Experimental Study Of Spinal Astrocyte Participating In The Modulation Of Neuropathic Pain

Part 1 The altervation of spinal astrocyte and pro-inflammatory cytokine in chronic constriction injury model of ratsStudy 1 Dynamic altervation of spinal astrocyte reaction in chronic constriction injury model of ratsObjective To observe the response of astrocytes in lumber spinal cord in chronic constriction injury (CCI) model of rats.Methods Sprague -Dawley rats were randomly divided into 2 groups, which received right sciatic nerve ligation (chronic constriction injury, CCI group) and sham-operation (Sham group) respectively. The mechanical and thermal pain threshold were measured at 1d before the operation and at 1d, 4d, 7d, 14d and 28d after the operation. The expression of GFAP was also assessed by immunohistochemical analysis at the same time point.Results CCI, and not sham surgery, produced significant mechanical allodynia and thermal hyperalgesia. The mechanical threshold began to decrease at 1d after the operation, peaked at 7d and lasted throughout the experiment. The thermal threshold began to decrease at 1d after the operation, peaked at 4d and lasted throughout the experiment. Immunoreactive(IR)-like GFAP protein in the ipsilateral spinal dorsal horn to the injury did not significantly increase until postoperative day 4, reached a peak level on postoperative day 7 and lasted over the duration of the study. Few positive astrocytes were distributed in the contralateral spinal cord or spinal cord of sham group.Conclusion The activation of lumber spinal astrocyte may be involved in the modulation of neuropathic pain arised from chronic constriction injury to the sciatic nerve.Study 2 Downregulation of TNF-alpha, IL-6 mRNA and protein expression in CCI model of rats after intrathecal administration of fluorocitrateObjective To investigate the effect of intrathecal administration of fluorocitrate (FC), an selective astrocyte metabolic inhibitor, on tumor necrosis factorα(TNF-α) and interleukin-6(IL-6) mRNA and protein expression in neuropathic pain model of rats.Methods The CCI model was established and FC was administrated intrathecally (1nmol/1μl) once daily for 6 consecutive days. Negative control for CCI was sham operation and that for FC was the vehicle. The mechanical and thermal pain threshold were measured at 1d before operation and at 1d, 3d, 5d, 7d after the operation. The expression of TNF-αand IL-6 mRNA was measured by reverse transcription polymerse chain reaction (RT-PCR) and the expression of TNF-αand IL-6 protein was measured by immunohistochemistry.Results CCI, not the sham operation, significantly induced the mechanical allodynia and thermal hyperalgesia, and markedly increased the expressions of TNF-αand IL-6 mRNA and immunoreactive(IR)-like TNF-α/IL-6 protein in the ipsilateral spinal dorsal horn to the injury. Intrathecal injection of FC suppressed the increased expressions of TNF-αand IL-6 induced by CCI in the spinal cord, and significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia. There were no effects on the mechanical allodynia and thermal hyperalgesia and the expressions of TNF-αand IL-6 by intrathecal FC on the sham operation group.Conclusion Fluorocitrate has the effect of analgesia on neuropathic pain. The activation of astrocyte and upregulation of the expression of TNF-αand IL-6 in the spinal cord may be involved in the modulation of neuropathic pain.Part 2 Study of NF-κB pathway in spinal astrocyte in neuropathic painSyudy 1 Co-expressions of GFAP and NF-κB in spinal cord of CCI model of ratsObjective To determine the possible intracellular signal transduction pathway associated with astrocyte, which have been activated by CCI and clarify the relationship of expression or activation of GFAP and NF-κB in the lumber spinal cord.Methods Sixteen male Sprague-Dawley rats were randomly divided into two groups: the CCI group which received the chronic constriction injury and the sham group which received the sham operation as control. The mechanical and thermal nociceptive thresholds were assessed with paw withdrawal latency (PWL) to von Frey filaments and radiant heat. The expressions of GFAP, NF-κBp65 and co-expressions of GFAP and NF-κB in spinal cord were examined 7d after CCI procedure by using immunocytochemical staining technique.Results At 7d post-lesion, CCI induced mechanical allodynia and thermal hyperalgesia and increased the numbers of GFAP and NF-κBp65 immunoreactive(IR) positive cells in the ipsilateral lumber spinal dorsal horn to the injury. Double immunocytochemical staining also showed the increase of GFAP/NF-κBp65-IR positive cells in the spinal dorsal horn in CCI group, while few positive cells were distributed in the sham group.Conclusion CCI activates NF-κBp65 in astrocytes in the dorsal horn and NF-κB pathways in astrocytes may be involved in the pathogenesis of neuropathic pain.Syudy 2 Alleviation of neuropathic pain by intrathecal injection of antisense oligonucleotides to p65 subunit of NF-κBObjective To investigate the effects of intrathecal administration of NF-κB p65 antisense oligodeoxynucleotides (ODN) on mechanical allodynia and thermal hyperalgesia and the expression of TNF-αand IL-6 mRNA in chronic constriction injury (CCI) model of rats.Methods Lumbar intrathecal catheters were implanted in male Sprague - Dawley rats. The chronic constriction injury (CCI) model was established and thermal and mechanical nociceptive thresholds were assessed with paw withdrawal latency (PWL) to radiant heat and von Frey filaments. The phosphorothioate -modified antisense oligodeoxynucleotides (ODNs) to p65 subunit of NF-κB were administered intrathecally on each of 5 successive days post-CCI. Nuclear NF-κB p65 expression was determined by Western blot. TNF-αand IL-6 mRNA were determined by RT-PCR. Results CCI induced mechanical allodynia and thermal hyperalgesia, and significantly increased the expression of NF-κB p65 protein, TNF-αand IL-6 mRNA. Intrathecal injection of antisense ODNs, but not missense ODNs, markedly suppressed the expression of NF-κB p65 protein, TNF-αand IL-6 mRNA, and significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia.Conclusion The activation of NF-κB pathway and the release of TNF-αand IL-6 may contribute to neuropathic pain in CCI rats. NF-κBp65 is likely to be an effective target for the treatment of neuropathic pain.