Name: repeated measurement of respiratory muscle activity and ventilation in mouse models of neuromuscular disease
Uploaded: 2017-04-17T14:00:00.000+00:00
Duration: 9 min 24 s
Description: Watch this Scientific Journal Video about Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease at JoVE.com

对这项工作的支持是由辛辛那提儿童医院医疗中心理事奖SAC和美国国立卫生研究院培训资助（T32NS007453）到VNJ提供

实验过程是由辛辛那提儿童医院医学中心的机构动物护理和使用委员会批准，并遵守NIH指南护理和使用实验动物进行。 1.准备遥测装置植入手术<ol><li>穿戴个人防护设备（ 即，磨砂，鞋套，袍，净毛，掩码和手术手套）。 注:此手术需要一个无菌区。 </li><li>打开培养箱（伺服控制加湿器/婴儿培养箱设定为29℃）并用干燥，白毛巾线它，以允许恢复正确变暖。 </li><li>在手术之前，所有消毒手术器械与环氧乙烷和在使用前的含酶洗涤剂和化学杀菌剂（或由制造商指定）消毒所述发射机。 注:手术器械应包括＃2椎板钳子（标准提示/直/ 12厘米）（×4），窄图案镊子（锯齿/弯曲/ 12厘米），组织分离剪刀（直/平钝/ 11.5厘米），和手术刀保持架和刀片。建议有一组独立的专门用于处理发射器的电线，以保持手术工具完好保留无菌器械（二＃2镊子和剪刀）的。 </li><li>消毒手术区域内的所有表面具有可接受的消毒剂。位置的立体声解剖显微镜，异氟烷麻醉机，手术工具，和指甲刀外科领域（参见材料的表 ）。 </li><li>为了保持鼠标体温而麻醉下，放置一个加热垫或水毯的无菌巾下在立体声解剖显微镜的前面。 </li><li>确保发射机是全功能使用之前。 注:一个小磁体中的发射器将打开和关闭设备的放置在2。当发射机上的RA密切举行DIO设定为500赫兹AM频率时，它将发出一个连续的，高声调的嗡嗡声。为了节省电池寿命，在动物植入前关闭电池断开。 </li><li>准备电极通过修整以用于处理电线使得存在的铅约3厘米（足以达到目标肌肉）（ 图1A）所保留的剪刀远侧引线手术前引线。可替代地，近侧线圈导线到设备并用缝合线一起配合他们使得存在的开卷铅大约3厘米。 注:保存电极的修整断部以制备"引线帽"（塑料绝缘外壳）导致，如在步骤1.9中描述。在步骤3中，发射机引线将通过肌肉被插入，所以与导线的远端，露出的部分必须保持在适当位置，并使用铅帽绝缘。 </li><li>使用解剖刀修剪过0.5厘米塑料覆盖物的不切断导线本身。使用FO保留的工具ř线操纵伸展的引线4的端部- 5倍其初始长度，使得它们很容易适应一个25号针头（ 图1B-B&#39;）的内部。修整引线的露出的线，使得它们为0.5厘米的长度。 </li><li>准备引线盖（塑料管，以覆盖导线端部）在手术前。使用解剖刀修剪过从围绕电极的各段的塑料外壳0.25厘米长管引线从步骤1.7保存。 注:四个铅帽需要为每个鼠标将要进行植入，但最好是准备所需瓶盖量的两倍。有额外的无菌准备铅帽是有帮助的情况下，确保铅帽是在第一次尝试失败。 </li><li>记录插入发射器的序列号和保存原包装与所述校准信息。每个发射机具有用于EMG记录不同频率的校准;这些状态输入采集软件，以获得可接受的EMG录音。 </li></ol> 2.准备鼠标外科手术<ol><li>选择所需的小鼠用于植入（ 即，SOD1（G93A）或对照）和称重动物。 注:鼠标（男性或女性）的推荐年龄和体重接受这种手术是P56 - P120和≥24克，分别。 </li><li>麻醉下3.5％异氟烷的小鼠用2升/分钟的氧气流率。执行脚趾捏和尾捏，以确保鼠标完全麻醉。 </li><li>一旦麻醉后，从下拉框中通过鼻锥通过设置异氟烷水平至1.5％，氧流量为1升/分移开鼠标和维持麻醉。执行捏脚趾和/或尾巴捏，以确保麻醉维持。 </li><li>涂上润滑剂眼药膏，以防止眼睛手术过程中干燥。 </li><li>剃鼠标以暴露耳朵和肩膀（ 图1C）之间的手术部位。 </li><l我&gt;剪辑同侧手术部位的脚趾甲，以减少鼠标拉出引线或在愈合过程中划伤造成伤口的机会。 </li><li>交替擦拭手术部位，先用消毒液，然后用异丙醇。重复2次以上。 </li></ol> 3.植入遥测设备记录斜角肌和斜方肌肌电活动<ol><li>放置动物解剖显微镜下，在其上的无菌覆盖垫加热垫的顶侧，并用胶带固定鼻锥到位。请注意，最好是植入的右侧，以减少从心脏起源ECG信号。 注:监测呼吸和，调节异氟烷水平如果必要的话，保持有规律的呼吸速率和麻醉的合适的手术平面。 </li><li>拉向沿着躯干同侧脚前肢。 注:此位置取代了肩胛骨尾端，提供手术进入到Scalene和斜方肌。 <ol><li>取钝弯钳，忍住前爪同侧到手术部位，以及在适当位置用胶带将爪（ 图1C）。使用强粘合医用胶带，以确保爪子是安全的程序的时间。 </li></ol></li><li>穿上一双崭新的外科手术手套。使用手术刀作出斜切口，约2厘米长，肩部和耳（红线在图1C）之间。 </li><li>使用两个＃2椎板钳，一个在每个手，拉回脂肪垫和分开斜方肌和颈阔肌肌肉以暴露覆盖胸锁乳突肌与斜角肌（ 图1D和E）的筋膜上。 </li><li>使用淡胸锁乳突肌和膈神经作为标志标识斜角肌。需要注意的是膈神经平行延伸到斜角肌，而胸锁乳突肌在于INFErior。该斜角肌从颈椎到斜方肌下排骨斜跑。生物电势引线将被插入到前斜角肌，其可以被识别为运行相邻于膈神经（ 图1F 和G）的肌肉。 