我们要感谢Vikram Darbhe和Patrick Franks的实验指导。这项工作得到了美国国家科学基金会研究生研究奖学金计划的支持。DGE-1324585以及NIH R01D084009和F31AR076920。本材料中表达的任何意见，发现和结论或建议均为作者的观点，并不一定反映美国国家科学基金会或NIH的观点。

西北大学的机构审查委员会（IRB）批准了这项研究的程序。所有参加这项工作的参与者在开始下面详述的方案之前都给予了知情同意。 注意:本研究中使用的特定超声系统具有EFOV-US功能，并且被采用，因为我们能够审查科学文献中有关该算法的详细信息和有效性评估22，26;还存在多个具有EFOV-US的其他系统18，20，30。使用了线性阵列传感器14L5（频率带宽5-14 MHz）。在该协议中成像的肌肉只是已捕获美国图像并测量了分筋膜长度的一小部分肌肉（例如，肱三头肌25，腕尺伸肌23，内侧腓肠10，外侧騥24，股二头肌8，31）。该协议旨在提供指针并描述必要的标准，以便它可以应用于我们提供的两个示例之外的肌肉。
1. 收集EFOV-US肌肉图像
制备
<ol><li>超声医师准备<ol>
<li>在操作超声系统之前，请通读系统手册，以熟悉系统安全性，维护系统，系统设置和控制等。此外，请查看系统获取 EFOV-US 图像的说明，并熟悉为获取 EFOV-US 图像而实现的方法。 注:不同的超声系统使用不同的术语命名EFOV-US模式。例如，在这里使用的系统中，EFOV模式被称为"全景成像"。虽然在各种商业系统中实现的算法的技术细节通常是知识产权，因此不是免费提供的，但从粗略的回顾来看，许多具有全景超声功能的商业系统描述了一种类似于Weng等人描述的方法。在开始涉及人类参与者的研究之前，建议通过直接从制造系统的公司，通过使用成像phantom26，32或其他方式（例如，与动物解剖24进行比较）来评估从任何系统获得的测量的一般有效性，作为开始涉及人类参与者的研究之前的重要步骤。</li>
		<li>花点时间熟悉感兴趣的肌肉的解剖结构以及周围的解剖结构。建议超声医师使用解剖学教科书或最好是交互式在线3D解剖模型来熟悉感兴趣的解剖学。</li>
	</ol>
</li>
	<li>参与者准备<ol>
<li>在开始成像方案之前，向参与者解释研究方案并获得IRB批准的同意。</li>
		<li>要求参与者穿着合适的衣服，以便能够接触到感兴趣的肌肉。例如，如果超声医师计划对前臂肌肉进行成像，则应要求参与者穿短袖衬衫。</li>
		<li>将参与者坐在可调节的椅子上，该椅子可以锁定到位。花点时间调整椅子，使参与者尽可能舒适，同时仍然提供对感兴趣肌肉的访问。 注意:如果没有可以完全平放的可调节椅子，某些研究设计可能需要使用桌子来接触感兴趣的肌肉（即腿筋）。</li>
		<li>将感兴趣的肌肉伸展的关节放在可以控制和重复的姿势中。使用临床指导33 定位解剖学标志物并实施测角测量;使用 ISB 标准定义联合坐标系34，35。通常，要测量关节角度，请使用皮肤安全标记标记解剖标志物（材料表），然后将手持式测角仪的中心与关节的旋转轴向上对齐，测角仪的手臂与关节段向上对齐。 注意:如果对被动肌肉进行成像，建议将感兴趣的肌肉置于相对较长的位置，以避免对松弛肌肉进行成像。<ol>
<li>为了复制本研究中拍摄的肱二头肌，坐下参与者双脚支撑，背部挺直，肩部外展85°，水平屈曲10°，肘部屈曲25°，前臂，手腕和手指中性。</li>
