这项工作得到了国家卫生研究院的资助 [R01 AR048179 和 R01 HL126204 到钢筋混凝土 W, T32 AR059033 和 F32 AR069469to 福斯特 G] 和肌肉萎缩症协会美国 [274143 和416364至 R.C.W.]。

这里描述的方法得到了加州大学洛杉矶分校动物保育和使用委员会的批准。野生类型 (C57BL/6J) 和mdx小鼠 (C57Bl/10ScSn 背景) 是从商业来源购买的。mdx: utrophin-Tg 小鼠是来自 Ervasti 的礼物, 得到了凯戴维斯的许可, 并在mdx背景上进行了维护。6月龄雄性小鼠进行了步行试验。老鼠在 Terasaki 生命科学 Vivarium 被维护了由动物看护和用途委员会建立的指南在加利福尼亚大学洛杉矶 (批准 #2000-029-43) 和批准为这些研究被授予由加州大学洛杉矶分校动物福利保证 (批准 #A3196-01)。
1. 分庭的筹备工作
<ol>
	<li>在一天的9的一致时间内, 在安静、温度控制的房间中执行数据收集。对被测试的小鼠进行基因型和治疗的盲测试。</li>
	<li>对任何开放的野外室系统进行适应性分析。 注意: 在这项研究中, 我们使用一个廉价的, 易于运输的记录室建造从一个大的垃圾箱。野外竞技场是一个托盘设置在笼或类似的高架平台。</li>
	<li>把相机放在房间上方的铁丝网上, 在整个竞技场上记录下来。</li>
	<li>每次试验前用消毒剂清洗记录室。</li>
</ol>
2. 演习前的协议和数据收集
<ol>
	<li>(可选) 在活动录制之前, 使用肌肉激活协议挑战每个鼠标。 注意: 这是可选的, 但在涉及mdx小鼠的实验中推荐使用。<ol>
		<li>允许鼠标握住数字力计的拉杆, 然后轻轻地向后拉动, 直到松开拉杆。每次试用重复此步骤五次。</li>
		<li>记录每项试验的峰值张力 (N)。 注意: 除了作为运动试验的挑战, 在这一握力测定过程中, 动物施加的力可以作为一个额外的功能结果测量2,10。</li>
		<li>对每只老鼠进行五次总试验, 每次试验1分钟休息。</li>
	</ol>
	</li>
	<li>紧跟握力测定或其他运动协议后, 将鼠标置于活动室。</li>
	<li>启动在会议厅竞技场的鼠标移动的视频记录。允许鼠标自由探索6分钟。</li>
	<li>停止录制在6分钟, 并将鼠标返回到其主笼。</li>
</ol>
3. 视频分析
<ol>
	<li>(可选) 通过降低帧速率为分析准备视频。为了减少跟踪分析的帧速率, 请在视频编辑软件 (例如、iMovie 或类似程序) 中使用以下协议, 将视频帧速率降低2倍。 注意: 根据视频的长度和帧速率, 在分析前降低视频帧速率可能是很有用的。在本研究中, 录像记录在30帧/秒 (6 分钟录音, 约10800帧总数)。<ol>
		<li>将视频加载到软件中。</li>
		<li>在 "速度" 菜单下, 选择 "速度: 快速", 并设置速度为2x。</li>
		<li>以. mp4 文件格式导出被破坏的视频以进行跟踪分析。</li>
	</ol>
	</li>
	<li>在软件程序中打开视频进行分析。使用 "线条" 工具设置视频录制的校准。沿着房间的一侧画一条线。右键单击该行, 然后选择 "校准测量..."以厘米为单位输入实际大小的腔室侧。</li>
	<li>要开始半自动跟踪鼠标位置, 请单击 "移动" 光标。 从视频的初始帧开始, 右键单击要跟踪的点;跟踪点用蓝色圆圈标记。 注意: 在本研究中, 每只动物的位置都通过跟踪尾巴的底部来跟踪。</li>
	<li>通过单击键盘上的右箭头来前进框架;跟踪点应根据尾部底座的位置自动移动。<ol>
		<li>如果跟踪位置与给定帧中的兴趣点不一致, 请手动将蓝色圆圈与尾部的底座对齐。 注意: 根据视频的质量和鼠标的速度, 跟踪可能需要不同级别的用户输入, 以保持与感兴趣点的对准。鼠标的路径应该在视频过程中前进。</li>
	</ol>
	</li>
	<li>完整视频的跟踪完成后, 保存视频和跟踪覆盖。通过选择 "导出到电子表格" 导出跟踪的位置数据。</li>
</ol>
4. 数据分析
<ol>
	<li>在电子表格软件程序中打开位置数据。移动分析软件在每个框架中报告鼠标的 X、Y 坐标位置。</li>
	<li>要计算帧移动的距离, 请使用以下公式 (其中第一帧的位置为 x1, y1 , 第二帧为 x2, y2): 
	<img alt="Equation" src="/files/ftp_upload/56772/56772eq1.jpg"> 注: 随着时间的推移, 累积的距离可以通过增加每个帧之间的距离来计算。</li>
</ol>