注意:注意。这个区域是高度血管，并且必须小心，避免切割锁骨下动脉。避免损伤膈神经和臂丛神经。 </li><li>一旦斜角肌和斜方肌已经确定（ 图1G），使皮下口袋对动物的背部，肩胛骨之间发射。 <ol><li>使用组织分离剪刀，只插入皮肤下面剪刀的钝顶和传播，直到一个口袋开口，其发射机的大约1.25倍的宽度形成（ 图1H）。 注:发射机应以最小的阻力插入，但麻子等不应该这么大，发射器可自行移动。如果口袋太小，发射机也可能摩擦皮肤，造成皮肤刺激，可能促使动物抓破皮肤和/或导线拔出。如果口袋太大，血清肿可能形成，或者发射机可以迁移到一个不利的位置。 </li></ol></li><li>温暖，无菌生理盐水冲洗并用抗肌肉的平坦的侧面插入发射器。使得其位于平面和导线从袋，出现平行于彼此而不是扭曲（ 图1I）的发送器的位置。卷曲丝的任何多余长度的装置和下把它平。 </li><li>运行从发射机到斜角肌和斜方肌引线，使得两组双电位引线平放并彼此平行。 </li><li>使用椎板镊子前斜角肌从周围的肌肉中分离，并通过斜角肌亩插入一个25号针CLE，垂直于肌肉纤维。 <ol><li>插入一个引线插入针的尖端，然后拔出针从肌肉，留下插入到肌肉到导线（ 图1J和K）的绝缘导线的后面。其色引线插入到肌肉记录。 </li></ol></li><li>放置氰基丙烯酸酯类粘合剂的一小滴在电线上，靠近在引线插入肌肉的暴露端，并使得没有导线引线盖和肌肉（ 图2A之间暴露快速滑过导线的导线帽和B）。 注:虽然这是一个公认的做法，以确保EMG氰基丙烯酸酯17，18引线，另一种方法是通过把它周围的丝线打结固定到位导线帽。 </li><li>修剪多余线远端的帽和应用氰基丙烯酸酯粘合剂的液滴到引线盖/导线的端部。给胶时间到释放（ 图2C和D）之前聚合。 </li><li>按照相同的步骤（步骤3.10-3.12）来插入平行相反的极性导致所述第一在同一肌肉，1 - 2毫米从第一引线程。 </li><li>重复步骤3.10 - 3.12插入导线进入斜方肌，位于只是前斜角肌（ 图1L和M）。 </li><li>确保引线是固定的地方，有刚够松弛引线的动物没有拉动导线进行身体运动。确保铅任何多余的长度不推向皮肤，因为这可能导致刺激，可能促使动物划伤或引线拉出。重新定位的线索，如果有必要，以防止任何可能发生的不适。 </li><li>轻轻取出磁带按住前肢。拉脂肪垫在背部肌肉，并用它来覆盖插入导线。用氰基丙烯酸酯粘合剂通过挑逗关闭切口皮瓣重新走到一起，使切口线了。与弯钳一起捏皮瓣的一部分，并且沿着这条线施加氰基丙烯酸酯类粘合剂的一小行。 </li><li>注入0.1毫升卡洛芬皮下的，以减轻术后疼痛，而动物是仍然麻醉下。 注: - 之后根据需要2天手术后，再继续管理0.1毫升卡洛芬一日一次1。 </li><li>从鼻锥中取出的动物，并将其放置在干净的笼中预温孵化，直到动物是清醒和周围的笼移动主动。保持动物在孵化至少15分钟后，监测它的运动和警觉性。 </li></ol> 4.术后护理<ol><li>房子动物单独手术。提供饮食凝胶和水瓶愈合的动物。 </li><li>监测手术后第一个30分钟的动物。检查动物至少每隔一小时˚F或5小时后手术。在手术后的日子里，每天检查至少两次。 </li><li>观看为坏死，感染沿切口和含有所述植入物（ 即，发热，肿胀，发红和）在体腔内，和血清肿形成。 注:第一个星期内出现这些迹象手术后。手术后愈合健康动物的一个月如图1N。虽然肌电图记录可以立即植入后进行的动物给予至少一周记录肌电图描记法之前医治，心电图信号可在植入后立即高。 </li></ol> 5.获取同步肌电和体积描记信号<ol><li>打开所有采集设备，包括偏流量。 注:对于小鼠的流速典型地设定为1.0升/分。 </li><li>校准使用流量计的体积描记法室（一个或多个）。 注:定期检查体积描记chambeRS以保证密封件不会破裂或损坏。涂层橡胶密封件与润滑剂诸如真空润滑脂每周一次，以保持其良好的状态。 </li><li>如制造商所指定的输入发射机校准。 </li><li>鼠标放置在体积描记室至少1个小时，以适应前它记录EMG和体积描记法。使用多个腔室，它是可能的，同时下一个鼠标在第二腔室驯化从一个鼠标记录。不要在发射机中的驯化期，以节省电池电力（ 图1O）转动。 </li><li>在此之前的记录（但校准后），通过将内1中的强磁体在植入动物的打开发射机;在接收器的前部的红光将指示当发射机上。 </li><li>使用标有"收购"的下拉菜单开始收购，并选择"开始采集"。虽然录音时间可通过实验而变化，一个典型的体积描记术和EMG记录持续1 - 3小时。 注:发射器具有240 Hz的固有采样率。 500 Hz的更快的速度在软件中设置点之间进行插值，并提供一个平滑的波形。低通滤波器（在作为抗混叠滤波器），并在植入物中的高通滤波器指定该遥测装置1- 50赫兹带宽。 60 Hz的A / C干扰没有贡献的过量噪声在EMG信号，因为植入物是电池供电的并且动物屏蔽植入物和从电场导致。体积描记法，肌电图和视频会自动实时通过收购软件同步。 </li><li>当采集结束时，关闭发射机有磁铁，并从腔室去除动物。 </li><li>如果启动另一个记录，清洁室，在从下一个动物新型变送器校准输入，并开始第二记录。如果翅片与收购当天ished，关闭发射机，清理体积描记室，并关闭所有采集设备和偏流。 </li></ol><img alt="图2" src="/files/ftp_upload/55599/55599fig1.jpg" /> 图1.遥测装置植入到测量呼吸肌EMG。有两对生物电势（A）的遥测发射器导致测量EMG。引线可以被修整至所需长度（底部）或盘绕和发射机（顶部）的下方卷起。 （B）变送器。 （B"）与引线修整过胶绝缘以暴露导线和让导线盖（插图）。 （B&#39;）与拉伸4线引线- 5倍它们的原始长度。引线应修剪，使得它们为0.5厘米长（未示出）。 （C）鼠标准备手术，用刮手术部位和正确定位前爪。红色虚线表示切口部位。 （D）表面的肌肉位于在初始切口脂肪垫和筋膜，可见下面。 T =斜方肌。 S =胸锁乳突肌。 P =颈阔肌。黄色箭头=膈神经。在（D）中所示的肌肉和膈神经的（E）卡通图。钳子应该被用来分开斜方肌和颈阔肌肌肉到达更深斜角肌，在（F）中示出和（G）。 （F）的地标用于识别斜角肌和斜方肌的位置。此图像显示锁骨下动脉（白色箭头），膈神经/臂丛（黑色箭头），和淡胸锁乳突肌（黄色箭头）。 （G）描绘卡通较深肌肉（ 即，中斜角肌，前斜角肌，和SCM），锁骨下动脉，和膈神经的位置。后斜角肌是不可见的。这些都可以访问只有当SUPERFICI人的肌肉（在D和E）被分开。 （H）制作用于使用钝尖剪刀发射器的口袋。 （I）在皮下袋中插入发射机中，而平行定位的引线从袋出现。 （J）的25号针插入斜角肌插入，垂直于肌肉纤维，以使该引线的隧道。 （K）插入斜角肌两个引线。引线帽被定位在端部和胶合就位。 （L）的25号针插入斜方肌插入，垂直于肌肉纤维，以使该引线的隧道。 （M）的所有四条引线插入斜方肌和斜角肌和平躺之前切口的闭合。 （N）充分回收鼠标，与背面发射机位于皮下。 （O）同时记录体积描记法，肌肉EMG活动，一个第二视频分别使用体积描记法室（黄色箭头），遥测接收垫（红色箭头），和照相机（黑色箭头）。一种多功能偏流经由塑料管（蓝色箭头）供给氧到鼠标连接到所述体积描记法室。 <a href="http://ecsource.jove.com/files/ftp_upload/55599/55599fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55599/55599fig2.jpg" /> 图2. 保护铅帽与瞬干胶。 （A）应用氰基丙烯酸酯（紫色圆圈）的一小滴在电极引线（E）丝邻近所述肌肉的暴露的导线。 （B）快速使得引线盖帽设置直接邻近肌肉所制备的引线帽（LC）滑到露出的线在氰基丙烯酸酯粘合剂。 （C）修剪过导线帽和导线的远端的一小部分，以便有暴露的电极本没有不与塑料绝缘。 （D）应用氰基丙烯酸酯粘合剂中的一个小水滴到引线盖的端。除去从动物导线帽的修整掉远端。 <a href="http://ecsource.jove.com/files/ftp_upload/55599/55599fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 6. ARM肌电图和体积描记法分析<ol><li>打开分析软件和审查感兴趣的文件（去"文件"，然后选择"打开文件审核"）。过滤使用30赫兹的高通滤波器通过对EMG跟踪右击EMG信号，选择"分析属性，"突出"高级属性1"标签，改变高通滤波器30赫兹。 注:该过滤步骤除去非判别，低频信息。 </li&gt; <li>通过基于在所述同步的视频文件中的缺少运动和缺乏大的，不规则的压力变化，由于在体积描记迹（ 图3A中的红色框）运动目测定位小鼠非活动区域;当鼠标睡着或清醒，但仍然出现不活动。 </li><li>独立识别EMG回合每块肌肉。 <ol><li>整流和滤波后的信号EMG在30毫秒（ 图4）相集成。 注意:由于小鼠呼吸在3赫兹的速率，每次呼吸是由大约11积分值来表示。 </li><li>通过当鼠标是不活动的和体积描记的轨迹显示正常呼吸（正常的呼吸）（ 图4）与平均的时间段3秒的EMG信号相关联的整流和积分值确定基线EMG振幅。 </li><li>识别由至少3个连续的整流和积分值定义的活动的"较量"是至少有50％增加基线EMG信号之上（步骤6.3.2确定）。 注:三个连续的值代表一个90毫秒的窗口，但一些较量将包含高于阈值大于3点的值，并会持续更长的时间超过90毫秒。 </li><li>使用同步的视频和体积描记法跟踪排除叹息（ 图3B）期间发生的发作;嗅探（ 图3C）;或意志鼠标动作，诸如头部转动或修饰。 </li><li>重复步骤6.3.1 - 6.3.4第二肌肉。 </li></ol></li><li>计算每个肌肉回合频率。录制）每个不活动时段的开始时间和结束时间，和b）使用上述标准发生的每个回合的时间。总结总的无效时间。通过的非活动时间超过了记录会话来计算回合频率的过程中，总分钟除以较量的总数。 </li><li>确定是否在通风变化与所记录米激活相关uscles。 <ol><li>选择呼吸参数被测量（ 例如，峰值吸气流量，潮气量，每分钟通气量，每分钟呼吸）。 注意:所有可能的选择都可以在P3设置下拉菜单中找到"导出参数"。 </li><li>识别EMG活动回合期间发生的呼吸和EMG基线活动期间发生的呼吸（ 图4）。 </li><li>创建解析器段跨越了与EMG回合活动相关联，并创建与基线肌电活动相关的独立解析器段体积描记法呼吸。务必将分析的类型"解析器赛格。" 注意:这样的选择是根据P3设置下拉菜单中找到"数据还原设置"。 </li><li>标志与体积描记法跟踪事件通过右键单击每个解析器段的开始。指定包含解析器段为"事件1"的下拉菜单和SP A回合ecify基线解析器段为"事件2"以区分两类段。 </li><li>在"功能"菜单，保存"标志节"和"导出数据的痕迹。"根据数据解析器菜单，保存"经分析审查文件"和"经分析导出的数据。" 注:对于每个单独的呼吸所选择的呼吸参数在标记导出的数据表中标记的标签被发现"导"。 </li><li>比较呼吸参数用于在ARM发作（标记为事件1）相对于基线活性（标记为事件2）过程中发生的，以确定是否肌肉活动与在通风变化相关的呼吸发生的呼吸。 </li></ol></li></ol>