			<li>为了复制本研究中拍摄的胫骨前部，膝盖屈曲60°，踝关节屈曲15°的受试者就座。</li>
		</ol>
</li>
		<li>使用布带固定参与者的肢体，以尽量减少成像方案期间的运动。</li>
	</ol>
</li>
</ol>图像采集
<ol start="3"><li>插入并打开超声波系统。确保检查设置为肌肉骨骼，选择正在使用的换能器（这里我们使用14L5），发射频率设置在5-17 MHz之间（这里使用11MHz），这是肌肉骨骼成像的典型频率范围。较高的频率通常用于更肤浅的成像，因为它们可以提高分辨率但降低波穿透力。</li>
	<li>进入系统设置以调整脚踏开关设置。出于本实验方案的目的，我们建议设置脚踏开关以启动/停止成像。如果正在使用的脚踏开关有多个踏板，请将其他踏板设置为"冻结"或"暂停"，以及"打印"或"存储"图像。</li>
	<li>在探头头部涂抹大量超声凝胶。</li>
	<li>将传感器放在参与者的皮肤上，在大致感兴趣的区域上。</li>
	<li>在肌肉的短轴平面上移动换能器。请注意，传感器的一侧有一个小的突起，称为指示器。具有指示器的换能器的一侧对应于超声图像的左侧。在短轴上成像时，让超声医师保持指示器横向指向，当超声医师处于长轴时，将指示器指向远端。</li>
	<li>识别短轴平面（垂直于肌纤维方向）中感兴趣的肌肉，并移动换能器远端和近端，以获得肌肉路径的完整可视化。<ol>
<li>使用皮肤安全墨水标记标记（材料表）标记重要的解剖学标志物（即肌肉的外侧和内侧边缘，肌肉肌腱连接处和肌肉插入）。</li>
	</ol></li>
	<li>一旦确定了肌肉的位置并进行了适当的标记，让超声医师在长轴平面（平行于肌肉纤维方向）中移动超声换能器。</li>
	<li>从肌肉的远端或近端开始，旋转和倾斜换能器以识别该点的分筋膜平面。当探头位置正确时，在皮肤上做一个标记。</li>
	<li>一旦沿着要扫描的整个所需长度建立了近似的分册平面，让超声医师按照此路径进行练习。</li>
	<li>要开始收集图像，请将超声系统置于EFOV-US模式。</li>
	<li>从肌肉的一端开始，单击脚踏开关开始图像采集，并在长轴上缓慢连续地移动超声换能器。到达肌肉末端后，单击脚踏开关以结束图像采集。</li>
	<li>练习并确保正确的传感器路径。在持续获得"高质量"EFOV-US图像之前，这可能需要几个练习图像（有关高质量图像的解释，请参阅第2节）。</li>
	<li>要优化图像可见性和清晰度，请考虑调整以下参数。<ol>
<li>深度:如果图像采集在捕获所需长度的肌肉之前结束，请增加图像的深度（在此处使用的系统中，增加图像深度会增加扫描的绝对长度）。</li>
		<li>对焦:将焦点箭头放在图像的下半部分，正好位于感兴趣的肌肉下方。</li>
		<li>增益:确保增益在图像深度之间保持平衡。</li>
		<li>速度:在指示器的指导下以最佳速度拍摄图像（在大多数系统中，全景成像期间，速度指示器显示在显示器上）。</li>
	</ol></li>
	<li>一旦收集了定性良好的图像（步骤2.1），点击 打印/存储 脚踏开关踏板或控制面板上的同义词按钮以保存图像。</li>
	<li>重复步骤1.13-1.16，直到获得3张高质量的EFOV-US肌肉图像。</li>
