Name: electrophysiological motor unit number estimation (mune) measuring compound muscle action potential (cmap) in mouse hindlimb muscles
Uploaded: 2015-09-25T14:00:00
Duration: 9 min 7 s
Description: Watch this Scientific Journal Video about Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles at JoVE.com

WDA is supported by grant funding from NIH-NICHD (5K12HD001097-17) and Cure SMA. SJK is supported by grant funding from NINDS (K08NS067282 and U01NS079163).

该协议被批准，并粘附在美国俄亥俄州立大学的Wexner医学中心的动物护理和伦理准则。 1.动物准备及麻醉<ol><li>在处理小鼠戴上手套。 </li><li>麻醉小鼠吸入异氟醚和发生在俯卧位。诱导使用3-5％异氟醚和每分钟O 2流量1升麻醉。继诱导麻醉，麻醉维持在2-3％，并每分钟O 2流量1升。 <ol><li>调节O 2流量和异氟烷百分比用于根据动物的疾病状态，年龄，和呼吸率充分麻醉。较小或较弱的动物可能需要适当的麻醉（ 即 1.5-2.5％异氟醚），异氟醚较少。 </li><li>通过施加光后肢脚掌的压力与物体如钳子证明缺乏戒断反应的确认充分麻醉。; </li></ol></li><li>保持温度在37℃的表面温度用的恒温加温板如温度变化​​可能会影响CMAP大小和持续时间。 </li><li>应用兽医石油基软膏的眼睛，以防止干燥。监测麻醉观察呼吸率水平并评估以下压力通过钳子施加到脚垫戒断反应。 </li><li>使用快船待研究后肢清除头发。去除毛发从后肢（S）以进行研究后，轻轻延伸后肢在膝盖处，绑架在臀部和固定到使用胶带的工作表面（ 见图1）。 </li><li>继CMAP和MUNE录音和麻醉停药，不要让动物无人看管，直到它重新获得足够的意识，以保持胸骨斜卧。不返回到动物的其他动物的公司直到完全恢复。 </li></ol> 2。RECORDING设置和设备<ol><li>地方的CMAP和MUNE记录电极如下图如图 1所示。 </li><li>用两个细环状电极的记录电极。 <ol><li>放置活性（E1）环上的皮肤覆盖在后肢的腓肠肌的近侧部分，在膝关节电极，和在皮肤上的脚的中间跖骨部基准（E2）环形电极。 </li><li>为了减少阻抗，外套皮肤环状电极底层用凝胶充分饱和残毛和最大化电极 - 皮肤接触。避免电极凝胶的过量施用，因为这可能会导致电极之间的桥梁，可以防止准确地记录。 </li></ol></li><li>对于坐骨神经的近端后肢刺激，使用两个绝缘28克单极针作为阴极和阳极。插入该阴极在近端后肢的区域并插入肛德更近侧在皮下组织覆盖骶骨。 <ol><li>避免插入刺激电极过于接近坐骨神经或太深，这将直接伤害坐骨神经或其它结构。 图1示出的电极放置。 </li></ol></li><li>为接地电极，放置在对侧后肢或尾部一次性表面电极。 </li></ol> 3.