我们感谢在埃尼动物设施的动物看护者帮助育种, Nuno 博士雷蒙对手稿有用的评论。通过合作研究中心 SFB-889 (项目 A8) 和 SFB-1190 (项目 P02) 和艾美奖诺特青年调查员奖 (1702/1), 德国研究基金会 (DFG) 提供的赠款支持了贝得通。C.M.R. 得到了来自神经、生物物理学和分子生物科学 (GGNB) 的哥廷根研究生院的研究员的支持。

这里报告的所有动物实验都是根据欧洲动物福利准则 (2010/63/欧盟) 进行的, 经 Niedersächsisches Landesamt Verbraucherschutz 和 Lebensmittelsicherheit (LAVES) 批准, 注册号 14/1701。
1. 研究设计
<ol><li>当动物行为工作需要周密规划时, 在设计实验时考虑以下参数。<ol>
<li>每组需要的动物数量。<ol>
<li>使用统计软件 (例如,通过、EDA 或 GPower) 计算所需的组大小。 注意: 组大小取决于动物之间的变化和表型的严重性。对于运动步态分析, 小鼠的数量通常为每组 10-20。</li>
		</ol>
</li>
		<li>实验动物的性别。<ol>
<li>根据动物的应变, 考虑雌激素水平对实验的影响。 注意: 许多行为研究关注雄性, 以避免雌激素水平对实验的影响。这些影响或多或少强烈取决于动物的背景应变。</li>
			<li>如果男女双方都将使用, 测试性别影响和评估两个性别独立时, 必要的。</li>
		</ol>
</li>
		<li>实验动物的年龄。<ol>
<li>使用成年动物 (2 月的年龄, 或更旧的), 如果只需要一个时间点。</li>
			<li>选择几个时间点时, 运动行为的变化, 以提高年龄是要研究。最早可能的时间点是1月后, 老鼠从他们的母亲断奶。定期对动物进行测试,例如每1、2或3个月。</li>
		</ol>
</li>
	</ol>
</li>
	<li>申请地方当局授权进行动物行为测试。</li>
	<li>制定采购试验动物的计划。<ol>
<li>制定一个育种计划或与动物经销商及时联系, 以便在实验开始的当天有足够的实验动物。</li>
		<li>允许动物习惯一个星期, 如果他们被保存在一个新的房间/设置在实验期间。</li>
	</ol>
</li>
</ol>2. 视频录制
注: 为了说明使用运动学步态分析, 这里有一个商业上可用的成像系统与它伴随的成像和分析软件 (见材料表) 使用。
<ol><li>启动计算机和成像软件。</li>
	<li>通过在家庭笼子里观察, 确定每种动物的健康状况和福祉, 并权衡它的平衡。</li>
	<li>需要时, 用刷子轻轻地将红色手指涂在动物的爪子上。允许油漆在备用清洁笼中干燥5分钟。 注意: 避免涂上动物的腹部, 因为油漆是用来增强爪子和身体的对比。这是有用的, 有黑色的手指油漆方便的更正。这一步骤是需要的动物棕色毛皮, 或如果爪子已经纹身的标识。如果选择画一只动物的爪子, 同一组和对照组中的所有动物都需要被涂上油漆。</li>