	<li>重复步骤1.6-1.17，直到获得所有感兴趣的肌肉。</li>
	<li>用毛巾轻轻擦拭参与者皮肤上的凝胶。然后让参与者冲洗掉皮肤区域或使用湿毛巾擦拭暴露于凝胶的皮肤。干。</li>
	<li>擦拭探头头部的凝胶并消毒。</li>
	<li>将映像作为未压缩的 DICOM 映像导出到 CD-DVD、闪存驱动器或通过本地网络导出到计算机上。</li>
</ol>2. 确定EFOV-US图像的"质量"
<ol><li>按照步骤1.13，让超声医师识别和评估感兴趣肌肉及其周围解剖结构的关键解剖学特征的质量。这是基于超声医师对解剖学和肌肉骨骼组织回声（组织反射超声波的能力）的了解的定性评估。要使EFOV-US图像在定性上被认为是"好的"，应满足以下条件:<ol>
<li>在任何肌肉的长轴图像中，检查超声医师是否可以清楚地将肌肉识别为低回声（深色）形状，具有代表深部和浅表肌肉筋膜的超回声（明亮）边界。</li>
		<li>在肌肉边界之间，检查超声医师是否可以将肌肉筋膜周围的结缔组织识别为超回声（明亮）线。 注意:当对多侧肌进行成像时，图像还应包含中央肌腱，这些肌腱显示在肌肉腹部，深部和浅表肌肉筋膜之间，作为高回声（明亮）结构。</li>
		<li>检查图像是否没有过度弯曲。这通常由图像中的阴影或间隙或图像上方的锯齿状柔性标尺线表示。</li>
	</ol>
</li>
	<li>如果图像缺少2.1中描述的一个或多个组织结构，则认为图像"定性较差"并返回实时2D模式。</li>
</ol>3. 肌束长度
<ol><li>要量化肌肉筋膜长度，请使用开源图像处理平台ImageJ。ImageJ可以在 https://imagej.net/Downloads 下载。 注意:虽然ImageJ经常实现24，25，31，36，37，38，但可以使用其他图像处理软件8，39 或自定义代码40，41测量肌肉束长度的量化。</li>
	<li>下载后，通过单击"文件"打开"图像J"中的DICOM图像作为DICOM 图像|打开 并选择要分析的图像。</li>
	<li>要确保保留 DICOM 图像属性，请单击"工具"菜单中的"直线"工具，然后在超声图像侧面的标尺上绘制一条从 0 到 1 cm 的直线。然后转到分析|测量以测量所制造的线。如果保留了图像属性，则直线的长度应为 1 cm。</li>
	<li>要测量图像中的分册长度，请完成以下操作。<ol>
<li>右键单击直线工具。</li>
		<li>选择 "分段线"。</li>
		<li>将光标移动到图像上，然后单击已选择要测量的分册的一端。 注意:只有在分册上进行测量，才能令人信服地看到整个筋膜路径（即，从一个腱膜病到下一个腱鞘炎或腱鞘炎到中央肌腱）。</li>
		<li>沿路径单击以确保捕获分册路径中的曲率。</li>
		<li>到达分册路径的末尾后，双击结束该行，然后转到 "分析|测量 以测量线的长度。 注意:首次进行测量时，将弹出一个新窗口"结果"。可以在"结果"窗口中通过转到"结果"|来管理显示的值 设置测量值。</li>
	</ol>
</li>
	<li>重复步骤3.4.3-3.4.5，直到在单个图像中进行多个分册测量。</li>
	<li>通过单击 "文件"|保存分册测量值保存 在结果选项卡上，或者可以将值复制并粘贴到另一个文档/电子表格中。</li>
</ol>