数据采集<ol><li>坐骨神经CMAP <ol><li>通过刺激坐骨神经为0.1毫秒持续时间方波脉冲和强度不等的1-10毫安获得坐骨神经CMAP响应。 </li><li>获得CMAP响应随刺激强度直到不再增加了响应的幅度。然后，为了确保超最大刺激，增加的刺激，以用于获得一个最大应答的刺激强度的〜120％，将获得的附加响应。如果没有进一步增加我n个CMAP大小，记录这个响应，最大CMAP。 </li><li>记录基线至峰和峰-峰振幅CMAP以mV（图2）。 </li></ol></li><li>平均单运动单位电位（SMUP）尺寸和MUNE计算<ol><li>确定平均单个运动单位电位（SMUP）大小的增量增产技术1。为了获得增量响应，提供0.1毫秒的持续时间最大刺激在1赫兹的频率，同时增加了强度0.03毫安步骤获得最小的全或无响应。获得与0.21毫安和0.70 mA的刺激强度的初步反应。 <ol><li>如果初始响应不与0.21 mA和0.70 mA的刺激强度发生，调整刺激阴极位置更近或远离坐骨神经的位置在近端大腿以减少或增加所需的刺激强度分别。 </li><li>如果初始我获得ncremental响应与0.21 mA和0.70 mA的之间的刺激强度，并满足下面指出（3.2.2），商店的标准并记录额外的增量而增加刺激强度是通过以下步骤0.03毫安调整，以获得总共9个附加增量是符合既定标准。 </li></ol></li><li>在增量响应的测量结果，确保每个增量满足以下条件。 <ol><li>确保增量响应的初始负峰值在时间上显示为CMAP说明的图 2中的阴影部分的最大CMAP响应的负峰值内对齐。 </li><li>确保每个增量响应是稳定的，没有分馏，通过观察三个重复反应确立。区分实时（叠加在先前记录单位）目视增量响应。 注：每个增量应该是视觉上不同并与前述的反应（图3）放大比较。分析实时允许识别较大振幅（增量值）的反应相比于现有的反应，和归属于背景噪声小的变化可以忽略不计。增量为10甲叠加图示于图4（B和D），以进一步说明这一点。 </li><li>后目视确认每个增量，确保所测量的振幅差（确认响应振幅的现有响应=振幅差）为至少25μV。 </li><li>如果增量小于25μV，丢弃和重新测量的响应。记录10增量后的反应，评估增量，以确保每个单独的增量响应的幅度不大于所有十个的增量的总和 （即最终响应的总幅度）的1/3。如果这个条件不满足，重新测量增量响应。 </li></ol></li><li>平均10个增量值，得到平均单个马达单元电位（SMUP）振幅的估计（图3）。注意： 图3详细说明了平均SMUP计算的基础，但平均SMUP振幅可以由最终增量响应的整个振幅除以增量的总数来简单地计算（即，10）。 实施例个别SMUP计算（图3中示出）： SMUP 1 =峰 - 峰值增量1的振幅 SMUP 1 = 0.050毫伏 SMUP 2 =（峰 - 峰振幅增量2） - （增量峰 - 峰振幅1） SMUP 2 = 0.150毫伏0.050 MV = 0.100毫伏<ol><li>计算每个后续的递增（最多总共10），并进行平均十个的增量。 </li></ol></li><li>计算MUNE由平均SMUP振幅除以最大CMAP波幅（峰 - 峰）（峰到峰）。 （MUNE = CMAP /平均SMUP）。在一些电系统中，SMUP增量测量μV，而CMAP在毫伏一般规定。必要时，转换CMAP和SMUP结果之前MUNE计算类似的单位。 </li></ol></li></ol>

