这项研究得到了发现格兰特AAW从加拿大自然科学和工程研究理事会。

1。生物力学步态分析的先决条件着手进行购买昂贵的运动功能分析设备，并规划将进行运动学和/或动能的运动功能评估的实验之前，它是实验者必须熟悉的技术和实践方面的生物力学分析，感觉的行为，操作性条件反射的动物和处理/存储/管理大量的数字化数据。虽然这些先决条件似乎是显而易见的许多的，它是后，才开始根据这些类型的实验，让学员认识到执行比较详细的运动功能分析技术和实践的复杂性。作者建议实验者参加在介绍生物力学的交易过程，熟悉或雇人熟悉数据管理所需的一种编程语言，并具有同等的重要性，花大量的时间进行交互，处理，并与实验动物工作。对于理解运动神经系统科学的分析和感觉运动行为分析，实验者被称为几个重要的参考（ 见1，4-7） 。 2。运动学和动力学测试仪器一个运动学和动力学的检测仪器，用于收集双边数据非常有用，是由以下几部分组成（见“表特定的试剂和仪器”更详细）组成： <ul><li>安静和足够大小的房间（不一定隔音，虽然在低流量区位于） </li><li>广播电台提供背景“噪音”，以限制从房间外的动物分心的噪音</li><li>从两端跑道等距离的中心位于透明有机玻璃封闭，平板表面跑道（180厘米长X宽20厘米x 60厘米高），方孔（11厘米× 11厘米） </li><li>这块丛玻璃（10.5厘米X 10.5厘米） </li><li>双面胶透明胶带</li><li> 3M反光胶带皮肤标记</li><li>创建皮肤标记打孔</li><li> Isofluorane anesthetizing动物麻醉机前先剃须，皮肤标记的位置</li><li>麦片为鼓励运动的动物，沿跑道的长度</li><li> AMTI HE6X6 forceplate </li><li>维科Motus定制的系统（软件，计算机等） </li><li> 4个摄像机（至少60 Hz的采样）与适当的连接电缆和三脚架</li><li> 4摄像机安装卤素灯提供最佳的照明动物主题的照明和皮肤反射标记</li><li>适当大小的标定架或卷威康Motus系统所必需的X，Y，Z位置校准成有意义的距离措施</li></ul> 3。动物训练收集数据之前，每个动物的，必须经过培训，跨平坦的表面，封闭跑道。从一个合适的动物供应商在收到大鼠，动物被驯化1周的新家。在此驯化的时间，几个麦片每天放置到老鼠的笼子。动物是限制其维护能源需求的食物，防止肥胖，并确保执行此任务的动力。此后，每个动物的处理10-15分钟，每天1周的实验者。在同一时期，每个动物被放置到位于任一端与麦片跑道。一旦动物变得熟悉他们的环境，他们将开始吃麦片。一旦动物的舒适，吃麦片内的跑道，然后实验者必须operantly条件的动物为食物奖励运行跑道的长度。这是通过折腾¼ cheerio大鼠是定位在跑道的另一端。一旦老鼠吃这个cheerio，另一荐cheerio被放置在跑道的另一端。这样做是为了每天15-20分钟，直到沿跑道的大鼠一贯（&gt; 90％），扔在一个恒定的速度移动（即没有启动，停止，探索，或在不改变步态）吃奔腾/边界的cheerio 。老鼠只应雇用小跑的步态。这一任务的动物在空调可导致动物奔腾和边界，这些步态是指示性动物旅行&gt; 90厘米/秒。边界和奔腾的步态，生物力学，更难以解释为由于各种原因（如腿铅列入标准等）。在我们的经验，一旦大鼠一贯采用奔腾或边界的步态，它是困难的，如果不是不可能的的，在跑道locomoting时，他们使用一个小跑步态。都是世所罕见的动物遭受从周边或中枢神经系统损伤后的速度&gt; 90厘米/秒。是变化的时间才能达到成功的培训BLE大鼠株和两性之间。只Wistar，刘易斯，埃文斯长，和只Sprague - Dawley株都能够始终如一地穿越跑道，2个星期内，从发病培训 8 。根据我们的经验，菲舍尔（F 344）大鼠往往需要4-6周以上，学习这方面的任务 8 。 4。关节位置标记前肢的运动学分析是不可靠的由于皮肤运动，置于皮肤上的物种加剧的前肢，像老鼠，有一个蜷缩的姿态9标记的工件。相反，前肢运动必须达到使用X射线摄影或透视10-13。因此，后肢关节的位置标志的位置仅仅是本文所述。 收集数据之前，各组大鼠麻醉后至少24小时在数据采集之前使用适当的吸入性麻醉剂（例如isofluorane，拨通了一个精确的vaporizo​​r 1.5-2％）和管理，通过面罩吸氧，关键地形解剖地标必须标明。鉴于简洁的程序，因为不使用长效麻醉剂，使用动物变暖设备不需要使用，以保持动物的体温。动物麻醉后，后肢和背部，髂脊水平是光头。动物，然后放置在胸骨斜卧和其后肢放置在一个近似的常设使用企业包装泡沫来支持它的位置。覆髂波峰最颅部分的皮肤，大粗隆股骨，胫骨粗隆外侧，跗关节，和第 5跖骨的远端和侧面标有一个无毒的永久性标记。该动物从麻醉中恢复。对于时间的研究，定期麻醉可能需要刮胡子的后肢从而允许随后的反射标记的位置（见下文）。此外，每日以前标记的解剖标志（使用相同的无毒标记）突出须老鼠会慢慢消除通过自然的修饰行为的标记。 5。数据记录所有摄像机的意见进行检查，以确保他们的立场是适当的，捕捉相同的视野。每个摄像机应放在对方约60-80度。视野应包括在中心forceplate和跑道的长度足以捕捉到两个大步。 校准量是放置在预先确定的跑道面积。一个每个相机，跑道内的校准量的单帧捕获。所有沿两极的长度校准标志是数字化。只有一次，一个令人满意的错误是在数字化完成，可以进行的运动数据收集实验者。此校准步骤是前收集数据的关键。