We wish to thank the study participants for making this work possible and the staff at the NIHR Southampton Biomedical Research Centre and the Wellcome Trust Clinical Research Facility, University Hospital Southampton for assistance with study measurements.
This work was supported by the NIHR Southampton Biomedical Research Centre, Nutrition, the MRC Lifecourse Epidemiology Unit and the University of Southampton. This report is independent research by the NIHR BRC Funding Scheme. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. AMB is supported by a NIHR Clinical Research Fellowship, HPP is supported by the NIHR Southampton Biomedical Research Centre.

伦理学声明:我们按照我们机构内部的肌肉活检过程的说明现在如下。肌肉活检过程已批准的赫特福德郡研究委员会的07 / Q0204 / 68赫特福德郡骨骼肌减少症研究中使用。所有参与者都签署知情同意书20。 注:单个操作员可以执行的程序，由一名助理谁可以以减少焦虑，并确保他们是舒适在任何时候都搞参与者的谈话帮助。处置室应配备一个接收器，可调节高度的沙发，干净的表面和程序手推车。 1.准备参加（图2） <ol><li>请参与者躺在舒适，仰卧在沙发上。从露出腹股沟折痕首选大腿。在露大腿下传播的一次性吸收片。确保腿保持整个过程中直接​​和轻松，但大腿瞬间紧张勾勒出股外侧肌 。 </li><li>标记过程现场大约三分之二向下从髂前上棘的髌骨的假想线。 </li></ol> 2.准备好皮肤（图2） <ol><li>剃皮肤至约4厘米周围拟议活检部位用无菌剃须刀片的直径和清洗用醇棉签皮肤。 注意:这是为了防止在愈合过程中的创伤任何后续毛发滞留。此步骤根据操作者的偏好是可选的。 </li><li>渗入皮肤和筋膜覆以局部麻醉。使用地下25针，以提高2％利多卡因的皮下疱然后渗入更深地进入皮下组织用23政针。旨在穿透到至少一半为一英寸取决于在活检部位皮下组织的内容。该深度应足以确保覆FASCIA被麻醉。允许2 - 麻醉剂才动作5分钟。通过与针或手术刀片末端轻轻探测皮肤确认麻醉。 </li></ol> 3.配制无菌场（图2） <ol><li>用肥皂洗手，保持干净的手，直到无菌礼服穿。在程序小车营造一个无菌区前盖上无菌手套的手。 </li><li>消毒活检部位用2％氯己定/异丙醇溶液或基于碘的皮肤消毒剂。应用的无菌披盖用粘合剂孔以暴露所述活检部位，但也保持无菌区域。 </li></ol> 4.执行程序（图2） <ol><li>随着大小11手术刀，做一个5 - 上表面10毫米的切口，下并通过覆筋膜。插入通过切口的conchotome的封闭咬前端以直角到股骨的长轴线，在深度平均半至一英寸。如果需要使用自由手握住并支持周围活检部位而conchotome的前端被打开和关闭的肌肉内的大腿。 </li><li>通过90旋转conchotome - 180°切割肌肉。撤回conchotome并打开尖到无菌生理盐水浸湿无菌纱布。重复该过程的单个伤口部位内，如果需要的话，无法获得足够的肌肉组织。转移纱布到置于冰上的容器。容器转移至制备实验室根据当地机构协议来处理肌肉。 </li></ol> 5.后步骤（图2） <ol><li>直接压住伤口长达5分钟。通过将它们平行于伤口，而不是直角，以避免潜在的去除瘢痕组织，如果免缝-条被无意中删除，重新打开伤口关闭与免缝，带伤口。广场上的免缝，带无菌剂敷料和领带两层绷带压缩并用胶带固定。 </li><li>解释伤口敷料方法到与会者，他们将必须执行一个换药3 - 手术后4天。确保参与者睡前那天晚上删除加压包扎。 </li></ol> 6.邮政活检咨询<ol><li>通知参与者，这是共同的经历一些大腿刚度可通过温和的运动得到缓解（ 例如 ，行走）。使用简单止痛，如对乙酰氨基酚。警告周围的活检部位，可能会持续长达两周时间以及伤口感染的潜在并发症瞬间麻木的参与者。 </li></ol> 7.说明主题后程序<ol><li>请参与者，以避免72小时剧烈活动（如爬山，跑步，举重）。请参与者，以避免在水中浸泡48 - 72小时。然而，指示participan吨至在淋浴，以保持敷料干燥时包裹保鲜膜或周围的活检部位相似。 </li><li>请参与者后3〜改变敷料 - 第4天，一周后取出免缝-条。 注:参加者应给予活检后护理，备用敷料和负责执行的程序团队的紧急联系方式批示。 </li></ol>

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None declared.

