中国神经再生研究(英文版) ›› 2020, Vol. 15 ›› Issue (6): 1160-1165.doi: 10.4103/1673-5374.270315

• 原著:周围神经损伤修复保护与再生 • 上一篇    下一篇

追踪神经再生轴突时程分析体内感觉轴突的再生

  

  • 出版日期:2020-06-15 发布日期:2020-07-05
  • 基金资助:

    中国国家自然科学基金项目(81460198,31260233),美国国家卫生研究院项目(R01NS064288,R01NS085176,R01EY027347),Craig H. Neilson基金会项目,BrightFocus基金会项目

Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons

Yan Gao1, Yi-Wen Hu2, Run-Shan Duan3 , Shu-Guang Yang4 , Feng-Quan Zhou4, Rui-Ying Wang2   

  1. 1 Guilin Medical University, Guilin, Guangxi Zhuang Autonomous Region, China
    2 Department of Orthopedic Surgery, Affiliated Hospital of Guilin Medical University, Guilin, Guangxi Zhuang Autonomous Region, China
    3 Department of Orthopedics, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, Zhengzhou, Henan Province, China
    4 Department of Orthopedic Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA
  • Online:2020-06-15 Published:2020-07-05
  • Contact: Rui-Ying Wang, PhD,77276533@qq.com; Feng-Quan Zhou, PhD,fzhou4@jhmi.edu.
  • Supported by:
    This work was supported by the National Natural Science Foundation of China, No. 81460198, 31260233; the National Institute of Health of the United States of American, No. R01NS064288, R01NS085176, R01EY027347 (to FQZ); the Craig H. Neilson Foundation, the BrightFocus Foundation (to FQZ).

摘要:

目前大多数研究通过免疫染色再生相关蛋白来检测轴突再生,这种方法可以间接地测量背根神经节中感觉神经元和脊髓中运动神经元的轴突长度。我们最近开发了一种以编码增强型绿色荧光蛋白的质粒DNA体内电穿孔转染成年背根神经节中感觉神经元的直接特异性测量整个神经再生感觉轴突长度的方法。(1)首先通过镊子挤压坐骨神经,建立坐骨神经挤压小鼠模型,并在损伤前2或3d时通过在同侧背根神经节电穿孔转染携带编码增强型绿色荧光蛋白的质粒DNA;(2)以共聚焦显微镜观察背根神经节或坐骨神经的荧光分布情况,损伤12,18h,1,2,3,4,5,6d时可通过表达绿色荧光的图像,测量坐骨神经挤压后的再生轴突长度;以Western blot检测背根神经节中凋亡相关蛋白Caspase-3的表达情况,发现体内电穿孔转染不影响背根神经节中凋亡相关蛋白Caspase-3的表达;接下来采用一种快速组织清除法成功清除了背根神经节和坐骨神经。清除的背根神经节的三维成像可提供表达增强型绿色荧光蛋白或荧光染料标记的微小RNA寡聚体的感觉神经元的高质量图像;(3)实验结果将为周围神经轴突再生直接时程分析提供帮助。实验于2014-3-7经桂林医学院动物伦理委员会批准(批准号:GLMC201503010)。

orcid: 0000-0002-2221-5032 (Rui-Ying Wang)

关键词: 轴突再生, 体内电穿孔, 组织清除, 背根神经节, 周围神经系统, 坐骨神经, 微小RNA, 细胞凋亡, 神经再生

Abstract: Most current studies quantify axon regeneration by immunostaining regeneration-associated proteins, representing indirect measure- ment of axon lengths from both sensory neurons in the dorsal root ganglia and motor neurons in the spinal cord. Our recently developed method of in vivo electroporation of plasmid DNA encoding for enhanced green fluorescent protein into adult sensory neurons in the dorsal root ganglia provides a way to directly and specifically measure regenerating sensory axon lengths in whole-mount nerves. A mouse model of sciatic nerve compression was established by squeezing the sciatic nerve with tweezers. Plasmid DNA carrying enhanced green fluorescent protein was transfected by ipsilateral dorsal root ganglion electroporation 2 or 3 days before injury. Fluorescence distribution of dorsal root or sciatic nerve was observed by confocal microscopy. At 12 and 18 hours, and 1, 2, 3, 4, 5, and 6 days of injury, lengths of regenerated axons after sciatic nerve compression were measured using green fluorescence images. Apoptosis-related protein caspase-3 expression in dorsal root ganglia was determined by western blot assay. We found that in vivo electroporation did not affect caspase-3 ex- pression in dorsal root ganglia. Dorsal root ganglia and sciatic nerves were successfully removed and subjected to a rapid tissue clearing technique. Neuronal soma in dorsal root ganglia expressing enhanced green fluorescent protein or fluorescent dye-labeled microRNAs were imaged after tissue clearing. The results facilitate direct time course analysis of peripheral nerve axon regeneration. This study was approved by the Institutional Animal Care and Use Committee of Guilin Medical University, China (approval No. GLMC201503010) on March 7, 2014.

Key words: axon regeneration, cell apoptosis, dorsal root ganglion, in vivo electroporation, microRNAs, peripheral nervous system, sciatic nerve, tissue clearing