Name: sagittal plane kinematic gait analysis in c57bl/6 mice subjected to mog35-55 induced experimental autoimmune encephalomyelitis
Uploaded: 2017-11-04T13:00:00.000+00:00
Duration: 13 min 2 s
Description: Watch this Scientific Journal Video about Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis at JoVE.com

我们要感谢希德 Chedrawe 的技术援助与拍摄。这项工作得到了加拿大 MS 协会 (EGID 2983) 的资助。

本议定书符合加拿大动物保育委员会的准则, 并获达尔豪大学实验室动物委员会批准. 
 1. 构造反射标记: <ol> <li> 使用 hand-held 孔冲床, 从一张反光纸上打出所需的小圆圈数。每只动物需要5个标记为一个单独录音;两个大的和三小的标记. </li>
	<li> 使用细剪刀, 使从周长延伸到圆心的直切口. </li>
	<li> 卸下标记的纸张背面, 以显示粘合剂表面。使用精细钳, 牢牢抓住标记和卷曲它本身使用你的手指形成一个锥形形状。做一个小的标记, 旋紧锥体。做一个大的标记, 卷曲的锥形松散. </li>
	<li> 使用 hand-held 胶枪, 用胶水将锥形标记的内侧填充, 同时用镊子夹紧锥尖, 并将标记粘附在一块扁平的纸板上。胶水将防止标记在录音过程中倒塌和弯曲, 以确保最佳反射光。当胶水是干的 (约10分钟), 从纸板上取下的标志与手术刀 ( 图 1A ). </li>
</ol> 2。将鼠标置于感应腔中, 准备记录 <ol> <li> 用异氟醚气体 (2.5%; 2 升/分 O 2 ) 麻醉鼠标。一旦鼠标是无意识的, 把它放在一个鼻子锥放置在循环水加热毯的顶部。麻醉的目的是使鼠标固定在标记位置;程序并不痛苦。因此, 不需要对麻醉深度进行评估. </li>
	<li> 对两只眼睛应用局部眼部润滑剂. </li>
	<li> 使用电动快船刮掉所需的后肢。从脚踝开始, 伸展到肋骨的脊柱和底部;确保没有毛皮留下, 因为这将损害标志的附着力. 
	注: 在这里, 右后肢被记录;然而, 任何一个后肢都可以使用. </li>
	<li> 使用永久性标记, 指示髂嵴和髋关节的位置。髂嵴是略低于肋骨底部, 很容易扪通过把膝盖在鼠标和 #39; s 的身体. 
	注意: 髋关节可以通过弯曲和伸展腿部找到骨盆和股骨之间的关节点. </li>
	<li> 使用细钳, 抓住 小 标记的尖端, 并在快速作用的粘合胶中浸出底座, 或等效的替代品。将标记放在第四位数的顶端, 并放置2-3 秒以使胶水干燥。以相同的方式将其他两个小标记放置在趾关节和踝关节上 ( 图 1B ). </li>
	<li> 放置 在髂嵴和髋关节 ( 图 1B ) 上的大 标记, 其方式与小标记相同. </li>
	<li> 从机头锥中取出鼠标, 然后使用转笼立即转到录音室。将鼠标放在固定式跑步机上, 并允许从麻醉中完全恢复. </li>
</ol> 3。步态记录 <ol> <li> 在记录鼠标和 #39 的步态之前, 请在跑步机上使用已知尺寸的校准块的图片. 
	注意: 这将允许将视频中的像素转换为实际的测量。相机应该放置大约120厘米从跑步机。