作者没有什么可透露的。

协议中的关键步骤。
获得高质量的EFOV-US图像有几个关键组成部分，可以产生有效和可靠的分册长度测量。首先，如方法1.1.2所示，超声医师必须花时间熟悉被成像肌肉的解剖结构以及周围的肌肉，骨骼和其他软组织结构。这将提高超声医师对正确肌肉进行成像的能力，并确定多个图像是否捕获了相同的肌肉平面。其次，超声医师在收集数据以供发表之前，应练习幻影和多个试点参与者的协议。已知，如果超声医师不能正确识别分筋平面，超声会导致测量误差，这是一项具有挑战性的任务，可以通过实践来改善。最后，强烈建议确保在所使用的超声系统中建立EFOV-US算法所做测量的有效性。如果该方法的准确性尚未得到证明，则可以使用超声幻影23，26 或通过与其他成像工具44 或尸体解剖45进行比较来轻松进行验证。
方法的修改和故障排除。
如果图像可见性较差或探头运动在动态扫描过程中不均匀，则添加超声凝胶可以通过改善探头与皮肤的耦合来提高图像质量。如果在捕获整个感兴趣对象之前被算法截断图像采集，则应增加图像的深度。增加图像的深度可扩展可用的扫描距离，从而可以在单个EFOV-US图像中捕获更长的物体。通常，在尝试改善或排除图像质量或图像采集问题时，最好参考超声系统的手册。
在这里，我们演示如何捕获从起源肌腱的肌肉肌腱连接处到插入肌腱的整个肌肉的EFOV-US图像。捕获整个肌肉对于某些肌肉是必要的，例如肱二头肌，其分束几乎跨越肌肉的整个长度。然而，对于其他肌肉，例如胫骨前部或其他侧状肌肉，不包括整个肌肉腹部的较短扫描仍可能捕获整个肌肉束。对于新手超声医师来说，从仍然捕获完整分束长度的较短扫描中获取图像可以降低探头与分束平面不对准的机会，并提高图像质量，从而降低分册测量误差的可能性。
该方法的局限性
值得注意的是，肌肉激活可以改变肌肉束的长度。由于扫描方法的性质，EFOV-US的主要局限性是它不能用于研究由于动态肌肉收缩（例如，在行走过程中）引起的肌肉筋膜变化46，47。此外，由于捕获EFOV-US图像所需的时间，由于肌肉疲劳，在最大收缩状态下对肌肉进行成像可能不可行。相反，EFOV-US方法有利于亚极大或被动成像。确保参与者，四肢或会话的肌肉活动恒定的一种方法是在成像过程中同时测量肌电图，并仅分析当肌肉处于某种所需活动水平时拍摄的图像。虽然建议这样做，特别是如果研究神经驱动改变的人群，但在这里研究的人群中没有进行肌电图测量。
虽然传统的超声已被证明对于测量体内肌肉筋膜长度是有效和可靠的，但如果超声检查员对超声换能器的对准偏离分筋膜平面，就会发生一些分形测量误差27，29，48。由于EFOV-US动态扫描的性质，人们担心EFOV-US方法可能比T-US21，24具有更多的误差。虽然最近的一项研究表明，在单腕肌肉中，EFOV-US中探针错位引起的分筋膜测量误差并不比成熟的T-US方法23更大，但B型超声的一般局限性是，您只能捕获相对较小的2D（2D）肌肉视图。单个分册的真正路径可能是3D;人们仍然担心，对于较长的分册，与从2D视图测量潜在3D路径长度相关的误差可能会更大。
该方法相对于现有/替代方法的重要性
静态B型超声是一种广泛接受的测量体内肌肉束长度的方法。然而，T-US探头的视野限制了可以直接测量的分册的长度。相反，测量比T-US视野更长的分册需要三角估计方法，扩散张量成像（DTI）或EFOV-US20。一般来说，超声成像比磁共振成像（MRI）技术（如DTI）更受青睐，因为MRI更昂贵且难以实施18。用EFOV-US捕获的分筋膜长度已被证明比三角估计方法更准确24，36，这是预期的，因为肌肉束定期遵循弯曲路径，但三角估计方法在计算肌肉束长度时假设线性。
应该注意的是，虽然大多数超声探头的长度为4-6厘米，但已使用高达10厘米的超声探头9，10。10 cm探头可实现更宽的视野，从而能够捕获更长，更直的分册。尽管如此，较长的探针长度会降低帧速率，需要成像表面（身体）也是直的，以避免成像组织的不均匀压缩，并且可能无法捕获更长的弯曲分册（不使用EFOV）20。
该方法的未来应用或方向
此处详细介绍了用于获取用于测量肌肉束长度的高质量EFOV-US图像的指南，旨在鼓励使用EFOV-US方法来扩展该领域具有体内肌肉结构数据的肌肉池。期望将这种方法应用于健康和受损人群（例如，中风后38，49 或骨科手术后的个体），以更好地了解肌肉功能和肌肉适应性。此外，这些体内数据对于开发更准确预测人体运动的模型以及开发特定于受试者的肌肉骨骼模型非常重要。
值得注意的是，EFOV-US方法不仅限于肌肉束长度的测量。该方法已用于测量肌腱长度50，51 和肌肉解剖横截面积，52，53 以及记录各种浅表病变54，55。因此，有机会开发类似于此处介绍的指南，用于使用EFOV-US方法为各种应用获取高质量图像。