<ol>
	<li>Johnson, R. A., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+mechanisms+of+compensatory+respiratory+plasticity+in+spinal+neurological+disorders.">Common mechanisms of compensatory respiratory plasticity in spinal neurological disorders.</a> Respir Physiol Neurobiol. 189 (2), 419-428 (2013).</li><li>Sieck, G. C., Gransee, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Respiratory Muscles: Structure, Function &amp; Regulation. , (2012).</li><li>Rizzuto, E., Pisu, S., Musaro, A., Del Prete, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Neuromuscular+Junction+Functionality+in+the+SOD1(G93A)+Animal+Model+of+Amyotrophic+Lateral+Sclerosis.">Measuring Neuromuscular Junction Functionality in the SOD1(G93A) Animal Model of Amyotrophic Lateral Sclerosis.</a> Ann Biomed Eng. 43 (9), 2196-2206 (2015).</li><li>Kennel, P. F., Finiels, F., Revah, F., Mallet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuromuscular+function+impairment+is+not+caused+by+motor+neurone+loss+in+FALS+mice:+an+electromyographic+study.">Neuromuscular function impairment is not caused by motor neurone loss in FALS mice: an electromyographic study.</a> Neuroreport. 7 (8), 1427-1431 (1996).</li><li>Pinto, S., Alves, P., Pimentel, B., Swash, M., de Carvalho, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+for+assessment+of+diaphragm+in+ALS.">Ultrasound for assessment of diaphragm in ALS.</a> Clin Neurophysiol. 127 (1), 892-897 (2016).</li><li>Stewart, H., Eisen, A., Road, J., Mezei, M., Weber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electromyography+of+respiratory+muscles+in+amyotrophic+lateral+sclerosis.">Electromyography of respiratory muscles in amyotrophic lateral sclerosis.</a> J Neurol Sci. 191 (1-2), 67-73 (2001).</li><li>Arnulf, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+disorders+and+diaphragmatic+function+in+patients+with+amyotrophic+lateral+sclerosis.">Sleep disorders and diaphragmatic function in patients with amyotrophic lateral sclerosis.</a> Am J Respir Crit Care Med. 161, 849-856 (2000).</li><li>Bennett, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+muscle+activity+during+REM+sleep+in+patients+with+diaphragm+paralysis.">Respiratory muscle activity during REM sleep in patients with diaphragm paralysis.</a> Neurology. 62 (1), 134-137 (2004).</li><li>Pinto, S., de Carvalho, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+responses+of+the+sternocleidomastoid+muscle+in+patients+with+amyotrophic+lateral+sclerosis.">Motor responses of the sternocleidomastoid muscle in patients with amyotrophic lateral sclerosis.</a> Muscle Nerve. 38 (4), 1312-1317 (2008).</li><li>Romer, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessory+respiratory+muscles+enhance+ventilation+in+ALS+model+mice+and+are+activated+by+excitatory+V2a+neurons.">Accessory respiratory muscles enhance ventilation in ALS model mice and are activated by excitatory V2a neurons.</a> Exp Neurol. 287 (Pt. 2, 192-204 (2017).</li><li>Moldovan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+excitability+changes+related+to+axonal+degeneration+in+amyotrophic+lateral+sclerosis:+Insights+from+the+transgenic+SOD1(G127X)+mouse+model.">Nerve excitability changes related to axonal degeneration in amyotrophic lateral sclerosis: Insights from the transgenic SOD1(G127X) mouse model.</a> Exp Neurol. 233 (1), 408-420 (2012).</li><li>Pagliardini, S., Gosgnach, S., Dickson, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+sleep-like+brain+state+alternations+and+breathing+characteristics+in+urethane+anesthetized+mice.">Spontaneous sleep-like brain state alternations and breathing characteristics in urethane anesthetized mice.</a> PLoS One. 8 (7), 70411 (2013).</li><li>Nicaise, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phrenic+motor+neuron+degeneration+compromises+phrenic+axonal+circuitry+and+diaphragm+activity+in+a+unilateral+cervical+contusion+model+of+spinal+cord+injury.">Phrenic motor neuron degeneration compromises phrenic axonal circuitry and diaphragm activity in a unilateral cervical contusion model of spinal cord injury.</a> Exp Neurol. 235 (2), 539-552 (2012).</li><li>Akay, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+measurement+of+muscle+denervation+and+locomotor+behavior+in+individual+wild-type+and+ALS+model+mice.">Long-term measurement of muscle denervation and locomotor behavior in individual wild-type and ALS model mice.</a> J Neurophysiol. 111 (3), 694-703 (2014).</li><li>Tysseling, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+evaluation+of+a+chronic+EMG+multichannel+detection+system+for+long-term+recordings+of+hindlimb+muscles+in+behaving+mice.">Design and evaluation of a chronic EMG multichannel detection system for long-term recordings of hindlimb muscles in behaving mice.</a> J Electromyogr Kinesiol. 23 (3), 531-539 (2013).</li><li>Weiergraber, M., Henry, M., Hescheler, J., Smyth, N., Schneider, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrocorticographic+and+deep+intracerebral+EEG+recording+in+mice+using+a+telemetry+system.">Electrocorticographic and deep intracerebral EEG recording in mice using a telemetry system.</a> Brain Res Brain Res Protoc. 14 (3), 154-164 (2005).</li><li>Pilla, R., Landon, C. S., Dean, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+potential+early+physiological+marker+for+CNS+oxygen+toxicity:+hyperoxic+hyperpnea+precedes+seizure+in+unanesthetized+rats+breathing+hyperbaric+oxygen.">A potential early physiological marker for CNS oxygen toxicity: hyperoxic hyperpnea precedes seizure in unanesthetized rats breathing hyperbaric oxygen.</a> J Appl Physiol. 114 (1985), 1009-1020 (1985).</li><li>Morrison, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+inhibitory+amino+acids+in+control+of+hypoglossal+motor+outflow+to+genioglossus+muscle+in+naturally+sleeping+rats.">Role of inhibitory amino acids in control of hypoglossal motor outflow to genioglossus muscle in naturally sleeping rats.</a> J Physiol. 552 (Pt. 3, 975-991 (2003).</li><li>Tscharner, V., Eskofier, B., Federolf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+the+electrocardiogram+signal+from+surface+EMG+recordings+using+non-linearly+scaled+wavelets).">Removal of the electrocardiogram signal from surface EMG recordings using non-linearly scaled wavelets).</a> J Electromyogr Kinesiol. 21 (4), 683-688 (2011).</li><li>Hof, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+remove+ECG+artifacts+from+trunk+muscle+EMG+signals.">A simple method to remove ECG artifacts from trunk muscle EMG signals.</a> J Electromyogr Kinesiol. 19 (6), e554-e555 (2009).</li><li>Lu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removing+ECG+noise+from+surface+EMG+signals+using+adaptive+filtering.">Removing ECG noise from surface EMG signals using adaptive filtering.</a> Neurosci Lett. 462 (1), 14-19 (2009).</li></ol>