	<li>在仪器的右上板设置跑步机的速度;如果将应用多个运行速度, 请先以最慢的速度开始。</li>
	<li>将动物放在试验室 (避免在关闭房间时夹紧尾巴或爪子)。用深色布盖住房间, 允许每只动物调整 1-2 分钟。</li>
	<li>打开测试室的灯, 把跑步机的灯旋转开关转向 "on" 位置。转动跑步机旋转开关到 "前进" 开始跑步机, 然后点击 "记录" 按钮在成像软件。 注意: 跑步机跑步的时候, 仔细地观察动物的性能是很重要的: 如果动物不能跟上跑步机的速度, 或者显示与运动无关的次要症状 (例如,癫痫发作)。测试条件可能需要重新调整。</li>
	<li>当动物运行稳定 (没有快速逃逸到两侧, 前面, 或背部), 记录至少5秒前停止跑步机。停止录音, 点击 "停止" 在成像软件, 并把跑步机旋转开关回到 "关闭" 的位置。 注意: 为了避免动物的不稳定运行, 它可以帮助他们运行几秒钟, 或允许他们跑在另一个方向 (通过转动跑步机旋转开关改为 "反向" 而不是 "前进")。</li>
	<li>单击成像软件中的 "处理" 按钮, 打开一个菜单, 在其中可以设置视频部分的开始和结束点 (用于分析)。为此, 请使用屏幕底部的滑块浏览视频。</li>
	<li>要选择当前时间点作为起始点或终点, 请分别单击 "从帧 #" 和 "到"。确保该部分至少包含7步/爪 (总共14步), 动物以恒定的速度稳定运行。</li>
	<li>输入动物身份、出生日期、体重和性别。将数据保存到计算机或服务器上所需的位置。单击 "照相机" 返回到录制界面。</li>
	<li>如果需要记录多个运行速度, 请使用所需的运行速度重复步骤 2.6-2.10。在录制下一个视频之前, 请确保红色颜料仍然存在于爪子上, 否则重复步骤2.3。</li>
	<li>录音后, 将动物放到其家庭笼子里。除去动物后, 用肥皂水彻底清洁跑步机皮带, 然后用消毒剂为下一个实验动物准备。</li>
</ol>3. 视频处理
<ol><li>启动分析软件并单击 "选择学习文件夹" 以选择带有录制视频的文件夹。</li>
	<li>选择一个视频或几个可以连续处理的视频, 然后单击 "转到"。</li>
	<li>使用 "重绘" 函数选择鼠标运行的区域;本节只应包含鼠标和白色背景。</li>
	<li>如果 "反向" 跑步机功能以前使用过, 选择 "检查主题的鼻子是您的权利 > > >" 镜像视频, 因为软件设计只分析动物跑到左边。单击 "接受" 继续。</li>
	<li>使用 "刷新" 功能可以查看软件检测到的默认掩码和爪子打印。 注意: 原来的视频显示在左边, 和黑白图像的建议爪印是在右边。</li>
	<li>在 "长度" 和 "宽度" 框中输入值, 以更改排除动物鼻子周围红色区域以进行分析的掩码;由于颜色类似的爪子, 不掩盖该区域可能导致软件意外分类的鼻子区域作为一个爪子。</li>