	<ol> <li> 将照相机放置在与跑步机相同的高度和级别上。为校准图像后的录制保持相同的相机位置. </li>
	</ol> </li> <li> 一旦鼠标完全从麻醉中恢复, 将跑步机转到低速 (5 厘米/秒), 让鼠标开始行走。确保跑步机皮带的方向是鼠标上的标记朝向相机. </li>
	<li> 将跑步机的速度逐渐提高到20厘米/秒; 这是大多数健康小鼠的理想的步态速度. 
	注意: 虽然所有的老鼠都以同样的速度行走是理想的, 但有些人可能无法始终如一地达到这个速度。
	<ol> <li> 如果鼠标无法在20厘米/秒内行走, 则根据需要降低速度, 并且一定要记下这一点。减少跑步机的速度直到达到一致的步骤周期. 
		注意: 以后的数据分析可以根据速度的差异进行调整. </li>
	</ol> </li> <li> 在鼠标稳步行走时开始录制视频 ( 即 , 以一致的速度行走, 不向一侧饲养或编织)。继续录制, 直到记录了8到12连续的步骤周期。对于每个视频, 记录跑步机的速度和记录的鼠标的侧面. </li>
	<li> 录制完成后, 关闭跑步机并将鼠标返回其笼子。在录音之间彻底清洁跑步机, 因为其他老鼠留下的气味可能会改变老鼠的行为。为了减少压力和皮肤损伤, 不要删除标记;允许老鼠自己移除它们. </li>
</ol> 4。分析 <ol> <li> 使用运动分析软件处理视频. 
	注意: 在我们的实验中, 我们使用了为成像和统计软件设计的自定义脚本 (参见 材料表 ), 这是由 Dr. 尼古拉斯 Stifani 写的。使用选定的运动分析软件执行以下步骤。
	<ol> <li> 从视频中提取标记的像素坐标, 并使用校准视频将像素值转换为厘米并计算每个帧的关节角度. </li>
		<li> 标识每个步骤周期的开始和结束, 从而获得有关步骤持续时间和长度的信息. </li>
		<li> 将步骤周期持续时间正常化为200规范化的帧, 这样 swing 和姿态分别由100帧表示. </li>
	</ol> </li> <li> 使用规范化帧, 使用电子表格软件计算数据分析的运动学参数 (请参见 材料表 )。
	<ol> <li> 若要建立特定节点的平均角度, 请将规范化框架内所有角度的平均值设置为: <img src="/files/ftp_upload/56032/56032eq1v4.jpg"/> 注意: 此处 x 表示给定规范化的角度值框架, n 表示规范化的帧编号. </li>
		<li> 要建立给定鼠标的特定关节的运动范围, 请从一组规范化帧中的最大角度减去最小角度, 如下所示: 
		 移动范围 = 角度 最大 角度 最小 。
		注意: 这里的角度 最大 和角度 最小 分别是在规范化的步骤周期内达到的最大和最小的角度. </li>
		<li> 要建立 RMS 差异, 首先从基线记录中减去每个实验时点的平均角度。其次, 平方的各差, 取所有平方值的平均值和平方根的平均值。等式如下: <img src="/files/ftp_upload/56032/56032eq3v4.jpg"/> 注意: 这里 <img src="/files/ftp_upload/56032/56032eq4.jpg"/> 表示基线记录的平均角度; y 代表每个实验时点的平均角度; n , 表示规范化帧的数目。根平均数平方 (RMS) 差是用于评估基线记录中步态偏差的测量方法. </li>
	</ol> </li> <li> 使用科学的绘图和统计软件分析和显示数据 (请参见 材料表 ). </li> </ol>

