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?...

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。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl (MW: 58.44)</td><td>Sigma-Aldrich</td><td>S7653-250G</td><td></td></tr><tr><td>KCl (MW: 74.55)</td><td>Sigma-Aldrich</td><td>P9333-500G</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O (MW: 156.01)</td><td>Chem Supply</td><td>471-500G</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;0 (MW: 246.48)</td><td>Chem Supply</td><td>MA048</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O (MW: 147.02)</td><td>Chem Supply</td><td>CA033</td><td></td></tr><tr><td>D-Glucose anhydrous (MW: 180.16)</td><td>Chem Supply</td><td>GA018-500G</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3 &lt;/sub&gt;(MW: 84.01)</td><td>Chem Supply</td><td>GA018-500G</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Two chambered organ bath&lt;br/&gt; Dimentions: 14 cm x 8 cm x 3 cm</td><td>Custom Made</td><td></td><td>Contact Laboratory Directly&amp;nbsp;</td></tr><tr><td>&amp;nbsp;732 MULTI -PURPOSE SEALANT CLEAR</td><td>Dow Corning Australia Pty Ltd</td><td>1890573</td><td></td></tr><tr><td>SYLGARD 184 SILICONE ELASTOMER KIT&amp;nbsp;</td><td>Dow Corning Australia Pty Ltd</td><td>1064291</td><td></td></tr><tr><td>STOPCOCK 3 WAY FEM-ML L/LOCK S</td><td>Terumo Medical Corporation</td><td>0912-2006</td><td></td></tr><tr><td>SYRINGES with Luer Lock Tips 50 ml, 20 ml, 10 ml</td><td>Terumo Medical Corporation</td><td>N/A</td><td></td></tr><tr><td>1.57 mm (ID) x 3.16 mm (OD) - Silastic Tubing</td><td>Masterflex</td><td>508-008</td><td></td></tr><tr><td>1.02 mm (ID) x 2.16 mm (OD) - Silastic Tubing</td><td>Masterflex</td><td>508-005</td><td></td></tr><tr><td>1.50 mm (ID) x 2.50 mm (OD) - Silastic Tubing</td><td>Masterflex</td><td>508-007</td><td></td></tr><tr><td>1.60 mm (ID) - Platinum cured silicone tubing&amp;nbsp;</td><td>Masterflex</td><td>96410 - 14</td><td></td></tr><tr><td>4.40 mm (ID) - Platinum cured silicone tubing&amp;nbsp;</td><td>Masterflex</td><td>96410 - 15&amp;nbsp;</td><td></td></tr><tr><td>3.10 mm (ID) - Platinum cured silicone tubing&amp;nbsp;</td><td>Masterflex</td><td>96410 -16</td><td></td></tr><tr><td>Graduated Laboratory Glass Bottles - 500 ml&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Thermofisher Scientific&amp;nbsp;</td><td>100-400</td><td></td></tr><tr><td>CHEMICAL RUBBER STOPPER 57 x 65 mm&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>CHEMICAL RUBBER STOPPER 29 mm x 32 mm</td><td></td><td></td><td></td></tr><tr><td>Water heater&amp;nbsp; (thermo regulator)&amp;nbsp;</td><td>Ratek&amp;nbsp;</td><td>TH7000&amp;nbsp;</td><td></td></tr><tr><td>Logitech Webcam</td><td>Logitech</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Virtual Dub - 1.9 11</td><td>virtualdub.org</td><td></td><td></td></tr><tr><td>MATLAB R2012a&amp;nbsp;</td><td>Graph Pad</td><td></td><td></td></tr><tr><td>Logitech Webcam Software</td><td>Logitech</td><td></td><td></td></tr></tbody></table>

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...

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

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

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an area of productive research with over 1,400 published animal studies in just the last 5 years. Numerous techniques have been developed for quantifying smooth muscle activity of the stomach, small intestine, and colon. In vitro and ex vivo techniques offer powerful tools for mechanistic studies of GI function, but outside the context of the integrated systems inherent to an intact organism. Typically, measuring in vivo smooth muscle contractions of the stomach has involved an anesthetized preparation coupled with the introduction of a surgically placed pressure sensor, a static pressure load such as a mildly inflated balloon or by distending the stomach with fluid under barostatically-controlled feedback. Yet many of these approaches present unique disadvantages regarding both the interpretation of results as well as applicability for in vivo use in conscious experimental animal models. The use of dual element strain gages that have been affixed to the serosal surface of the GI tract has offered numerous experimental advantages, which may continue to outweigh the disadvantages. Since these gages are not commercially available, this video presentation provides a detailed, step-by-step guide to the fabrication of the current design of these gages. The strain gage described in this protocol is a design for recording gastric motility in rats. This design has been modified for recording smooth muscle activity along the entire GI tract and requires only subtle variation in the overall fabrication. Representative data from the entire GI tract are included as well as discussion of analysis methods, data interpretation and presentation.