<ol><li>Nagaraju, K., Carlson, G., De Luca, A. <a target="_blank" href="http://www.treat-nmd.eu/downloads/file/sops/dmd/MDX/DMD_M.2.1_002.pdf">Behavioral and locomotor measurements using open field animal activity monitoring system.</a> TREAT-NMD SOP Number M2.1.002. 2. 2, (2010).</li>
<li>Tatem, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+and+locomotor+measurements+using+an+open+field+activity+monitoring+system+for+skeletal+muscle+diseases%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Behavioral and locomotor measurements using an open field activity monitoring system for skeletal muscle diseases.</a> J Vis Exp. (91), e51785(2014).</li>
<li>Kobayashi, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sarcolemma-localized+nNOS+is+required+to+maintain+activity+after+mild+exercise%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Sarcolemma-localized nNOS is required to maintain activity after mild exercise.</a> Nature. 456 (7221), 511-515 (2008).</li>
<li>Belanto, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microtubule+binding+distinguishes+dystrophin+from+utrophin%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Microtubule binding distinguishes dystrophin from utrophin.</a> Proc Natl Acad Sci U S A. 111 (15), 5723-5728 (2014).</li>
<li>McDonald, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+6-minute+walk+test+in+Duchenne%2FBecker+muscular+dystrophy%3A+longitudinal+observations%5BTitle%5D)+AND+%22Muscle+Nerve%22%5BJournal%5D)">The 6-minute walk test in Duchenne/Becker muscular dystrophy: longitudinal observations.</a> Muscle Nerve. 42 (6), 966-974 (2010).</li>
<li>Mazzone, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Star+Ambulatory+Assessment%2C+6-minute+walk+test+and+timed+items+in+ambulant+boys+with+Duchenne+muscular+dystrophy%5BTitle%5D)+AND+%22Neuromuscul+Disord%22%5BJournal%5D)">Star Ambulatory Assessment, 6-minute walk test and timed items in ambulant boys with Duchenne muscular dystrophy.</a> Neuromuscul Disord. 20 (11), 712-716 (2010).</li>
<li>Kobayashi, Y. M., Rader, E. P., Crawford, R. W., Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endpoint+measures+in+the+mdx+mouse+relevant+for+muscular+dystrophy+pre-clinical+studies%5BTitle%5D)+AND+%22Neuromuscul+Disord%22%5BJournal%5D)">Endpoint measures in the mdx mouse relevant for muscular dystrophy pre-clinical studies.</a> Neuromuscul Disord. 22 (1), 34-42 (2012).</li>
<li>Acosta, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+the+six-minute+walk+test+to+characterize+golden+retriever+muscular+dystrophy%5BTitle%5D)+AND+%22Neuromuscul+Disord%22%5BJournal%5D)">Use of the six-minute walk test to characterize golden retriever muscular dystrophy.</a> Neuromuscul Disord. 26 (12), 865-872 (2016).</li>
<li>Sousa, N., Almeida, O. F., Wotjak, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+hitchhiker%27s+guide+to+behavioral+analysis+in+laboratory+rodents%5BTitle%5D)+AND+%22Genes+Brain+Behav%22%5BJournal%5D)">A hitchhiker's guide to behavioral analysis in laboratory rodents.</a> Genes Brain Behav. 5, Suppl 2. 5-24 (2006).</li>
<li>Gibbs, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+levels+of+sarcospan+are+well+tolerated+and+act+as+a+sarcolemmal+stabilizer+to+address+skeletal+muscle+and+pulmonary+dysfunction+in+DMD%5BTitle%5D)+AND+%22Hum+Mol+Genet%22%5BJournal%5D)">High levels of sarcospan are well tolerated and act as a sarcolemmal stabilizer to address skeletal muscle and pulmonary dysfunction in DMD.</a> Hum Mol Genet. 25 (24), 5395-5406 (2016).</li>
<li>Gillis, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multivariate+evaluation+of+the+functional+recovery+obtained+by+the+overexpression+of+utrophin+in+skeletal+muscles+of+the+mdx+mouse%5BTitle%5D)+AND+%22Neuromuscul+Disord%22%5BJournal%5D)">Multivariate evaluation of the functional recovery obtained by the overexpression of utrophin in skeletal muscles of the mdx mouse.</a> Neuromuscul Disord. 12, Suppl 1. S90-S94 (2002).</li>
<li>Tinsley, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+full-length+utrophin+prevents+muscular+dystrophy+in+mdx+mice%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Expression of full-length utrophin prevents muscular dystrophy in mdx mice.</a> Nat Med. 4 (12), 1441-1444 (1998).</li>
<li>Song, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Suite+of+clinically+relevant+functional+assays+to+address+therapeutic+efficacy+and+disease+mechanism+in+the+dystrophic+mdx+mouse%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Suite of clinically relevant functional assays to address therapeutic efficacy and disease mechanism in the dystrophic mdx mouse.</a> J Appl Physiol. 122 (3), 593-602 (2017).</li>
<li>Bolivar, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intrasession+and+intersession+habituation+in+mice%3A+from+inbred+strain+variability+to+linkage+analysis%5BTitle%5D)+AND+%22Neurobiol+Learn+Mem%22%5BJournal%5D)">Intrasession and intersession habituation in mice: from inbred strain variability to linkage analysis.</a> Neurobiol Learn Mem. 92 (2), 206-214 (2009).</li>
</ol>