	<li>调整滑块 "过滤器噪音" 和 "过滤毛皮和深色补丁", 以优化黑白爪子打印。设置 "过滤噪声" 滑块到 ~ 800-950 为黑动物和 ~ 700-800 为褐色或白色动物, 取决于动物的确切的毛皮颜色。当设置令人满意时, 选择 "确定"。 注: "过滤毛皮和深色补丁" 滑块取决于如何 "红色" 的爪子。对于手绘的爪子, 该值通常约为 100-120, 而对于非手绘爪子, 最佳值是大约 50-100。这些设置取决于毛皮和爪子的颜色阴影, 需要为每种动物进行优化。黑和白色爪打印应该有清楚的表示法爪子以尽可能小的背景噪声尽可能。</li>
	<li>选择一个或多个通过第一次调整的视频 (在视频名称前标有 "@@"), 然后选择 "转到" 函数开始分析这些视频。 注意: 每个视频的分析需要 2-5 分钟。因为这个步骤不需要实验者的输入, 所以可以在一夜之间运行对几个视频的分析。</li>
	<li>选择分析后的视频 (用 "@@@" 标记), 然后单击 "转到"。注意, 爪子区域 (在 cm2) 与传送带的接触随着时间的推移 (步态动力学) 为每一个单独的爪子现在可以看到。要比较原始视频和所选区域的计算爪打印, 请使用 "播放视频" 功能。</li>
	<li>使用以下 (三) 工具纠正软件所犯的小错误。<ol>
<li>使用 "正确" 选项删除错误的信号,例如,当软件记录信号时, 即使相应的爪子与皮带没有接触。单击一次可缩放到相关区域, 并将对象的左边框标记为在第二次单击时删除, 右边框与第三次单击。</li>
		<li>使用 "连接" 选项结合两个信号,例如,当没有信号记录几个帧, 即使爪子是与皮带接触。单击一次放大相关区域, 双击两个对象中间的组合。</li>
		<li>使用 "删除" 选项可以完全删除分析中的时间点。仅当错误不能用 "正确" 或 "连接" 功能固定时,例如,当左前肢爪的信号被意外记录为左后肢爪时, 才使用此选项。单击一次可缩放到相关区域, 并将区域的左边框标记为在第二次单击时删除, 右边框与第三次单击。 注: 这些工具只能用来改正小错误;系统故障 (例如,如果从一只爪子的信号是极端微弱的) 不能被改正: 录影应该被排除从分析和各自的动物的录音重复, 当可能时。请注意, 在使用 "正确"、"连接" 或 "删除" 选项后, "播放视频" 选项不再可用, 单击 "撤消" 按钮将重置所有3编辑工具。</li>
	</ol>
</li>
	<li>选择 "下肢体" 进行4肢体;当 "下肢体" 被点击最后的爪子后, 软件完成分析, 并显示该动物在4屏幕上的结果。</li>
</ol>4. 步态分析
<ol><li>当分析来自一个实验的所有视频时, 选择所有视频并单击 "重新组织结果" 以导出结果 (电子表格文件中的参数列表)。</li>
	<li>打开具有 "reorganized_stride_info" 结尾的文件, 并添加此电子表格中未包括的信息: 组信息 (例如,基因型、治疗)、年龄和动物长度和宽度的测量, 保存在另一个具有 "SFI_TFI_PFI_reorganized_stride_info" 结尾的电子表格文件。</li>
	<li>在必要时, 将步态参数规范化到动物的宽度或长度,例如, SL 到动物的长度和 SW 到动物的宽度。</li>
