目的 探讨用川芎嗪预处理冷保存的大鼠坐骨神经修复周围神经缺损的可行性.方法 40只Wistar大鼠和40只SD大鼠分别为供体和受体,取供体双侧坐骨神经长15 mm,随机在川芎嗪溶液(0、50、100、200 mg/L,A、B、C、D组,n＝20)中4 ℃冷保存6周,透射电镜观察坐骨神经超微结构,Calcein-AM荧光染色,激光扫描共聚焦显微镜(laser scanning confocal microscope,LSCM)观察;受体大鼠随机分为A'、B'、C'、D'组(n＝10,对应A、B、C、D组),用冷保存6周的供体神经修复对应组受体坐骨神经10 mm缺损.术后分期行大体观察,检测坐骨神经功能指数(SFI),术后20周行电生理检测、再生神经图像分析和透射电镜观察.结果 大鼠坐骨神经冷保存6周,A组神经纤维严重脱髓鞘变化和轴索萎缩变性、蜂窝状改变,而B、C、D组变化较轻;LSCM结果显示,B、C、D组荧光强度高于A组,B组高于C、D组,差异均有统计学意义(P< 0.05),C、D组间差异无统计学意义(P> 0.05).术后动物存活至实验结束,各期SFI,20周电生理检测,再生有髓神经纤维数目、髓鞘厚度,B'、C'、D'组与A'组比较,B'组与C'、D'组比较差异均有统计学意义(P< 0.05),C'、D'组间差异无统计学意义(P> 0.05);术后20周透射电镜观察显示,A'组再生有髓神经纤维少、髓鞘薄,结缔组织增生明显,B'、C'、D'组有髓神经纤维较多、髓鞘较厚,结缔组织增生不明显.结论 一定质量浓度的川芎嗪对冷保存大鼠坐骨神经具有保护作用,并能促进异体移植后神经的再生.

Objective To investigate the feasibility of repairing nerve defect of sciatic nerve in rats by the pretreatment of cold preservation with tetramethylpyrazine. Methods Forty Wistar rats and 40 SD rats were selected as donors and recipients, respectively [3 months, SPF male rat (200 ± 20) g], the sciatic nerve segments of 15 mm were harvested from the donors, then randomly preserved in 4 ℃ tetramethylpyrazine solution (0, 50, 100, and 200 mg/L, in A, B, C, and D groups, n = 20) for 6 weeks, the ultrastructure of sciatic nerve was observed by transmission electron microscope (TEM). Calcein-AM staining was used to observe the change of fluorescence intensity with laser scan confocal microscope (LSCM). The recipients were randomly divided into A ', B', C', and D' groups (n = 10, corresponding to A, B, C, and D groups), 10 mm defect of sciatic nerve in recipient was repaired by the nerve preserved for 6 weeks. The gross appearance and sciatic nerve function index (SFI) were detected at different period after the surgery. The electrophysiological changes, regenerated nerve image analysis, and TEM were performed in 20 weeks after transplantation. Results The nerve preserved for 6 weeks, there were severe demyelination and neuraxis denaturation in group A, but these changes in groups B, C, and D were more gentle than those in the group A. The fluorescence intensity in the groups B, C, and D was higher than that in group A, and in group B higher than that in groups C and D (P <0.05). There was no significant difference in groups C and D (P >0.05). All the rats survived to the end of the experiment. There were significant differences (P <0.05) between groups B', C', D' and group A', group B' and groups C', D' while there were no significant differences between groups C' and D' (P >0.05) in different times of SFI after transplantation, electrophysiological detection, the number of myelinated nerve fibers, and thickness of myelin at 20 weeks, The observation of TEM in 20 weeks showed that myelinated nerve fibers and thickness of myelin sheath in group A' was thinner than those in groups B', C', and D', and that the hyperplasia of connective tissues in group A' was more serious than that in groups B', C', and D'. Conclusion Tetramethylpyrazine plays a protective role on the cold preservation of sciatic nerve, and could promote the nerve regeneration after allogeneic transplantation.

国家自然科学基金面上项目(81373668);重庆市渝中区科技计划项目(20120219)