如果不进行准确校准，如果校准不会立即发生之前记录会话，所有产生的数据将不准确，无法使用。重要的是，如果任何相机触摸或移动，它是最安全的承担，需要重复校准系统。 之前放置在跑道上的动物，它的重量记录，并预先制作的锥形反光皮肤标记（使用3M反光胶带，见附表）坚持在预先确定的感觉后肢地形地标标志。记录动物的体重将允许追溯地面反作用力 - 体重正常化的一个重要方面，如果想使群体之间的比较。此外，测量体重，有利于在实验期间监测动物的总体健康。标记的位置，只需要适当的动物处理和不需要麻醉的动物。如果反光胶带粘合剂不足，坚持以动物的皮肤，极少量的无毒胶水（如3M VetBond组织粘合剂）可以用来促进动物的身体上的标记的坚持。一旦后肢上的标记上，实验者应舒适的位置，电脑的键盘附近上，已在手通过威康Motus系统连接事件标记。使用校准文件作为模板，有几个文件是在预先录制。通常情况下，25至30个文件需要保存。每个文件应命名为唯一。每个文件都将代表一个记录被记录动物的运行。二十五个30个文件，以确保收集足够数量的符合纳入标准的速度（即60-90厘米/秒）的运行。足够数量的文件被创建后，实验者可以开始收集数据。电子xperimenter必须提示跑道内穿梭的老鼠扔在跑道两端麦片荐件。认真协调和时序，在启动触发事件标记和前完成沿跑道成功完成了一通大鼠。原油地面反作用力跟踪研究后，录制后的左侧或右侧肢体是否打力板，该文件是保存和关闭。平等的左，右侧肢体forceplate点击数应记录在案。从一个给定的运行记录数据的过程是反复进行，直到有足够数量的运行已记录。 6。数据分析运动学和地面反作用力完成数据收集后，每一种动物从每个的运行需要评估的速度。使用两个相对固定的标志物（如翅膀的iliums），可以评估一个虚拟点之间的标记（创建威康Motus文件模板时之前完成）。在这个“虚拟”的点的速度计算，髂标记的标记必须数字化。使用威康motus软件，这个虚点在X方向（水平方向运动）的速度计算。在这样做时，只运行一个给定范围内的速度内（确定先验 ）在最后的分析。我们发现，动物60-90厘米/秒之间移动使用的是一致的小跑步态。最小的10次（5运行，左侧肢体接触与力平台和5运行右侧肢体的力量接触的平台）是必需的。一旦确定每个动物可接受的运行，其余皮肤标记的数字化工作必须完成。 为了弥补皮肤超过膝盖的运动伪影，膝盖的位置的估计是计算三角（两圆相交的二维运动学;或交叉点之间的2球三维运动学），先前已描述。现在可以决定的，髋关节，膝关节和飞节的角度，速度和加速度。立场和摆动时间也可以进行评估，但其准确性是有限的，根据被雇用的摄像机的采样速度。这些以及其他可以直接计算（即无出口）使用威康Motus KineCalc软件或数据可以导出为ASCII数据和分析软件如Matlab使用自定义的例程。 地面反作用力的数据测量和放大AMTI测力台，并在1200赫兹威康Motus收集。因此，一旦地面反作用力的数据收集，适当的数字滤波器适用于使用维康Motus的数据。由于实验者已经确定适当的皮肤标志，地面反作用力同时作为动态数据收集的数据，可以使用维康Motus KineCalc直接或间接使用一些其他的定制软件进行分析后，数字化的旅行，并确定了可接受的运行速度例程。各种力量的变量，在三个正交方向，可以计算的。这些变量包括峰值力，面积UND曲线（即脉冲）等，然而，更重要的是，实验者必须为每个单独的每种动物的运行保持左，右肢数据。从右侧或左侧肢体提取出的数据是平均为每个动物和动物的有代表性的数据。数据，然后使用适当的统计程序进行分析。 7。代表性的成果为了表示这种形式的运动分析的实用工具，运动学和地面反作用力被确定为青年，中年和老年的雌性Wistar大鼠。从这种分析中，年龄有关的差异，发现雌性Wistar大鼠。特别是，地面反作用力的分析表明，减少老年大鼠前肢的制动能力，往往使用横向稳定的后肢比其他群体的动物（图1）。运动学分析没有透露各组间的统计差异，虽然表明，运动可以很容易地从几乎任何年龄大鼠（图2）记录。 <img src="/files/ftp_upload/2138/2138fig1tmb.jpg" alt="图1" /> 图地面反作用力的跟踪取自年轻左侧肢体（4月龄; N = 7），中年（13-14月龄; N = 7）和老年（24月龄; N = 5 ）雌性Wistar大鼠。右侧肢体相似。这是显而易见的老年大鼠，使用它们的前肢制动（* = P &lt;0.05）相比，青年和中年大鼠，老年大鼠幼鼠相比，横向稳定倾向于使用他们的后肢(**).实线表示的意思是，虚线表示平均值± SE;虚线线代表平均东南<a href="http://www.jove.com/files/ftp_upload/2138/2138fig1.jpg">，请点击这里看到一个更大的</a>数字。 <img src="/files/ftp_upload/2138/2138fig2tmb.jpg" alt="图2" /> 图2，青年，中年和老年大鼠的左侧肢体运动坚持数字。棒的数字代表的左侧肢体各组的平均运动。有动物群体之间无显着性关节角度的差异，在<a href="http://www.jove.com/files/ftp_upload/2138/2138fig2.jpg">步骤</a>周期<a href="http://www.jove.com/files/ftp_upload/2138/2138fig2.jpg">，请点击这里看到一个更大</a>的数字。

