Name: 视频: Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice
Uploaded: 2016-02-03T15:00:00.000+00:00
Duration: 7 min 41 s
Description: 13.9K 次观看. The University of Melbourne. This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

JCB和ELH-Y分别由国防部CDMRP孤独症研究计划的美国能源部（AR11034）的支持。 NHMRC（1047674）到墨尔本的信任ELH-Y.The月斯图尔特助学金，资助大学奖学金MS。我们感谢阿里·塔希尔，法蒂玛Ramalhosa和格拉西亚塞格的技术贡献。

动物处理和动物颈椎脱臼之前，所有实验均严格按照由动物实验委员会墨尔本大学批准的方案进行（伦理ID:1212494.7） 1.组织收集和解剖<ol><li>通过颈椎脱位安乐死成年小鼠。如果可能避免麻醉，以防止对肠道功能的影响通过位于感兴趣的神经元群体受体。 </li><li>记录动物的总体重，针体用注射针头（尺寸:20 G）（通过四只爪子，腹侧暴露实验者）紧紧的夹层板。 </li><li>使用解剖镊子和剪刀;使通过表皮覆盖下腹部肌肉层的切口。继续使用镊子和剪刀沿腹部到胸骨中线打开腹腔。 </li><li>为了防止脱水组织，倒physiological盐水（Krebs溶液:118氯化钠，4.6氯化钾，2.5 氯化钙 ，1.2 MgSO 4上，1的NaH 2 PO 4，25的NaHCO 3，11 D-葡萄糖以mM -在RT与卡波金气体鼓泡; 95％ 的 O 2和5 ％CO 2的至少20分钟）到在在解剖过程定期（ 即每30-60秒）腹腔内容。 </li><li>以分离结肠而附着到盲肠和直肠，保持轻轻使用动物的身体上方约4-5厘米解剖镊，同时使用细清扫剪刀修整肠系膜盲肠（位于邻近结肠的近端）。 </li><li>注意不要处理，拉伸或削减胃肠组织，同时消除相邻的肠系膜。 </li><li>去除多余的组织（如膀胱和睾丸）。使用粗解剖剪，使两个垂直切口（即从中线约为0.5厘米），以削减盆骨上EA结肠的通道侧。 </li><li>用细剪刀直肠毗邻，从组织盆腔分离和修剪从结肠周围的肌肉。 </li><li>放置全长结肠（从盲肠到直肠）到含有生理盐水的烧杯中（预先用卡波金的氧化），并继续在RT充氧。取出盲肠和直肠用细解剖剪刀，并在烧杯中进行更换。 </li><li>空粪粒/通过使用附连到填充有生理盐水200μl的移液管尖5毫升注射器在口头端施加轻柔正压力从结肠制剂肠内容。为了定向的组织在未来的，使用昆虫销来标记结肠，在那里在粘膜条纹是可见的最基端。 <ol><li>修改塑料枪头通过用刀片除去大约1cm的最宽/近端以适合5mL注射器。以增加流速，通过切割上昂加宽远端/较小端勒（约45℃）用剃刀刀片。 </li></ol></li><li>握结肠组织轻轻在其与解剖钳近端，插入枪头成通过内腔结肠和冲洗生理盐水的腔。 </li></ol> 2.结肠组织和实验设置了视频成像的制备<ol><li>通过附连到设置两个腔式器官浴（图1和图3），使用连接到200μl的移液管尖的注射器所有管路冲洗生理盐水。此步骤除去，可以阻止的溶液的流动管内的任何碎屑。油管规格指的是材料表。 </li><li>确保该器官浴的腔室连续地与卡波金鼓泡生理盐水灌流（95％O 2和5％的CO 2，流速为5毫升分钟-1率）。使用定期温度探头，确保TEMPERAT洗澡的URE保持33°C -37℃之间。 </li><li>填充通过3路阀连接到所述入口管与使用50毫升注射器大约20-30毫升生理盐水流入储存器。 </li><li>在实验过程中，使用arubber塞子与玻璃管（内径5毫米）的通过其中心的插入保持在流入容器内的压力。确保流入贮存器的上部开口被牢固密封的（与内玻璃管剩余​​启封）。 </li><li>放置结肠（长5-7厘米）放入器官浴室，确保它在生理盐水中完全浸没。 </li><li>导管插入结​​肠段在两个浸没入口/出口管的每个端部（入口/不同直径的出口管可以根据组织管腔如的尺寸而改变。产后组织，成年小鼠和豚鼠）在浴中使用标准棉缝纫线。结肠的近侧端连接到所述入口管和结肠到出口管的末端（通过3路阀连接到一个垂直流出管; 6厘米高）。确保该组织是不超负荷或过于放松后插管，​​以避免与以后的测量和分析的干扰。 </li><li>通过测量近端和使用15厘米的标尺远侧插管之间的距离记录结肠的长度。这是解释在收缩长，传播速度和收缩起始点的变化非常重要。 </li><li>确保稳定的基线管腔压力（厘米H 2 O）在开始实验之前被建立。通过测量到的生理盐水流入贮存器（口服）和垂直流出管（肛门）的玻璃管内的弯液面从组织的垂直距离计算管腔的压力。 </li><li>维持组织筹备工作在恒定的腔内压力（如1-2厘米H 2 O）通过确保流入塞密封pressu重新被保持恒定，并且没有堵塞存在于集合起来。 </li><li>离开结肠记录肠运动之前在生理盐水中平衡30分钟。检查中符合或流出管产生的任何障碍，并通过流入水库施加压力或通过重新cannulating结肠的口腔和肛门的两端将其删除。 </li></ol> 3.图像采集和实验方案<ol><li>记录肠运动作为使用摄像机的视频文件（30帧/秒，640×840像素）位于10-15厘米上的标准实验室蒸煮器官浴上方放置（图1）。 </li><li>打开从桌面上的图标虚拟配音（视频捕捉软件）。从"文件"选项卡，选择"捕捉AVI&#39;查看摄像机图像。在"音频"选项卡中取消"启用音频捕获"禁用声音。 <ol><li>在视频选项卡中选择"压缩"和"DivX6.9.2。编解码器＆＃39;压缩视频文件以最小化的存储空间使用。在安装压缩软件"的DivX"的这一功能将变得可用。 </li></ol></li><li>手动调整照相机的位置，以便使整个插管结肠段是可见的（与紧邻所述视频帧的垂直边缘插管区域）经由视频捕获软件窗口（ 参见图1）。为了提高图像质量和减少反射，附加一个纸/纸板屏蔽相机支撑到方框外来光到器官浴。通过使用照相机软件接口调节亮度，对比度和曝光设置优化视频质量。 <ol><li>拍摄前，设定一个特定的文件名;在"文件"选项卡，选择"设置捕获文件"并为视频指定一个唯一的名称。 </li><li>首先从"捕捉"工具栏按下"捕获文件"捕获视频。要停止recording选择了"停止捕获"按钮或键盘上的"ESC"键。 </li></ol></li><li>取代包含用新鲜的生理盐水，每30分钟的流入贮存器内的生理盐水。 <ol><li>记录管腔压力（见2.9），并使用温度探头，注意器官浴的温度。重复这些步骤，在整个实验（即，每30分钟），以确保这些变量是恒定的。 </li></ol></li><li>总共3小时（包括药物的应用和清洗时间）记录结肠活动。用于数据存储的目的，在15分钟的视频段记录数据。一个完整的实验通常会由12×15分钟录像。 <ol><li>控制的条件下1小时（生理盐水）记录活性。 </li><li> 30分钟，应用药物（通过流入水库灌流进入浴缸或管理进入管腔）到1小时。 注:不同的给药途径可以ÿield不同的效果23。 </li><li>在1小时的清除期应用新鲜生理盐水浴或内腔。 </li></ol></li></ol> 4.数据处理和生成时空地图<ol><li>过程中使用的内部软件写在MATLAB每个视频录制脱机（R2012a，版本7.4;应要求提供），以产生时空地图说明结肠动力。 <ol><li>从桌面上的图标开放分析软件。 </li><li>在命令窗口中，依次打开分析控制面板输入"Analyse2"。 </li></ol></li><li>利用边缘检测功能，通过点击控制面板窗口".avi文件边缘检测"产生时空地图。此功能将使分析软件来读取音频视频交错（AVI）格式的重新编码的视频。 </li><li>执行以下步骤依次使用ADA产生时空地图ptive边缘检测算法。 <ol><li>通过选择"添加文件"选项卡中打开录制的视频文件，选择"打开视频"，选择视频文件的位置。 </li><li>从分析软件中列出的视频文件，选择感兴趣的文件。 </li><li>视频，例如，结肠的整个长度的范围内选择感兴趣的矩形区域，从在该结肠口服插管到肛门插管的点。确保近端和远端插管点相邻的感兴趣的区域的垂直边界。边缘检测线（红色和绿色）会自动出现叠加在实时图像上。 </li><li>确保该边缘检测阈值线是紧邻同时在结肠组织的上限和下限的结肠（这可以为每个视频通过控制面板上的改变检测阈值进行优化）。确保该边缘检测升分级表是通过调节对比度和使用控制面板亮度沿结肠图像的长度是连续的。 </li><li>进入结肠（毫米）插入宽度对话框的全长（记录在步骤2.7）。然后选择"生成热图"。 </li><li>在弹出的窗口中，选择一个位置要保存的文件和时空地图指定一个文件名。 </li><li>选择".su2"格式，并选择"添加到队列"。 注意:.su2格式压缩数据到较小的文件大小和多个视频文件可被添加到队列。 </li><li>选择"开始检测"产生时空地图。 </li></ol></li></ol> 5.时空地图分析<ol><li>利用时空映射来估计参数，例如频率，传播速度，长度和CMMCs的持续时间，肠直径和起始和收缩的方向的点。 <ol><li>开始分析时空地图，在命令窗口中键入"analyse2&#39;在上一节中介绍。相反，"边缘检测"，请选择"热图分析"按钮。 </li><li>打开时空地图文件（.su2文件）通过选择"添加文件"选项卡，并指定以前获得.su2文件的位置。 </li><li>一旦时空图是在屏幕上可见，指定在控制面板上的颜色范围，确保了最小的被设置为1。 注意最大可从5-10根据组织的收缩性的范围内。 </li><li>选择"锁定颜色范围"，以确保从一个单一的实验相同的条件下进行了分析的所有地图。 </li><li>确保时限设置是从给定的实验中，所有时空地图不变。 </li></ol></li><li>为了确定CMMC频率，手动计数遍历结肠的长度的50％以上的收缩。这些收缩可以visualized为红色/橙色条纹（标准HSV色指数）的时空地图上（参见图2的例子）。是较短或不传播其他收缩也可鉴定和定量。 </li><li>如果需要的话，在从这些地图更高的分辨率进行进一步的分析。例如，详​​细性质，包括速度和各收缩的持续时间可以被检查。 <ol><li>为了测量的速度和持续时间，选择热图分析控制面板上的"批注收缩浪"按钮。 </li><li>接着，通过点击"放大"按钮并选择感兴趣的区域放大到每CMMC。然后选择"手动标注"按钮使用鼠标从最初始的位置每次收缩的末尾画一条线标注每一个收缩。该方法可用于测量出现传播胀缩的表观传播速度和持续时间（对于例如，见一个在图2A，2B）的地图的人端。 注:速度和持续时间的数据将在结果窗口下。这些值可以被转移到通过选择"导出数据"标签感兴趣的电子表格。不需要的注释可以通过点击"删除选中的注释"按钮删除。 </li></ol></li><li>如果需要的话，利用（分别为时间和位置沿结肠，）x和时空映射的y坐标，以确定​​小收缩或CMMCs开始的位置。 </li><li>同样地，为了确定收缩之间的时间间隔，用"注释"功能来测量收缩之间的持续时间。正如以前，这些结果可以通过选择"导出数据"选项卡导出到一个电子表格。 </li><li>为了测量静息肠道宽度，选择热图分析面板上的"拿横截面"按钮。 <ol><li>选择"添加9;颞截面板上。在地图中的任何位置双击产生叠加在热图上的水平线。食道直径数据现在将显示在一个新的窗口。 </li><li>为了测量直径肠，放在一个波峰和波谷相邻光标获得平均宽肠为特定的实验条件。肠道直径的变化可以实验条件之间进行比较。 </li></ol></li></ol>

