自噬抑制剂3-甲基腺嘌呤可提高小鼠同种异体坐骨神经移植效率

3-Methyladenine improves the efficiency of sciatic nerve allograft in mice

背景:近年最新研究表明,华勒氏变性的发生与许旺细胞的自噬活动密切相关,对许旺细胞自噬活动进行调控,可以显著影响华勒氏变性的发生发展,从而改变后续的轴突再生及髓鞘化过程。目的:在同种异体神经移植中对移植片段的细胞自噬过程进行抑制,观察是否影响移植后的修复效率。方法:获取8只雌性C57BL/6J小鼠(购自北京维通利华)坐骨神经片段16条,分2组,分别于含自噬抑制剂3-甲基腺嘌呤的培养基及普通培养基中处理72 h。取16只雌性C57BL/6J小鼠,建立左侧坐骨神经缺损模型,实验组(n=8)植入含自噬抑制剂3-甲基腺嘌呤培养基处理过的坐骨神经片段,对照组(n=8)植入普通培养基处理的坐骨神经片段,术后2,4,6,8周,记录坐骨神经指数;术后8周取再生坐骨神经段,分别进行苏木精-伊红染色、免疫荧光染色、甲苯胺蓝染色、透射电镜观察等。动物实验通过北京协和医学院动物伦理委员会批准。结果与结论:①实验组术后8周的坐骨神经指数高于对照组(P<0.05),其余时间点两组间比较差异无显著性意义(P>0.05);②苏木精-伊红染色显示,实验组神经组织完整,对照组神经组织可见大面积空洞;③免疫荧光染色显示,实验组可见较完整的神经束结构,对照组未见完整的神经束结构;④甲苯胺蓝染色显示,实验组可见有髓神经纤维和部分再生无髓神经纤维,对照组仅见少量有髓神经纤维与新生无髓轴突;⑤透射电镜显示,实验组髓鞘厚度及有髓纤维直径均大于对照组(P<0.05);⑥结果表明应用3-甲基腺嘌呤处理移植前神经片段,可抑制许旺细胞自噬,有助于保留移植物髓鞘结构完整性,促进轴突再生及功能的恢复。

BACKGROUND:Recent studies have shown that the occurrence of Wallerian degeneration is closely related to autophagy in Schwann cells.The regulation of autophagy in Schwann cells can significantly affect the occurrence and development of Wallerian degeneration,subsequently altering axon regeneration and myelination.OBJECTIVE:To clarify whether sciatic nerve allograft can achieve higher efficiency when the cell autophagy is inhibited by 3-methyladenine.METHODS:We harvested 16 sciatic nerve segments from 8 female C57BL/6J mice that were purchased from Beijing Vital River Laboratory Animal Technology Co.,Ltd.in China.All the segments were equally divided into experimental and control groups and cultured in 3-methyladenine culture medium and normal culture medium for 72 hours,respectively.Another 16 female C57BL/6J mice were taken to make animal models of left sciatic nerve defects.After modeling,the sciatic nerve segments were grafted to repair sciatic nerve defect through microsurgery:3-methyladenine-treated nerve segments in the experimental group and normally treated nerve segments in the control group.Sciatic nerve index in each mouse was recorded at 2,4,6,and 8 weeks after modeling.At 8 weeks after modeling,the regenerated nerve segments were histologically analyzed using hematoxylin-eosin staining,immunofluorescent staining,toluidine blue staining,and transmission electron.The animal experiment was approved by the Animal Ethics Committee of Peking Union Medical College.RESULTS AND CONCLUSION:There were no differences in the sciatic nerve index between the two groups(P>0.05)except at 8 weeks after modeling(P<0.05).Hematoxylin-eosin staining revealed an intact nerve structure in the experimental group but a large area of voids in the control group.Immunofluorescent staining indicated that there were nerve tracts with more complete structures in the experimental group than the control group.Toluidine blue staining revealed some myelinated and unmyelinated nerve fibers regenerated in the experimental group and only a few of myelinated nerve fibers and unmyelinated axons newly formed in the control group.Under the transmission electron microscope,myelin sheath thickness and myelinated fiber diameter were significantly higher in the experimental group than the control group(P<0.05).Therefore,3-methyladenine-treated nerve allografts could inhibit autophagy in Schwann cells,maintain the myelin sheath structure of the allograft,and promote axonal regeneration and functional recovery.