	<li>按组、年龄和运行速度对结果进行排序: 独立分析所有这些条件。 注意: 不同的年龄或运行速度不能在同一组中组合。</li>
	<li>计算所有实验条件下每个参数平均值 (平均值) 值、标准偏差和标准误差。</li>
	<li>根据实验设计进行统计分析,例如,使用2尾t检验, 将突变体/被处理动物与野生型 (体重)/对照组进行比较, 或对方差进行比较。</li>
	<li>查看所有测量的参数: 绘制每个参数以更好地可视化结果是很有帮助的。如果给定参数中存在统计差异, 请检查其他相关参数是否相应变化。 注: 例如, 如果在某一测试组中 SL 明显减少, 这也会导致更高的步频 (因为运行速度相同), 并可能导致增加的 SW (以保持姿态稳定)。</li>
	<li>选择与模型最相关的参数, 并且/或与人类疾病中的观察相媲美。对于演示文稿, 为每个组创建具有代表性的视频, 并通过图形显示相关参数的读数来补充它们, 因为微妙的步态变化通常不明显的视频。</li>
</ol>5. 故障排除
注意: 有些动物, 特别是有焦虑表型的老鼠模型, 即使是在跑步机上跑步之类的简单任务, 也可能有困难。以下是可以采取的步骤, 以降低焦虑水平, 并鼓励跑步。
<ol><li>适应性和积极的执行。<ol>
<li>在第一次测试前 2-3 天, 把鼠标放在测试室里, 用深色布盖住, 然后把灯关掉。让鼠标调整到新的环境为 ~ 5 分钟. 将周或巧克力/坚果黄油 (如花生酱) 添加到测试室中, 这样就可以形成一个正的关联。</li>
	</ol>
</li>
	<li>由空气喷/后边界的负执行。<ol>
<li>老鼠不喜欢空气喷吹或在他们身后的运动, 并将逃离的干扰。为了激励跑步, 使用温和的空气喷吹, 或有节奏的运动的柔性杆, 形成的后方边界的测试室, 鼓励鼠标跑向前部的测试室。</li>
	</ol>
</li>
	<li>慢点开始<ol>
<li>在测试快速运行速度时, 以较低的速度启动跑步机, 然后慢慢地将跑步机速度提高到所需的测试条件。</li>
	</ol>
</li>
	<li>尽量减少自由运动。<ol>
<li>测试室长度受两个可调节的杆在前面和后面的限制。如果一个试验动物跟上运行速度, 但不稳定运行, 限制室的长度, 使运行更稳定。</li>
	</ol>
</li>
	<li>如果上述测量不成功, 则在第二天运行记录。如果动物在三天的测试后仍拒绝运行, 请将其记录为发现, 并将该动物排除在进一步测试之外。 注意: 步态分析的结果取决于高质量的视频记录。如果视频已被仔细记录, 在分析过程中没有理由排除视频。如果视频质量不足, 在步骤3.6 中, 当设置数字爪打印的创建参数时, 它将变得明显。如果其他身体部位, 除了爪子和鼻子出现红色 (例如,由于在生殖器周围缺少毛皮或手指油漆 sprinklings 在腹部), 质量下降显著。步骤3.6 中的调整只允许更正小问题, 如果这不能使视频达到可接受的信号/噪声比, 则需要从分析中排除视频, 并且需要重复录制。因此, 建议在执行录制后不久对视频进行分析。</li>
</ol>

