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...

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 border="0" cellpadding="0" cellspacing="0" width="893">
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			<td style="width:344px;">Nikon D750</td>
			<td style="width:167px;">Used to film the video</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Reflective tape</td>
			<td>B&#38;L Engineering</td>
			<td>MKR-Tape-2</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fine scissors</td>
			<td>Fine Science Tools</td>
			<td>15023-10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glue gun</td>
			<td>Craftsmart</td>
			<td>E231647</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">scalpel handle #4</td>
			<td>Roboz</td>
			<td>R5-9884</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Scalpel Blade No.10</td>
			<td>Feather</td>
			<td>2020-12</td>
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			<td height="40" style="height:40px;">C57BL/6 mice</td>
			<td>Charles River Laboratories</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Anesthetic machine</td>
			<td>EZ Anesthesia</td>
			<td>EZ-AF9000 Auto Flow System</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Recirculating water heating blanket</td>
			<td>Androit</td>
			<td>HTP-1500</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">topical eye lubricant</td>
			<td>Refresh</td>
			<td>DIN00210889</td>
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		<tr height="40">
			<td height="40" style="height:40px;">Shaver</td>
			<td>Oster</td>
			<td>78997-010</td>
			<td></td>
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			<td height="40" style="height:40px;">High speed camera</td>
			<td>Fastec</td>
			<td>Fastec IL3-100</td>
			<td></td>
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		<tr height="40">
			<td height="40" style="height:40px;">High power light</td>
			<td>Smith Victor Corporation</td>
			<td>Model 700 SG (600 Watt quartz light, 120 Volts)</td>
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			<td height="40" style="height:40px;">Light Stand</td>
			<td>Promaster</td>
			<td>LS1</td>
			<td style="width:167px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;">Treadmill</td>
			<td colspan="2">Custom built at the Zoological Institute, University of Cologne</td>
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			<td height="40" style="height:40px;">Microsoft Excel 2016</td>
			<td>Microsoft</td>
			<td>Version 2016</td>
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		<tr height="40">
			<td height="40" style="height:40px;">KinemaJ</td>
			<td>Nicolas Stifani</td>
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			<td>This is a script generated for use with ImageJ</td>
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		<tr height="40">
			<td height="40" style="height:40px;">KinemaR</td>
			<td>Nicolas Stifani</td>
			<td></td>
			<td>This is a script generated for use with Rstudio</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vicon Motus</td>
			<td>Vicon Motus</td>
			<td>Version 9.00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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?...

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

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

Assessing Burrowing, Nest Construction, and Hoarding in Mice

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening of possible treatments for AD currently largely focuses on cognitive testing, which frequently demands expensive equipment and lots of experimenter time. However, human episodic memory (the most severely affected aspect of memory in AD) is different to rodent memory, which seems to be largely non-episodic. Therefore the present ways of screening for new AD treatments for AD in rodents are intrinsically unlikely to succeed. A new approach to preclinical screening would be to characterise the ADL of mice. Fortuitously, several such assays have recently been developed at Oxford, and here the three most sensitive and well-characterised are presented.
Burrowing was first developed in Oxford13. It evolved from a need to develop a mouse hoarding paradigm. Most published rodent hoarding paradigms required a distant food source to be linked to the home cage by a connecting passage. This would involve modifying the home cage as well as making a mouse-proof connecting passage and food source. So it was considered whether it would be possible to put the food source inside the cage. It was found that if a container was placed on the floor it was emptied by the next morning., The food pellets were, however, simply deposited in a heap at the container entrance, rather than placed in a discrete place away from the container, as might be expected if the mice were truly hoarding them. Close inspection showed that the mice were performing digging ("burrowing") movements, not carrying the pellets in their mouths to a selected place as they would if truly hoarding them.6



Food pellets are not an essential substrate for burrowing; mice will empty tubes filled with sand, gravel, even soiled bedding from their own cage. Moreover, they will empty a full tube even if an empty one is placed next to it8.



Several nesting protocols exist in the literature. The present Oxford one simplifies the procedure and has a well-defined scoring system for nest quality5. 



A hoarding paradigm was later developed in which the mice, rather than hoarding back to the real home cage, were adapted to living in the "home base" of a hoarding apparatus. This home base was connected to a tube made of wire mesh, the distal end of which contained the food source. This arrangement proved to yield good hoarding behaviour, as long as the mice were adapted to living in the "home base" during the day and only allowed to enter the hoarding tube at night.

Burrowing, nesting, and hoarding are species-typical activities that mice readily perform in the laboratory. This article describes how they can be easily and cheaply assessed. These protocols are extremely sensitive to mouse strain, brain lesions and diseases. Moreover they constitute &#x201C;environmental enrichment&#x201D; for the mice, and embody the &#x201C;Refinement&#x201D; aspect of the &#x201C;3 Rs&#x201D;18.

评估挖洞，在小鼠的鸟巢建设和囤积

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening ...

科研

神经科学

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

测量小鼠运动协调

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This is mostly due to the difficulty of microdissection of fetal brains and their sectioning for determination of apoptotic cells caused by prenatal exposure to alcohol. The experiments described here provide visualized techniques from mice breeding to the identification of cell death in fetal brain tissue. This study used C57BL/6 mice as the animal model for studying fetal alcohol exposure and the role of trophic peptide against alcohol-induced apoptosis. The breeding consists of a 2-hr matting window to determine the exact stage of embryonic age. An established fetal alcohol exposure model has been used in this study to determine the effects of prenatal alcohol exposure in fetal brains. This involves free access to alcohol or pair-fed liquid diets as the sole source of nutrients for the pregnant mice.
The techniques involving dissection of fetuses and microdissection of fetal brains are described carefully, since the latter can be challenging. Microdissection requires a stereomicroscope and ultra-fine forceps. Step-by-step procedures for dissecting the fetal brains are provided visually. The fetal brains are dissected from the base of the primordium olfactory bulb to the base of the metencephalon.
For investigating apoptosis, fetal brains are first embedded in gelatin using a peel-away mold to facilitate their sectioning with a vibratome apparatus. Fetal brains embedded and fixed in paraformaldehyde are easily sectioned, and the free floating sections can be mounted in superfrost plus slides for determination of apoptosis or cell death.
TUNEL (TdT-mediated dUTP Nick End Labeling; TdT: terminal deoxynucleotidyl transferase) assay has been used to identify cell death or apoptotic cells. It is noteworthy that apoptosis and cell-mediated cytotoxicity are characterized by DNA fragmentation. Thus, the visualized TUNEL-positive cells are indicative of cell death or apoptotic cells.
The experimental designs here provide information about the use of an established liquid diet for studying the effects of alcohol and the role of neurotrophic peptides during pregnancy in fetal brains. This involves breeding and feeding pregnant mice, microdissecting fetal brains, and determining apoptosis. Together, these visual and textual techniques might be a source for investigating prenatal exposure of harmful agents in fetal brains.

The experimental designs proposed here focus on studying the effects of alcohol exposure in apoptosis and the application of neurotrophic peptide during pregnancy in fetal brain. A detailed description from the breeding to the collection of fetal brains is described. Techniques for determination of apoptosis are also described in detail.

实验方法测试的影响，在怀孕期间对酒精诱导的细胞凋亡在C57BL / 6小鼠神经营养肽，ADNF-9

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用<em&gt;在子宫内</em&gt;电穿孔的C57BL / 6小鼠

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

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 ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

C57BL/6 小鼠自身免疫性神经炎的功能和病理评价的简易方法

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

c57bl6 小鼠耳蜗植入术与电唤起脑干反应记录

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

小鼠体内合成蛋白的定量测量

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

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 ...