脊髓背角补体异常活化与神经病理性疼痛关系的实验研究

目的 观察神经病理性疼痛(neuropathic pain,NPP)模型大鼠脊髓背角补体C3变化,并探索脊髓补体异常活化在NPP形成中的作用.方法 实验分两部分:①84只SD大鼠随机分为正常对照组、假手术后1、3、7 d组及坐骨神经结扎后1、3、7 d组,每组12只.分别在建模后1、3、7 d测定大鼠机械痛阈值.并按照分组在规定时间处死大鼠,采用RT-PCR、免疫组化和免疫比浊等测定脊髓中补体C3的表达情况.②48只SD大鼠随机分假手术+生理盐水组、CCI+生理盐水组、CCI+CVF组(持续尾静脉注射CVF组)和CCI+生理盐水+CVF组(单次尾静脉注射CVF组),每组12只.分别在术前和术后1、3、7、14 d测定大鼠机械痛阈值,两周后处死大鼠,用免疫组化和免疫比浊技术测定大鼠L4～L6脊髓中补体C,含量;并用分光光度法测定L4～L6脊髓匀浆中超氧化物歧化酶(superoxide dismutase,SOD)和丙二醛(malondialdehyde,MDA),同时透射电镜观察L4～L6脊髓背角神经元内部结构变化.结果 CCI大鼠坐骨神经结扎侧后1、3、7 d脊髓L4～L6背角补体C,蛋白及mRNA都显著表达,并呈进行性升高趋势.而假手术组无明显变化;Pearson相关分析显示,CCI大鼠脊髓背角补体异常活化状况与大鼠痛觉过敏程度相关.单次尾静脉注射CVF可一过性提高模型大鼠痛觉阈值,并随着药物的代谢作用逐渐减弱;持续给予CVF可抑制大鼠痛觉过敏的发生.给予CVF可逆转模型大鼠脊髓中SOD活力并降低MDA产物含量.给予CVF可减轻模型大鼠脊髓背角神经元线粒体肿胀及核膜损伤程度(P<0.05).结论 外周神经损伤诱发的NPP大鼠脊髓背角发生了补体异常活化现象,并且该处的补体活化参与NPP的痛觉过敏形成.

Relationship between abnormal complement activation in the spinal dorsal horn and neuropathologic pain in rats

Objective To observe the change of complement 3 in dorsal horn of spinal cord and to investigate the role of ab-normal activation of complement protein in pathogenesis of NPP. Methods This study comprises two parts. In part 1, 84 healthy male Sprague-Dawley rats were divided randomly into seven groups:normal control group, sham I d group, sham 3 d group, sham 7 d group, CCI 1 d group, CCI 3 d group, CCI 7 d group (n=12). The mechanical pain threshold were measured in different time ac-cording to the group, while the mRNA and protein of Complement 3 were determined by RT-PCR, immunoturbidimetry and immuno-histochemistry. In part 2, 48 healthy male Sprague-Dawley rats were divided randomly into sham operation + saline group, CCI + saline group, CCI + CVF group(continual injection CVF group) and CCI + saline + CVF group(single injection CVF group)(n=12). Pain thresholds by mechanical stimulation were recorded at preoperation and 1, 3, 7, 14 d after operation in each group. Comple-ment 3 expression in the spinal dorsal horn was determined immunohistochemically to confirm the efficacy of CVF. The SOD activity and MDA content in the spinal cord homogenate were determined, and intracellular changes of spinal dorsal horn neurons were ob-served under electronmicroscopy. Results The expression of mRNA and protein of complement 3 in spinal dorsal horn increased at 1, 3, 7 d after CCI, but those were not shown in rats in sham operation group and normal control group. The pearson analysis demonstrated that there was an initate relation between complement 3 content and pain threshold. Hyperalgesia was obviously alleviat-ed in CCI + saline + CVF group after administration of CVF, and the threshold revived following attenuation of the role of CVF. Hy-peralgesia was not evoked after sciatic nerve ligation in CCI + CVF group. Compared with CCI + saline group, the SOl) activity in-creased sinificianfly and MDA content decreased obviously in CCI + CVF group (P<0.05). Electronmicroscopy demonstrated mito-chondrial swelling and cytomembrane breakdown of spinal dorsal horn neurons in CCI + saline group while mitochondrial swelling was mitigated in spinal dorsal horn neurons in CCI + CVF group. Conclusion Abnormal complement activation occurred in the spinal dorsal horn of rats with peripheral nerve injury induced NPP and participated in the development of hyperalgesia.