作者声明没有竞争的财政利益。

研究运动协调是神经退行性疾病模型的一种有用的方法, 特别是对运动协调受到严重影响的 PD 等疾病的描述。在运动步态分析功能检测的帮助下, 我们可以确定在运动问题开始时动物步态的细微变化, 或者在弱变性的模型中, 因此相对温和的表型。鉴于各种神经退行性疾病模型中的表型广泛, 包括小步态异常和严重的运动损伤, 这种方法很适合根据动物的年龄和移动能力来评估步态参数。严重受损的...

神经退行性疾病给病人、家庭和社会带来了沉重的负担, 随着预期寿命的增加, 世界人口继续老龄化, 这将变得更加令人担忧。神经退行性疾病最常见的症状之一是平衡和流动性问题。因此, 衰老哺乳动物 (如啮齿动物) 模型和/或模型显示神经退行性表型的运动行为的表征, 是证明特定动物模型在体内相关性的重要工具, 或治疗旨在改善疾病症状的治疗方法。几乎每一种治疗神经退行性疾病的方法最终都需要在动物模型中进行试验, 然后才开始对人类进行临床试验。因此, 重要的是要有可靠的, 可重复的行为测试, 可以用来始终如一地量化疾病相关的表型沿年龄的发展, 以确保候选药物, 这表明潜在的体外模型, 可以有效改善活体动物的表型。
啮齿类动物运动行为评估的一个方面是运动学步态分析, 它可以由录像 (也称为腹面平面的)1,2进行。这种建立的方法利用了连续记录的啮齿动物的底部行走在一个透明和机动的跑步机皮带1,2,3,4。对视频提要数据的分析创建了所有四肢体的 "数字爪印", 它能动态、可靠地重述啮齿类动物的行走模式, 最初是由羽衣甘蓝等所描述的。2和 Amende等。3。
基于成像的步态分析的原理是测量与跑步机带接触的爪面积, 随着时间的推移, 每个单独的爪子。每种姿态都代表了爪面积 (在制动阶段) 的增加和爪面积 (推进阶段) 的减少。接下来是没有检测到信号的摆动相位。摇摆和姿态在一起形成一大步。除了步态动力学参数, 还可以从录制的视频中提取姿势参数。示范性参数及其定义列于表 1中, 包括立场宽度 (SW; 从前爪或后爪子到鼻尾轴的组合距离), 步幅长度 (SL; 同一爪两大步之间的平均距离), 或爪子放置角度 (爪子的角度到鼻子-尾巴轴)。姿势和步态动力学数据允许得出关于动物平衡的结论 (通过姿势参数及其在几个步骤中的变异性) 和协调 (通过步态动力学参数)。其他参数, 如共济失调系数 (由 [(最大) 计算的 SL 变异性。SL−min。sl)/平均 SL]), 后肢共享的姿势时间 (两个后肢都与皮带接触的时间), 或爪子拖拉 (从完全立场到爪的所有的爪子在皮带的总面积) 也可以提取, 并已报告在各种神经退行性 di 改变星瑞机型5、6、7、8 (见表 1)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>参数</td>
			<td>单位</td>
			<td>定义</td>
		</tr>
<tr>
<td>摇摆时间</td>
			<td>女士</td>
			<td>爪不与皮带接触的时间长短</td>
		</tr>
<tr>
<td>姿态时间</td>
			<td>女士</td>
			<td>爪与皮带接触的时间长短</td>
		</tr>
<tr>
<td>% 刹车</td>
			<td>% 的立场时间</td>
			<td>脚爪在刹车阶段所占的姿势时间百分比</td>
		</tr>
<tr>
<td>% 推进</td>
			<td>% 的立场时间</td>
			<td>在推进阶段爪的比例时间百分比</td>
		</tr>
<tr>
<td>姿态宽度</td>
			<td>厘米</td>
			<td>前后爪与鼻尾轴的组合距离</td>
		</tr>
<tr>
<td>步幅长度</td>
			<td>厘米</td>
			<td>同一爪两大步之间的平均距离</td>
		</tr>
<tr>
<td>步幅频率</td>
			<td>步幅/秒</td>
			<td>每秒完成步长数</td>
		</tr>
<tr>
<td>爪子放置角度</td>
			<td>度</td>
			<td>爪的角度关于动物的鼻尾轴</td>
		</tr>
<tr>
<td>共济失调系数</td>
			<td>天文单位</td>
			<td>由 [(最大 sl-最小 sl]/平均 sl] 计算的 SL 变异性</td>
		</tr>
<tr>
<td>% 共享立场</td>
			<td>% 的立场</td>
			<td>后肢共用姿势时间;两个后肢同时接触皮带的时间</td>
		</tr>
<tr>
<td>爪拖</td>
			<td>mm2
</td>
			<td>从完全立场到爪子升降的皮带上的爪子的总面积</td>
		</tr>
<tr>
<td>肢体装载</td>
			<td>cm2
</td>
			<td>最大 dA/dT;破断阶段爪面积最大变化率</td>
		</tr>
<tr>
<td>步进角度可变性</td>
			<td>度</td>
			<td>后爪作为 SL 和 SW 函数的角度的标准偏差</td>
		</tr>
</tbody></table>表1。可通过腹面成像测试的关键步态参数的定义。
对神经退行性疾病的啮齿动物模型的运动行为进行评估, 取决于某一特定年龄模型的表型的严重性。一些疾病, 最突出的 PD, 表现出强烈的运动行为 (运动) 赤字, 无论是在病人和动物模型。pd 的四个关键症状之一是运动迟缓, 随着年龄的增长, 在 pd9早期已经出现严重的步态损伤。对 1-甲基4苯基-12、36-tetrahydropyridin (MPTP) 的急性 PD 模型进行研究, 已采用了 "新品步态分析10、11、12"。然而, 鉴于这种模式的急性性质, 这些研究并没有解决与年龄有关的运动缺陷的进展。最近的几项研究已经对衰老的小鼠进行了步态分析, 如13、14、15, 强调了了解疾病进展与进步年龄的相关性。.
除运动缺陷外, 神经退行性疾病的动物模型往往难以集中于检查任务, 显示显著的认知障碍, 特别是随着年龄的提高。这种表型可以影响运动行为测试的结果。即, 最广泛使用的测试, 以检查电机赤字, rotarod 测试16, 依赖于认知, 关注, 和压力17,18。虽然在机动跑步机上行走的意愿也取决于这些因素, 但记录的读出是运行的, 这是一个更标准化的特点, 而且远没有被改变的认知影响。压力和注意力的影响可能在特定的参数, 如摆动/立场时间的压力, 和 SL 的注意19,20, 但不是在整体运行能力。
运动学步态分析方法进一步提供了选择调整啮齿动物模型挑战的优势。可调角度和速度的跑步机允许步行速度从 0.1-99.9 厘米/秒, 使严重行走障碍的啮齿动物仍然能够以慢速运行 (~ 10 厘米/秒)。非受损动物可以以更快的运行速度 (30-40 厘米/秒) 来测量。观察测试过的动物是否能够以某种速度运行, 本身就能产生结果。此外, 啮齿类动物还可以在量角器的帮助下, 通过将跑步机倾斜到所需的角度, 或者附加一个加权的雪橇到老鼠或老鼠的后腿上, 来进行倾斜或下降。
除了对患者中变异的单个蛋白质的大量研究之外, 最近对有缺陷的吞过程与变性13、21、22之间的联系有了越来越多的认识, 23,24,25,26,27,28。小鼠模型与减少的水平 endophilin a (从今以后 endophilin), 一个关键的球员在网格蛋白介导的吞13,21,29,30,31,32,33,45和网格蛋白独立吞34, 被发现显示变性和年龄依赖性的损伤在运动活动13,21。三基因编码 endophilin 蛋白家族: endophilin 1, endophilin 2, endophilin 3。值得注意的是, 因 endophilin 蛋白的耗尽而导致的表型差异很大, 这取决于缺失 endophilin 基因13、21的数量。虽然在出生后几个小时内, 所有 endophilin 基因的三重敲出 (高) 都是致命的, 而没有 endophilin 1 和2的老鼠在出生后3周内无法生长和死亡, 那么三 endophilins 中的任何一个都没有显示出明显的表型供测试。情况21。其他 endophilin 突变基因型显示寿命缩短, 并发展13岁以上的运动障碍。例如, endophilin 1 高-2 ht-3 高老鼠显示步行改变和马达协调问题 (由运动步态分析和 rotarod 测试) 已经在3月的年龄, 而他们的窝, endophilin 1 高-2-3 高动物, 显示一个重要的仅在15月13岁时, 电机协调减少。由于这些模型中的表型的多样性很大, 因此有必要确定和应用一种能够结合动物的运动和认知能力以及年龄的各种挑战的测试。在这里, 我们详细的实验程序, 利用运动学步态分析, 以评估运动损伤的发生和进展的小鼠模型, 显示神经退行性变化 (即endophilin 突变体)。这包括测量不同年龄的步态参数和运动损伤的不同严重性。