筋膜长度是骨骼肌结构的一个重要参数，总体上表明肌肉产生力的能力1，2。具体而言，肌肉的筋膜长度可以深入了解肌肉可以产生活动力的绝对长度范围3，4。例如，给定除筋膜长度外，所有等轴测力生成参数（即平均肌节长度、下角、生理横截面积、收缩状态等）具有相同值的两块肌有较长筋膜的肌肉将在较长的长度上产生其峰值力，并且将产生比具有较短束的肌肉更宽的长度范围的力 3.肌肉筋膜长度的量化对于了解健康的肌肉功能和肌肉的力产生能力的变化非常重要，这些变化可能是由于肌肉使用改变（例如，固定5，6，运动干预7，8，9，高跟鞋佩戴10）或肌肉环境的变化（例如，肌腱转移手术11，肢体分心12）而发生的).肌肉束长度的测量最初是通过离体尸体实验获得的，该实验允许直接测量解剖的筋膜13，14，15，16。这些离体实验提供的宝贵信息导致人们有兴趣实施体内方法17，18，19以解决尸体无法回答的问题;体内方法允许在天然状态以及不同的关节姿势，不同的肌肉收缩状态，不同的加载或卸载状态以及具有不同条件（即健康/受伤，年轻/年老等）的人群中量化肌肉参数。最常见的是，超声波是用于获得体内肌肉束长度的方法18，19，20;它比其他成像技术（如扩散张量成像（DTI））更快，更便宜，更容易实现18，21。
扩展视野超声（EFOV-US）已被证明是测量体内肌肉束长度的有效且可靠的方法。虽然通常采用传统超声（T-US），但其视场受超声换能器阵列长度的限制（通常在4至6厘米之间，尽管有些探头延伸至10 cm10）18，20。为了克服这一限制，Weng等人开发了一种EFOV-US技术，该技术可以从动态的长距离扫描中自动获取复合的二维"全景"图像（长达60厘米）22。图像是通过实时拟合一系列传统的B型超声图像而创建的，因为换能器动态扫描感兴趣的物体。由于连续的 T-US 图像具有较大的重叠区域，因此一个图像与下一个图像之间的微小差异可用于计算探头运动，而无需使用外部运动传感器。一旦计算出两个连续图像之间的探头运动，"当前"图像将连续与前面的图像合并。EFOV-US方法允许直接测量长而弯曲的肌肉束，并且已被证明在肌肉，试验和超声医师中是可靠的23，24，25 并且对平坦和弯曲的表面都有效23，26。
实施超声波来测量体内肌肉束长度并非易事。与其他涉及更多自动化方案（即MRI，CT）的成像技术不同，超声依赖于超声医师的技能和解剖学知识27，28。有人担心探头与分册平面的不对中可能导致分册措施出现重大误差。一项研究表明，使用超声和 DTI MRI 测量的分束长度测量几乎没有差异（平均< 3 mm），但也表明测量精度较低（差的标准偏差约为 12 mm）29。尽管如此，已经表明，新手超声医师在经验丰富的超声医师的实践和指导下，可以使用EFOV-US23获得有效的测量值。因此，应努力证明适当的协议，以减少人为错误并提高使用EFOV-US获得的测量的准确性。最终，开发和共享适当的方案可能会扩大实验者和实验室的数量，这些实验者和实验室可以从文献中重现分册长度数据，或者在尚未在体内研究的肌肉中获得新的数据。
在该协议中，我们展示了如何实施EFOV-US方法以获得可用于量化肌肉束长度的高质量肌肉骨骼图像。具体而言，我们解决了（a）收集单个上肢和单个下肢肌肉的EFOV-US图像（b）实时确定EFOV-US图像的"质量"，以及（c）离线量化肌肉结构参数。我们提供此详细指南，以鼓励采用EFOV-US方法在由于长筋膜而未在体内研究的肌肉中获取肌肉束长度数据。

<table>
	<tbody>
		<tr>
			<td>14L5 linear transducers</td>
			<td>Siemens</td>
			<td>10789396</td>
			<td></td>
		</tr>
		<tr>
			<td>Acuson S2000 Ultrasound System</td>
			<td>Siemens</td>
			<td>10032746</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable chair (Biodex System)</td>
			<td>Biodex Medical Systems</td>
			<td>System Pro 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Skin Marker Medium Tip</td>
			<td>SportSafe</td>
			<td>n/a</td>
			<td>Multi-color 4 Pack recommended</td>
		</tr>
		<tr>
			<td>Ultrasound Gel - Standard 8 Ounce Non-Sterile Fragrance Free Glacial Tint</td>
			<td>MediChoice, Owens &#38;Minor</td>
			<td>M500812</td>
			<td></td>
		</tr>
	</tbody>
</table>

obtaining quality extended field-of-view ultrasound images of skeletal muscle to measure muscle fascicle length

