DMK是由一个IRACDA津贴从NIGMS（K12GM093857）到弗吉尼亚联邦大学的支持。这项工作是由NIDDKD授予DK34153约翰R. Grider支持。

弗吉尼亚联邦大学的机构动物护理和使用委员会（IACUC）批准了该协议中使用的所有动物安乐死程序。 1.准备解决方案<ol><li>制备4L的Krebs缓冲液（[以mM]组成:118氯化钠，氯化钾4.75，1.19 KH 2 PO 4，1.2 MgSO 4干燥，2.54 氯化钙 ，25的NaHCO 3，11葡萄糖）。称出各固体化学，投入去离子水的适当体积的适当的量和混合用涡旋直至溶液澄清。 </li><li>然后，充气与carboxygen（95％O 2，5％CO 2）30分钟，同时加热至37℃的溶液中。 </li><li>确定溶液的pH值，同时在37℃下（相同的温度，在器官浴），并在必要时使用的HCl调节至pH 7.4。保持Krebs缓冲液连续carboxygenated并在37℃下整个实验的长度。 </li><li>放从蠕动泵入缓冲储水并打开蠕动泵的流入和流出管，以开始在整个管道和器官浴系统中的缓冲器的灌注。 注:缓冲器最初可以抽回到原来的容器，直到除了任一组织的成器官浴或化学物质进入灌注缓冲液中。这减少了缓冲的需要进行实验的总量。随后传出缓冲区灌注线应该放到一个空的容器，这样的迁入和传出缓冲区不要混用。 </li><li>校准加热系统的温度对循环器浴使得器官浴内的缓冲区变，并保持37.0±0.5℃下进行的实验的持续时间。确认，并把组织片段在浴在步骤2.8之前至少一个更多的时间，如果必要修改，克雷布斯缓冲液的pH。 </li></ol> 2.准备工作组织的<ol><li>安乐死豚鼠通过在密封腔或经当地动物护理和使用委员会另一种安乐死​​方法使用吸入二氧化碳/窒息。 </li><li>确认动物安乐死后，纵向切开与剥离剪刀打开腹壁并找到肠子。剪下贴在小肠，帮助揭露盲肠肠系膜。 注:要确认所批准的动物护理和使用委员会安乐死执行辅助程序如双侧开胸手术。 </li><li>定位盲肠的远端和横切近端结肠只是远端到其盲肠连接。然后横切近端结肠再次〜8厘米远离第一横断。 </li><li>将结肠组织到温暖和carboxygenated克雷布斯溶液（第1节）为进一步解剖。引脚上的大肠放入夹层浴和使用一套剪刀小去除尽可能多的肠系膜和脂肪越好。 </li><li>以heated夹层托盘，轻轻灌注通过使用耦合到平端的导管的注射器内腔Krebs缓冲液中删除所有腔内含量（以帮助防止组织的穿孔）。在此过程中灌注避免过度扩张段。 </li><li>获得两个火抛光玻璃管导管（3-毫米直径），并放置一段短的聚乙烯管（约3毫米长和〜3毫米的直径）周围进入组织腔的玻璃管的一部分。 注意:这将允许缝线放置组织周围和玻璃管保持安全和不滑，作为准备放入器官浴中，并在改变的组织的长度。 </li><li>插入一个导管进入组织制剂的口腔端和领带围绕小片管周围使用外科医生的结导管的缝合。轻轻尝试将导管退了出去，确保结舒适。然后，执行另一导管进入T中的同一个动作他反口组织的结束。 </li><li>一旦导管固定，移动从解剖盘的整备（组织加导管）到器官浴之一。将制备成朝向远离用户的口头端浴。 </li><li>未来由两个建议的方法之一安装/固定玻璃管导管向器官浴。 注:保护玻璃管防止所述组织段从实验期间改变长度或未来上述Krebs缓冲液，在浴的表面水平。 <ol><li>选项​​A:通过使用模塑的粘土或塑料夹子的固定玻璃管到塑料器官浴的一侧的顶部的顶部部分。 </li><li>选项​​B:固定玻璃管子，通过使用塑料夹守稳器官浴准备相机的金属杆。 </li></ol></li><li>一旦导管被固定到浴，附加一个5厘米的管件的每个导管的开口端。对于口服猫海特，附上该管连接到一个10毫升的注射器，其将用于缓冲注入近侧结肠段的内腔中。为反口导管，该管件将允许管腔流出到被引导到一个收集容器（50毫升烧杯中，水库等 ）。 </li><li>使用连接到口腔端的注射器，慢慢冲洗穿过组织管腔温热Krebs缓冲液，以确保液体可以流过所述组织。流由液体离开所述反口导管证实。校准视频记录系统（协议第3节）而组织达到平衡30分钟。 </li></ol> 3.校准视频记录系统<ol><li>校准摄像机高度放置，视频设置为亮度和对比度，并使用该软件的水平距离。 注:对于腔内灌注设置的最佳设置比来确定小球推进13的速度的设定不同。 </li><li> CLI放摄像机的实验片上进行校准。然后在"文件"菜单中，单击"校准"。 </li><li>一旦视频出现在"校准工作站"窗口中，将相机对准一个高度，其中图像包括插入到结肠腔导管的末端。 </li><li>然后，校准用附在每个浴半透明标尺贴纸图像中观察到的水平距离。 注:此校准是至关重要的管腔直径和收缩波的速度更新版本的软件计算。 </li><li>在相同的"校准工作站"窗口中，将红色垂直指引，让他们10毫米的间隔根据相机图像中的统治者。然后，键入适当的距离（10毫米）到"远方光标"窗口，点击"CAL"按钮。 </li><li>接着，调整增益，亮度，和"校准工作站&#39;窗口内活门滑块以修改，以使摄像机图像组织段显示为黑色的剪影在浅色背景。 注: 图4显示了这种校准过程中好的和坏的例子。 </li><li>要适当调整图像，也可以调整相机本身的对焦和光圈旋钮。 </li><li>校准现已完成。点击"保存"，然后点击"确定"，在弹出窗口中，最后点击"退出"按钮。 </li></ol> 4.一般实验方法<ol><li>经过摄像机标定程序和组织平衡30分钟完成，命名中的每个实验方案的实验。试用名称可以根据名称和化合物和液体体积的浓度的那部分在实验过程中被intraluminally灌注到组织片段。 </li><li>然后，闭塞管从反口导管通过使用管夹的突出。 注意:这可以防止管腔液体离开福在反口端rther实验，允许在管腔使用特定体积的挑起各级腹胀的（ 图1）。 </li><li>注入约的Krebs缓冲液0.7 ml，在近端结肠腔，以提供足够的腹胀启动推进收缩在豚鼠离体的准备。 注意:此卷可能略有不同（0.1 - 0.2毫升）根据完好分段的总长度和拉伸的施加于分段在器官浴中的量。 </li><li>一旦该段被管腔液 ​​胀得，打开相机，然后记录运动的时间预先确定的时间段（如10分钟）。 <ol><li>具体来说，双击适当命名的审判开了实验相机视图，然后单击切换开关置于"ON"位置，查看相机领域。然后，点击录音键开始录音（录音键仍为红色，而相机记录）一第二再次单击录制按钮停止录制。 </li></ol></li><li>在这一初始控制腹胀试验结束时，松开/拆下钳闭塞反口导管，以允许所述组织段来推进肿胀液体流出腔。 </li><li>后10分钟的再平衡时间，重复此过程与任何各种营养素，生物活性剂，药物，或激动剂/拮抗剂的管腔液 ​​的修改段（例如，短链脂肪酸或癸的推进运动酸）10,18。 <ol><li>为了帮助轨距当新的实验流体进入结肠段，离开气泡靠近注射器的端口，以便于新溶液进入结肠腔的气泡的进展的灌注将允许用户知道实验流体时已到达内腔。 </li><li>继续腔内灌注与反口导管打开，直到泡沫完全推通过腔内灌注系统（可能需要2-3灌注液毫升）。然后，让所述组织段通过打开的反口导管排出流体达5分钟。 注意:在这个程序之后，管腔将包含流体的可忽略的量，允许已知体积的液体进入腔的灌注。 </li></ol></li><li>再闭塞使用管夹的反口管腔导管管（如在步骤4.3）注入肿胀流体在通过注射器的控制条件相同体积。记录实验期间购买的分析和比较，以控制克雷布斯条件的运动模式（如在步骤4.5）。 </li><li>重复第4.4 - 4.7与不同的腔化合物或不同浓度相同的管腔化合物的协议。 </li></ol> 5，建设时空地图的（STmaps） <ol><li>记录实验完成后，双击一个特定试验的名称以打开分析风流建设的STMAP的。 </li><li>在视频播放方面，调节对比度和亮度滑块在分析窗口，使图像适合于分析（黑色组织剪影背景光; 图4）。 注:如果在开始实验的图像只能在这一点上进行修改，以在较小的程度之前不进行照相机校正适当。 </li><li>一旦图像被适当对比，在视频图像窗口内设置的水平和垂直红色准则来隔离组织区域进行分析，并除去含有伪影区域。另外，通过确定启动时选择从记录在适当的时间段，并停止的时间点进行分析。 <ol><li>具体而言，使用分析软件中绿色的"A"和黄色的"B"按钮选择视频中的起点和终点。将时间滑块移动到视频片段的起点来分析和n点击绿色的"A"按钮。然后，将滑块移动到段的末尾来分析和点击黄色的"B"按钮。 </li></ol></li><li>接下来，单击"马达分析"选项卡打开STMAP窗口，然后单击"秒表"按钮。点击秒表后，点击旁边的录制的电影中的组织的黑色剪影中的十字光标开始STMAP产生。 </li><li>点击在组织轮廓的十字线后，软件生成并显示STMAP视频的该区域。 注意:这可能需要根据视频选择用于分析的长度几分钟。 </li><li>点击&#39;放大&#39;，以查看STMAP和控制的放大图像来调整图像。 <ol><li>单击新建窗口中的"颜色"选项，以查看STMAP颜色代替灰度。同样，点击"启用"选项，可以将地图和v中的光标移动的具体像素IEW管腔直径的变化对于特定组织区域的跟踪。完成后，单击"退出"。 </li></ol></li><li>导出STMAP用于论文或演讲，或通过选择"文件"菜单，然后选择"导出数据"，然后"元文件"进一步分析ImageJ的。然后命名STMAP，将被保存为的.emf文件，然后单击"保存"按钮。 </li><li>或者，通过捕捉的屏幕截图保存STMAP的图像。这是必要的保存STMAP的伪彩色版本。 </li></ol>收缩波速在STmaps 6.分析<ol><li>分析收缩传播或收缩持续时间的速度，首先STMAP的.emf文件转换为.tiff，.gif注意，.jpg或.BMP格式都可以接受的ImageJ通过选择的文件转换程序。 注:ImageJ的不开从软件的STmaps的.EMF格式输出。 <ol><li>可选择性伊利，使用截屏抓图软件采取在屏幕上STMAP的画面并将其保存在适当的文件格式。 </li></ol></li><li>然后打开ImageJ的重新格式化STMAP，然后打开"插件"菜单并选择"GIMMProcessor打开GIMMProcessor插件"。这将打开两个"GIMMProcessor"和"测量表"窗口。 </li><li>点击ImageJ的窗口中的"矩形选择工具"，然后用它通过点击并拖动比例尺的整个区域的STMAP的右上角勾勒。之后选择该区域，单击插件的"校准设置"按钮。最后，插入适当的距离（mm）和时间（秒）进入校正盒根据对比例尺的值，然后单击"完成"。 </li><li>接下来，单击ImageJ的窗口中画线工具。通过一传播收缩沿着第角的中心吸取STMAP的线Ë斜坡通过点击并拖动STMAP形象。另外，画一条垂直线，通过非传播收缩带，以确定收缩持续时间。 </li><li>之后该行适当绘制（只有一条线可以在同一时间内得出），点击"开始测量"按钮，在插件生成一个读出的水平距离的线，垂直距离和斜的;它们分别对应于长度（mm），时间（秒）和速度（毫米/秒）。此数据显示在"测量表"的窗口。 </li><li>重复的画线分析步骤给定STMAP中多次和数据保存为.gmd文件。点击插件"保存"按钮，并命名该文件。然后点击"保存"再次以完成该过程。这些数据随后可以与其它试验的数据进行比较，或用于重新画线上的ST​​MAP。 </li></ol>

Catamount Research and Development, Inc., creator of the GIMM system, covered publication fees for this manuscript.