为了保证参与者的安全，高效地进行肌肉活检，至关重要的是要注意协议中的关键步骤。 参加者必须在认真考虑排除标准，以避免并发症，如出血，伤口愈合不良选择。整个手术过程中严格无菌是必不可少的。正确的技术的应用将保证参加者的舒适性，最大限度地减少不必要的创伤参与者的组织，并允许分析足够的样本产量。有时候，可能很难获得肌肉样品，特别是如果参与者都有过度皮下脂​​肪组织。即使参与者精心选择在这种情况下应该做了一个继续进行的程序的决定，可能会出现出血过多。直接压到伤口部位长达5分钟可能是必要的，以鼓励止血和减少瘀伤。以减少出血的另一种方法是将使用肾上腺素利多卡因，这将导致一个局部血管收缩。然而，肾上腺素可以与随后的分析和谨慎干扰必须被使用。这是笔者的偏好不能在此过程中使用肾上腺素。 多个分析可以肌肉组织中的给定的在显微镜，图像分析，组织化学和分子分析方法的进步来执行。这些包括评估肌肉形态学例如 ，纤维组合物，纤维的横截面面积，血管，单纤维收缩活性，RNA的分析，蛋白质以及酶活23,24,25,19,26,27。虽然用于肌病的临床诊断过程中经常使用，肌肉活检越来越多的研究完成。例如，我们曾表明，在肌肉形态发育的影响可以解释低出生体重和肌肉减少19之间的关联。这种技术，西米的潜在局限性拉尔其他半开的技术，包括被限制为一个部位，其中的形态，即，肌肉纤维类型可能不能代表整个肌肉群的。 与韦尔Blakesley conchotome肌肉活检是一种可行的和可接受的过程。此过程提供了另一种方法的的Bergstrom和其他活检方法。这是很容易掌握，并给出用于分析的良好样品的产率。并发症的发生率是低和不适保持最低的方法是使用标准化的技术来执行时，如上所述，维持严格的无菌条件下和上后活检护理全面的建议中给出。

获得骨骼肌的肌病和其他神经肌肉退化性疾病的诊断依赖于安全和有效的方法这是可以接受的，而不是失能向患者1。从历史上看，获得的肌肉组织进行分析的方法包括在全身麻醉或验尸进行开放式活检。这些技术使肌肉的直接可视化，并允许更大的一块肌肉进行活检。给定这些技术必须在患者住院和恢复方面的潜在的缺点，经皮"半开放"肌肉活组织检查方法被引入作为替代。最初是由杜氏（1806年至1875年），谁使用用套针自行建造针，并能获得来自活受试者的肌肉样品没有全身麻醉2中执行了这种技术。自那时以来，各种形式的活组织检查针已经使用3,4,5。然而，活组织检查Ñ通过贝里斯特伦在1962年开发的eedle已在临床实践中最常用的;无论是在儿童和成人，以及在研究6,7,8,9,10。它拥有锐利的套管针，切割套管和推杆驱逐组织后活检。从该过程获得的肌肉的产量已报告从25 -293毫克的范围内重复采样11,9之后。 HENRIKSSON介绍了1979年韦尔- Blakesley conchotome作为替代半开放式的肌肉活检的方法（ 图1）12。该仪器是不像贝里斯特伦结构单一的结构和设计就像一个锋利的尖咬钳子。针尖大小的范围可以从4毫米到6毫米的宽度。当刺骨尖端的两个边缘相对，中空形成确保肌肉的捕获。该conchotome通过5插入 - 10毫米皮肤切口并且避免需要一个套管针穿透肌肉和上覆筋膜作为scalpel可被用来使一个轨道下降到肌肉。 此方法允许受控的组织穿透具有高度的机动性13,14。它是网站，不当的压力可能损坏的神经血管或底层骨结构如特别有用的。在胫骨前15,14的部位。使用conchotome肌肉收益率是可变的，并在我们的实践20 - 200毫克的肌肉已经多次取样后获得。该conchotome过的Bergstrom针的一个潜在的优势，此外，以高度的机动性它提供的，是该咬尖端并不需要被削尖或作为贝里斯特伦的套管针构造16取代经常。 使用韦尔Blakesley conchotome肌肉活检的总体目标是为了获得足够量的肌肉组织，以使能对诊断和resear组织化学和分子分析CH研究。这是一个简单而安全的过程，可以很容易地了解到。利用这种技术，适合于活检肌肉包括二头肌，三头肌，三角肌，腓肠肌，胫骨前肌，比目鱼和sacrospinals 17,15,7,18,14。 股外侧的最外部分是用于活检最常见的部位，因为它避免了大腿内的主要神经血管结构。它被识别大约三分之二向下的假想线接合髂前上棘和髌骨。肌肉形态学数据，例如肌纤维区域，肌纤维的比例，毛细血管密度从股衍生外侧采样是在文献中允许研究11,19之间的比较广泛的应用。