本文件提供评估运动使用连续定量运动学和地面反作用力的决心的方法。重要的是这种方法的一项有兴趣的人在运动生物力学，感觉动物的行为，数据管理和操作的强大背景。虽然运动学和地面反作用力的决心需要更多的时间和专业知识，一些其他形式的运动分析（如端点措施，有序评定量表）相比，所获得的数据是敏感的，客观的和定量的各种14-22骨科和神经1-3，23-32模型的疾病，在不同的物种。 我们所提供的数据描述应变匹配的大鼠的信息不能收集的各种年龄使用简单和较不敏感的措施，在运动之间的差异。此外，运动的运动学和动力学分析已用于描述在多种神经系统的条件下，其他形式的评价是不成功的中的运动变化，8日，23日至25日，27使用敏感的措施进行评估时，就显得尤为重要潜在therapeutants各种型号的疾病。如果测试是不够敏感，辨别一个潜在的therapeutant运行实验者犯了II型统计错误的风险的影响（即结论有没有治疗效果时，其实有效果）。此外，因为端点措施和评估运动的主观测试中，存在一种偏见的潜力。运动学和动力学的评价在这纯粹是客观的，提供适当的纳入/ 排除的标准是先验的，实验者简单的收集，审查和适当的统计数据（即有没有主观成分测定数据）。 运动学和动力学分析，也能用于众多物种的能力。事实上，运动学，地面反作用力的决心，或两者已被用于多种物种，如大象33-35，牛36，马37-40，狗4，41-45，猫21，46-49，各种啮齿类动物3，8，50，51，52-55，鸟类4，鱼56，57（这个列表并不详尽）。然而，在作者的经验，对小鼠的使用是有问题的的老鼠不容易operantly沿跑道条件。鉴于此，小鼠不会旅行在一个相对恒定的转速的，而加快和减慢时，穿越跑道。部分，此行为可能是运行在一台跑步机和视频在跑步机上的录音动物locomoting 58小鼠克服实验仪器的地面反作用力的决心，如果是跑步机的使用进行修改，地面反作用力的决心可能只有很容易获得垂直地面反作用力作为跑步机的皮带会干扰前船尾和德尔梅迪奥侧向力的决心。 总而言之，运动的运动学和动力学分析是一种可靠，灵敏，客观的方法，可用于各种型号的整形外科和神经系统的条件聘用。此外，所有的设备已成为在啮齿类动物中使用，从而否定任何不执行这种形式感觉行为分析的相关原因。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Low load miniature force platform</td>
<td>&nbsp;</td>
<td>AMTI</td>
<td>HE6X6</td>
<td>http://amti.biz/PDFDownloads/0908HE6x6.pdf</td>
</tr>
<tr>
<td>Vicon Motus</td>
<td>&nbsp;</td>
<td>Vicon</td>
<td>Vicon Motus software</td>
<td>http://www.vicon.com/applications/animal.html 