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The authors have nothing to disclose

使用此视频成像技术，CMMC频率测量为结肠运动的野生型的指示和NL3 R451C小鼠，自闭症谱系障碍17的小鼠模型。我们的结果表明在CMMCs中相比，在5HT 3/4受体拮抗剂托烷司琼表明NL3 R451C小鼠表现出增加的敏感性托烷司琼的存在野生型小鼠突变NL3 R451C小鼠数量的减少。因此，我们建议，人类neuroligin -3- R451C突变改变血清素通路，可能通过调节在肠神经元，粘膜或两者任5HT 3或5HT 4受体功能。这凸显了该方法对基因型之间并确定具体目标后来的研究表型差异鉴定价值。 该方法可以被修改以通过获取视频，以提高的空间分辨率配有相机的立体显微镜装入。这种方法使得能够从在胚胎的时间点在胃肠道的小制剂早E12.5 31制作的记录。神经调节可通过内腔或器官浴外部结肠制剂加以应用。此外，这种方法是在一系列物种，包括小鼠，大鼠和豚鼠评估大型和小型肠胃蠕动有用。 此方法一般故障处理步骤包括验证经由管溶液的流动，该组织制备的可行性，恒定管腔压力的维护并确保结肠段位于从器官浴的壁离开。管道内堵塞，可改变腔压力，防止收缩的发生;因此，所有的管必须彻底清洗插管前除去盐晶体或碎屑/排泄物。空气必须从管道线中移除<html>直接与之前的实验（即，通过用生理盐水灌注管）插管相关联。此外，组织标本必须小心，以防止导致在结肠的固定性损伤处理。为了避免组织损伤，保证了结肠是牢固（但不是紧紧）附接到所述套管在录制过程中，保持恒定的温度和CO 2 + O 2的浴连续供应。还要确保腔内的压力保持不变，没有收缩是手动记录期间调整流入水库启动。确保结肠组织不接触器官浴的壁收缩期间，因为这将阻止相关时空映射的边缘检测分析。这可以通过在平衡周期监测收缩和调节结肠的位置，以防止这种情况的实验过程中发生的被避免。 _content"&gt;与此技术相关的若干限制，应该分析和解释的数据，包括该方法的低吞吐量性质时，应考虑到考虑。虽然该方法可有效地识别迁移运动模式的变化，它不能确定期间是否发生胀缩一个CMMC的进展被神经介导的或仅仅被动反应的收缩活动（即，从流体的运动引起的）。为在整个结肠壁扩散的浓度梯度允许luminally施加药物的作用被归因于内的动作粘膜，但在长时间的实验粘膜变性，可能会发生，从而改变这些药物在记录期间的动作的位点。此外，药物是否具有在肌间和粘膜下丛不同效果不能使用这种方法来确定。与此相反，该方法使对肠道NE的整体评估通过测量运动模式的全面改变rvous系统。进一步的考虑包括需要考虑到数据的性质（即，计数数据为CMMC频率，要求非参数分析）和CMMCs的低频，设计实验和适当的数据分析策略时。 最近，巴恩斯及其同事提出了结肠组织需要为了观察CMMCs 32的刺激，但是出版从我们的实验室证明自发CMMCs可以通过简单地通过肠系膜7钉扎所述组织的器官浴观察结果。在这些条件下CMMCs的存在不仅展现CMMCs的自发性的，但这种技术的有用性进一步阐述建立在结肠运动的变化。尽管这种方法适用于胃肠道外结肠区域，小肠能动性的复杂性，需要更DETailed比那些用于定量CMMCs 33分析的策略。 此实验方法具有非常高的空间和时间分辨率，并包括药物输送的两个外部和内腔调查肠神经系统上不同的浓度梯度的影响内的选项。此外，这种方法适用于进食状态6,23时分析小肠分割。该方法的离体性质使肠神经系统的作用，在不存在的中枢神经系统的输入进行评估，因此是一种理想的方式来调查胃肠运动中的各种模式，包括疾病的遗传模型（见图2）6,34。 这种方法也可以用来比较生理数据到运动活动23,33,35的计算机模拟。这种模拟可以预测运动模式中与生理实验33,35直接比较时空图的形式。使用快速傅立叶变换和小波分析36，平滑肌心脏起搏器的贡献（按Cajal间质细胞生成），以运动也可以提取。此外，该视频成像技术，可与电活动的细胞外记录在肌肉3相结合，使神经和肌码型发生器的贡献加以区别。注意，外记录方法在不存在的平滑肌收缩或松弛解析抑制接点电位。 虽然这种技术是对胃肠能动性的在大范围的准备和物种的分析确立，它也有潜力在其他系统中使用，如血管收缩的肠系膜（研究先前通过更简单的直径跟踪系统进行分析37）和骨骼肌。