作者什么都没有透露。

这里展示的程序允许呼吸肌肉活动和通风的无创（发射机的初始手术植入后）测量了在相同的动物数月。该技术具有超过在麻醉的小鼠标准EMG技术的若干优点:1）在实验中需要更少的小鼠，并提供在跨越疾病阶段的单个小鼠（以记录的，而不是使用多个小鼠在不同的疾病阶段）来自同一个站点数据的能力; 2）数据分析可以与更强大的统计测试（ 即，使用重复测量的，而不是单独的比较实验组）来执行，; 3）EMG和WBP的同时记录允许的上通风ARM活动的影响的直接评估;和4）实验可以在睡眠/觉醒的不同状态的小鼠来进行。此外，由于健康小鼠都在休息ARM较量的频率非常低，这种技术是CA中的疾病10的早期症状阶段检测在ALS模型小鼠ARM活动的频率甚至微小的变化pable。但是，由于该技术测量大但未知数量的肌肉纤维的自然行为期间在实验上控制强度的活性，而不是遵循神经刺激，所以不适合用于估计电动机单元大小或数量。另一个限制是，适合于在小鼠植入基于遥测发射机目前只限于两组生物电势引线;因此，只有两个网站可以从相同的鼠标记录。对于需要在相同的鼠标多于两个肌肉的同时记录实验中，多个引线EMG可以植入并连接到使用导线系绳的采集系统，如前所述14，15。然而，修改的体积描记法腔室或密封件将被要求允许肌肉活动和通风的同时记录如果鼠标束缚。 当应用这种技术，该协议的某些步骤必须小心进行。生物电位导线必须放置，使它们不会妨碍运动或刺激覆皮肤。此外，发射器必须被定位成使得它不影响小鼠的正常运动或姿势。建议将引线（ 即，完全嵌入在正确肌肉和不接触相邻的肌肉）和缺乏肌肉损伤或感染的正确位置是通过尸检证实的实验已经终止之后。另外，至关重要的是，所述发射机被每个记录会话之后关闭以节省电池寿命。 在胸部和颈部区域从肌肉EMG测量的不可避免的结果是记录心电图（ECG）信号，其表现为重的高可能性gular的EMG迹（ 图3B，箭头）内的尖峰。 ECG信号可通过使得所有金属的肌肉完全嵌入引线的精心布局和避免靠近大血管位置最小化。植入引线插入身体，而不是左边，也就是靠近心脏的右侧的肌肉，也可减少ECG信号。尽管ECG信号可以被滤出EMG迹线用的计算算法的或减去一个独立地记录的ECG信号19，20，21，它通常不是必要的。所述ECG信号可以从由它的规则的形状，频率和振幅的信号EMG可以容易地区别。 所描述的技术已经被用于测量静止在SOD1（G93A）小鼠ALS 10的模型中ARM活性的变化。辅助呼吸肌也被招募OT她的神经肌肉疾病（ 例如，肌营养不良，脊髓性肌萎缩，周围神经病变等 ）和以下神经或脊髓损伤。因此ARM活性可以充当代理，以测量膜片的功能障碍和评估疾病的严重性，监视从损伤的恢复，或评估潜在治疗益处，以提高呼吸在多种动物疾病或损伤的模型。

在高需求（ 如运动）的时间辅助呼吸肌（ARMS）加大通风量，并有助于在隔膜功能受损以下损伤或疾病1，2，保持通风。虽然在光圈功能变化在肌萎缩侧索硬化（ALS）的患者和小鼠模型已经很好地描述3，4，5，6，较少被知道关于在ALS臂的活性或功能。然而，一项研究表明，招募ARM的ALS患者比那些具有类似隔膜功能障碍不7预后较好。此外，ARM活性足以在隔膜麻痹8的情况下呼吸。这些研究表明，战略，以增强ARM功能可以改善breathi纳克在从神经肌肉疾病，脊髓损伤，或在其中振动板功能受损的其它条件的患者。然而，控制ARM招聘呼吸的机制在很大程度上是未知的。方法来测量呼吸功能，并研究组是如何招募需要在疾病或损伤的动物模型时间ARM活动的变化，以及评价疗法来改善ARM招聘和通风。此外，手臂与光圈功能的渐进性丧失一致的增加的活性可以是用于神经肌肉疾病如ALS 7，9，10的疾病进展的有用的生物标志物。 该方案描述于非侵入性的方法（以下初始手术）和重复地测量在清醒，表现小鼠呼吸肌和通风的活性。肌电图的同步录音Y（EMG），全身体积描记法（WBP）和视频允许研究者评估ARM活动冲击通风如何变化，并确定当被摄体处于静止或移动的。这种方法的一个主要优点是，它可以在清醒，表现小鼠中执行，而一些可替代的方法来测量的EMG需要麻醉和/或是终端的程序11，12，13。 EMG活动的对清醒小鼠随时间的记录，也可以通过EMG的长期植入引线，其中，所述鼠标通过导线拴到采集系统14，15来完成的。因为栓系小鼠可与正常运动或行为干扰和可能不与一个标准的体积描记法室兼容，所描述的方法使用遥测装置以无线方式传输的EMG信号的采集系统。变送器可以上或关闭用磁铁以节省电池电量，并允许重复EMG活动的测量超过几个月。该协议可以很容易地适应通过将EMG测量的额外呼吸或非呼吸肌活性通向不同的肌肉。可替代地，两个引线中的一个可以被用于测量脑电活动来评估睡眠状态或识别发作活动16。该技术已成功用于测量在休息在整个疾病进展的ALS小鼠模型中ARM活性的变化，并确定关键的神经元驱动臂活动健康小鼠10。

