中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2017, Vol. 12 ›› Issue (8): 1357-1364.doi: 10.4103/1673-5374.213560

• 原著:周围神经损伤修复保护与再生 • 上一篇    下一篇

三重免疫酶染色：一种能同时鉴定正常、去神经支配和神经再支配大鼠单个骨骼肌冷冻切片全部肌纤维类型的新方法

  

  • 收稿日期:2017-07-19 出版日期:2017-08-15 发布日期:2017-08-15
  • 基金资助:

    美国武装部队再生医学研究所资助；美国国家推进转化医学中心项目；美国Mayo Clinic神经外科项目；美国Mayo Clinic神经再生医学中心基金

A novel triple immunoenzyme staining enables simultaneous identification of all muscle fiber types on a single skeletal muscle cryosection from normal, denervated or reinnervated rats

Peng Wu1, 2, 3, Shuya Zhang4, Robert J. Spinner1, Michael Torres Lizardi5, Yudong Gu2, 6, Cong Yu2, 6, Michael J. Yaszemski7, Anthony J. Windebank4, Huan Wang1   

  1. 1 Department of Neurological Surgery, Mayo Clinic, Rochester, MN, USA;
    2 Department of Hand Surgery, Huashan Hospital, Fudan University, Shanghai, China;
    3 Shanghai Tenth People’s Hospital, Tongji University, Shanghai, China;
    4 Department of Neurology, Mayo Clinic, Rochester, MN, USA;
    5 Department of Medicine, New York Presbyterian Weill Cornell Medical Center, New York, NY, USA;
    6 Shanghai Key Laboratory of Peripheral Nerve and Microsurgery, Shanghai, China;
    7 Departments of Orthopedic Surgery and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA
  • Received:2017-07-19 Online:2017-08-15 Published:2017-08-15
  • Contact: Huan Wang, M.D., Ph.D.,wang.huan@mayo.edu.

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摘要：

最近开发出的三重免疫荧光染色（TIF）可在单个骨骼肌冷冻切片上同时鉴定所有肌纤维，但该技术存在诸如快速光褪色和肌纤维轮廓不清楚的缺点，而三重免疫酶染色（TIE）可能避免这些缺点。实验旨在建立灵敏、且具有特异性的三重免疫酶染色技术，以鉴定正常、去神经支配和神经再支配的大鼠骨骼肌纤维类型，并开发肌肉纤维定量的系统取样方法。取正常、去神经支配和神经再支配的Lewis大鼠后肢胫骨前肌和比目鱼肌，每块肌肉取5个连续的冷冻切片，其中1个切片用于TIE，4个切片用于单一免疫酶染色（SIE）。三重免疫酶染色系统包括两种抗原的聚合报告基因酶染色系统（A4.74，为BA-F8分别用于鉴定MyHC-I和MyHC-IIA型肌纤维）和一种抗原（BF-F3用于鉴定MyHC-IIB型肌纤维）的碱性磷酸酶染色系统，以及相应的检测系统和各自的色原体。定量检测12.5％，25％，33％和50％的肌纤维类型，并将其与从所有纤维（100％）获得的纤维类型定量进行比较。在单个三重免疫酶染色冷冻切片上可同时发现包括单一型和混合型在内的所有肌纤维类型，且轮廓清晰。三重免疫酶染色切片上的肌纤维类型与连续的SIE切片观察到的肌纤维类型匹配性非常高，匹配率高达95％。12.5％肌纤维的系统取样可以代表整个肌肉的真实纤维类型分布。实验证明三重免疫酶染色是一种新颖的、可以有效显现正常、去神经支配和神经再支配的大鼠骨骼肌纤维类型的染色方法。

orcid:0000-0002-5540-0648(Huan Wang)

 

关键词: 神经再生, 骨骼肌, 肌纤维, 免疫组化, 三重免疫酶染色, 肌球蛋白重链, 大鼠

Abstract:

Triple immunofluorescence staining has recently been developed to simultaneously identify all muscle fibers on a single cryosection which is helpful for clinical and basic research, but it has disadvantages such as fast photobleaching and unclear outlines of muscle fibers. Triple immunoenzyme staining (TIE) is likely to avoid these disadvantages. In this study, we aimed to establish a sensitive and specific TIE technique to identify fiber types in normal, denervated, and reinnervated rat muscles, and to develop a systematic sampling method for muscle fiber quantification. Tibialis anterior and soleus from normal, denervated, and reinnervated Lewis rat hind limbs were used. Five consecutive cryosections were cut from each muscle, including one for TIE and four for single immunoenzyme staining (SIE). The TIE was performed using the polymerized reporter enzyme staining system for the first two antigens (A4.74 for MyHC-IIA, BA-F8 for MyHC-I) and alkaline phosphatase staining system for the third antigen (BF-F3 for MyHC-IIB), followed by corresponding detective systems and respective chromogens. The type of muscle fibers was quantified by systematic sampling at 12.5%, 25%, 33% and 50% of all muscle fibers, and was compared with that acquired from counting all the fibers (100%). All muscle fiber phenotypes, including pure and hybrid, could be simultaneously identified on a single TIE cryosection with clear outlines. The fiber types on TIE slides matched well with their respective counterpart on the consecutive SIE slides with a 95% match rate. Systematic sampling of 12.5% fibers could represent the true fiber type distribution of the entire muscle section. Our results suggest that novel TIE can effectively visualize fiber types in normal, denervated or reinnervated rat muscles.

Key words: nerve regeneration, muscle fiber phenotyping, immunohistochemistry, triple immunoenzyme staining, myosin heavy chain, rats, neural regeneration