单骨骼肌肉纤维解剖，用于全山神经肌肉结的免疫荧光和形态分析
Summary
准确检测神经肌肉结状组件的能力对于评估其结构中的修改至关重要，因为病理学或发育过程。在这里，我们提出了一个简单明了的方法，以获得全安装神经肌肉交汇点的高质量图像，可用于执行定量测量的完整描述。
Abstract
神经肌肉结 （NMJ） 是运动神经和骨骼肌之间的一个专门接触点。此外围突触具有高形态和功能可塑性。在许多神经系统疾病中，NMJ是导致神经转移衰竭、虚弱、萎缩，甚至肌肉纤维死亡的早期病理靶点。由于其相关性，对 NMJ 组件之间关系的某些方面进行定量评估的可能性有助于了解与其装配/拆解相关的流程。与肌肉配合时，第一个障碍是获得技术专长，在不损害肌肉纤维的情况下快速识别和解剖肌肉。第二个挑战是利用高质量的检测方法获取可用于进行定量分析的 NMJ 图像。本文提出了一个分步协议，从大鼠中解剖外周数字龙骨和鞋底肌肉。它还解释了使用免疫荧光来可视化全安装NMJ的预和后合成元素。 获得的结果表明，该技术可用于建立突触的微观解剖，并识别其某些成分在生理或病理条件下状态的微妙变化。
Introduction
哺乳动物神经肌肉结（NMJ）是由运动神经元神经末梢、骨骼肌肉纤维上的后关节膜和末梢施万细胞1、2、3组成的大型胆碱三方突触。这种突触表现出高形态和功能可塑性4，5，6，7，8，即使在成年时，NMJs可以进行动态结构修改。例如，一些研究人员已经表明，运动神经末梢在微米尺度9时不断改变其形状。另据报道，NMJ的形态响应功能要求，改变使用，老化，锻炼，或变化的运动活动4，10，11，12，13，14，15。因此，训练和缺乏使用是改变NMJ的一些特征的基本刺激，如其大小，长度，突触囊泡和受体的分散，以及神经末梢分支14，16，17，18，19，20。
此外，已经表明，任何结构变化或退化这个重要交汇点可能导致运动神经元细胞死亡和肌肉萎缩21。人们还认为，神经和肌肉之间交流的改变可能是生理年龄相关NMJ变化的原因，也可能是其在病理状态下的破坏。神经肌肉结拆解在肌萎缩性侧索硬化症（ALS）的发病中起着至关重要的作用，这是一种神经退行性疾病，是肌肉神经相互作用受损的最佳例子之一。尽管对运动神经元功能障碍进行了大量研究，但ALS中观察到的恶化是否由于运动神经元的直接损伤而发生，然后延伸到皮质-脊柱预测22：在ALS中观察到的恶化是否由于运动神经元的直接损伤而发生，以及是否延伸到皮质-脊柱预测22：是否由于运动神经元的直接损伤而恶化，仍有待讨论。或者，如果它应该被认为是一个离散轴突，其中退化开始在神经末梢和进展到运动神经元 Somas23，24。鉴于ALS病理学的复杂性，考虑独立过程的混合是合乎逻辑的。由于NMJ是肌肉和神经之间生理病理相互作用的核心作用，其不稳定性是有关疾病起源的关键点，有待分析。
哺乳动物神经肌肉系统在功能上被组织成离散的运动单元，由运动神经元和肌肉纤维组成，这些肌肉纤维完全由神经末梢内侧组成。每个电机单元都有具有相似或相同的结构和功能特性的纤维25。运动神经元选择性招募允许优化肌肉响应功能需求。现在很明显，哺乳动物的骨骼肌肉由四种不同的纤维类型组成。有些肌肉是根据他们最丰富的纤维类型的特征命名的。例如，鞋底（后肢后肢的后肢肌肉参与身体姿势的维护）具有大多数慢抽搐单位（类型 1），并被识别为慢肌肉。相反，扩展数字长久（EDL）基本上由具有类似快速抽搐特性的单位（2型纤维）组成，被称为用于运动所需的相位运动的快速肌肉。换句话说，虽然成人肌肉是塑料的性质，由于荷尔蒙和神经的影响，其纤维组成决定了执行不同活动的能力，如鞋底经历持续低强度活动和EDL，表现出更快速的单抽搐。不同类型的肌肉纤维中可变的其他特征与它们的结构有关（线粒体含量、肉瘤视网膜的延伸、Z线的厚度）、肌素ATPase含量和肌素重链组成26、27、28、29。
对于啮齿动物NMJ，肌肉之间有显著的差异28，29。从大鼠对鞋底和EDL进行的形态学分析显示突触区域与纤维直径之间呈正相关关系（即鞋底慢纤维的突触区域大于EDL快速纤维），但NMJ区域与纤维大小的比例在肌肉30、31两个肌肉中相似。此外，在神经末梢方面，1型纤维的端板绝对区域低于2型纤维，而纤维直径的正常化使1型纤维中的神经末梢区域成为最大的32。
然而，很少有研究侧重于形态学分析，以显示一些NMJ组件33，34的变化的证据。因此，由于NMJ在生物体功能中的相关性，其形态和生理学在各种病理中都发生了变化，因此，必须优化不同类型肌肉的解剖方案，使其质量足以使整个NMJ结构的可视化。也有必要评估在不同的实验情况或条件，如老化或运动35，36，37，38发生预或后合成变化。此外，它可以帮助证明NMJ成分的更微妙的变化，如改变神经丝磷化在终端神经末梢，如ALS39报道。
Protocol
所有动物程序均按照国家法律 N+ 18611 的准则执行，该法律旨在保护用于实验目的的动物。该议定书已得到机构道德委员会（CEUA IIBCE，第004/09/2015号议定书）的批准。 1. 肌肉解剖（第1天） 注：在开始之前，在杜尔贝科的磷酸盐盐水（DPBS）中制作40 mL的0.5%的副甲醛（PFA），pH 7.4。可选，使 20 mL 的 4% PFA。准备 5 mL 阿利库特，并在 -20 °C 下冻结。 解剖当天，?…