<table><tbody><tr><td>B6.Cg-Tg (SOD1*G93A)1 Gur/J</td><td>Jackson Laboratory</td><td>4435</td><td></td></tr><tr><td>Plethysmography Chamber</td><td>Buxco Respiratory Products/ Data Sciences International</td><td>601-1425-001</td><td></td></tr><tr><td>Telemetry Receivers (Model RPC-1)</td><td>Data Sciences International</td><td>272-6001-001</td><td></td></tr><tr><td>Bias Flow Pump (Model BFL0500)</td><td>Data Sciences International</td><td>601-2201-001</td><td></td></tr><tr><td>ACQ-7000 USB</td><td>Data Sciences International</td><td>PNM-P3P-7002XS</td><td></td></tr><tr><td>Dataquest A.R.T. Data Exchange Matrix</td><td>Data Sciences International</td><td>271-0117-001</td><td></td></tr><tr><td>New Ponemah Analysis System</td><td>Data Sciences International</td><td>PNM-POST-CFG</td><td></td></tr><tr><td>Ponemah Physiology Platform Acqusition software v5.20</td><td>Data Sciences International</td><td>PNM-P3P-520</td><td></td></tr><tr><td>Ponemah Unrestrained Whole Breath Plethysmography analysis package v5.20</td><td>Data Sciences International</td><td>PNM-URP100W</td><td></td></tr><tr><td>Configured Ponemah Software System</td><td>Data Sciences International</td><td>PNM-P3P-CFG</td><td></td></tr><tr><td>Analysis Module (URP)</td><td>Data Sciences International</td><td>PNM-URP100W</td><td></td></tr><tr><td>Universal Amplifier</td><td>Data Sciences International</td><td>13-7715-59</td><td></td></tr><tr><td>Sync Board</td><td>Data Sciences International</td><td>271-0401-001</td><td></td></tr><tr><td>Sync Cable</td><td>Data Sciences International</td><td>274-0030-001</td><td></td></tr><tr><td>Transducer-Pressure Buxco</td><td>Data Sciences International</td><td>600-1114-001</td><td></td></tr><tr><td>Flow Meter</td><td>Data Sciences International</td><td>600-1260-001</td><td></td></tr><tr><td>Magnet and Radio included in F20-EET Starter Kit</td><td>Data Sciences International</td><td>276-0400-001</td><td></td></tr><tr><td>Axis P1363 Video Camera&amp;nbsp;&amp;nbsp;</td><td>Data Sciences International</td><td>275-0201-001</td><td></td></tr><tr><td>Terg-A-Zyme</td><td>Fisher Scientific</td><td>50-821-785</td><td>Enzyme Detergent</td></tr><tr><td>Actril</td><td>Minntech Corporation</td><td>78337-000</td><td>Chemical Sterilant</td></tr><tr><td>Stereo Dissecting Microscope (Model MEB126)</td><td>Leica</td><td>10-450-508</td><td></td></tr><tr><td>Servo-Controlled Humidifier/Infant Incubator</td><td>OHMEDA Ohio Care Plus</td><td>6600-0506-803</td><td></td></tr><tr><td>TL11M2-F20-EET Transmitters</td><td>Data Sciences International</td><td>270-0124-001</td><td></td></tr><tr><td>Dumont #2 Laminectomy Forceps - Standard Tips/Straight/12 cm (x2)&amp;nbsp;</td><td>Fine Scientific Instruments</td><td>11223-20</td><td>For handling wires</td></tr><tr><td>Dumont #2 Laminectomy Forceps - Standard Tips/Straight/12 cm (x2)</td><td>Fine Scientific Instruments</td><td>11223-20</td><td>For surgery</td></tr><tr><td>Narrow Pattern Forceps- Serrated/Curved/12 cm</td><td>Fine Scientific Instruments</td><td>17003-12</td><td></td></tr><tr><td>Spring Scissors - Tough Cut/Straight/Sharp/12.5 cm/6 mm Cutting Edge</td><td>Fine Scientific Instruments</td><td>15124-12</td><td></td></tr><tr><td>Tissue Separating Scissors - Straight/Blunt-Blunt/11.5 cm</td><td>Fine Scientific Instruments</td><td>14072-10</td><td></td></tr><tr><td>Fine Scissors - Tough Cut/Curved/Sharp-Sharp/9 cm&amp;nbsp;</td><td>Fine Scientific Instruments</td><td>14058-11</td><td>For cutting wires and clipping nails</td></tr><tr><td>Scalpel Handle #3</td><td>World Precision Instruments</td><td>500236</td><td></td></tr><tr><td>Scalpel Blade</td><td>Fine Scientific Instruments</td><td>10010-00</td><td>For preparing lead caps</td></tr><tr><td>Polysorb Braided Absorbable suture</td><td>Coviden</td><td>D4G1532X</td><td>For coiling transmitter leads</td></tr><tr><td>Gluture&amp;nbsp;</td><td>Zoetis Inc.</td><td>6606-65-1</td><td>Cyanoacrylate adhesive</td></tr><tr><td>3 mL Syring Slip Tip - Soft</td><td>Vitality Medical</td><td>118030055</td><td></td></tr><tr><td>25 G Needle (x2)</td><td>Becton Dickinson and Co.</td><td>305-145</td><td></td></tr><tr><td>Cotton Tipped Applicators</td><td>Henry Schein Animal Health</td><td>100-9175</td><td></td></tr><tr><td>Andis Easy Cut Hair Clipper Set</td><td>Andis</td><td>049-06-0271</td><td>Electrical Razor sold at Target</td></tr><tr><td>Isoflurane</td><td>Henry Schein Animal Health</td><td>29404</td><td>Anesthetic&amp;nbsp;</td></tr><tr><td>Isopropyl Alcohol 70%</td><td>Priority Care 1</td><td>MS070PC</td><td></td></tr><tr><td>Dermachlor 2% Medical Scrub (chlorohexidine 2%)</td><td>Butler Schein</td><td>55482</td><td></td></tr><tr><td>Artificial Tears</td><td>Henry Schein Animal Health</td><td>48272</td><td>Lubricant Opthalmic Ointment</td></tr><tr><td>Vacuum grease</td><td>Dow Corning Corporation</td><td>1597418</td><td></td></tr><tr><td>Water Blanket</td><td>JorVet</td><td>JOR784BN</td><td></td></tr></tbody></table>

repeated measurement of respiratory muscle activity and ventilation in mouse models of neuromuscular disease

辅助呼吸肌帮助时膈肌功能受损保持通风。以下方案描述了一种用于在数周或辅助呼吸肌肉活动的个月，同时测量在非麻醉，自由地表现小鼠通风重复测量的方法。该技术包括一个无线电发射器的外科植入和电极的插入通向斜角肌和斜方肌测量这些吸气肌肉的肌电活动。通风是通过全身体积描记术测量的，和动物运动由视频评估，并与肌电活动同步。肌萎缩侧索硬化症的小鼠模型中的肌肉活动和通风的测量都显示这个工具如何可以用来研究肌肉活动如何随时间变化的呼吸和评估肌肉活动对通风的影响。所描述的方法可以通过电子邮件asily适合于测量其它肌肉的活动或疾病或损伤的额外的鼠标模型来评估辅助呼吸肌活动。