肌肉束长度通常在体内使用传统超声测量，是定义肌肉力产生能力的重要参数。然而，超过 90% 的上肢肌肉和 85% 的下肢肌有比普通传统超声（ T - US ）探头的视野更长的最佳分束长度。一种较新、不太常用的称为扩展视场超声（EFOV-US）的方法，可以直接测量比单个T-US图像的视场更长的薄片。这种方法可以自动将来自动态扫描的T-US图像序列拟合在一起，已被证明对于在体内获得肌肉束长度是有效和可靠的。尽管有许多具有长筋膜的骨骼肌，并且EFOV-US方法对这种筋膜进行测量的有效性，但很少有已发表的研究使用这种方法。在这项研究中，我们展示了如何实施EFOV-US方法来获得高质量的肌肉骨骼图像，以及如何量化这些图像的分筋膜长度。我们预计，该演示将鼓励使用EFOV-US方法来增加健康和受损人群中的肌肉池，我们有体内肌肉束长度数据。

实施扩展视野超声（EFOV-US）以从4名健康志愿者的肱二头肌长头和胫骨前部获得图像（表1）。 图1 显示了在这个代表性成像会议中两块肌肉的EFOV-US图像，并突出显示了每个图像的重要方面，例如肌肉腱鞘炎，中心肌腱，筋膜路径等。成像过程结束后，对每个个体中每块肌肉的3张定性"良好"图像（图2）进行了分析。实施ImageJ以测量每张图像4个分册。在每张图像中，测量了从起源到插入的路径可以令人信服地可视化并且位于所选肌肉不同部分的分册。本研究获得的肱二头肌（14.6 ±1.7 cm）和胫骨前部（7.3 ± 0.6 cm）的平均分筋膜长度在先前报告的分形肌长度范围内25，42 （表1）。
由于该协议最具挑战性和主观性的部分是决定因素，这些因素导致正确将图像视为定性"好"或定性"坏"。我们提供了几个"好"和"坏"图像的示例（图 2），以及图像地标和质量如何因人而异（图 3）。此外，我们还突出显示了图像中特别"不良"的部分。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>主题</td>
			<td>性</td>
			<td>高度（m）</td>
			<td>年龄</td>
			<td>二头肌侧</td>
			<td>二头肌束长度（厘米）</td>
			<td>胫骨前侧</td>
			<td>胫骨前分筋膜长度（厘米）</td>
		</tr>
<tr>
<td>1</td>
			<td>M</td>
			<td>1.78</td>
			<td>24</td>
			<td>L</td>
			<td>16.4 ± 0.3</td>
			<td>L</td>
			<td>7.6 ± 0.1</td>
		</tr>
<tr>
<td>2</td>
			<td>F</td>
			<td>1.8</td>
			<td>23</td>
			<td>R</td>
			<td>12.2 ± 0.2</td>
			<td>L</td>
			<td>7.5 ± 0.2</td>
		</tr>
<tr>
<td>3</td>
			<td>M</td>
			<td>1.82</td>
			<td>24</td>
			<td>L</td>
			<td>14.9 ± 0.2</td>
			<td>R</td>
			<td>7.7 ± 0.1</td>
		</tr>
<tr>
<td>4</td>
			<td>F</td>
			<td>1.79</td>
			<td>28</td>
			<td>R</td>
			<td>14.7 ± 0.2</td>
			<td>L</td>
			<td>6.4 ± 0.3</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>平均</td>
			<td>14.6</td>
			<td></td>
			<td>7.3</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>标清</td>
			<td>1.7</td>
			<td></td>
			<td>0.6</td>
		</tr>
</tbody></table>表1:参与者人口统计和数据。 分册长度的测量值表示为平均±标准偏差。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61765/61765fig01.jpg"> 图 1:两个示例肌肉的示意图和 EFOV 图像。 （左）正在研究的肌肉的插图。（右）顶部的"良好"图像示例，以及轮廓为整个肌肉（深蓝色），中央肌腱（浅蓝色）和肌肉束（白色）的相同图像。每个图像在图像的右下角都有一个相应的 1 厘米比例尺（白色）。 <a href="https://www.jove.com/files/ftp_upload/61765/61765fig01large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61765/61765fig2v2.jpg"> 图 2:图像质量演示。 演示从参与者1和2的肱二头肌和胫骨前部获得的三个定性"好"和三个定性"坏"图像。（Top A & B）在所有定性的"良好"图像中，可以从内部肌腱延伸到肌肉腱膜的分册可视化。我们说明了定性上"坏"的图像，不应该进行分析。图像中将其限定为"坏"的部分被强调（蓝色方框和箭头），包括锯齿状或断裂的图像，过度或非解剖学相关的弯曲，排除整个分册的图像以及具有模糊中央肌腱的图像。每个图像都有一个比例尺（白色垂直线），代表1厘米。该图的这一部分突出了图像之间的可变性，这主要是由于超声医师在单独的成像扫描之间不一致。（底部 A 和 B）显示一个"好"的肱二头肌和一个"好"的胫骨前肌。然后，原始图像上的橙色框被放大，以更准确地说明在ImageJ中测量分册时看到的变焦。下图显示了具有代表性的轮廓分册（白色虚线）。这些图像被认为是"好的"，因为可以从原点到插入跟随分册，并且图像的缩放部分没有实质性的失真或伪影。 <a href="https://www.jove.com/files/ftp_upload/61765/61765fig2v2large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61765/61765fig03v5.jpg"> 图 3:个人图像质量的差异。 参与者之间存在图像质量和可见性的差异，主要是由于解剖学变化（即肌肉大小，肌肉长度，皮下脂肪含量）和肌肉含量的差异（即肌内脂肪，结缔组织，纤维化的数量）。具体而言，肌肉含量和肌肉上方组织层的变化会影响成像肌肉的回声强度43。个体之间的自然解剖学差异将导致不同个体的美国图像在位置和/或相对大小上有所不同的肌肉结构特征。这种不同参与者肌肉的展示强调了对解剖学的透彻理解和充分练习获得不同个体图像的重要性，以便对所获得图像的质量和准确性充满信心。 <a href="https://www.jove.com/files/ftp_upload/61765/61765fig03v5large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>