作者没有什么可透露的。

野外试验提供了一种有效而非侵入性的方法来检测肌肉疾病的小鼠模型中的运动功能, 而这种检测的变异可以作为临床前研究的一项重要的终点措施。这类化验的一个主要限制是活动监测系统的高成本和有限的可及性。在这项研究中, 我们展示了一个低成本的开放现场活动分析系统, 可以产生类似的结果, 现有的商业软件和设备。该系统可以在 vivaria 的地方使用, 从而避免了将动物转移到核心设施的需要。这一分析的多功能性也使得在各种研究和教育设置中使用视频跟踪, 包括以动物生理学和行为为重点的本科生实验课程。
在这项研究中, 我们发现, 野生类型和mdx小鼠之间的自愿运动的差异, 可以很容易地发现后, 通过运动的恶化。这一强大的差异已经报告了其他团体的研究类似的活动结果, 包括 hour-long 开放现场化验和隔夜饲养化验。虽然我们观察到未野生类型和mdx小鼠之间的自愿移动没有区别, 但这与其他组13的结果是一致的。一些研究人员报告了未野生类型和mdx鼠标之间在自愿移动距离上的差异,7, 而其他报告没有差异13。我们的发现支持在测试之前使用肌肉激活, 以便可靠地检测mdx活动中的统计学意义差异。
在这项研究中, 我们提出了一个新的开放领域环境的小鼠。使用这一新的环境协议, 我们观察到小组内部很少有变化。但是, 当鼠标放置在不熟悉的环境中时, 它们的活动往往会提高14。如果同样的动物在多个疗程中被测试, 动物应该习惯于记录室并且在记录数据之前多次接受完整的测试协议。
从野外试验中评估数据的一个重要的考虑因素是它对动物的行为和情绪状况非常敏感, 尤其是焦虑样的行为。mdx可能对新环境具有不同的行为响应, 而不是野生类型的控制动物。然而, 我们另外测试了肌肉特异的抢救, mdx: utr-Tg 老鼠, 表达 utrophin 在肌肉特定促进者之下。这些小鼠的表现和野生类型的动物一样, 表明在mdx小鼠的任何行为差异都不会推动自愿移动的改变。
野外试验广泛应用于行为范式以及运动功能的研究中, 有多种参数可以从活动监测系统中记录。在商业活动分析软件中常用的一些测量方法包括自动检测特定行为、转向频率、垂直移动以及特定感兴趣区域的偏好。本研究采用的6分钟步行距离法, 其输出量主要限于水平移动距离和速度。
此外, 由于在本协议中半自动跟踪所需的用户输入级别, 这种检测可能不太适合在较长时间内进行活动监视或同时测量多个动物。在这项研究中, 我们发现, 训练有素的用户可以处理和提取数据的视频约15-30 分钟每记录。虽然这只代表了 small-scale 实验的适度时间负担, 但对于在较长时间内观察大型样本集的研究而言, 这一水平的分析将很快变得望而却步。虽然更多的全自动商业跟踪系统通常成本超过2万美元, 这些解决方案可能是更成本-和时间为实验室的移动经常测试。然而, 对于只测试少量动物的实验室和教育环境, 这种方法的简单设置和重现性为评估运动行为提供了一个有价值的工具。
可利用的数据分析类型是在确定临床前研究的适当终点化验中的重要考虑因素。当我们能够通过6分钟移动距离 (野生类型、 mdx和mdx: utr-tg) 来一致地检测出三种基因型之间的差异时, 对 longer-term 活动的更复杂的分析可能是可取的, 当看微妙自愿行动的变化。但是, 这种低成本设置的易用性和简单性使它成为mdx鼠标模型中前临床研究的有用和高度相关的功能测试。