肠神经系统（ENS）是胃肠道的固有的神经元网络，并调制各种功能，诸如肠内容消化，营养吸收和分泌和流体的重吸收。在ENS的神经元位于肌间和粘膜下神经丛。肌间神经丛在调节肠胃蠕动1，而黏膜下神经丛的一大作用是主要参与分泌2,3的控制。肌间神经丛坐落于胃肠壁的纵向和环形肌层之间。肠壁的平滑肌层的收缩活性通过混合和推进沿着肠管3的长度肠内容便于胃肠道的主要功能。虽然非本征神经供应到胃肠道从中枢神经系统有助于体内胃肠功能中，ENS是能够独立调节胃肠功能。这种独特的特性使肠神经回路的功能调查，他们对肠胃蠕动体外贡献。 结肠移行络合物（CMMCs）是在缺乏粪粒4-9的离体小鼠结肠中观察到的主要电机图案自发的，神经性的事件。 CMMCs被定义为沿着水平距离，该距离至少结肠的总长度的一半传播（即，从盲肠到直肠）10节律性收缩。 CMMCs并且推进粪粒的收缩型态之间的关系尚未清楚地确定，但是一些药理差异已经报道11。尽管如此，ENS的中枢神经系统独立工作的能力和神经介导的运动模式在IS中存在olated结肠提供了一个理想的检测系统来检查的运动障碍，从根本ENS功能障碍所致。胃肠运动模式的自发性允许响应于药理刺激功能的改变进行评估。 使用视频成像和时空映射首次制定定量检查豚鼠12小肠道蠕动。在这里， 离体技术进行说明，使小鼠结肠动力模式的研究利用视频成像和这些录音的分析，构建高分辨率（〜100微米，33毫秒）结肠直径地图作为位置沿结肠功能和时间（时空地图）的。利用内部边缘检测软件（Analyse2;应要求提供），由全长结肠段实时承包数据进行处理，生成每个实验时空地图。在此步骤中，视频格式（AVI）文件是最优等授权的，并转换成使用Analyse2时空映射。时空映射（图2）描述了收缩随时间和使多个参数，包括传播速度，大小，长度和持续时间的测量。肠直径也被记录在整个实验作为组织段的整体收缩的量度的持续时间。这种方法可以适用于识别在收缩配合这可能表明改变的肠神经连接的起始点的差别。 旨在评估在豚鼠颗粒推进类似的视频成像协议已经报道13但是在这里我们概述了自发结肠运动的量化的视频成像方法的应用程序（即在没有颗粒）。我们还提供了详细的资料，以协助清扫和准备胃肠组织上的视频成像的方法。本协议提供了用于分析在疾病的动物模型包括遗传小鼠模型肠胃功能的肠溶神经控制的可访问和容易复制工具的研究人员。 视频成像技术使得结肠运动的响应于各种药理学药剂的分析。药物可通过肠腔或器官浴外部结肠制剂给药。鼠标胃肠道的不同区域表现出特定运动模式，如在结肠小肠分割和CMMCs。 此技术已经被用来识别在小肠功能应变的差异;与5-HT 3和5-HT 4拮抗剂差动灵敏度在Balb / c和C57 / BL6小鼠的空肠，观察由于在两种菌株6表示的TPH2基因的多态性。动力上5-HT的抑制的作用仍然CONtroversial，作为冲突的数据已报道内源性5-HT对结肠的蠕动和CMMCs 14,15的重要性。改建动力前和出生后发育7中，以及在疾病10的动物模型肠胃蠕动基因突变的影响，也可以通过利用视频成像检查。在这里，我们说明了结肠运动的自闭症的NL3 R451C小鼠模型，它体现在NLGN3基因编码突触粘附蛋白人类neuroligin-3 16的错义突变研究使用的方法。这种突变是首次确诊患有自闭症谱系障碍（ASD）17，这是强烈胃肠道功能障碍相关的18-22患者识别。我们调查了NL3 R451C突触突变是否会影响使用视频成像技术在ENS神经输出。我们在基线和响应于血清素能5H呈现数据表征CMMCsŧ3/4受体拮抗剂托烷司琼 自闭症的NL3 R451C小鼠模型。