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作者声明他们没有什么可透露的。

在小鼠与 EAE, 两种最常用的测量运动缺陷的方法是临床评分和秋季潜伏期从 rotarod27,28。这些技术有几个局限性。虽然方便和广泛使用, 临床评分是有限的只产生序号水平的数据, 这意味着, 临床评分之间的差异的大小是未知的。临床评分也因无法提供有关电机缺陷性质的精确信息而遭受损失。rotarod 试验提高了临床评分的一些局限性, 但只测量一般的运动协调, 不衡量步行的具体方面。
相比之下, 运动学步态分析提供了有关运动的具体方面的敏感措施, 包括不同关节的运动范围和平均角度。MOG35-55 EAE 小鼠在髋关节和膝关节运动中的细微缺陷已在 DPI9 发现, 大约5-9 天前出现临床症状或 rotarod 缺陷26。尽管临床症状完全缓解, 但在缺乏 rotarod 赤字的情况下, 这些缺陷仍然存在26。重要的是, 受损的踝关节运动测量的 RMS 差异非常好与白物质损失在脊髓26。
有几个方法论上的要点值得特别提及: 1) 准确和一致地放置关节标记是至关重要的-髋关节和髂嵴必须仔细识别心悸;2) 有必要从8-12 步周期获得录音。平均这些步骤周期产生一个代表性的平均步骤周期, 可以进一步分析;3) 必须建立最佳照明条件, 以确保标记在录音中清晰可见。如果标记没有被正确地照亮, 这可能使数字化录影一个费力的过程, 因为许多运动分析节目将无法跟踪标记, 需要手工跟踪。
这项技术的另一个限制是劳动密集型。例如, 要记录和分析来自一组10只老鼠的数据, 我们估计整个过程大约需要 7.0-9.0 小时 (h)。制作50标记 (每只老鼠 5) 需要大约2.0 小时. 记录鼠标行走行为可以单独完成或在一对。单独工作时, 每只老鼠大约需要25分钟, 而对每只老鼠的工作需要大约10分钟;因此, 记录10只老鼠可能需要从 1.5 h (对) 到 4.0 h (独奏)。最后, 数据分析和绘图大约需要3.5 小时。虽然这项技术是劳动密集型, 我们认为, 潜在的洞察力的疾病机制提供的运动学步态分析证明这一投资。有良好的行为相关性疾病病理学是有用的, 因为串行测量可以采取从活老鼠性。鉴于踝关节运动与腰椎脊髓白质损耗的近乎完美的相关性26, 该方法可用于确定实验过程中 EAE 小鼠鞘和髓的时间分布, 从而要评估的恢复。
步态分析是复杂的严重瘫痪, 限制运动的后肢。然而, 即使是严重瘫痪的小鼠 (临床评分和 #62; 3.0) 在某种程度上也能走动。在这些情况下, 前肢是用来拉动动物向前, 和一些后肢运动发生, 可以通过运动学步态分析测量。即使在这些严重的情况下, 仍然可以测量后肢功能的恢复时间。只有在非常严重的情况下 (20% 的动物的临床评分和 #62; 3.5 在峰值疾病, DPI 16-23), 我们一直无法获得有用的录音后肢运动。然而, 这些动物通常恢复一些后肢功能的 DPI 30, 允许有意义的录音, 以获得在那个时间点。
这项技术的未来应用是耦合运动学数据与同时肌录音的后肢在运动。该技术已在小鼠 ALS 和 SCI 模型中进行, 可用于阐明肌肉活动、神经支配和步态之间的关系。这项技术还可以与更多的 MS 和鞘的目标模型结合, 这可能产生更多的离散步态缺陷, 包括焦点 EAE 模型29,30或 cuprizone 诱导的鞘31。
我们所描述的测量 EAE 小鼠关节运动的技术也可以应用于其他损害步态的疾病。不同的步态变化已经报告了小鼠 PD, SCI, ALS, 和中风8,9,10,11,13,.例如, 帕金森病模型的特点是减少步幅长度和速度, 导致提高节奏, 以保持步行速度32。因此, 运动学步态分析提供了强有力的行为工具, 以阐明疾病的机制和识别潜在的治疗使用这些模型。