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The authors have nothing to disclose.

MUNE和CMAP经常用在调查研究和监测患者的神经肌肉疾病如ALS和脊髓性肌萎缩（SMA）9,10临床相关的措施，例如，在形状记忆合金，CMAP和MUNE很好地相关与年龄，严重性和临床功能10-14措施。两个措施是微创和允许的功能评估纵向在同一个人。重要的是，这些措施不能测量活化或招募电动机单元的皮质运动神经元，但它们提供的运动神经元的完整性和其功能对应，电动机单元一个临床相关的评估。 神经肌肉疾病的动物模型是人类疾病的发病机制的理解以及对潜在的有效治疗剂的临床前的发展是至关重要的。翻译结果的措施和生物标志物，可以的能力跨物种的利用可以促进和加快有前途的临床前研究结果对人类临床试验的翻译。几个研究小组先前已经利用两个电和力（机械）的测量来估算运动单位的功能在小鼠模型2-4，15-22。由于措施的相对复杂性，我们还改进了直观的格式，这些技术允许更广泛地使用和实施的小鼠。视频演示和指令的格式，允许强调过程的关键步骤和潜在的缺陷加以解决。这些技术在运动神经元疾病的潜在疗法的临床前试验中的应用可能会提高翻译公认的治疗方法，从小鼠到人类疾病。 有收购的CMAP和MUNE反应的过程中的几个关键步骤。正确的和一致的记录电极放置和用后腿足够的电极接触肢是幅度和减小背景噪声重现的测量是至关重要的。因此，后肢皮肤和电极之间的密切接触，应始终如一地证实。我们已发现，表面电极提供更加一致和CMAP MUNE录音比针电极。由于非常薄的皮下组织，针记录表面的小的移动可导致在CMAP的幅度很大的差异。此外，针状电极更侵入性是不是最佳的新生小鼠或纵向研究，由于潜在的肌肉破坏和伤害。的非选择性，表面电极记录一个潜在缺点涉及的减弱表现型分辨率的可能性，如果一个特定肌肉是或多或少参与与另一个相比，这已被报道的ALS小鼠模型21。 相比于CMAP获取平均SMUP尺寸在技术上是更具挑战性。由于较小responsE尺寸（以μV而非毫伏的范围）的背景噪声可以更成问题。背景噪声可以通过调整接地电极，阴极，阳极，和检查等电气设备的实验装置附近被减小。法拉第笼，通常用于细胞内电应用中，并不需要。视觉确定个人SMUP反应是最困难的技巧掌握，并采取实践有足够的可重复性一致的结果。以确保正在记录SMUPs最大CMAP响应的持续时间内启动是重要的。我们已经确定的标准验收的各个增量反应，使这个过程更易于执行，增加区域内和跨信度。 增量MUNE技术的一个潜在的缺点包括高估的功能的运动单位的数量，由于摩托车的交替的可能性R单元。我们已经使用类似于Shefner 等人的技术。在每个反应，应再现地看出，共3次，以减少这种现象3的影响。 根据我们的经验，临床电生理系统本文所述由于改善考官，电学系统接口的人体工程学设计，允许容易控制的研究进行了优化。在我们的实验室中使用的双通道系统配备有使用的放大器具有24比特模拟到数字转换器和每个通道48 kHz的采样率的两个非切换放大器通道。硬件增益可以从10nV调整到100 mV /分。低频滤波器具有范围从0.2赫兹-5千赫和高频滤波器设置范围从30赫兹-10千赫。恒定电流刺激时（强度：0〜100毫安，持续时间：0.02-1毫秒）。大多数临床系统具有类似的适当的功能，并且可以进行调整，以充分地记录和CMAP MUNE响应。一个dditionally，标准电生理钻机可组装到适当记录和CMAP MUNE，但接口可能需要调整为便于刺激调整和快速识别和CMAP SMUP响应。 我们以前曾利用CMAP的技术和MUNE这里描述以允许在产后早期到成年5在小鼠后肢的坐骨神经神经支配的肌肉的快速和可重复的评估。这些技术允许评估在小鼠模型中当行为测试为运动功能是不可行的或者是不可靠的。促进这种技术新生小鼠应用运动单位发展的研究，并有扩大我们的运动神经元的神经支配和修剪理解的潜力。例如，我们已经表明，记录有MUNE官能马达单元的数量将在头两个个星期从polyneuronal修剪到mononeuronal神经支配期间增加生活S在新生小鼠5。测试小鼠过长时间使用这种技术的能力适合于电机单元响应于外周神经损伤，遗传性神经肌肉疾病和老化的研究。