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			<td>NaCl (MW: 58.44)</td>
			<td>Sigma-Aldrich</td>
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			<td>Sigma-Aldrich</td>
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			<td>MgSO4.7H20 (MW: 246.48)</td>
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			<td>CA033</td>
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			<td>D-Glucose anhydrous (MW: 180.16)</td>
			<td>Chem Supply</td>
			<td>GA018-500G</td>
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			<td>NaHCO3 (MW: 84.01)</td>
			<td>Chem Supply</td>
			<td>GA018-500G</td>
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			<td>Name</td>
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			<td>Two chambered organ bath 
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			<td>Custom Made</td>
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			<td>Contact Laboratory Directly&#160;</td>
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			<td>&#160;732 MULTI -PURPOSE SEALANT CLEAR</td>
			<td>Dow Corning Australia Pty Ltd</td>
			<td>1890573</td>
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			<td>Terumo Medical Corporation</td>
			<td>0912-2006</td>
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			<td>1.50 mm (ID) x 2.50 mm (OD) - Silastic Tubing</td>
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			<td>508-007</td>
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			<td>1.60 mm (ID) - Platinum cured silicone tubing&#160;</td>
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			<td>4.40 mm (ID) - Platinum cured silicone tubing&#160;</td>
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			<td>3.10 mm (ID) - Platinum cured silicone tubing&#160;</td>
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			<td>96410 -16</td>
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			<td>Graduated Laboratory Glass Bottles - 500 ml&#160;&#160;&#160;&#160;&#160;</td>
			<td>Thermofisher Scientific&#160;</td>
			<td>100-400</td>
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			<td>CHEMICAL RUBBER STOPPER 57 x 65mm&#160;</td>
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			<td>Water heater&#160; (thermo regulator)&#160;</td>
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			<td>Logitech Webcam</td>
			<td>Logitech</td>
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			<td>Name</td>
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			<td>Virtual Dub - 1.9 11</td>
			<td>virtualdub.org</td>
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			<td>MATLAB R2012a&#160;</td>
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			<td>Logitech Webcam Software</td>
			<td>Logitech</td>
			<td></td>
			<td></td>
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video imaging and spatiotemporal maps to analyze gastrointestinal motility in mice

肠神经系统（ENS）在调节胃肠（GI）运动中起重要作用，并且可以独立于中央神经系统的作用。在ENS函数变化的胃肠道症状和疾病的主要原因，并可能有助于在神经精神障碍包括自闭症报道胃肠道症状。它是公认的离体结肠段产生被称为结肠移植电机配合（CMMCs）自发的节律性收缩。一个过程来分析前说明小鼠结肠的体内筹备CMMCs的肠道神经调节。结肠从动物解剖并冲洗以在器官浴中插管之前除去粪便内容。数据经由定位在器官浴上方的摄像机获取的，并通过一个内部软件包转换成高分辨率的时空图。使用这种技术，基线收缩模式和结肠S于ENS功能的药理作用egments可以在3-4小时进行比较。此外，CMMCs的传播长度和速度可被记录，以及在肠道中直径和收缩频率的变化。这种技术是用于表征在转基因小鼠模型（以及在其它物种，包括鼠和豚鼠）胃肠运动模式是有用的。以这种方式，在CMMCs药理学诱导的变化被记录在野生型小鼠和孤独症的人类neuroligin-3 R451C小鼠模型。此外，这种技术可应用于胃肠道的其他区域，包括十二指肠，空肠和回肠，并在小鼠中不同发育年龄。