步态是四肢的一系列重复运动, 用来实现运动。步态由阶梯周期组成, 分为两个阶段: 姿态阶段, 即脚在地面向后移动以推动身体向前;和摆动阶段, 脚下是离地和向前移动。步态紊乱是许多神经退行性疾病的特征, 如脊髓损伤 (SCI)、多发性硬化 (MS)、肌萎缩侧索硬化 (ALS)、帕金森病 (PD) 和中风;这些疾病的临床前鼠模型经常重述他们各自的步态损伤1。对小鼠运动的基本控制机制进行了深入的研究2,3。此外, 还有许多人类神经系统疾病的小鼠模型4。因此, 在小鼠的步态分析是一个很有吸引力的方法, 以测量多个方面的运动赤字已知的解剖关联。研究的步态在小鼠模型可能提供洞察到病理基地的运动赤字的神经退行性疾病, 并使识别的潜在治疗。
一些用于测量啮齿类动物步态的技术包括目视检查 (例如、低音鼠标缩放5和开放字段测试6) 和从腹面的步态分析7。最近, 测量后肢运动矢状面运动学的方法越来越受欢迎, 因为它们提供了有关运动执行的更多信息, 因此对步态的细微变化更敏感8,9,10,11. 为研究矢状面后肢运动而开发的运动学技术在跑步机上的9,12在 SCI、ALS、外伤性皮质损伤、中风和亨廷顿氏病8,9,10,11,13,14,15,16。相比之下, 这些技术在研究多发性硬化症小鼠模型的运动缺陷方面的应用是有限的17。
实验性自身免疫脑脊髓炎 (EAE) 是 MS18最常用的小鼠模型。诱导 EAE 的两种主要方法是通过主动或被动接种。在主动 EAE, 小鼠用髓鞘抗原免疫, 导致脊髓和小脑性 T 细胞介导的 neuroinflammation 和鞘。另一方面, 被动 EAE 是通过将性 T 细胞从具有活性 EAE 的鼠标转移到一个天真的鼠标19而诱导的。如其他地方所述, 疾病病程和神经病受中枢神经系统 (CNS) 抗原和小鼠的影响20,21,22,23,24 ,25。在 EAE 实验中, 控制小鼠注射完整的弗氏的佐剂 (CFA) 没有髓鞘抗原。EAE 的特点是上升麻痹, 从尾部的弱点开始, 并可能涉及前肢, 导致共济失调和瘫痪20。我们最近的特点是 C57Bl/6 小鼠的步态变化, 受髓鞘胶质糖蛋白 35-55 (MOG35-55) 诱导的 EAE。这些研究表明, 步态分析优于经典行为分析, 因为偏离正常踝关节运动是高度相关的程度, 白物质损失在腰椎脊髓的 EAE 小鼠26。相比之下, 白物质损失与其他两种传统行为方法 (临床评分和 rotarod) 的相关性较弱,26更弱。
我们在这里描述了运动步态分析, 以检测 EAE 小鼠在跑步机上行走时矢状面的运动缺陷。五反射标记被放置在后肢, 以确定运动的臀部, 膝盖, 和踝关节在高速录像。运用运动分析软件对联合游览的运动学数据进行提取。这些技术的效用, 以量化移动赤字的 MOG35-55模型的 EAE 讨论。这些技术也适用于研究其他小鼠神经退行性疾病模型的步态缺损。

<table><tbody><tr><td>Camera</td><td>Nikon</td><td>Nikon D750</td><td>Used to film the video</td></tr><tr><td>Reflective tape</td><td>B&amp;amp;L Engineering</td><td>MKR-Tape-2</td><td></td></tr><tr><td>Fine scissors</td><td>Fine Science Tools</td><td>15023-10</td><td></td></tr><tr><td>Forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Glue gun</td><td>Craftsmart</td><td>E231647</td><td></td></tr><tr><td>scalpel handle #4</td><td>Roboz</td><td>R5-9884</td><td></td></tr><tr><td>Scalpel Blade No.10</td><td>Feather</td><td>2020-12</td><td></td></tr><tr><td>C57BL/6 mice</td><td>Charles River Laboratories</td><td></td><td></td></tr><tr><td>Anesthetic machine</td><td>EZ Anesthesia</td><td>EZ-AF9000 Auto Flow System</td><td></td></tr><tr><td>Recirculating water heating blanket</td><td>Androit</td><td>HTP-1500</td><td></td></tr><tr><td>topical eye lubricant</td><td>Refresh</td><td>DIN00210889</td><td></td></tr><tr><td>Shaver</td><td>Oster</td><td>78997-010</td><td></td></tr><tr><td>High speed camera</td><td>Fastec</td><td>Fastec IL3-100</td><td></td></tr><tr><td>High power light</td><td>Smith Victor Corporation</td><td>Model 700 SG (600 Watt quartz light, 120 Volts)</td><td></td></tr><tr><td>Light Stand</td><td>Promaster</td><td>LS1</td><td></td></tr><tr><td>Treadmill</td><td>Custom built at the Zoological Institute, University of Cologne</td><td></td><td></td></tr><tr><td>Microsoft Excel 2016</td><td>Microsoft</td><td>Version 2016</td><td></td></tr><tr><td>KinemaJ</td><td>Nicolas Stifani</td><td></td><td>This is a script generated for use with ImageJ</td></tr><tr><td>KinemaR</td><td>Nicolas Stifani</td><td></td><td>This is a script generated for use with Rstudio</td></tr><tr><td>Vicon Motus</td><td>Vicon Motus</td><td>Version 9.00</td><td></td></tr><tr><td>GraphPad Prism</td><td>GraphPad</td><td>Version 6.00</td><td></td></tr></tbody></table>

sagittal plane kinematic gait analysis in c57bl/6 mice subjected to mog35-55 induced experimental autoimmune encephalomyelitis