<table><tbody><tr><td>DigiGait</td><td>Mouse Specifics, Inc., Framingham, Massachusetts, USA</td><td></td><td>DigiGait Imager and Analysis Software are included with the hardware</td></tr><tr><td>non-transparent blanket or dark cloth</td><td></td><td></td><td>cover the test chamber to reduce the animal&#039;s feeling of exposure/stress</td></tr><tr><td>balance</td><td>e.g. Satorius</td><td></td><td>balance with 0.1 g accuracy and a maximum load of at least 100 g</td></tr><tr><td>red finger paint</td><td>e.g. Kreul or Staedtler</td><td></td><td>for increasing the contrast between paws and animal&amp;rsquo;s body</td></tr><tr><td>small paint brush</td><td></td><td></td><td>soft brush to apply finger paint to the animal paws</td></tr><tr><td>diluted detergent</td><td></td><td></td><td>for cleaning</td></tr><tr><td>disinfectant, e.g. Meliseptol or 70% ethanol</td><td>e.g. B.Braun</td><td></td><td>for desinfection</td></tr></tbody></table>

gait analysis of age-dependent motor impairments in mice with neurodegeneration

运动行为测试通常用于确定啮齿动物模型的功能相关性, 并测试这些动物新开发的治疗方法。具体来说, 步态分析允许在人类患者中观察到的与疾病相关的表型, 特别是在影响运动能力的神经退行性疾病中, 如帕金森病 (PD)、阿尔茨海默病 (AD)、肌萎缩侧索硬化症 (ALS) 和其他。在这条线的早期研究中, 步态参数的测量是费力的, 取决于难以控制的因素 (例如,运行速度, 连续运行)。腹面成像系统的发展使步态分析成为可能, 使该方法成为评估啮齿动物运动行为的有效工具。在这里, 我们提出了一个深入的协议, 如何使用运动学步态分析, 以检查年龄依赖性进展的运动缺陷的小鼠模型的变性;endophilin 的小鼠线, 神经退行性损伤随着年龄的增长逐渐增加, 被用作例子。

为了说明运动学步态分析的应用, 我们对 C57BL/6J 小鼠的步态分析, 以及几个 endophilin 突变线, 使用商用仪器和软件 (请参阅表材料)。在此设置中, 在透明跑步机下的高速摄像机记录鼠标的运行情况 (图 1A)。软件然后识别红色的爪子和白色或黑色毛皮的对比。由于我们的实验动物有深褐色的毛皮颜色, 我们用红色的手指画了所有主题的爪子。...

Watch this Scientific Journal Video about Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration at JoVE.com

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

在本研究中, 我们演示了基于腹面成像的运动步态分析方法, 以监测运动协调的细微变化以及小鼠模型中变性的进展 (如 endophilin 突变体) 。鼠标线)。

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. ...

gait-analysis-age-dependent-motor-impairments-mice-with

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

11.8K 次观看.  European Neuroscience Institute (ENI). 在本研究中, 我们演示了基于腹面成像的运动步态分析方法, 以监测运动协调的细微变化以及小鼠模型中变性的进展 (如 endophilin 突变体) 。鼠标线)。

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

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.

Deficits in fine motor coordination can be assessed with the balance beam test. Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the beam.

在小鼠体内的汽车平衡和协调的评估使用的平衡木

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

科研

神经科学

Battery of Behavioral Tests Assessing General Locomotion, Muscular Strength, and Coordination in Mice

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the animal&#39;s wellbeing and health. There are numerous behavioral tests that may be applied. We selected a test for general locomotion, the open field test (OFT); a test for muscular strength, the mesh test (MT); and a test for coordination, the rotarod test (RR). Testing can be accomplished on a weekly or monthly basis. As a test for general locomotion, the OFT works by objectively monitoring movement parameters while the mouse is in an open field apparatus. The field is generally a 2&#39; x 2&#39; box, and the movements are recorded through laser sensing or through video capture. The mouse is placed in the center of the open field and allowed to move freely for the test. The MT uses the latency for a mouse to fall off an inverted screen as a measure of muscular strength. A mouse is placed on a screen, which is inverted over a clear box, and is timed for their latency to fall. Three trials are performed, with the best of the three trials scored for that day. A score of 60 s is the maximum time a mouse is left inverted. Mice are given a 5-min rest period between mesh test trials. Lastly, an accelerated protocol on the RR assesses motor coordination and endurance. During a trial, a mouse walks on a rotating rod as it increases in speed from 4 rpm to 40 rpm over 5 min. The trial ends when the mouse touches the magnetized pressure sensor upon falling. Each mouse undergoes three trials, and the best trial is scored for that day. This combined behavioral data allows for the global assessment of mobility, coordination, strength, and movement of the test animals. At least two out of the three behavioral testing measures must show improvement for an animal to qualify as having overall improved motor function.