所描述的方案用于植入遥测设备，并记录和斜角肌斜方肌EMG，WBP和SOD1（G93A）ALS模型小鼠的视频。时段，其中所述动物是无活性的（ 例如，不动）用的视频记录确定并通过在微量WBP（ 图3A）缺乏运动相关的活动的确认。不活动时段包括在REM或非REM睡眠所花费的时间，以及时间花费清醒，但仍（ 图3A）。在这个非激活时间EMG活动记录为一个回合当至少3个连续的整流和集成（超过30毫秒）值有幅度与至少增加了50％以上的基线EMG水平（ 图4）。叹息或嗅探（体积描记法通过测定）过程中发生的活动的发作，或意志运动（由视频评估）从分析中排除（ 图3B-C）。在EA SOD1（G93A）小鼠rly-到阶段中期症状（ 见表1）增加的ARM活性表现出较量静止那个持续一到几个呼吸（ 图4）。 ARM活性的发作是在症状前SOD1（G93A）（ 图3A）或野生型小鼠的10罕见。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 阶段 </td><td> 州 </td><td> 发病期 </td><td> 后肢介绍 </td></tr><tr><td> 0 </td><td>症状前</td><td> &lt;P100 </td><td>无显着差异相比wildtypes。 </td></tr><tr><td> 1 </td><td>发病</td><td> 〜P100 </td><td>当小鼠从尾悬挂后肢崩溃。 </td></tr><tr><td> 2 </td><td>麻痹</td><td> 〜P120 </td><td>全部或部分坍塌后肢震颤的外观。 </td> &lt;/ TR&gt; <tr><td> 3 </td><td>麻痹发作</td><td> 〜P140 </td><td>行走困难，脚趾卷曲和/或脚拖动。 </td></tr><tr><td> 4 </td><td>高级瘫痪</td><td> 〜P150 </td><td>最小关节运动，后肢不能被用于向前运动。 </td></tr><tr><td>五</td><td>终末期</td><td> 〜P160 </td><td>鼠标无法从侧面30秒内权利本身。 </td></tr></tbody></table> 表1. ALS样疾病进展的神经学评分在SOD1（G93A）小鼠。 <img alt="图3" src="/files/ftp_upload/55599/55599fig3.jpg" /> 图3. 代表WBP和肌电图的痕迹。 （A - C）斜角肌和斜方肌的从预有症状的SOD1（G93A）小鼠（年龄P98）WBP和EMG。 （A）期间当t<html>他的动物处于休息状态（红色框）用于分析。红外箱跟踪显示在体积描记迹线和肌肉活动大的和不规则的峰在EMG迹线，典型当动物正在移动，如由同步视频记录确定（未示出）。红色框显示EMG迹线缺乏EMG较量，症状前小鼠的特性。 （B）EMG活动经常发生叹息直接前面（如图体积描记迹线）的较量。叹息由高振幅灵感随后戏剧性期满表征。黑箭头指向特性ECG信号。 （C）的EMG活动的较量而鼠标嗅探频繁地发生。嗅探是通过在频率和振幅在多个呼吸（与EMG活动的突发共同存在的）延长的增加反映在体积描记法跟踪。颜色="＃0066CC"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/55599/55599fig4.jpg" /> 图4. EMG活动的评分较量 。 （A和B）WBP的两个例子，过滤斜方肌EMG痕迹，并整流，并从有症状的SOD1（G93A）小鼠（年龄P126）集成斜方肌的EMG信号。蓝色虚线表示基线水平EMG，通过在3秒的时间段平均整流和积分信号来确定。红色虚线表示的幅度增长比基线EMG活动了50％。当至少3个连续的整流和积分值超过50％的基线阈活性的回合进行评分。 <a href="http://ecsource.jove.com/files/ftp_upload/55599/55599fig4large.jpg" target="_blank">请点击此处查看人</a>arger版本这个数字。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td> PIF（毫升/秒） </td><td> TV（毫升） </td><td> MV（mL /分钟） </td><td> 呼吸频率（次/ min） </td></tr><tr><td> 幼稚（N = 5） </td><td> 4.4±0.7 </td><td> 0.27±0.04 </td><td> 58±13 </td><td> 223±41 </td></tr><tr><td> 植入（N = 4） </td><td> 4.1±0.2 </td><td> 0.27±0.11 </td><td> 56±29 </td><td> 201±32 </td></tr><tr><td> P值 </td><td> 0.439 </td><td> 1.000 </td><td> 0.893 </td><td> 0.410 </td></tr><tr><td colspan="5">所示的值反映平均值±SD。 P值用学生t检验来计算。 </td></tr></tbody></table>幼稚（未注入）并植入阶段4 SOD1（G93A）小鼠之间的呼吸的表2的比较。无显著差异在峰值吸气流量（PIF），潮气量（TV），每分钟通气量（MV），或呼吸每分钟两组间被发现。示出的值反映了平均值±SD。 P值用学生t检验来计算。 EMG和/或WBP的重复测量可以在相同的鼠标在几个月内进行，与手术后的1到2周的恢复期后肌电信号或基线变化很小。的时间过程通常是由电池寿命的限制，因此将通过各个记录的频率和持续时间来确定。研究人员应该知道，偶尔可能会发生不良事件，由于植入的装置。鼠标可能拔出从植入的肌肉或划痕/咀嚼导线在皮肤如果导线或发射之三是放置不当。在大多数情况下，道德因素决定了这些动物被牺牲掉。发射器可被移除，灭菌，并且在另一个小鼠再植入。 要验证装置植入并不影响呼吸，幼稚SOD1（G93A）小鼠（未注入）在ALS阶段4和植入SOD1（G93A）小鼠在ALS阶段4之间体积描记术的测量进行比较。无显著差异在峰值吸气流量（PIF），潮气量（TV），每分钟通气量（MV），或呼吸每分钟两个组（ 表2）之间被发现了。斜角肌和斜方肌是彼此相邻的，并且直接与彼此接触。虽然同时EMG较量在两个肌肉有时观察到的，强EMG较量也在当EMG较量处于斜角肌（反之亦然）缺席斜方肌检测，表明存在最小植入我电极之间的串扰n各自的肌肉。 （数据未显示），当引线被放置在斜方肌和胸锁乳突肌还观察到EMG活动的独立的较量。

Watch this Scientific Journal Video about Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease at JoVE.com

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

本文介绍了通过植入遥测装置在整个疾病进展全身体积描记术和肌电自由表现肌萎缩侧索硬化症（ALS）小鼠模型中的通风和呼吸肌肉活动的重复测量的方法。

在神经肌肉疾病的模型小鼠呼吸肌肉活动和通风的重复测量

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...

repeated-measurement-respiratory-muscle-activity-ventilation-mouse

Universidad San Sebastian

Research

JoVE Journal

Medicine

12.9K 次观看.  University of Cincinnati. 本文介绍了通过植入遥测装置在整个疾病进展全身体积描记术和肌电自由表现肌萎缩侧索硬化症（ALS）小鼠模型中的通风和呼吸肌肉活动的重复测量的方法。 

视频: Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

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.