运动步态分析在矢状面经常被用来表征运动缺陷的多发性硬化 (MS)。我们描述了这些技术的应用, 以确定步态缺陷的 MS, 称为实验性自身免疫脑脊髓炎 (EAE) 的小鼠模型。瘫痪和运动缺陷的小鼠受 EAE 通常评估使用临床评分规模。然而, 这个规模只产生的序号数据, 提供了很少的信息的确切性质的电机赤字。EAE 疾病的严重性也评估了 rotarod 的性能, 这提供了一个一般的运动协调的措施。相比之下, 矢状面后肢的运动学步态分析产生了关于运动如何受损的高度精确的信息。为了执行这一过程, 反射标记被放置在后肢, 以检测关节运动, 而鼠标在跑步机上行走。运动分析软件用于测量行走过程中标记物的运动。然后从结果数据中推导出运动学步态参数。我们展示了这些步态参数是如何用来量化 EAE 的髋、膝和踝关节运动损伤的。这些技术可以用来更好地了解疾病的机制, 并确定潜在的治疗 MS 和其他神经退行性疾病, 损害移动性。

图 1是用于运动学步态分析的过程的示意图表示。首先, 在5解剖点上制作并放置在鼠标上的反射标记。当鼠标在跑步机上行走时, 步态会被记录下来。运动分析软件用于提取运动学数据进行后续分析。
图 2A-c表示控制 CFA 鼠标对臀部、膝盖和踝关节角度的步进周期, 记录在相隔一周的三连续记录会话中。波形之间的重叠显示1-3 节的步骤周期中的最小偏差。图 2D-f表示第二个控制 CFA 鼠标的步骤周期, 它显示了从记录会话1-3 的更大的行走可变性。虽然步周期沿 y-axis 移动, 波形的形状在录音之间保持一致。这种变化的水平是典型的鼠标行走。
图 3A-c表示 EAE 在三连续记录会话中记录的鼠标的步骤周期。从第一次到第二次录音, 步态的变化极小, 但在第三次会议上, 步态在所有三关节上都发生了深刻的改变。对于臀部, 一个显着的扁平化的步骤周期已经发生, 表明大量的运动损失。膝盖变得更弯曲, 更不能够伸展和支持动物的体重。踝关节的运动也有极大的改变。脚背和跖屈在摆动 (白色面板) 和立场 (绿色面板) 阶段, 分别延迟。这些缺陷表明, 在这个关节肌肉薄弱, 因为它的能力, 以提高其脚在摆动阶段, 并推动身体向前在立场阶段。
在图 4中显示的以下数据是从 Fiander et al.中重新发布的(2017)具有权限的26 。利用 one-way 重复测量方差分析栎-Sidak 多次比较试验, 将所有时间点与基线26进行比较。在每个时点计算平均角度 (图 4A和图 4D)、运动范围 (图 4B和图 4E) 和 RMS 差异 (数字 4C和数字 4F), 以量化步态赤字 (每组 n=8)。在目前的 EAE 实验中, 临床评分的开始是 DPI 14, 这是在第二周的录音之后。CFA 小鼠的平均膝关节角度 (图 4A) 或膝关节 RMS 差异 (图 4C) 没有变化, 但没有表现出一个小的增加膝关节运动范围 [F (27) = 5.871, p = 0.0083], DPI 16 和30相对于基线 (图 4B)。这种细微的变化可能反映了 CFA 注射的痛苦。与 CFA 动物对比, 有大变动在膝盖关节为 EAE 动物为平均角度 [F (67) = 11.08, p和 #60; 0.0001] (图 4D), 运动范围 [F (67) = 14.42, p 和 #60; 0.0001] (图 4E) 和 RMS差异 (图 4F)。平均角度明显减少, 表明 EAE 小鼠的膝盖在行走时更有弯曲。这可能表明肌肉无力, 因为动物无法伸展他们的膝关节, 以支持他们的体重。运动的范围也减少了, 又可能是由于动物无法伸展膝关节。膝关节有效值差异显著增加, 表明 EAE 小鼠膝关节的运动与基线记录基本不同。
使用 one-way 重复测量分析了图 5中的数据, 栎-Sidak 多次比较测试将步态参数值与临床评分 0.5-3.5 的结果进行了比较, 并将临床分值0。相关分析也进行了使用长矛 (ρ)。平均膝关节角度 (图 5A)、运动范围 (图 5B) 和 RMS 差异 (图 5C) 与临床评分 (p和 #60; 0.001) 密切相关。这些关节运动与经典临床评分的相关性证实了运动步态分析对 EAE 小鼠的运动缺陷进行评估的有效性。膝关节运动范围 (图 5A) 和 RMS 差异 (图 5C) 从临床评分 2.0 (p和 #60; 0.05) 开始显著减少。这些发现表明, 受损的膝部运动不会导致因临床评分低于2.0 而发现的运动缺陷。然而, 膝关节平均角度 (图 5B) 从临床评分 1.0 (p和 #60 0.05) 开始减少。这表明, 对于膝关节运动, 平均角度是最敏感的三措施。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56032/56032fig1v2.jpg"> 