Watch this Scientific Journal Video about Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length at JoVE.com

Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length

本研究描述了如何使用扩展视场超声（EFOV-US）方法获得高质量的肌肉骨骼图像，以进行肌肉束长度测量。我们将这种方法应用于具有束状物的肌肉，这些肌肉延伸到普通传统超声（T-US）探针的视野之外。

获得高质量的骨骼肌扩展视野超声图像，以测量肌肉束长度

Muscle fascicle length, which is commonly measured in vivo using traditional ultrasound, is an important parameter defining a muscle&#8217;s force ...

obtaining-quality-extended-field-view-ultrasound-images-skeletal

Universidad San Sebastian

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Medicine

Unit 1

The Muscular System

SCIENTISTS IN ACTION

3.6K 次观看.  Northwestern University. 本研究描述了如何使用扩展视场超声（EFOV-US）方法获得高质量的肌肉骨骼图像，以进行肌肉束长度测量。我们将这种方法应用于具有束状物的肌肉，这些肌肉延伸到普通传统超声（T-US）探针的视野之外。

视频: Obtaining Quality Extended Field-of-View Ultrasound Images of Skeletal Muscle to Measure Muscle Fascicle Length

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

离体骨骼肌肌的力学分析

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

科研

医学

Manual Muscle Testing:  A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, and persistent nerve and muscle impairments after hospital discharge.1-6 Using an explicit protocol with a structured approach to training and quality assurance of research staff, manual muscle testing (MMT) is a highly reliable method for assessing strength, using a standardized clinical examination, for patients following ARDS, and can be completed with mechanically ventilated patients who can tolerate sitting upright in bed and are able to follow two-step commands. 7, 8 
This video demonstrates a protocol for MMT, which has been taught to &ge;43 research staff who have performed &gt;800 assessments on &gt;280 ARDS survivors. Modifications for the bedridden patient are included. Each muscle is tested with specific techniques for positioning, stabilization, resistance, and palpation for each score of the 6-point ordinal Medical Research Council scale.7,9-11 Three upper and three lower extremity muscles are graded in this protocol: shoulder abduction, elbow flexion, wrist extension, hip flexion, knee extension, and ankle dorsiflexion. These muscles were chosen based on the standard approach for evaluating patients for ICU-acquired weakness used in prior publications. 1,2.

Survivors of acute respiratory distress syndrome (ARDS) and critical illness frequently develop long-lasting muscle weakness. Manual muscle testing (MMT) is a standardized clinical examination commonly used to measure strength of peripheral skeletal muscle groups. This video demonstrates MMT using the 6-point Medical Research Council scale.