Representative Results
此协议提供了一个简单的方法来隔离和免疫污渍肌肉纤维从两种不同类型的肌肉（快速和慢抽搐肌肉，见 图1）。使用正确的标记和 /或探针，可以检测和评估 NMJ 组件，因为从定量角度来评估疾病进展或特定药物治疗可能产生的一些形态变化。在本研究中，NMJ的早熟和后合成成分分别使用抗Nf或抗磷Nf抗体和Btx（图2，上面板）进行荧光标记。免疫染色?…
Discussion
在本文中，我们提出了一个详细的协议，用于解剖两个大鼠骨骼肌（一个慢抽搐，另一个快速抽搐），纤维肌肉隔离和免疫荧光检测的预和后合成标记，以定量评估NMJ的变化以及组装/拆解过程。这种协议可用于啮齿动物模型41，42评估NMJ在生理或病理过程中，如这里例证的运动神经元退化模型，如在ALS hSOD1G93A大鼠发现。
为了获得成…
Declarações
The authors have nothing to disclose.
Acknowledgements
非常感谢CSIC和PEDECIBA为这项工作提供的财政支持：娜塔莉亚罗萨诺为她的手稿更正;马塞洛卡萨库贝塔， 使视频和尼科拉斯博拉托借给他的声音。
Materials
| Stereomicroscope with cool light illumination | Nikon | SMZ-10A | |
| Rocking platform | Biometra (WT 16) | 042-500 | |
| Cover glasses (24 x 32 mm) | Deltalab | D102432 | |
| Premium (Plus) microscope slides | PORLAB | PC-201-16 | |
| Tweezers | F.S.T | 11253-20 | |
| Uniband LA-4C Scissors 125mm | E.M.S | 77910-26 | |
| Disponsable surgical blades #10 | Sakira Medical | 1567 | |
| Disponsable sterile syringe (1 ml) | Sakira Medical | 1569 | |
| Super PAP pen | E.M.S | 71310 | |
| 100 μl or 200 μl pipette | Finnpipette | 9400130 | |
| Confocal microscope | Zeiss | LSM 800 – AiryScan | |
| NTac:SD-TgN(SOD1G93A)L26H rats | Taconic | 2148-M | |
| 1X PBS (Dulbecco) | Gibco | 21600-010 | |
| Paraformaldehyde | Sigma | 158127 | |
| Triton X-100 | Sigma | T8787 | |
| Glycine | Amresco | 167 | |
| BSA | Bio Basic INC. | 9048-46-8 | |
| Glycerol | Mallinckrodt | 5092 | |
| Tris | Amresco | 497 | |
| Purified anti-Neurofilament H (NF-H), Phosphorylated Antibody | BioLegend | 801601 | Previously Covance # SMI 31P |
| Purified anti-Neurofilament H (NF-H), Nonphosphorylated Antibody | BioLegend | 801701 | Previously Covance # SMI-32P |
| Alexa Fluor 488 goat anti-Mouse IgG (H+L) | Thermo Scientific | A11029 | |
| α-Bungarotoxin, biotin-XX conjugate | Invitrogen | B1196 | |
| Streptavidin, Alexa Fluor 555 conjugate | Invitrogen | S32355 | |
| Diaminophenylindole (DAPI) | Sigma | D8417 |
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Citar este artigo
Bolatto, C., Olivera-Bravo, S., Cerri, S. Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions. J. Vis. Exp. (174), e62620, doi:10.3791/62620 (2021).