马达单元数估计（MUNE）最初是由麦科马斯等人所述 。超过三十年以前1。原来的技术是，使用的刺激逐渐增加，以获得次最大增量的复合肌肉动作电位（CMAP）记录技术的改进。这些增量进行相加并进行平均，以确定一个马达单元电位（SMUP）的估计大小。这个大小被分为CMAP响应来估计电动机单元的数目支配被测试的肌肉。以下的原始描述，同时使用电生理反应和增量力许多变化（机械）测量已用于人类研究和动物模型2。该MUNE技术是由Shefner和同事改性调查肌萎缩性侧索硬化症（ALS）3，4的小鼠模型。 在当前的描述中，我们详细介绍simplif的MUNE技术是快速执行IED修改。重要的是，CMAP和MUNE允许在这两个新生和成年小鼠5-8可靠的措施。有经验的人可以在每头牲畜10-20分钟执行这些措施，并重复措施是允许收购纵向数据5可行的。在目前的研究中，我们采用了临床电诊断系统。根据我们的经验，临床电生理系统在体内电生理数据的快速，高效地捕捉优化，但标准的电钻机可以很容易地适应这种应用。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
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			<td>Pro trimmer Pet Grooming Kit</td>
			<td>Oster</td>
			<td>078577-010-003</td>
			<td>clippers for hair removal</td>
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			<td>Natus Neurology</td>
			<td>Model no longer available</td>
			<td>portable electrodiagnostic system</td>
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			<td>monopolar needles 28 gauge</td>
			<td>Teca</td>
			<td>017K121</td>
			<td>cathode and anode stimulating electrodes</td>
		</tr>
		<tr>
			<td>Alpine Biomed Digital Ring Electrode with twisted wires and 1.5 mm TP connectors.</td>
			<td>Alpine Biomed</td>
			<td>9013S0312</td>
			<td>recording electrodes</td>
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			<td>Helping Hands alligator clip with iron base</td>
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			<td>64-079</td>
			<td>Maintaining recording electrode placement&#160;</td>
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			<td>Parker Laboratories</td>
			<td>9013G5012</td>
			<td>applied to reduce skin impedance</td>
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		<tr>
			<td>monoject curved tip irrigating syringe</td>
			<td>Covidien</td>
			<td>81412012</td>
			<td>utilized for application of electrode gel</td>
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			<td>EMG needle cable</td>
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			<td>902-RLC-TP&#160;</td>
			<td>to connect monopolar electrodes to electrodiagnostic stimulator</td>
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			<td>Carefusion</td>
			<td>019-415000&#160;</td>
			<td>ground electrode</td>
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			<td>Small Heating Plate with built-in RTD sensor, 15x10cm</td>
			<td>World Precision Instruments</td>
			<td>61830</td>
			<td>warming plate used with animal temperature controller to transmit heat to animal</td>
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			<td>Silicone pad for use with ATC2000</td>
			<td>World Precision Instruments</td>
			<td>503573</td>
			<td>conductive removable pad to cover warming plate for easy cleaning</td>
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		<tr>
			<td>Animal temperature controller</td>
			<td>World Precision Instruments</td>
			<td>ATC2000</td>
			<td>low noise animal heating system for maintaining animal temperature</td>
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			<td>Veterinarian petroleum-based ophthalmic ointment&#160;</td>
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			<td>applied during anesthesia to avoid corneal injury</td>
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</table>

electrophysiological motor unit number estimation (mune) measuring compound muscle action potential (cmap) in mouse hindlimb muscles

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the functional status of a motor unit pool in vivo. These measures can provide insight into the normal development and degeneration of the neuromuscular system. These measures have clear translational potential because they are routinely applied in diagnostic and clinical human studies. We present electrophysiological techniques similar to those employed in humans to allow recordings of mouse sciatic nerve function. The CMAP response represents the electrophysiological output from a muscle or group of muscles following supramaximal stimulation of a peripheral nerve. MUNE is an electrophysiological technique that is based on modifications of the CMAP response. MUNE is a calculated value that represents the estimated number of motor neurons or axons (motor control input) supplying the muscle or group of muscles being tested. We present methods for recording CMAP responses from the proximal leg muscles using surface recording electrodes following the stimulation of the sciatic nerve in mice. An incremental MUNE technique is described using submaximal stimuli to determine the average single motor unit potential (SMUP) size. MUNE is calculated by dividing the CMAP amplitude (peak-to-peak) by the SMUP amplitude (peak-to-peak). These electrophysiological techniques allow repeated measures in both neonatal and adult mice in such a manner that facilitates rapid analysis and data collection while reducing the number of animals required for experimental testing. Furthermore, these measures are similar to those recorded in human studies allowing more direct comparisons.