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

The in vivo measurement of smooth muscle contractions along the gastrointestinal tract of laboratory animals remains a powerful, though underutilized, technique. Flexible, dual element strain gages are not commercially available and require fabrication. This protocol describes the construction of reliable, inexpensive strain gages for acute or chronic implantation in rodents.

的微型双元应变计测量加工和植入<em&gt;体内</em&gt;胃肠收缩鼠害。

Gastrointestinal dysfunction remains a major cause of morbidity and mortality. Indeed, gastrointestinal (GI) motility in health and disease remains an ...

科研

生物工程

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

在髓系细胞动态实时成像<em&gt;装甲运兵车<sup&gt;最小/ +</sup</em&gt;肠道肿瘤通过旋转盘共聚焦显微镜

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

免疫与感染

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

鼠内镜<em&gt;体内</em&gt;发生和肠道的评价多模式成像伤口愈合和炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

医学

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

在实时麻醉小鼠成像中性粒细胞和单核细胞在肠系膜静脉用活体显微镜

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

新的治疗肠道病变的疗效，使用活体小鼠的串行内镜成像的非侵入性评估

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Gastrointestinal Motility Monitor (GIMM)

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility in isolated segments of guinea pig distal colon. The complete system consists of a computer, video camera, illuminated organ bath, peristaltic and heated water bath circulating pumps, and custom GIMM software to record and analyze data. Compared with traditional methods of monitoring colonic peristalsis, the GIMM system allows for continuous, quantitative evaluation of motility. The guinea pig distal colon is bathed in warmed, oxygenated Krebs solution, and fecal pellets inserted in the oral end are propelled along the segment of colon at a rate of about 2 mm/sec. Movies of the fecal pellet proceeding along the segment are captured, and the GIMM software can be used track the progress of the fecal pellet. Rates of propulsive motility can be obtained for the entire segment or for any particular region of interest. In addition to analysis of bolus-induced motility patterns, spatiotemporal maps can be constructed from captured video segments to assess spontaneous motor activity patterns. Applications of this system include pharmacological evaluation of the effects of receptor agonists and antagonists on propulsive motility, as well as assessment of changes that result from pathophysiological conditions, such as inflammation or stress. The guinea pig distal colon propulsive motility assay, using the GIMM system, is straightforward and simple to learn, and it provides a reliable and reproducible method of assessing propulsive motility.

Gastrointestinal Motility Monitor GIMM

Evaluation of colonic motility in the guinea pig distal colon with the Gastrointestinal Motility Monitor (GIMM) is a straightforward and simple to learn approach to quantitatively evaluate propulsive motility in the gastrointestinal tract.

肠胃蠕动监视器（GIMM）

The Gastrointestinal Motility Monitor (GIMM; Catamount Research and Development; St. Albans, VT) is an in vitro system that monitors propulsive motility ...

生物学

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

卡哈尔（ICC）的网络的间质细胞在小鼠体内的可视化

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

毛里小肠内皮淋巴细胞的内质成像

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system.

运动性的时空制图<em&gt;离体</em肠子&gt;准备工作

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

Studying Murine Small Bowel Mechanosensing of Luminal Particulates

Gastrointestinal (GI) motility is critical for normal digestion and absorption. In the small bowel, which absorbs nutrients, motility optimizes digestion and absorption. For this reason, some of the motility patterns in the small bowel include segmentation for mixing of luminal contents and peristalsis for their propulsion. Physical properties of luminal contents modulate the patterns of small bowel motility. The mechanical stimulation of GI mechanosensory circuits by transiting luminal contents and underlying gut motility initiate and modulate complex GI motor patterns. Yet, the mechanosensory mechanisms that drive this process remain poorly understood. This is primarily due to a lack of tools to dissect how the small bowel handles materials of different physical properties. To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit. We gavage live mice with fluorescent liquid or tiny fluorescent beads. After 30 minutes, we dissect out the bowels to image the distribution of fluorescent contents across the entirety of the GI tract. In addition to high-resolution measurements of the geometric center, we use variable size binning and spectral analysis to determine how different materials affect small bowel transit. We have explored how a recently discovered "gut touch" mechanism affects small bowel motility using this approach.

To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit.

研究小鼠小肠对管腔颗粒的机械传感

Gastrointestinal (GI) motility is critical for normal digestion and absorption. In the small bowel, which absorbs nutrients, motility optimizes digestion ...

视频成像和时空地图来分析胃肠动力小鼠

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的微型双元应变计测量加工和植入

在髓系细胞动态实时成像

鼠内镜