The force platform can be purchased as an integrated package with the Vicon Motus system.</td>
</tr>
<tr>
<td>Vicon Motus KineCalc</td>
<td>&nbsp;</td>
<td>Vicon</td>
<td>Vicon Motus KineCalc software</td>
<td>http://www.vicon.com/applications/animal.html Proprietary software compatible with Vicon Motus data, thus enabling direct and customizable data calculations.
</td>
</tr>
<tr>
<td>Video Cameras</td>
<td>&nbsp;</td>
<td>Vicon</td>
<td>Varies depending upon needs</td>
<td>http://www.vicon.com/applications/animal.html A minimum of 4 cameras are required for bilateral 2D or 3D imaging of hindlimb kinematics during rat locomotion. Cameras that sample at 60 Hz are sufficient for rats locomoting between 60 and 90 cm/s. High speed cameras will permit identification of very small, yet possibly important, differences in interlimb coordination.</td>
</tr>
<tr>
<td>Calibration volume or frame</td>
<td>&nbsp;</td>
<td>Vicon</td>
<td>Custom</td>
<td>http://www.vicon.com/applications/animal.html An appropriate calibration volume must be purchased and used for calibrating the coordinates prior to performing videography each and every time of new data collection or if any of the cameras are moved. This dimensions of this calibration volume are based upon the field of view being examined.
</td>
</tr>
<tr>
<td>Reflective tape</td>
<td>&nbsp;</td>
<td>3M</td>
<td>NA</td>
<td>Can be purchased bulk through a 3M distributor or on an as-needed basis from VICON
 http://solutions.3m.com/en_US/?WT.mc_id=www.3m.com/us http://www.vicon.com/applications/animal.html</td>
</tr>
<tr>
<td>Cheerios</td>
<td>&nbsp;</td>
<td>General Mills</td>
<td>Cheerios</td>
<td>http://www.cheerios.com/</td>
</tr>
<tr>
<td>Tissue adhesive</td>
<td>&nbsp;</td>
<td>3M</td>
<td>&nbsp;</td>
<td>Can be purchased through a 3M distributor 
http://solutions.3m.com/en_US/?WT.mc_id=www.3m.com/us</td>
</tr>		</tbody>
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kinematics and ground reaction force determination: a demonstration quantifying locomotor abilities of young adult, middle-aged, and geriatric rats