可靠和可重复的测量肌肉功能是关键的评估潜在的治疗 DMD 的疗效。DMD 是由肌营养不良蛋白基因突变引起的遗传性疾病, 导致渐进性肌肉无力, 移动丧失, 最终心肺衰竭。最广泛使用的 DMD 的动物模型是肌营养不良蛋白-空mdx鼠标。在mdx鼠标以及其他营养和病的类似动物模型中, 一组功能测试已成为评估疾病进展的常规方法。通常使用的在体内测试包括测量前肢握力, 导线悬挂时间, rotarod 最大, 在跑步机跑步的时间和运动活动跟踪的疲劳。在这一领域, 为了使这些测试标准化, 有了很大的努力, 目的是减少前临床研究的变异性, 提高在小鼠体内测试的治疗的转化潜能1,2。
临床前测试的一个重要类别是测量自愿运动, 这一参数在小鼠肌肉萎缩症模型中经常被改变。这通常是通过基于野外活动监测的分析来测试的, 并且可以在几分钟或数小时的过程中评估水平 (行走) 或垂直 (饲养) 运动,2,3,4。一些研究表明, 在mdx小鼠中, 特别是在运动之后, 自愿改变的运动, 这些测量结果对药物治疗和疾病进展很敏感。执行这些化验的一个主要限制是需要专门的、高成本的设备。在这里, 一个低成本的方法, 跟踪鼠标移动使用现成的资源。
6分钟步行距离是一种常用的标准作为临床评估工具的个人杜氏肌营养不良症5,6。这项措施的修改已被用来评估杜氏动物模型的结果, 包括mdx小鼠7和金毛猎犬肌肉萎缩症 (GRMD) 狗8。在这项研究中, 我们记录自愿开放野外运动在6分钟后, 紧接着一个温和的运动挑战。然后使用自由开源软件计算移动距离, 以测量随时间推移的水平移动。
这种方法的主要优点是可以在不同的环境中测试动物, 而无需专门的设备或昂贵的商业软件进行分析。这一分析的一个重要方面是, 它可以在一个基本的实验室设置, 而无需移动或转移到一个专门的核心设施 vivarium 小鼠。这里描述的视频跟踪协议非常适合于对相对较短的青年进行移动评估, 并且能够检测出野生类型和mdx小鼠之间的活动差异, 并揭示了救援中的功能改进模型的 DMD。