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects of a disease or treatment on an animal's wellbeing. In order to globally assess motor functioning, we selected tests for general locomotion, muscular strength, and coordination: the open field test, the mesh test, and the rotarod test, respectively.

用于评估一般运动、肌肉力量和小鼠协调性的行为测试电池

Behavioral testing is used in pre-clinical trials to assess the phenotypic effects and outcomes that a particular disease or treatment has on the ...

行为学

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 ...

使用行为测试验证 Angelman 综合征的小鼠模型

Behavioral Characterization of an Angelman Syndrome Mouse Model

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of AS. We use the rotarod learning paradigm, detailed gait analysis, and nest building test to detect and characterize animal motor impairments. We test animal emotionality in the open field and elevated plus maze tests, as well as the affect in the tail suspension test. When AS mice are tested in the open field test, the results should be interpreted with care, since motor dysfunctions influence mouse behavior in the maze and alter activity scores.
The reproducibility and effectiveness of the presented behavioral tests has already been validated in several independent Uba3a mouse lines with different knockout variants, establishing this set of tests as an excellent validation tool in AS research. Models with the relevant construct and face validity will warrant further investigations to elucidate the pathophysiology of the disease and grant the development of causal treatments.

作者聚焦:用于验证 Angelman 综合征小鼠模型的一系列高度可重复的行为测试 

This manuscript presents a set of highly reproducible behavioral tests to validate an Angelman syndrome mouse model.

安格曼综合征小鼠模型的行为表征

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of ...

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the application of these techniques to identify gait deficits in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). Paralysis and motor deficits in mice subjected to EAE are typically assessed using a clinical scoring scale. However, this scale yields only ordinal data that provides little information about the precise nature of the motor deficits. EAE disease severity has also been assessed by rotarod performance, which provides a measure of general motor coordination. By contrast, kinematic gait analysis of the hind limb in the sagittal plane generates highly precise information about how movement is impaired. To perform this procedure, reflective markers are placed on a hind limb to detect joint movement while a mouse is walking on a treadmill. Motion analysis software is used to measure movement of the markers during walking. Kinematic gait parameters are then derived from the resultant data. We show how these gait parameters can be used to quantify impaired movements of the hip, knee, and ankle joints in EAE. These techniques may be used to better understand disease mechanisms and identify potential treatments for MS and other neurodegenerative disorders that impair mobility.

Kinematic gait analysis in the sagittal plane yields highly precise information about how movement is executed. We describe the application of these techniques to identify gait deficits for mice subjected to autoimmune-mediated demyelination. These methods may also be used to characterize gait deficits for other mouse models featuring impaired locomotion.

MOG35-55 诱发自身免疫性脑脊髓炎 C57BL/6 小鼠矢状面运动步态分析

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

慢性压迫性损伤小鼠步态的自动分析

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

自发交替评估，淀粉样蛋白β和脑神经病理学转基因小鼠模型中的新物体识别和肢体紧扣

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

医学

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

自动步态分析，评估带外周神经或脊髓挫伤的啮齿动物的功能恢复

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...

变性小鼠年龄依赖性运动障碍的步态分析

摘要

探索更多视频

此视频中的章节

在小鼠体内的汽车平衡和协调的评估使用的平衡木

用于评估一般运动、肌肉力量和小鼠协调性的行为测试电池

神经肌肉疾病小鼠模型行为花型的低成本盖特分析

安格曼综合征小鼠模型的行为表征

安格曼综合征小鼠模型的行为表征

安格曼综合征小鼠模型的行为表征

MOG35-55 诱发自身免疫性脑脊髓炎 C57BL/6 小鼠矢状面运动步态分析

慢性压迫性损伤小鼠步态的自动分析

自发交替评估，淀粉样蛋白β和脑神经病理学转基因小鼠模型中的新物体识别和肢体紧扣

自动步态分析，评估带外周神经或脊髓挫伤的啮齿动物的功能恢复