行为，在其最广泛的定义，可以定义为电机的生理过程体现。因此，所有的行为表现，通过电机系统。运动神经科学和骨科等领域，是为多种疾病模型的常用评估行为。例如，外伤后神经系统的运动恢复是最常用的评估行为 1-3之一。虽然运动可以使用的各种端点测量（例如，所需的时间来完成的运动任务等），半定量运动的措施（如序号评定量表（如巴索贝蒂和布雷斯纳汉运动功能（BBB）的评级规模等））和替代评估行为的措施（如肌肉力量，神经传导速度等），只有动力学（测力）和运动学（细分的身体在太空测量）提供了一个战略的动物是能够locomote 1的详细描述。虽然不是新的，运动学和动力学locomoting啮齿类动物的测量现在更容易获得由于为此目的而设计的市售设备的可用性。然而，更重要的是，实验者需要与生物力学分析的理论非常熟悉和了解分析这些形式的好处和限制，会变成什么样一个相对劳动密集型的研究展开前。本文件旨在描述一个使用市售设备的运动学和地面反作用力的数据收集方法。设备和仪器设置的详细信息，培训前动物，纳入和排除标准，可接受的运行，并收集数据的方法进行了阐述。我们通过描述应变匹配的年轻成年，中年，老年大鼠的运动学和动力学的行为分析技术的实用。

Watch this Scientific Journal Video about Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats at JoVE.co

Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

运动往往是研究在各种型号的疾病，如神经科学和骨科等领域的行为的结果。此视频文件，打算来形容收集老鼠在奔放的运动地面反作用力和运动学的方法。

运动学与地面反作用力的测定：一个示范量化青年，中年和老年大鼠的运动能力能力

Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes.  As such, all behaviors manifest through the ...

kinematics-ground-reaction-force-determination-demonstration

Research

JoVE Journal

Neuroscience

19.6K 次观看.  Riverview, NB. 运动往往是研究在各种型号的疾病，如神经科学和骨科等领域的行为的结果。此视频文件，打算来形容收集老鼠在奔放的运动地面反作用力和运动学的方法。

视频: Kinematics and Ground Reaction Force Determination: A Demonstration Quantifying Locomotor Abilities of Young Adult, Middle-aged, and Geriatric Rats

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

成人北极黄鼠海马神经干细胞的生长和分化

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

科研

神经科学

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

成年鼠脑铸造三维成像和形态分析

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

糖尿病大鼠的热痛觉阈值的快速测定

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Microdialysis of Ethanol During Operant Ethanol Self-administration and Ethanol Determination by Gas Chromatography

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. 
Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.

A method to determine the time course of ethanol concentration in the brains of rats during operant ethanol self-administration is described. Gas chromatography with flame ionization detection is used to quantify ethanol in the dialysate samples, because it has the sensitivity required for the small volumes that are generated.

微透析的乙醇操作性乙醇的自我管理和乙醇的测定气相色谱

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol.  ...

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

The spinal motoneuron has long been a good model system for studying neural function because it is a neuron of the central nervous system with the unique properties of (1) having readily identifiable targets (the muscle fibers) and therefore having a very well-known function (to control muscle contraction); (2) being the convergent target of many spinal and descending networks, hence the name of "final common pathway"; and (3) having a large soma which makes it possible to penetrate them with sharp intracellular electrodes. Furthermore, when studied in vivo, it is possible to record simultaneously the electrical activity of the motoneurons and the force developed by their muscle targets. Performing intracellular recordings of motoneurons in vivo therefore put the experimentalist in the unique position of being able to study, at the same time, all the compartments of the "motor unit" (the name given to the motoneuron, its axon, and the muscle fibers it innervates1): the inputs impinging on the motoneuron, the electrophysiological properties of the motoneuron, and the impact of these properties on the physiological function of the motoneurons, i.e. the force produced by its motor unit. However, this approach is very challenging because the preparation cannot be paralyzed and thus the mechanical stability for the intracellular recording is reduced. Thus, this kind of experiments has only been achieved in cats and in rats. However, the study of spinal motor systems could make a formidable leap if it was possible to perform similar experiments in normal and genetically modified mice.
For technical reasons, the study of the spinal networks in mice has mostly been limited to neonatal in vitro preparations, where the motoneurons and the spinal networks are immature, the motoneurons are separated from their targets, and when studied in slices, the motoneurons are separated from most of their inputs. Until recently, only a few groups had managed to perform intracellular recordings of motoneurons in vivo2-4 , including our team who published a new preparation which allowed us to obtain very stable recordings of motoneurons in vivo in adult mice5,6. However, these recordings were obtained in paralyzed animals, i.e. without the possibility to record the force output of these motoneurons. Here we present an extension of this original preparation in which we were able to obtain simultaneous recordings of the electrophysiological properties of the motoneurons and of the force developed by their motor unit. This is an important achievement, as it allows us to identify the different types of motoneurons based on their force profile, and thereby revealing their function. Coupled with genetic models disturbing spinal segmental circuitry7-9, or reproducting human disease10,11, we expect this technique to be an essential tool for the study of spinal motor system.