<table><tbody><tr><td>Video camera</td><td>Apple Inc.</td><td>ME553LL/A</td><td>For recording ambulation video, iPhone 5S Plus (or equivalent)</td></tr><tr><td>Kinovea software (version 0.8.15)</td><td>Kinovea Association</td><td></td><td>Open source video analysis software. Free download, PC compatible (Version 0.8.15, www.kinovea.org)</td></tr><tr><td>iMovie (version 10.0.6)</td><td>Apple Inc.</td><td></td><td>Any similar software can be used to reduce video frame rate (optional)</td></tr><tr><td>Roughneck 32 Gallon Black Round Trash Bin (Open field chamber)</td><td>Rubbermaid</td><td># 1778013</td><td>Any open field chamber system can be adapted for recording. This study uses a recording chamber constructed out of a tray on a platform, at the bottom of a large trash bin.</td></tr><tr><td>Avant White Plastic Tray 15&quot;W x 10&quot;D x 1.45&quot;H (Open field chamber)</td><td>US Acrylic, LLC</td><td></td><td>Any open field chamber system can be adapted for recording. This study uses a recording chamber constructed out of a tray on a platform, at the bottom of a large trash bin.</td></tr><tr><td>C57BL/6J</td><td>Jackson Laboratory</td><td>#000664</td><td>Male 6 month mice</td></tr><tr><td>C57BL/10ScSn-Dmd/J (&lt;em&gt;mdx&lt;/em&gt;)</td><td>Jackson Laboratory</td><td>#001801</td><td>Male 6 month mice</td></tr><tr><td>&lt;em&gt;mdx:&lt;/em&gt; utrophin-Tg (fiona)</td><td>Gift from from James Ervasti, with permission from Kay Davies</td><td></td><td>Male 6 month mice</td></tr></tbody></table>

a simple and low-cost assay for measuring ambulation in mouse models of muscular dystrophy

在肌营养不良症的治疗中, 测量功能性结局是临床前测试的一个重要方面。在小鼠模型中自愿移动的评估是一种非侵入性的和可重复的活性测定方法, 与患者步行6分钟的测量和相关的移动评分直接类似。许多测试鼠标移动速度和距离的常用方法都是基于野外试验, 在这个测试中, 动物在竞技场中的自由运动是随着时间的推移而测量的。这种方法的一个主要缺点是, 用于高分辨率运动跟踪的商用软件和设备成本高昂, 可能需要将鼠标转移到专门的设备进行测试。在这里, 我们描述了一个低成本, 视频的测量鼠标移动的系统, 利用自由和开源软件。使用这个协议, 我们证明, 自愿移动在肌营养不良蛋白-无效的假mdx小鼠模型的杜氏肌肉萎缩症 (DMD) 是减少相对于野生类型的鼠标活动。在表达 utrophin 转基因的mdx小鼠中, 没有观察到这些活动的缺陷, 并且总的移动距离与野生型小鼠是无法区分的。这种方法是有效的测量变化的自愿步行与不良病理学, 并提供了一个多才多艺的平台, 可以很容易地适应不同的研究设置。