图 1: 用鼠标进行运动学步态记录的示意图.一旦反射标记被做, 他们被放置在髂嵴, 髋关节, 踝关节, 趾关节, 和尖端的第四位数。当鼠标在跑步机上行走时, 用高速摄影机记录步态。运动分析软件用于提取步态参数, 用于后续分析。<a href="//ecsource.jove.com/files/ftp_upload/56032/56032fig1v2large.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/56032/56032fig2v2.jpg"> 
图 2: 接收 CFA 的两个控制小鼠的步进周期波形示例 白色和绿色背景分别代表摇摆和姿态阶段。对于鼠标 1, 臀部 (A)、膝部 (B) 和踝关节 (C) 步进周期波形在间隔一周的3连续录制会话中相互重叠。对于鼠标 2, 臀部 (D)、膝部 (E) 和踝关节 (F) 的步进周期波形因行走行为的固有变异性而稍有偏离。<a href="//ecsource.jove.com/files/ftp_upload/56032/56032fig2v2large.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_uploa alt="Figure 3" class="xfigimg" src=""></img alt="Figure 3" class="xfigimg" src="/files/ftp_uploa>d/56032/56032fig3v2 
图 3: EAE 小鼠的步进周期波形.白色和绿色背景分别代表摇摆和姿态阶段, 连续三个录音会议间隔一个星期分开。在 3rd记录会话中, 髋关节 (A)、膝部 (B) 和踝关节 (C) 波形因 EAE 疾病进展而大大改变。<a href="//ecsource.jove.com/files/ftp_upload/56032/56032fig3v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56032/56032fig4v2.jpg"> 
图 4: 平均角度、运动范围和根均方根用于分析运动学数据.计算平均角度、运动范围和 RMS 差异, 以量化 EAE 小鼠的运动缺陷。平均膝关节角度 (A)、运动范围 (B) 和 RMS (C) 对 CFA 小鼠的影响仍然相对恒定。EAE 的小鼠显示膝关节平均角 (D)、运动范围 (E) 和 RMS (F) 受损。数据以平均±标准差表示;* p和 #60; 0.05, ** p和 #60; 0.01, *** p和 #60; 0.001, 日后免疫接种 (DPI)-2 的差异;# p & #60; 0.05, 与峰值赤字的区别。转载于参考文献 26, 经原出版商许可。<a href="//ecsource.jove.com/files/ftp_upload/56032/56032fig4v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56032/56032fig5v2.jpg"> 
图 5.平均膝关节角度、运动范围和 RMS 差异与临床评分相关 对膝关节运动的三运动学指标与临床评分进行相关分析, 比较两种方法。平均膝关节角度 (A)、运动范围 (B) 和 RMS 差异 (C) 与临床评分密切相关。膝关节的运动和 RMS 的差异减少开始的临床评分为 2.0, 而平均膝关节角下降较早的临床评分为1.0。数据以平均±标准差表示;* p和 #60; 0.05 与临床评分的差异0.0。对于ρ, ** p和 #60; 0.001。<a href="//ecsource.jove.com/files/ftp_upload/56032/56032fig5v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis at JoVE.com