CMAP和MUNE的技术本报告中所述的记录允许利用微创电极放置（图1）的坐骨神经支配后肢肌肉的神经肌肉功能的。超最大CMAP大小，其表示从一肌群的输出总，可以使用振幅和区（图2）的参数进行描述，但是，在目前的方法中，我们使用振幅量化CMAP和SMUP尺寸。由于CMAP应对措施累加肌纤维的去极化肌肉内，病理从运动神经元的肌纤维随时随地都可以导致减少CMAP大小。因此，CMAP给人总的功能状态极好的措施。正如预期的那样，CMAP规模将发展5期间增加。由于可能发生以下去神经（即侧支出芽）代偿性改变，CMAP大小可被保持，尽管马达neur的流程或运动性轴索损失。因此，MUNE的技术中是必需的，以确定运动神经元轴突或输入到肌肉或肌肉群的被测试。个别增量记录（ 图3）允许估计单电机单元（SMUP尺寸）的平均输出的，得到约运动单位的功能状态的更详细信息。 CMAP和MUNE可以用来测量在神经肌肉疾病的各种小鼠模型神经肌肉功能。 在图4中，在成年对照小鼠和成年。鼠标11周后坐骨神经的调查结果进行了对比。继坐骨神经，宗严重降低50估计功能的运动单位与278功能的运动单位在对照组小鼠正常的结果进行比较。与此相反，CMAP波幅在压碎的动物（39.6毫伏基线至峰，74.9毫伏峰 - 峰值）是表示仅轻微减少与对照相比（49.0毫伏基线到- 峰值，84.2毫伏峰 - 峰）由于侧支出芽。 <img alt="图1" src="/files/ftp_upload/52899/52899fig1.jpg" /> 图1.电极放置的黑（E1）“激活”电极（A）和红色（E2）“参考”记录电极（B）的放置在腓肠肌在腓肠肌在膝盖处的近端部分。上述刺激阴极（黑色）（C）和阳极（红色）（D）中被插入皮下近端到记录电极，以产生远端响应。一次性盘电极（D）放置在后肢，尾骶骨或作为地面，以减少工件。 <a href="https://www.jove.com/files/ftp_upload/52899/52899fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图2.复合肌肉动作电位，图为是代表性的CMAP响应的示图：（A）基准-峰振幅是从等电基线测量到初始负峰值（负电压基线以上描绘）。（ B）中的峰-峰振幅是从负峰 ​​值电压到正峰值电压测量。灰色阴影区为负峰面积。 <a href="https://www.jove.com/files/ftp_upload/52899/52899fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52899/52899fig3.jpg" /> 图3.增量反应。两个有代表性的增量响应如图叠加和伊索拉化。对于每个增量的MUNE计算幅度被测定的峰 - 峰值。增量＃1是初始全或无响应记录和表示单个马达单元电位（SMUP）。每个后续增量（＃2-10）代表增加量子叠加在之前的回应。因此，为了获得SMUP幅度的递增2-10，现有响应的幅度从获得的增值幅度减去。 <a href="https://www.jove.com/files/ftp_upload/52899/52899fig3large.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/52899/52899fig4.jpg" /> 图4.实施例坐骨神经CMAP和MUNE。（A）的坐骨神经复合肌肉动作电位（CMAP）的成人（6月龄）对照小鼠与基线至峰振幅的49.0毫伏和峰到84.2 mV的峰值。屏幕灵敏度=每格10毫伏和屏幕持续时间10毫秒。（B）的十个相应的增量的反应（对照小鼠）与3.028毫伏总幅度被除以10，以确定平均SMUP尺寸（0.3028毫伏）。屏幕的灵敏度= 0.5毫伏和每格1毫秒的扫描速度。计算MUNE = 278（MUNE = CMAP /平均SMUP（84.2毫伏/ 0.3028毫伏））（C）坐骨神经CMAP11周以下的成年小鼠坐骨神经（6个月以下），显示轻度降低基线到峰值（ 39.6毫伏）和峰 - 峰幅度（74.9毫伏）。屏幕灵敏度=每分和每格1毫秒扫描速度为10mV。（D）的十个相应的增量响应（在小鼠与神经压迫）与14.923毫伏总峰-峰振幅除以10获得平均SMUP的1.4923 mV的大小。屏幕的灵敏度= 2每分毫伏和每格1 ms的扫描速度。计算MUNE = 50（MUNE = CMAP/平均SMUP（74.9毫伏/ 1.4923毫伏））。 （**注不同的灵敏度坐骨神经挤压鼠标的增量答复）。 <a href="https://www.jove.com/files/ftp_upload/52899/52899fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles at JoVE.com

Electrophysiological Motor Unit Number Estimation MUNE Measuring Compound Muscle Action Potential CMAP in Mouse Hindlimb Muscles

我们提出精致的协议，允许在电动机单元功能的小鼠体内监测。技术测量复合肌肉动作电位（CMAP）和电动机单元数估计（MUNE）在坐骨神经支配鼠标后肢肌肉进行说明。

电电机组数估计（MUNE）在小鼠后肢肌肉测量复合肌肉动作电位（CMAP）

Compound muscle action potential (CMAP) and motor unit number estimation (MUNE) are electrophysiological techniques that can be used to monitor the ...

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Electrophysiological Motor Unit Number Estimation (MUNE) Measuring Compound Muscle Action Potential (CMAP) in Mouse Hindlimb Muscles

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