使用透射电子显微镜定量骨骼肌纤维中的亚细胞糖原分布
Summary
改进的固定后程序增加了组织中糖原颗粒的对比度。本文提供了一个分步方案，描述了如何处理组织，进行成像，并使用立体学方法获得骨骼肌中纤维类型特异性亚细胞糖原分布的无偏和定量数据。
Abstract
通过使用透射电子显微镜，可以获得含有单个肌肉纤维的固定样品的高分辨率图像。这样可以量化超微结构方面，例如体积分数，表面积与体积比，形态测量和不同亚细胞结构的物理接触位点。在20世纪70年代，开发了一种用于增强细胞中糖原染色的方案，并为使用透射电子显微镜对糖原和糖原粒度的亚细胞定位的一系列研究铺平了道路。虽然大多数分析将糖原解释为它均匀分布在肌肉纤维内，仅提供单个值（例如，平均浓度），但透射电子显微镜显示糖原作为离散糖原颗粒储存在不同的亚细胞区室中。在这里，描述了从组织收集到定量测定单个骨骼肌纤维的不同亚细胞区室中糖原的体积分数和粒径的分步方案。考虑如何1）收集和染色组织标本，2）执行图像分析和数据处理，3）评估估计的精度，4）区分肌肉纤维类型，以及5）方法学陷阱和局限性。
Introduction
糖原颗粒由葡萄糖和各种相关蛋白的支化聚合物1 组成，在高代谢需求期间构成重要燃料2。虽然糖原颗粒尚未得到广泛认可，但它也构成了一种局部燃料，其中一些亚细胞过程优先利用糖原，尽管还有其他更持久的燃料如血浆葡萄糖和脂肪酸3，4。
几篇综述5，6主要基于透射电子显微镜（TEM）7，8对糖原亚细胞分布的一些最早文献，讨论了将糖原储存为亚细胞特异性局部燃料的重要性。第一项研究使用不同的方案来增加糖原从组织化学染色技术到阴性和阳性染色的对比度9，10。一个重要的方法论发展是用亚铁氰化钾还原锇11，12，13，14改进了固定后方案，显著改善了糖原颗粒的对比度。这种改进的方案并未用于一些关于运动诱导的糖原消耗的开创性工作15，但由Graham及其同事重新引入16，17。
基于二维图像，糖原的亚细胞分布最常被描述为位于三个池中的糖原颗粒：构膜下（就在表面膜下），肌原间（肌原纤维之间）或肌原纤维内（肌原纤维内）。然而，糖原颗粒也可以被描述为与例如肌质网7 或细胞核18相关。除了亚细胞分布外，TEM估计的糖原含量的优点还在于可以在单纤维水平上进行定量。这允许研究纤维间的变异性，并对纤维类型和细胞成分（如线粒体和脂滴）进行相关性分析。
这里，描述了TEM估计的骨骼肌纤维中三种常见的糖原亚细胞池（构膜下，肌原间和肌原内）的纤维类型特异性体积含量的方案。该方法已应用于人类19，大鼠20和小鼠21的骨骼肌;以及鸟类和鱼类22;和来自大鼠的心肌细胞23。
Protocol
使用人类活检骨骼肌样本的本方案已获得南丹麦卫生研究伦理区域委员会（S-20170198）的批准。在皮下给予局部麻醉（每个切口1-3mL利多卡因2%）后，使用Bergström针从 蚴蚴外侧 肌的皮肤切口进行肌肉活检。如果使用孤立的整只大鼠肌肉，根据丹麦欧登塞大学医院动物伦理委员会的指导方针，在获得肌肉活检之前，通过颈椎脱位处死动物。 1. 原发性固定、后固定、嵌…
Representative Results
使用该协议，糖原颗粒看起来是黑色和不同的（图1 和 图2）。糖原的正常值如图 3所示。这些数据基于在不同的先前研究中收集的来自41名健康年轻男性的362种纤维19，24，29，30，31。在这里，可以看出肌原?…
Discussion
该方法的关键步骤是在固定后使用亚铁氰化钾还原锇。这种修饰的固定剂用于糖原检测的选择性不能用化学完全解释，但也包括实验结果，表明在已知不含糖原的组织中或细胞外空间中没有检测到这种颗粒11。
关键参数是估计值的精度和光纤间的变化。通过遵循本成像方案，获得每根纤维不同糖原池估计值的0.1至0.2之间的误差系数。此误差水平远低于单个光…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了瑞典奥林匹克委员会的支持。
Materials
| 1,2-Propylene oxide | Merck | 75-56-9 | |
| Embedding 812 resin medium kit | Taab | T031 | |
| Glutaraldehyde solution 25% | Merck | 1.04239.0250 | |
| ITEM | Olympus | Imaging software | |
| Leica EM AC20 | Leica | Automatic contrasting system | |
| OSIS Veleta digital camera | Olympus | ||
| Osmium tetroxide 4% solution | Polysciences | 0972A | |
| Philips CM 100 Transmission EM | Philips | ||
| Potassium hexacyanoferrate (II) trihydrate | Sigma-Aldrich | 455989-245G | |
| Sodium cacodylatbuffer 0,2 M ph 7.4 | Ampliqon.com | AMPQ40989.0500 | |
| Ultra-microtome Leica UC7 | Leica | ||
| Ultrostain lead citrate 3%, stabilised solution | Leica | 16707235 | |
| Uranyl acetate dihydrate | Polysciences | 6159-44-0 |
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Cite This Article
Jensen, R., Ørtenblad, N., di Benedetto, C., Qvortrup, K., Nielsen, J. Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy. J. Vis. Exp. (180), e63347, doi:10.3791/63347 (2022).