达的患者患有ASD 90％遇到胃肠功能紊乱，包括腹泻和便秘18,24,25的阵列。然而，这些胃肠问题的根本原因是未知的。患者识别与ASD许多突变与突触蛋白在突触传递或功能有助于改变和干扰有关。一种这样的突变，在编码基因的细胞粘附分子人类neuroligin-3（NL3 R451C），是在两兄弟鉴定患有ASD 17。在在人类neuroligin蛋白的451位的精氨酸残基这一突变导致被替换为半胱氨酸。表达这种突变秀NL3 R451C小鼠躯体感觉皮层16,26海马25,27内沿着增加AMPA增加和NMDA受体介导的活性GABA介导的传输。 28-30。作为肠神经系统在调节胃肠功能的重大作用，我们推测，R451C突变会影响蠕动。因此，为了研究在胃肠道的功能，由于突触异常可能改变我们力求审查关于这些小鼠CMMC频率R451C突变的影响。 因为血清素（5-HT）作用在ENS调制小鼠6胃肠功能我们响应于从WT和NL3 R451C小鼠结肠制剂对5-HT 3/4受体拮抗剂托烷分析运动模式。 为了评估突触突变是否改变CMMCs当肠神经系统药理学扰动，所述5HT 3/4受体拮抗剂 托烷司琼（特罗普;10μM，哪些块既5HT3和5HT 4受体）加入到含有结肠制剂（图2）的浴中。使用从20年龄匹配的雄性小鼠（11 WT和8 NL3 R451C）结肠组织。在托烷的存在下，NL3 R451C小鼠显示与WT相比同窝在CMMC频率的降低。时空映射表示在WT和NL3 R451C结肠制剂CMMCs和收缩活性的代表性的例子分别示于图2A至 2E。虽然在对照条件WT和NL3 R451C之间没有观察到差异，托烷司琼显著在WT和NL3 R451C小鼠（ 图2C，2F）降低CMMC频率。在野生型小鼠，CMMCs的中位数为控制条件相比，23至15日在托烷司琼（P = 0.023）。在NL3小鼠控制条件CMMCs的中位数为19.5在托烷司琼的存在（p值= 0.022）2相比较。此外，托烷司琼不得不与WT相比（p值= 0.047）上CMMCs的在NL3 R451C小鼠的频率更大的效果。 <img alt="图1" src="/files/ftp_upload/53828/53828fig1.jpg" /> 图1.器官浴设置和产生时空地图。（A）新鲜解剖胃 ​​肠段放入装有生理盐水的器官浴（横截面图），并在口腔和肛门插管结束。口腔插管连接到填充有生理盐水并连接到流出管肛门插管流入储存器。一种摄像机，以便记录结肠的收缩活动定位在器官浴的上方。 （B）的运动被转换成以蓝色和缩区域扩张的结肠高分辨率时空映射的标签区域In减轻。 （C）表示从成年小鼠WT结肠运动（CMMCs）时空地图。个人CMMCs表示为地图中的红色垂直区域。 X轴表示时间增加（0-15分钟）。 Y轴表示（在基部，口腔顶部肛门）沿结肠段的空间位置。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53828/53828fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53828/53828fig2.jpg" /> 图2.时空地图显示增加了NL3 R451C 小鼠结肠 托烷司琼的敏感性时空地图显示，从WT对照组（A）结肠段和托烷司琼的存在CMMC频率。（特罗普; B）。特罗普在WT冒号（C）降低CMMC频率。 Spatiote<html>从NL3结肠mporal地图 在控制条件下（D）和特罗普（E）的存在。特罗普引起的结肠NL3（F）CMMC频率的强还原性。响应于特罗普CMMC频率NL3被显著降低与WT结肠相比（p值= 0.047，未示出）。肠道宽度（像素的颜色）被指示在Y轴（任意单位，范围1-6）。在电子商务比例尺适用于所有地图。特罗普;托烷司琼。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53828/53828fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53828/53828fig3.jpg" /> 图3.示意图器官浴。（A）顶视图，（B）底面，成立了两个腔器官浴（三）前视图，（D）侧面视图。尺寸mm。REF ="htt​​ps://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53828/53828fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice at JoVE.com

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

视频成像和时空地图来分析胃肠动力小鼠

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

video-imaging-spatiotemporal-maps-to-analyze-gastrointestinal

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

13.9K 次观看.  The University of Melbourne. This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

视频: Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔:鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

科研

神经科学

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

视频oculography小鼠

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

在视神经纤维通过诚信小鼠对比增强MRI 活体成像

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying neonatal seizures and hypoxia ischemia. This manuscript describes a method for continuous video electroencephalogram (EEG) monitoring in neonatal mice to detect seizures and analyze EEG background during hypoxia ischemia. Use of video and EEG in conjunction allows description of seizure semiology and confirmation of seizures. This method also allows analysis of power spectrograms and EEG background pattern trends over the experimental time period. In this hypoxia ischemia model, the method allows EEG recording prior to injury to obtain a normative baseline and during injury and recovery. Total monitoring time is limited by the inability to separate pups from the mother for longer than four hours. Although, we have used a model of hypoxic-ischemic seizures in this manuscript, this method for neonatal video EEG monitoring could be applied to diverse disease and seizure models in rodents.

This manuscript describes a method for continuous video EEG recordings using multiple depth electrodes in neonatal mice undergoing hypoxia-ischemia.

新生小鼠缺氧缺血期间的连续视频脑电图

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying ...

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

This protocol describes the quantification of volumetric cerebral hemodynamic variations in the mouse brain using functional ultrasound (fUS). Procedures for 3D functional activation map following sensory stimulation as well as resting-state functional connectivity are provided as illustrative examples, in anesthetized and awake mice.

使用经颅功能超声成像的小鼠全脑3D激活和功能连接映射

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ...