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 border="0" cellpadding="0" cellspacing="0" width="684">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">DigiGait</td>
			<td style="width:123px;">Mouse Specifics, Inc., Framingham, Massachusetts, USA</td>
			<td style="width:123px;"></td>
			<td style="width:224px;">DigiGait Imager and Analysis Software are included with the hardware</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">non-transparent blanket or dark cloth</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td style="width:224px;">cover the test chamber to reduce the animal&#39;s feeling of exposure/stress</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">balance</td>
			<td style="width:123px;">e.g. Satorius</td>
			<td style="width:123px;"></td>
			<td style="width:224px;">balance with 0.1 g accuracy and a maximum load of at least 100 g</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">red finger paint</td>
			<td style="width:123px;">e.g. Kreul or Staedtler</td>
			<td style="width:123px;"></td>
			<td>for increasing the contrast between paws and animal&#8217;s body</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">small paint brush</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td style="width:224px;">soft brush to apply finger paint to the animal paws</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">diluted detergent</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td style="width:224px;">for cleaning</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">disinfectant, e.g. Meliseptol or 70% ethanol</td>
			<td style="width:123px;">e.g. B.Braun</td>
			<td style="width:123px;"></td>
			<td style="width:224px;">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 San Sebastian

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

细胞聚集法评价果蝇切口与反式配体的结合及其对顺式配体的抑制作用

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Immunolabeled, 澄清人胎盘长柔毛血管网络的三维渲染与分析

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

种系线虫与胸苷模拟教育的细胞周期分析

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

人类诱导多能干细胞衍生神经细胞治疗移植小鼠脑损伤控制皮质影响模型

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

从外周单核细胞生成诱导神经干细胞,分化至多巴胺能神经元前体,用于移植研究

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

在免疫缺陷小鼠中表演人类骨骼肌异种移植物

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...