Simultaneous Intracellular Recording of a Lumbar Motoneuron and the Force Produced by its Motor Unit in the Adult Mouse In vivo

This new method permits the simultaneous intracellular recording of a single adult mouse motoneuron and the measurement of the force produced by its muscle fibers. The combined investigation of the electrical and mechanical properties of motor units in normal and genetically modified animals is a breakthrough for the study of the neuromuscular system.

同时细胞内记录的车组在成年小鼠的腰椎运动神经元产生的力<em在体内</em

The  spinal motoneuron has long been a good model system for studying neural  function because it is a neuron of the central nervous system with the ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such behavior is spontaneous locomotor activity. Here we describe our protocol that utilizes Drosophila population monitors and a tracking system that allows continuous monitoring of the spontaneous locomotor activity of flies for several days at a time. This method is simple, reliable, and objective and can be used to examine the effects of aging, sex, changes in caloric content of food, addition of drugs, or genetic manipulations that mimic human diseases.

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Drosophila melanogaster are useful in studying genetic or environmental manipulations that affect behaviors such as spontaneous locomotor activity.&#160; Here we describe a protocol that utilizes monitors with infrared beams and data analysis software to quantify spontaneous locomotor activity.&#160;

在自发活动的测定<em&gt;果蝇</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Quantifying Microglia Morphology from Photomicrographs of Immunohistochemistry Prepared Tissue Using ImageJ

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As the first responders, microglia have important functions to mitigate neuron and glia dysfunction, and in this process, they undergo a broad range of morphologic changes. Microglia morphologies can be categorized descriptively or, alternatively, can be quantified as a continuous variable for parameters such as cell ramification, complexity, and shape. While methods for quantifying microglia are applied to single cells, few techniques apply to multiple microglia in an entire photomicrograph. The purpose of this method is to quantify multiple and single cells using readily available ImageJ protocols. This protocol is a summary of the steps and ImageJ plugins recommended to convert fluorescence and bright-field photomicrographs into representative binary and skeletonized images and to analyze them using software plugins AnalyzeSkeleton (2D/3D) and FracLac for morphology data collection. The outputs of these plugins summarize cell morphology in terms of process endpoints, junctions, and length as well as complexity, cell shape, and size descriptors. The skeleton analysis protocol described herein is well suited for a regional analysis of multiple microglia within an entire photomicrograph or region of interest (ROI) whereas FracLac provides a complementary individual cell analysis. Combined, the protocol provides an objective, sensitive, and comprehensive assessment tool that can be used to stratify between diverse microglia morphologies present in the healthy and injured brain.

Microglia are brain immune cells that survey and react to altered brain physiology through morphologic changes which may be evaluated quantitatively. This protocol outlines an ImageJ based analysis protocol to represent microglia morphology as continuous data according to metrics such as cell ramification, complexity, and shape.

应用 ImageJ 对免疫组化制备组织显微照片小胶质细胞形态学的定量研究

Microglia are brain phagocytes that participate in brain homeostasis and continuously survey their environment for dysfunction, injury, and disease. As ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

定量自发 Ca2+ 通量及其在原发性小鼠中脑神经元中的下游效应

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

A Metric Test for Assessing Spatial Working Memory in Adult Rats Following Traumatic Brain Injury

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of human studies, animal models provide suitable alternatives to test treatment methods, and to study the mechanisms and related complications of the condition. Experimental rodent models have historically been the most widely used due to their accessibility, low cost, reproducibility, and validated approaches. A metric test, which tests the ability to recall the placement of two objects at various distances and angles from one another, is a technique to study impairment in spatial working memory (SWM) after TBI. The significant advantages of metric tasks include the possibility of dynamic observation, low cost, reproducibility, relative ease of implementation, and low stress environment. Here, we present a metric test protocol to measure impairment of SWM in adult rats after TBI. This test provides a feasible way to evaluate physiology and pathophysiology of brain function more effectively.

Traumatic brain injury (TBI) is commonly associated with memory impairment. Here, we present a protocol to assess spatial working memory after TBI via a metric task. A metric test is a useful tool to study spatial working memory impairment after TBI.

创伤性脑损伤后评估成年大鼠空间工作记忆的指标测试

Impairments to sensory, short-term, and long-term memory are common side effects after traumatic brain injury (TBI). Due to the ethical limitations of ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.