<table><tbody><tr><td>Weil-Blakesley conchotome</td><td>Gebr&amp;uuml;der Zepf Medizintechnik, D&amp;uuml;rbheim, Germany</td><td>None</td><td>6 mm biting tip</td></tr><tr><td>5 ml 2% lidocaine</td><td>Generic use</td><td>None</td><td>One 5 ml vial should suffice for adequate anaesthesia</td></tr><tr><td>Alcohol swab</td><td>Generic use</td><td>None</td><td></td></tr><tr><td>5 ml syringe</td><td>Generic use</td><td>None</td><td></td></tr><tr><td>25 G and 23 G sterile needles</td><td>Generic use</td><td>None</td><td></td></tr><tr><td>Sterile scalpel size 11</td><td>Generic use</td><td>None</td><td></td></tr><tr><td>Sterile gauze squares</td><td>Generic use</td><td>None</td><td></td></tr><tr><td>Sodium chloride 0.9% 10 ml</td><td>Fannin</td><td>1122/002/001</td><td></td></tr><tr><td>Steri-Drape&amp;trade; Small Drape with Adhesive Aperture</td><td>3M</td><td>1020</td><td></td></tr><tr><td>ChloraPrep 2% w/v /70% v/v cutaneous solution&amp;nbsp;</td><td>CareFusion</td><td>270400</td><td>Two</td></tr><tr><td>Steri-Strip&amp;trade; 1/4 inch x 3 inch Reinforced Adhesive Skin Closures</td><td>3M</td><td>R1541</td><td></td></tr><tr><td>PRIMAPORE sterile dressing 15 cm x 8 cm</td><td>Smith &amp;amp;&amp;nbsp;&lt;br/&gt; Nephew</td><td>66000318</td><td></td></tr><tr><td>Cotton crepe bandage Hospicrepe 233 7,5 cm x 4,5 m</td><td>Hartmann</td><td>915562</td><td></td></tr><tr><td>Flexible cohesive bandage Co-Plus LF</td><td>BSN medical</td><td>2904565</td><td></td></tr><tr><td>Disposable absorbent sheet</td><td>Generic use</td><td>None</td><td></td></tr><tr><td>Micropore or transpore tape</td><td>Generic use</td><td>None</td><td></td></tr><tr><td>Sterile gown with sterile paper towels</td><td>Generic use</td><td>None</td><td></td></tr><tr><td>Sterile gloves</td><td>Generic use</td><td>None</td><td></td></tr></tbody></table>

human vastus lateralis skeletal muscle biopsy using the weil-blakesley conchotome

使用韦尔 - Blakesley conchotome经皮肌活检是公认的在临床和科研实践。它是一种安全，有效和耐受性良好的技术。在韦尔 - Blakesley conchotome有一个尖锐的针尖刺骨用4 - 第6毫米宽的空洞。它是通过一个5插入 - 10毫米皮肤切口，并且可以被操纵用于受控的组织穿透。尖端被打开，在组织内关闭，然后通过90 -180°旋转切割肌肉。下面重复获得的肌肉量采样可以从20毫克至290毫克，可以为组织学和分子生物学研究进行处理而变化。伤口需要保持干燥和剧烈的身体活动保持最小为大约72小时，虽然活动的正常水平可以立即重新启动之后的过程。当接近注重的是主体，全无菌和后期护理程序选择此过程是安全有效的。右和left-component左侧astus外侧适于活检取决于参与者偏好。