开放野外试验的变化已被证明是一种有效的方法来测试疾病进展的模型肌肉萎缩症。在这里, 我们演示了一种通用的视频跟踪方法, 用于分析 two-dimensional 鼠标移动使用现成的记录设备和开源软件 (图 1A-B)。与类似分析报告的结果一致, 我们显示mdx小鼠在测试之前, 在一个6分钟的野外任务中, 比野生型小鼠更少的自愿运动挑战 (图1C,图 2)。随着握力挑战的轻度消耗, mdx鼠标在移动协议的最初几分钟内通常仍然保持不动, 并且在移动时的动作量会适度增加4到 5 (图 3A)。在测试野生类型和mdx动物时, 如果没有运动挑战, 我们检测到的总距离没有差异 (图 2)。
这是与其他组的报告相一致的, 研究类似的运动分析, 未mdx显示与野生型小鼠没有区别或小的减少。我们还对表达高水平 utrophin 的转基因小鼠进行了评估, 以测试我们的检测灵敏度 (mdx: utr-tg, 菲奥娜线)11,12。mdx: utr-tg 是一种具有少量不良特征的mdx表型的健壮的救援模型。在练习之后, 表达 utrophin 的mdx小鼠与野生类型是无法区分的, 在总的距离 (图 3B) 或所测量的任何累计时间点 (图 3A) 上没有显著差异。
视频跟踪依赖于帧的可变性, 通过手动观察评分或自动软件检测来衡量。自由和开放源码运动分析软件允许对动物运动进行半自动检测, 在这种情况下, 在生成活动轨迹时, 可以手动纠正未被察觉或高估的运动。为了最大限度地减少数据处理在这个分析, 我们缩减原始的录影从被记录的 30 fps 到 15 fps。为了确定降低采样率的效果, 我们测量了1分钟全帧速率视频的总距离, 然后比较了从缩减版本的数据集 (图 4A) 导出的距离测量。我们发现, 将帧速率降低一半 (抽取 2) 将测量的距离降低了 5.0%, 并将帧速率降低为 7.5 fps 保留了近90% 的原始距离计算 (89.2% fps 视频的距离)。在这一点之后, 测量距离的精确度急剧下降, 虽然即使是高度毁灭的数据集仍然接近动物的路径 (图 4B)。这些观察强调了在评估动物活动时考虑帧速率和信号处理的重要性, 并证明了高空间分辨率可以保留在缩减的视频数据中。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56772/56772fig1.jpg"> 
图 1: 具有代表性的野外移动距离6分钟的痕迹.(A) 在本研究中使用的活动记录室的设置。握力仪是用于运动的挑战之前的化验, 然后自由移动记录在开放的野外平台。(B) 在运动跟踪软件中使用跟踪分析的6分钟视频录制的复合图像。(C) 覆盖两个代表性的痕迹 (重量, 灰色, n = 1;mdx, 红色, n = 1)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56772/56772fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56772/56772fig2.jpg"> 
图 2: 运动对mdx移动的影响。6 分钟的移动距离被记录在6月的年龄有或没有事先锻炼 (non-exercised mdx n = 5, 重量n = 4; 行使了mdx n = 5, 重量n = 5)。在未时, 小波变换和mdx之间没有显著差异, 但在检测前的小鼠中有显著差异 (无运动: 体重 1077.0±106.4 厘米, mdx 971.0 ±36.16 厘米; 运动: 重量770.2 ±30.75 厘米,mdx 127.8 ±36.16 厘米)。数据代表的意思是± SEM. 统计数据使用 two-way 方差分析, 其次是 Tukey 的多个比较的测试 (*p和 #60; 0.05, ** p 和 #60; 0.01, *** p 和 #60; 0.001 和 ***p和 #60; 0.0001)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56772/56772fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56772/56772fig3.jpg"> 
图 3: utrophin 的转基因表达改善了mdx鼠标的移动.6分钟的移动距离被记录在6月的年龄使用开放的野外活动室 (重量n = 5, mdx n = 5, mdx: utr-Tg n = 4)。(A) 累计距离 (分钟)。在小波和mdx之间没有发现明显的差异: utr-tg 在任何一点。统计数据使用 two-way 方差分析, 其次是 Tukey 的多个比较的测试 (*p和 #60; 0.05, **p和 #60; 0.01, ***p和 #60; 0.001 和 ***p和 #60; 0.0001)。(B) 总的开放字段距离超过6分钟 (重量770.2 ± 30.75 cm, mdx 127.8 ± 36.16 cm, mdx: utr-Tg 701.3 ±33.54 厘米)。数据代表的意思是± SEM. 统计数据使用 one-way 方差分析, 其次是 Tukey 的多个比较的测试 (*p和 #60; 0.05, ** p 和 #60; 0.01, *** p 和 #60; 0.001 和 ***p和 #60; 0.0001)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56772/56772fig3large.jpg" target="_blank">请点击这里查看更大版本的这个数字。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56772/56772fig4.jpg"> 
图 4: 采样率对移动跟踪的影响.在一个野生类型的鼠标, 1 分钟的移动距离记录和跟踪使用运动跟踪软件在全帧速率 (30.0 fps, 1819 总帧)。确定了鼠标在每个帧中的 two-dimensional 位置, 然后这些数据被摧毁以在较低帧速率的视频中模拟跟踪 (30、22.5、20、15、10、7.5、6、3、1.5 和 0.75 fps)。(A) 从每个缩减数据集中的坐标计算出的总距离。虚线显示了从全帧速率视频计算出的距离的5% 以内的值。这项研究的数据是从视频中被摧毁的 15 fps (红色箭头) 获得的。(B) 覆盖的跟踪比较全帧速率跟踪与 1.5 fps (上面) 和 15 fps (下)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56772/56772fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Simple and Low-cost Assay for Measuring Ambulation in Mouse Models of Muscular Dystrophy at JoVE.com

