中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2022, Vol. 17 ›› Issue (10): 2316-2320.doi: 10.4103/1673-5374.337055

• 原著:神经再生技术 • 上一篇    

霍乱毒素亚单位B结合荧光素在大鼠腓肠肌注射后3-7d时可观察到标记神经元的最佳形态

  

  • 出版日期:2022-10-15 发布日期:2022-03-16
  • 基金资助:

    中国中医药学会创新基金项目(CI2021A03407)；国家重点研发计划项目(2019YFC1709103)；国家自然科学基金项目(81774432，81774211，82004492，81801561)；中央公益事业研究院基本科研业务(ZZ13-YQ-068，ZZ14-YQ-032，ZZ14-YQ-034)

Alexa Fluor 488-conjugated cholera toxin subunit B optimally labels neurons 3–7 days after injection into the rat gastrocnemius muscle

Jing-Jing Cui, Jia Wang, Dong-Sheng Xu, Shuang Wu, Ya-Ting Guo, Yu-Xin Su, Yi-Han Liu, Yu-Qing Wang, Xiang-Hong Jing, Wan-Zhu Bai   

  1. Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences, Beijing, China
  • Online:2022-10-15 Published:2022-03-16
  • Contact: Wan-Zhu Bai, PhD, wanzhubaisy@hotmail.com.
  • Supported by:
    This study was supported by the CACMS Innovation Fund, No. CI2021A03407 (to WZB); the Project of National Key R&D Program of China, No. 2019YFC1709103 (to WZB); the National Natural Science Foundation of China, Nos. 81774432 (to JJC), 81774211 (to WZB), 82004492 (to JW), 81801561 (to DSX); and the Fundamental Research Funds for the Central Public Welfare Research Institutes of China, Nos. ZZ13-YQ-068 (to JJC), ZZ14-YQ-032 (to JW), ZZ14-YQ-034 (to DSX).

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摘要：

神经示踪方法是神经解剖学领域中研究神经通路/环路以及评估神经损伤后修复的有效手段，但如何获取最佳的标记效果仍然是一个难题。实验将霍乱毒素亚单位B结合荧光素488注射到大鼠腓肠肌后，其脊髓腹角、背根神经节和交感神经链中分别可见标记的运动神经元、感觉神经元和交感神经元，且同时在胫前肌注射霍乱毒素亚单位B结合荧光素594也获得了类似的结果。进一步评估霍乱毒素亚单位B结合荧光素488注射后不同时间点的神经元数量和形态，结果显示最早可在注射后12h观察到标记的运动和感觉神经元，且并随着时间延长的荧光标记加强。在注射后3-7d时，标记的神经元形态结构最为清晰和完整，可明确辨别运动神经元的树突和轴突。而在第14天时神经元的形态结构已经变得模糊和不完整。继而通过尼氏染色以及荧光免疫组织化学检测发现，霍乱毒素亚单位B结合荧光素488标记的神经元可保持正常的形态和生化特征，也不会引起其周围的小胶质细胞的形态改变。表明霍乱毒素亚单位B单独或联合荧光素从肌肉组织逆行示踪可在注射后3-7d时观察到标记神经元的最佳形态，且不会影响神经元及周围细胞的形态，因而这种示踪剂适用于评估损伤神经的再生或退行变化。

https://orcid.org/000-0001-6285-7788 (Wan-Zhu Bai)

关键词: 神经示踪, 霍乱毒素亚单位B结合荧光素, 小胶质细胞, 降钙素基因相关肽, 感觉神经元, 运动神经元, 交感神经元, 最佳时间窗, 胫前肌, 局在性分布

Abstract: Neural tract tracing is used to study neural pathways and evaluate neuronal regeneration following nerve injuries. However, it is not always clear which tracer should be used to yield optimal results. In this study, we examined the use of Alexa Fluor 488-conjugated cholera toxin subunit B (AF488-CTB). This was injected into the gastrocnemius muscle of rats, and it was found that motor, sensory, and sympathetic neurons were labeled in the spinal ventral horn, dorsal root ganglia, and sympathetic chain, respectively. Similar results were obtained when we injected AF594-CTB into the tibialis anterior muscle. The morphology and number of neurons were evaluated at different time points following the AF488-CTB injection. It was found that labeled motor and sensory neurons could be observed 12 hours post-injection. The intensity was found to increase over time, and the morphology appeared clear and complete 3–7 days post-injection, with clearly distinguishable motor neuron axons and dendrites. However, 14 days after the injection, the quality of the images decreased and the neurons appeared blurred and incomplete. Nissl and immunohistochemical staining showed that the AF488-CTB-labeled neurons retained normal neurochemical and morphological features, and the surrounding microglia were also found to be unaltered. Overall, these results imply that the cholera toxin subunit B, whether unconjugated or conjugated with Alexa Fluor, is effective for retrograde tracing in muscular tissues and that it would also be suitable for evaluating the regeneration or degeneration of injured nerves.

Key words: Alexa Fluor-conjugated cholera toxin subunit B, calcitonin gene-related peptide, microglia, motor neurons, neural tract tracing, optimal time window, sensory neurons, somatotopic organization, sympathetic neurons, tibialis anterior muscle