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

We present refined protocols that allow in vivo monitoring of motor unit function in the mouse. Techniques to measure compound muscle action potential (CMAP) and motor unit number estimation (MUNE) in the mouse hind limb muscles innervated by the sciatic nerve are described.

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 ...

科研

行为学

A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation in mouse models is a non-invasive and reproducible activity assay that is directly analogous to measures of patient ambulation such as the 6-minute walk test and related mobility scores. Many common methods for testing mouse ambulation speed and distance are based on the open field test, where an animal&#39;s free movement within an arena is measured over time. One major downside to this approach is that commercial software and equipment for high-resolution motion tracking is expensive and may require transferring mice to specialized facilities for testing. Here, we describe a low-cost, video-based system for measuring mouse ambulation that utilizes free and open-source software. Using this protocol, we demonstrate that voluntary ambulation in the dystrophin-null mdx mouse model for Duchenne muscular dystrophy (DMD) is decreased relative to wild-type mouse activity. In mdx mice expressing the utrophin transgene, these activity deficits are not observed and the total distance traveled is indistinguishable from wild-type mice. This method is effective for measuring changes in voluntary ambulation associated with dystrophic pathology, and provides a versatile platform that can be readily adapted to diverse research settings.

This protocol describes a flexible, low-cost system for measuring mouse ambulation in an open field activity assay. We show that a 6-minute ambulation assay based on this system detects a decrease in voluntary movement in mdx mice, and accurately distinguishes improvement in a muscle-specific rescue of these animals.

一种简便、低成本的肌营养不良小鼠步行模型的测定方法

Measuring functional outcomes in the treatment of muscular dystrophy is an essential aspect of preclinical testing. The assessment of voluntary ambulation ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

评价呼吸肌慢性脊髓损伤个人使用呼吸电机控制评估（RMCA）

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

医学

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

生物学

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

使用强迫振动技术在小鼠呼吸系统力学评价

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

Assessment of Respiratory Function in Conscious Mice by Double-chamber Plethysmography

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to non-invasively assess some features of the respiratory function in humans as well as in laboratory animals. The present article focuses on the application of the double-chamber plethysmography (DCP) in small animals. It provides background information on the methodology as well as a detailed step-by-step procedure to successfully assess respiratory function in conscious, spontaneously breathing animals in a non-invasive manner. The DCP can be used to monitor the respiratory function of multiple animals in parallel, as well as to identify changes induced by aerosolized substances over a chosen time period and in a repeated manner. Experiments on control and allergic mice are used herein to demonstrate the utility of the technique, explain the associated outcome parameters, as well as to discuss the related advantages and shortcomings. Overall, the DCP provides valid and theoretically sound readouts that can be trusted to evaluate the respiratory function of conscious small animals both at baseline and after challenges with aerosolized substances.

The objective of the present article is to provide a detailed description of the recommended procedures to evaluate respiratory function in conscious mice by double-chamber plethysmography.

双室 Plethysmography 对清醒小鼠呼吸功能的评价

Air volume changes created by a conscious subject breathing spontaneously within a body box are at the basis of plethysmography, a technique used to ...

Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

The assessment of respiratory physiology has traditionally relied upon techniques, which require restraint or sedation of the animal. Unrestrained whole-body plethysmography, however, provides precise, non-invasive, quantitative analysis of respiratory physiology in animal models. In addition, the technique allows repeated respiratory assessment of mice allowing for longitudinal studies.

用奔放的整体体积描记测量呼吸功能的影响

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative ...

<meta charset="utf8"></head><body>量化神经退行性啮齿动物模型中的膈运动单位功能

Assessing Rat Diaphragm Motor Unit Connectivity Outcome Measures as Quantitative Biomarkers of Phrenic Motor Neuron Degeneration and Compensation

Loss of ventilatory muscle function is a consequence of motor neuron injury and neurodegeneration (e.g., cervical spinal cord injury and amyotrophic lateral sclerosis, respectively). Phrenic motor neurons are the final link between the central nervous system and muscle, and their respective motor units (groups of muscle fibers innervated by a single motor neuron) represent the smallest functional unit of the neuromuscular ventilatory system. Compound muscle action potential (CMAP), single motor unit potential (SMUP), and motor unit number estimation (MUNE) are established electrophysiological approaches that enable the longitudinal assessment of motor unit integrity in animal models over time but have mostly been applied to limb muscles. Therefore, the objectives of this study are to describe an approach in preclinical rodent studies that can be used longitudinally to quantify the phrenic MUNE, motor unit size (represented as SMUP), and CMAP, and then to demonstrate the utility of these approaches in a motor neuron loss model. Sensitive, objective, and translationally relevant biomarkers for neuronal injury, degeneration, and regeneration in motor neuron injury and diseases can significantly aid and accelerate experimental research discoveries to clinical testing.

<meta charset="utf8"></head><body>作者聚焦：研究神经退行性疾病中的神经肌肉反应和运动神经元可塑性

In this study, we present an in vivo method for estimating motor unit number and size to quantify rat diaphragm motor unit connectivity. A step-by-step approach to these techniques is described.

评估大鼠膈肌运动单元连接结果测量作为膈运动神经元变性和代偿的定量生物标志物

Loss of ventilatory muscle function is a consequence of motor neuron injury and neurodegeneration (e.g., cervical spinal cord injury and amyotrophic ...

在神经肌肉疾病的模型小鼠呼吸肌肉活动和通风的重复测量

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此视频中的章节

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）

一种简便、低成本的肌营养不良小鼠步行模型的测定方法

评价呼吸肌慢性脊髓损伤个人使用呼吸电机控制评估（RMCA）

一个可逆的，非侵入性方法，气道阻力测量和支气管肺泡灌洗小鼠流体取样

使用强迫振动技术在小鼠呼吸系统力学评价

双室 Plethysmography 对清醒小鼠呼吸功能的评价

用奔放的整体体积描记测量呼吸功能的影响

评估大鼠膈肌运动单元连接结果测量作为膈运动神经元变性和代偿的定量生物标志物

评估大鼠膈肌运动单元连接结果测量作为膈运动神经元变性和代偿的定量生物标志物

评估大鼠膈肌运动单元连接结果测量作为膈运动神经元变性和代偿的定量生物标志物