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

一种简便、低成本的肌营养不良小鼠步行模型的测定方法

该协议描述了一个灵活的, 低成本的测量鼠标移动在野外活动的测试系统。我们显示, 基于这个系统的6分钟的步行试验检测到在mdx小鼠中自愿运动的减少, 并准确地区分了这些动物的肌肉特异性抢救的改进。

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

a-simple-low-cost-assay-for-measuring-ambulation-mouse-models

Nanyang Technological University

Research

JoVE Journal

Behavior

9.9K 次观看.  University of California. 该协议描述了一个灵活的, 低成本的测量鼠标移动在野外活动的测试系统。我们显示, 基于这个系统的6分钟的步行试验检测到在mdx小鼠中自愿运动的减少, 并准确地区分了这些动物的肌肉特异性抢救的改进。

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

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

帕金森氏症小鼠模型的感觉功能评估

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

科研

行为学

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing locomotive impairment in animal models of neuromuscular disease and efficacy of therapeutic drugs that may improve locomotion and/or muscle function. The open field activity measurement provides a different measure than muscle strength, which is commonly assessed by grip strength measurements. It can also show how drugs may affect other body systems as well when used with additional outcome measures. In addition, measures such as total distance traveled mirror the 6 min walk test, a clinical trial outcome measure. However, open field activity monitoring is also associated with significant challenges: Open field activity measurements vary according to animal strain, age, sex, and circadian rhythm. In addition, room temperature, humidity, lighting, noise, and even odor can affect assessment outcomes. Overall, this manuscript provides a well-tested and standardized open field activity SOP for preclinical trials in animal models of neuromuscular diseases. We provide a discussion of important considerations, typical results, data analysis, and detail the strengths and weaknesses of open field testing. In addition, we provide recommendations for optimal study design when using open field activity in a preclinical trial.

Open field activity levels are used to assess locomotive and behavioral activity levels. This protocol provides a well-designed, standardized protocol to use in preclinical trials for neuromuscular disorders.

行为和运动测量使用一个开放的领域活动监控系统的骨骼肌疾病

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing ...

Low-cost Protocol of Footprint Analysis and Hanging Box Test for Mice Applied the Chronic Restraint Stress

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important behavioral test to determine whether the mice mimic the symptoms of patients. Motor deficits are often induced by stress when no spontaneous motor phenotype is observed in the mouse models. Therefore, gait analysis followed by stress loading would be a sensitive method for evaluating the motor phenotype in mouse models. However, researchers face the requirement of an expensive apparatus to obtain quantitative results automatically from gait analysis. For stress, stress loading by simple methods without expensive apparatuses required for electric shock and forced running is desirable. Therefore, we introduce a simple and low-cost protocol consisting of footprint analysis with paper and ink, hanging box test to evaluate motor function, and stress loading defined by restraint with a conical tube. The motor deficits of mice were successfully detected by this protocol.

The low-cost protocol consisting of footprint analysis and hanging box test after restraint stress is useful for evaluating the movement disorders of mouse model.

小鼠应用慢性约束应激的低成本足迹分析和吊箱试验协议

Gait disturbance is frequently observed in patients with movement disorders. In mouse models used for movement disorders, gait analysis is important ...

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

变性小鼠年龄依赖性运动障碍的步态分析

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

发育生物学

Low-Cost Gait Analysis for Behavioral Phenotyping of Mouse Models of Neuromuscular Disease

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method of gait analysis demonstrated here is a simple but effective measure of gait abnormalities in murine models. Footprints are analyzed by painting a mouse&#8217;s feet with non-toxic washable paint and allowing the subject to walk through a tunnel on a sheet of paper. The design of the testing tunnel takes advantage of natural mouse behavior and their affinity for small dark places. The stride length, stride width, and toe spread of each mouse is easily measured using a ruler and a pencil. This is a well-established and reliable method, and it generates several metrics that are analogous to digital systems. This approach is sensitive enough to detect changes in stride early in phenotype presentation, and due to its non-invasive approach, it allows for testing of groups across life-span or phenotypic presentation.