上述过程是安全的，在临床和研究设置接受的。当活检作为诊断过程的一部分来执行，肌肉应根据患者的症状和肌肉无力的迹象来选择。活检部位应该从以前的受伤，挛缩或仪器21.1自由。在研究中，在标准条件如禁食或排斥的糖尿病患者可能需要19。抗凝血剂如华法林需要停止并且过程前凝血时间进行检查。作为我们的经验表明，它不显著增加过量出血22的风险，可以继续以剂量为75mg的简单阿司匹林。此外，应考虑到参与者采取任何影响伤口愈合的药物。从本质上讲，停药的风险，应该对程序的各利益进行权衡案件。期间参加者的选择，应取得潜在过敏局部麻醉剂，碘/洗必泰和伤口敷料的一个详细历史记录，并根据需要使用的替代品。参与者应警告过敏反应可表现为增加了皮肤发红，局部肿胀或起泡的罕见的，但潜在的发生。在这些情况下去除敷料（S）和治疗用抗组胺/类固醇可能是必要的。 该过程大约需要15 - 20分钟。它以抓紧无菌，以尽量减少伤口感染的危险性是必不可少的。在手术过程中的参与者可能会遇到一些不适。任何剧烈的疼痛发生可能需要局部麻醉的进一步渗透。手术后，大腿僵硬通常是经验丰富，可以通过温和的运动得到缓解（ 例如 ，步行）。在赫特福德郡骨骼肌减少症研究（HSS），与疼痛视觉模拟评分（VAS）（刻度从0模 - "不痛"到100mm - "痛那么糟糕，因为可以&#39;）测量的疼痛评分是典型的低配为7毫米的中位数的程序和4mm在一天表明受试者在本研究的过程后发现该过程微创痛苦22（材料数据表）的文献报道伤口并发症的.The率范围为1％至3％，并主要包括卷绕血肿形成13,9,14。一项研究描述了一个严重的一个子筋膜血肿和随后的深静脉血栓形成13。在HSS卷绕并发症（描述为瘢痕3周下方的硬块）的比率为1％22。与会者可能会遇到麻木给周围需要通过皮肤和皮下组织切开小切口部位。在我们的经验，这麻木一直是短暂的，一个或两个凌晨内已完全解决<html> KS。 相对于代表组织学的结果，我们先前研究发展的影响与肌肉形态学19之间的关联。在这项研究中的意思是在48下出生体重（≤3.18千克）男性为4903微米2（1354微米2）和4644微米2（1022微米2）在47更高出生体重（≥3.63千克）男性I型肌纤维区域（SD）而平均II型肌纤维面积是4046 平方微米（1166微米2）及分别3859微米2（1127微米2）在较低和较高的出生体重的人。 <img alt="图1" src="/files/ftp_upload/53075/53075fig1.jpg" /> 图1.韦尔- Blakesley Conchotome的图像。该韦尔- Blakesley conchotome用6毫米咬尖在这项研究中使用。5 / 53075fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53075/53075fig2.jpg" /> 图2.肌肉活检技术使用韦尔- Blakesley Conchotome。（A）的腿从腹股沟折痕曝光。在股外侧肌活检面积剃光头发，打上，渗透利多卡因和用消毒清洗。活检区域分离。皮肤和覆盖筋膜然后用一个大小11手术刀刺破。 （B）中的conchotome被插入由手术刀制成，并通过90°的旋转，以切除肌肉的轨道。光盘。 5 - 10毫米缝合伤口用免缝-条和打扮（此前公布的22）， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53075/53075fig2large.jpg" target="_blank">请点击这里查看LAR</a>GER版本这个数字。 <img alt="图3" src="/files/ftp_upload/53075/53075fig3.jpg" /> 图3.免缝带钢的应用。该steri-条平行于小切口的应用。此方法与愈合良好的速率相关联;通常在一周之内。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53075/53075fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53075/53075fig4.jpg" /> 图4. 股外侧肌肉活检。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53075/53075fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td><stroNG&gt;视觉模拟评分疼痛评分（毫米） </td><td> 中位数（IQR [IQR]） </td></tr><tr><td>在手术过程中</td><td> 7（1 - 34） </td></tr><tr><td>一天后程序</td><td> 4（0 - 16） </td></tr><tr><td>七天后程序</td><td> 1（0 - 4） </td></tr><tr><td></td><td></td></tr><tr><td> 每日活动已恢复 </td><td> N（％） </td></tr><tr><td>一天后</td><td> 60（65％） </td></tr><tr><td>两天后</td><td> 26（28％） </td></tr><tr><td>二级以上天数</td><td> 7（7％） </td></tr></tbody></table> 表1.疼痛视觉模拟评分（VAS）得分和Conchotome肌肉活检赫特福德郡骨骼肌减少症在研究93后研究参与者的活动恢复 。疼痛规模范围为0模 - "不痛"来最大100毫米 - "痛那么糟糕，因为可以"。大多数与会者通常恢复日常活动一天后的过程。

Watch this Scientific Journal Video about Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome at JoVE.com

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

人的<em&gt;股外侧肌</em&gt;骨骼肌活检使用韦尔 - Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

human-vastus-lateralis-skeletal-muscle-biopsy-using-weil-blakesley

Nanyang Technological University

Research

JoVE Journal

Medicine

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

16.6K 次观看.  University of Southampton, University Hospital Southampton. This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

视频: Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

科研

发育生物学

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

胫骨神经切断术 - 一个标准化的模型失神经诱导小鼠骨骼肌

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

医学

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

从骨骼肌活检程序分离、培养、表征和分化人类肌肉祖细胞

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

人骨骼肌活检手术使用修改伯格斯坦技术

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

小鼠骨骼肌形态及纤维型成分的半自动化分析

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

神经刺激器引导注射自体干细胞近马左喉返神经

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.

肌肉活检中的免疫标记肌纤维退化

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. ...

生物学

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

从人类肌肉提比利斯前部高级隔间收集骨骼肌肉活检，用于机械评估

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

从小鼠骨骼肌中分离线粒体用于呼吸测定

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

人的

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