危重病人中的应用手册肌力测试:下肢肌力测量方法

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, ...

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

骨骼肌蛋白质组学性别异形

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Method to Measure Tone of Axial and Proximal Muscle

The control of tonic muscular activity remains poorly understood. While abnormal tone is commonly assessed clinically by measuring the passive resistance of relaxed limbs1, no systems are available to study tonic muscle control in a natural, active state of antigravity support. We have developed a device (Twister) to study tonic regulation of axial and proximal muscles during active postural maintenance (i.e. postural tone). Twister rotates axial body regions relative to each other about the vertical axis during stance, so as to twist the neck, trunk or hip regions. This twisting imposes length changes on axial muscles without changing the body's relationship to gravity. Because Twister does not provide postural support, tone must be regulated to counteract gravitational torques. We quantify this tonic regulation by the restive torque to twisting, which reflects the state of all muscles undergoing length changes, as well as by electromyography of relevant muscles. Because tone is characterized by long-lasting low-level muscle activity, tonic control is studied with slow movements that produce "tonic" changes in muscle length, without evoking fast "phasic" responses. Twister can be reconfigured to study various aspects of muscle tone, such as co-contraction, tonic modulation to postural changes, tonic interactions across body segments, as well as perceptual thresholds to slow axial rotation. Twister can also be used to provide a quantitative measurement of the effects of disease on axial and proximal postural tone and assess the efficacy of intervention.

We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

测量方法轴向和近端肌肉音

The control of tonic muscular activity remains poorly understood.  While abnormal tone is commonly assessed clinically by measuring the passive resistance ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

高通量流式细胞仪检测细胞为基础的试验检测抗体，以N-甲基-D-天冬氨酸受体或多巴胺2受体在人血清

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

右心室结构和功能的肺动脉缩窄经胸超声小鼠模型的评估

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

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 ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

骨骼肌疾病的定量磁共振成像

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Early Detection of Drug-Induced Renal Hemodynamic Dysfunction Using Sonographic Technology in Rats

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is estimated to contribute to 19- 25% of all clinical cases of acute kidney injury (AKI) in critically ill patients. AKI detection has historically relied on metrics such as serum creatinine (sCr) or blood urea nitrogen (BUN) which are demonstrably inadequate in full assessment of nephrotoxicity in the early phase of renal dysfunction. Currently, there is no robust diagnostic method to accurately detect hemodynamic alteration in the early phase of AKI while such alterations might actually precede the rise in serum biomarker levels. Such early detection can help clinicians make an accurate diagnosis and help in in decision making for therapeutic strategy. Rats were treated with Cisplatin to induce AKI. Nephrotoxicity was assessed for six days using high-frequency sonography, sCr measurement and upon histopathology of kidney. Hemodynamic evaluation using 2D and Color-Doppler images were used to serially study nephrotoxicity in rats, using the sonography. Our data showed successful drug-induced kidney injury in adult rats by histological examination. Color-Doppler based sonographic assessment of AKI indicated that resistive-index (RI) and pulsatile-index (PI) were increased in the treatment group; and peak-systolic velocity (mm/s), end-diastolic velocity (mm/s) and velocity-time integral (VTI, mm) were decreased in renal arteries in the same group. Importantly, these hemodynamic changes evaluated by sonography preceded the rise of sCr levels. Sonography-based indices such as RI or PI can thus be useful predictive markers of declining renal function in rodents. From our sonography-based observations in the kidneys of rats that underwent AKI, we showed that these noninvasive hemodynamic measurements may consider as an accurate, sensitive and robust method in detecting early stage kidney dysfunction. This study also underscores the importance of ethical issues associated with animal use in research.

Early stage hemodynamic dysfunction is critical to the development of kidney disease. Yet, detection methodologies are limited. Recent advances in sonography provide a noninvasive, accurate option for early detection of kidney injury. This study outlines a step-by-step, sonographic methodology for detecting kidney dysfunction using a drug-induced nephrotoxicity rat model.

大鼠使用超声技术药物引起的肾血流动力学障碍的早期检测

The kidney normally functions to maintain hemodynamic homeostasis and is a major site of damage caused by drug toxicity. Drug-induced nephrotoxicity is ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

冠脉流量和冠状动脉血流储备用小鼠压力超负荷基于模型的超声评估

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...