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

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

矢状面的运动学步态分析获得了关于如何执行运动的高度精确的信息。我们描述了这些技术的应用, 以确定小鼠的步态缺陷受自身免疫介导的鞘。这些方法也可以用来表征步态缺陷的其他小鼠模型的运动障碍。

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

sagittal-plane-kinematic-gait-analysis-c57bl6-mice-subjected-to-mog35

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

8.5K 次观看.  Dalhousie University. 矢状面的运动学步态分析获得了关于如何执行运动的高度精确的信息。我们描述了这些技术的应用, 以确定小鼠的步态缺陷受自身免疫介导的鞘。这些方法也可以用来表征步态缺陷的其他小鼠模型的运动障碍。

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

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

髓鞘少突胶质细胞糖蛋白（MOG<sub&gt; 35-55</sub&gt;）诱导实验性自身免疫性脑脊髓炎（EAE）的C57BL / 6小鼠

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

科研

免疫与感染

3D Kinematic Gait Analysis for Preclinical Studies in Rodents

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of complex algorithms and mathematical modeling that accompany 3D data collection and analysis procedures. This work provides a simple, user-friendly, step-by-step detailed methodology for 3D kinematic gait analysis during treadmill locomotion in healthy and neurotraumatic rats using a six-camera motion capture system. Also provided are details on 1) calibration of the system in an experimental set-up customized for quadrupedal locomotion, 2) data collection for treadmill locomotion in adult rats using markers positioned on all four limbs, 3) options available for video tracking and processing, and 4) basic 3D kinematic data generation and visualization and quantification of data using the built-in data collection software. Finally, it is suggested that the utility of this motion capture system be expanded to studying a variety of motor behaviors before and after neurotrauma.

Presented here is a protocol to collect and analyze three-dimensional kinematics of quadrupedal locomotion in rodents for preclinical studies.

3D运动性步态分析,用于啮齿动物的临床前研究

The utility of three-dimensional (3D) kinematic motion analysis systems is limited in rodents. Part of the reason for this inadequacy is the use of ...

行为学

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 ...

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 ...

神经科学

Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with embedded pericytes, and the glia limitans composed by the parenchymal basement membrane and astrocytic end-feed embracing the abluminal aspect of central nervous system (CNS) microvessels. In addition to maintaining CNS homeostasis the NVU controls immune cell trafficking into the CNS. During immunosurveillance of the CNS low numbers of activated lymphocytes can cross the endothelial barrier without causing BBB dysfunction or clinical disease. In contrast, during neuroinflammation such as in multiple sclerosis or its animal model experimental autoimmune encephalomyelitis (EAE) a large number of immune cells can cross the BBB and subsequently the glia limitans eventually reaching the CNS parenchyma leading to clinical disease. 
Immune cell migration into the CNS parenchyma is thus a two-step process that involves a sequential migration across the endothelial and glial barrier of the NVU employing distinct molecular mechanisms. If following their passage across the endothelial barrier, T cells encounter their cognate antigen on perivascular antigen-presenting cells their local reactivation will initiate subsequent mechanisms leading to the focal activation of gelatinases, which will enable the T cells to cross the glial barrier and enter the CNS parenchyma. Thus, assessing both, BBB permeability and MMP activity in spatial correlation to immune cell accumulation in the CNS during EAE allows to specify loss of integrity of the endothelial and glial barriers of the NVU. 
We here show how to induce EAE in C57BL/6 mice by active immunization and how to subsequently analyze BBB permeability in vivo using a combination of exogenous fluorescent tracers. We further show, how to visualize and localize gelatinase activity in EAE brains by in situ zymogaphy coupled to immunofluorescent stainings of BBB basement membranes and CD45+ invading immune cells.

