| Background: There is growing recognition that glia in spinal cord could contribute to neuropathic pain powerfully and complement proteins, secreted by glia, could play an important role in the pathogenesis of diseases in central nervous system. Study on the effect of complements activation on the invasion mechanism of neuropathic pain has been educing a new research field. Recently, Milligan, an American notable professor, blocked the activation of the membrane attack pathway of the complement cascade by intrathecal injection of soluble complement receptor 1(sCR1) in rats. He found that intrathecal sCR1 showed no effect on the behavioral responses of contral groups but abolished the expression of mechanical allodynia induced by peripheral nerve inflammation (sciatic inflammatory neuropathy model), partial sciatic nerve injury (chronic constriction injury model) and intrathecal injection of human immunodeficiency virus type 1 gp120, a viral envelope protein that activates glia. The fact that enhanced nociceptive effect of nerve injury was prevented or reversed in all 3 paradigms suggests that complement system might be broadly involved in spinally mediated pain enhancement. Nevertheless, they did not further investigate whether a complement reaction cascade occurred in the spinal dorsal horn during NPP.Objective: The aim of this study was to observe the mRNA and protein of Complement 3 in dorsal horn of spinal cord and the effects of cobra venom factor(CVF) on pain threshold in rats with neuropathic pain(NPP), to investigate the roles of abnormal activation of complement proteins in invasion mechanism of NPP.Methods: This study was composed of three parts as follows.(1) Establishment and evaluation of CCI model in rats. Thirty adult male Sprague- Dawley rats were divided into three groups (traditional CCI group, improved CCI group and sham group) randomly. The threshold in nerve injured hind paw to thermal stimulus and mechanical stimulus and behavioral changes of rats were recorded at pre-operation and 2, 4, 6, 8, 10, 12, 14days after operation.(2) The expression of mRNA and protein of Complement 3 in spinal dorsal horn in rats with neuropathic pain. Eight-four healthy male Sprague-Dawley rats were divided randomly into seven groups as normal control group, sham 1,3,7d group and CCI 1,3,7d group. The left sciatic nerve was ligated loosely in rats in CCI groups, and that was not in rats in sham-operation groups. The mechanical and thermal pain thresholds were measured in different time according to the groups, while the mRNA and protein of Complement 3 were determined by methods of RT-PCR, immunoturbidimetry and immunohistochemistry.(3) Effects of cobra venom factor on the model of neuropathic pain in rats. Sprague-Dawley rats were divided randomly into sham operation + saline group (Group A), CCI + saline group (Group B), CCI + CVF group (Group C) and CCI +saline+ CVF group (Group D). Sciatic nerve ligature was performed classically in Group B, C, and D to construct CCI model. The sciatic nerve of rats in Group A was exposured only with no ligation. The saline was administrated preoperation and every day after operation in Group A and B. In the same times, CVF was administrated in Group C. CVF was administrated at 4 day after ligation, in other times, saline was administrated in group D. Pain threshold by mechanical was recorded at pre-operation and 1,3, 7, 14days after operation in each group. The rats were sacrificed on day 14 and expression of Complement 3 in the spinal dorsal horn was determined immunohistochemically to confirm the efficacy of CVF. Meanwhile, SOD activity and MDA content in the spinal cord homogenate were determined, and intracellular morphologic changes in spinal dorsal horn neurons were observed under electron microscopy.Results: (1) Rats in traditional CCI group and improved CCI group exhibited typical hyperalgesia phenomena, and the pain threshold in both groups decreased significantly. Compared to traditional CCI group, the hyperalgesia phenomena in rats of the improved CCI group existed in a longer time. (2) The mechanical and thermal hyperalgesia were observed in rats one day after the sciatic nerve ligation, and the state persisted at 3 and 7days after CCI comparing to those in sham operation group rats. The expressions of mRNA and protein of Complement 3 in spinal dorsal horn increased at 1, 3, 7days after CCI but those changes did not show in sham operation group and normal control group. Pearson analysis demonstrated that there was negative relationship between the content of Complement 3 relation and pain threshold. (3) Compared with the Group A, thermal pain sensitivity in the rats of Group B and Group C were detected higher at 3 day after operation (P<0.01). Hyperalgesia was obviously alleviated in Group D after administration of CVF compared with that in Group B (P<0.01), and attenuation of intrathical CVF revived the threshold. Hyperalgesia was not evoked after ligation in Group C. Compared to Group B, SOD activity decreased sinificiantly and MDA content increased obviously in Group C. Electron microscopy demonstrated intact structures and no swelling of the organelles such as mitochondria in spinal dorsal horn neurons of the Group A, but mitochondrial swelling, crista disruption, and cytomembrane breakdown of spinal dorsal horn neurons showed in Group B, while mitochondrial swelling was mitigated in spinal dorsal horn neurons in Group C.Conclusions: (1) The improved CCI model showed more excellent stability, and the characteristics of pain in rats in the improved CCI group seemed to be more similar to those in NPP patients. It seemed that the improved CCI model should be useful for studies, particularly in the aspect of immune mechanism. (2) Expressions of mRNA and protein of Complement 3 in spinal dorsal horn were high in rats with NPP,these reflected that the characteristic of the dynamic changes of Complement 3 could contribute to the establishment and maintenance of neuropathic pain. (3) CVF could effectively alleviate hyperalgesia in NPP model rats by exhausting the spontaneous pain and evoked pain if administration before operation. The activation state of Complement 3 may be in intimate relationship with pain and analgesia in NPP model rats.
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