Footprint analysis is a low-cost alternative to digitized gait analysis programs for researchers quantifying movement abnormalities in mice. Because of its speed, simplicity, and longitudinal potential, it is ideal for behavioral phenotyping of mouse models.

神经肌肉疾病小鼠模型行为花型的低成本盖特分析

Measurement of animal locomotion is a common behavioral tool used to describe the phenotype of a given disease, injury, or drug model. The low-cost method ...

A Simple Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

一种测定果蝇运动活性的简单技术

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

Home-Based Monitor for Gait and Activity Analysis

Current outcomes in neuromuscular disorder clinical trials include motor function scales, timed tests, and strength measures performed by trained clinical evaluators. These measures are slightly subjective and are performed during a visit to a clinic or hospital and constitute therefore a point assessment. Point assessments can be influenced by daily patient condition or factors such as fatigue, motivation, and intercurrent illness. To enable home-based monitoring of gait and activity, a wearable magneto-inertial sensor (WMIS) has been developed. This device is a movement monitor composed of two very light watch-like sensors and a docking station. Each sensor contains a tri-axial accelerometer, gyroscope, magnetometer, and a barometer that record linear acceleration, angular velocity, the magnetic field of the movement in all directions, and barometric altitude, respectively. The sensors can be worn on the wrist, ankle, or wheelchair to record the subject&#8217;s movements during the day. The docking station enables data uploading and recharging of sensor batteries during the night. Data are analyzed using proprietary algorithms to compute parameters representative of the type and intensity of the performed movement. This WMIS can record a set of digital biomarkers, including cumulative variables, such as total number of meters walked, and descriptive gait variables, such as the percentage of the most rapid or longest stride that represents the top performance of patient over a predefined period of time.

This innovative device uses magneto-inertial sensors to permit gait and activity analysis in uncontrolled environments. Currently in the qualification process as an outcome measure in the European Medical Agency, one of the applications will be to serve as a clinical endpoint in clinical trials in neuromuscular diseases.

基于家庭的盖特和活动分析监视器

Current outcomes in neuromuscular disorder clinical trials include motor function scales, timed tests, and strength measures performed by trained clinical ...

生物工程

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

体内运动神经元变性小鼠前肢肌作用电位的电生理测量

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

神经科学

Isometric and Eccentric Force Generation Assessment of Skeletal Muscles Isolated from Murine Models of Muscular Dystrophies

Critical to the evaluation of potential therapeutics for muscular disease are sensitive and reproducible physiological assessments of muscle function. Because many pre-clinical trials rely on mouse models for these diseases, isolated muscle function has become one of the standards for Go/NoGo decisions in moving drug candidates forward into patients. We will demonstrate the preparation of the extensor digitorum longus (EDL) and diaphragm muscles for functional testing, which are the predominant muscles utilized for these studies. The EDL muscle geometry is ideal for isolated muscle preparations, with two easily accessible tendons, and a small size that can be supported by superfusion in a bath. The diaphragm exhibits profound progressive pathology in dystrophic animals, and can serve as a platform for evaluating many potential therapies countering fibrosis, and promoting myofiber stability. Protocols for routine testing, including isometric and eccentric contractions, will be shown. Isometric force provides assessment of strength, and eccentric contractions help to evaluate sarcolemma stability, which is disrupted in many types of muscular dystrophies. Comparisons of the expected results between muscles from wildtype and dystrophic muscles will also be provided. These measures can complement morphological and biochemical measurements of tissue homeostasis, as well as whole animal assessments of muscle function.

Muscle function measurements contribute to the evaluation of potential therapeutics for muscle pathology, as well as to the determination of mechanisms underlying physiology of this tissue. We will demonstrate the preparation of the extensor digitorum longus and diaphragm muscles for functional testing. Protocols for isometric and eccentric contractions will be shown, as well as differences in results between dystrophic muscles, representing a pathological state, and wildtype muscles.

等距和骨骼肌产生偏心力评估隔离肌营养不良症的小鼠模型

Critical to the evaluation of potential  therapeutics for muscular disease are sensitive and reproducible physiological  assessments of muscle function. ...

生物学

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

小鼠肌功能的无创评价

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...