Here, we present protocols to investigate impairment of the neurovascular unit during experimental autoimmune encephalomyelitis in vivo. We specifically address how to determine blood-brain barrier permeability and gelatinase activity involved in leukocyte migration across the glia limitans.&#160;

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

The neurovascular unit (NVU) is composed of microvascular endothelial cells forming the blood-brain barrier (BBB), an endothelial basement membrane with ...

使用 MouseWalker 量化脊髓损伤小鼠的运动功能障碍

Using the MouseWalker to Quantify Locomotor Dysfunction in a Mouse Model of Spinal Cord Injury

The execution of complex and highly coordinated motor programs, such as walking and running, is dependent on the rhythmic activation of spinal and supra-spinal circuits. After a thoracic spinal cord injury, communication with upstream circuits is impaired. This, in turn, leads to a loss of coordination, with limited recovery potential. Hence, to better evaluate the degree of recovery after the administration of drugs or therapies, there is a necessity for new, more detailed, and accurate tools to quantify gait, limb coordination, and other fine aspects of locomotor behavior in animal models of spinal cord injury. Several assays have been developed over the years to quantitatively assess free-walking behavior in rodents; however, they usually lack direct measurements related to stepping gait strategies, footprint patterns, and coordination. To address these shortcomings, an updated version of the MouseWalker, which combines a frustrated total internal reflection (fTIR) walkway with tracking and quantification software, is provided. This open-source system has been adapted to extract several graphical outputs and kinematic parameters, and a set of post-quantification tools can be to analyze the output data provided. This manuscript also demonstrates how this method, allied with already established behavioral tests, quantitatively describes locomotor deficits following spinal cord injury.

作者聚焦:使用 MouseWalker 量化脊髓损伤小鼠模型中的运动功能障碍  

An experimental pipeline to quantitatively describe the locomotor pattern of freely walking mice using the MouseWalker (MW) toolbox is provided, ranging from initial video recordings and tracking to post-quantification analysis. A spinal cord contusion injury model in mice is employed to demonstrate the usefulness of the MW system.

使用MouseWalker量化脊髓损伤小鼠模型中的运动功能障碍

The execution of complex and highly coordinated motor programs, such as walking and running, is dependent on the rhythmic activation of spinal and ...

小鼠实验性自身免疫性脑脊髓炎的诱导和评价

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

作者聚焦：创建与雄性和雌性小鼠相关的多功能实验性自身免疫性脑脊髓炎模型 

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

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

摘要

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此视频中的章节

髓鞘少突胶质细胞糖蛋白（MOG

3D运动性步态分析,用于啮齿动物的临床前研究

变性小鼠年龄依赖性运动障碍的步态分析

神经肌肉疾病小鼠模型行为花型的低成本盖特分析

慢性压迫性损伤小鼠步态的自动分析

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

使用MouseWalker量化脊髓损伤小鼠模型中的运动功能障碍

使用MouseWalker量化脊髓损伤小鼠模型中的运动功能障碍

使用MouseWalker量化脊髓损伤小鼠模型中的运动功能障碍

两性多发性硬化症建模：MOG_35-55 诱导的实验性自身免疫性脑脊髓炎

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

摘要

探索更多视频

此视频中的章节

髓鞘少突胶质细胞糖蛋白（MOG

3D运动性步态分析,用于啮齿动物的临床前研究

变性小鼠年龄依赖性运动障碍的步态分析

神经肌肉疾病小鼠模型行为花型的低成本盖特分析

慢性压迫性损伤小鼠步态的自动分析

体内自身免疫性脑髓炎实验性神经血管内障碍和胶质屏障的可视化

使用MouseWalker量化脊髓损伤小鼠模型中的运动功能障碍

使用MouseWalker量化脊髓损伤小鼠模型中的运动功能障碍

使用MouseWalker量化脊髓损伤小鼠模型中的运动功能障碍

两性多发性硬化症建模：MOG35-55 诱导的实验性自身免疫性脑脊髓炎

两性多发性硬化症建模：MOG_35-55 诱导的实验性自身免疫性脑脊髓炎