antibody was continuously injected every3days to neutralize the effect of TNF-a in CCI model. The GRK6expression and behavior test were detected afterward.Results (1) The unilateral chronic constriction injury model of neuropathic pain induced persistent bilateral mechanical allodynia and heat hyperalgsesia, which started from1d after CCI and lasted for more than21days. Maximal mechanical allodynia and heat hyperalgseia were observed at day7.(2) After CCI, a clear reduction in GRK6protein expression was detected in both bilateral DRG and L4.5spinal segments. The expression of GRK6progressively decreased from3day after injury, declined to the lowest at day5, and then gradually increased. The expression of GRK6was predominantly in the superficial layers of lumba spinal cord which was confirmed by the localtion of substrance P and IB4. GRK6immunoreactivity was mostly co-localized with neurons, partly with IB4positive neurons, and rarely with astrocyte and microglia. Immunohistochemistry staining showed a clear bilateral reduction in GRK6in the spinal dorsal horn at5day after CCI, particularly in laminae Ⅰ-Ⅱ.(3) After injecton of over-expressed GRK6-lentiviral, the results of immunoflourense and Western Blot showed up-regulation of GRK6in spinal cord. Over-expression GRK6attenuated the mechanical allodynia and heat hyperalgesia induced by CCI.(4) The TNF-a level in bilateral sides of spinal cord were increased in day1after injury, and reached the peak at day3, then decreased. After the isolated spinal dorsal horn neurons were treated with TNF-a, the decreased expression of GRK6in the neurons was detetected by Western Blot. Intrathecal injection of TNF-a antibody up-regulated GRK6and attenuated neuropathic pain induced by CCI.Conclusions Unilateral CCI induced marked bilateral neuropathic pain and GRK6downregulation in the DRG and lumba spinal cord, and the expression of GRK6was mostly co-localized with neurons. The up-reguation of GRK6partly attenuated the pain response of CCI. The expression of TNFa was increased after CCI and TNF-a significantly modulated GRK6expression in ex vitro and in vivo. All these data indicated cytokine TNFa induced decreased neuronal GRK6expression may be the potential mechanism which contributes to the regulation of neuropathic pain. |
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