肠运动已被查看和从许多基于被记录的参数的性质观点说明。视频录制和时空映射已被证明的有价值的工具，允许通过肠道的长链段整体运动和/或推进的分析以及活性分析在沿区段的特定点。该方法采取视频记录和时空映射可以是双重的，并是反射检查的区域和的腔内容物的性质。在肠段其中管腔内容是多个流体和在近端结肠，其中内容是更半固体的活性是通过推注或输注腔内引入流体的诱导。从这些视频记录作出时空映射被设计成如上所述代表整个段的运动。与此相反，在中期到远端结肠，其中内容是更牢固，活性是通过插入一个粪便PELLE的发起吨（环氧涂层自然沉淀或人工丸）和时空映射被设计通过结肠，以反映如图Hoffman等13的 JOVE文章中的丸粒的移动。因此，实验和分析的设置是很关键的取决于刺激和区域的种类正在研究中。因此，对于产生和液诱导的肠能动性时空映射的分析的关键步骤是:1）适当的除去从解剖组织肠系膜; 2）拍摄之前，正确的图像校准; 3）适当的去除STMAP生成和分析过程中的文物; 4）在分析系统的适当的设置; 5）获得的手巧，以导尿和缝合部分而不损坏它们。 虽然使用管腔直径STmaps的具有改进的可视化和分析充分蠕动模式随着肠的区域的能力，最好用结合时的技术中的压力或肌肉功能测量收缩2,15,20。例如，尽管一些肌肉收缩可能改变管腔直径略和是对一些STmaps（即，生肌涟漪）可见它们实际上可能不引起的肠内容物25中任推进或混合。这不能被未经这种技术对其他功能测量的耦合已知的。而且，许多组织标本在这种类型的系统（即，一个封闭的腔系统或恒定腔灌注通过泵系统）的性质导致内STmaps工件。因此，用户必须知道他们的特定器官的制备和实验如何导致在数据和方法，以避免或排除这些工件在数据分析 （例如，肠系膜诱导的垂直线或暗像素化伪影是由于组织的无能从系统处于关闭管腔制备排出流体）。有多种方法的一个中腔灌注圆通肠段除了一个封闭的系统。一种方法是代替使用开放系统，该系统保持恒定的腔内/背压力通过在制备8-10,30的肛门端使用一个凸起管和/或单向阀。这允许流体期间推进收缩移出制剂。 由于该系统是建立主要是为了检测变化管腔直径，这些收缩或运动模式，不严重影响管腔直径往往难以通过该协议的可视化。自变化STMAP中的像素着色是基于变化的腔直径，运动模式，不会导致在大直径的改变不会被可视化以及在这种方法中，如果强收缩也相同记录中存在。所描述的可视化和波纹型收缩分析（图3），设定记录接近吨视频中的分析线Ø组织壁能避免这个问题。这种方法减少了STMAP中显示的最大直径，所以收缩，仅最低限度改变组织直径可以被可视化。另一种选择来解决这个问题是改变所分析的视频段的持续时间，要排除收缩极大影响管腔直径，从而使较小的收缩更容易显现。这导致运动性最低程度改变管腔直径寻找类似一个单独的STMAP其中收缩极大地改变管腔直径的潜在问题。这是因为，在地图上的白色像素的确定是基于最小直径在给定的视频。如果没有在视频（很少或没有收缩环形肌的）非常小的收缩，不改变制剂的直径大大可以类似于从另一个视频蠕动收缩内直径多变性。因此，要考虑这个数字是非常重要的传说中的地图的右上角。如果最大直径和最小直径之间的差小到STMAP比较它从以确定象素色调变化的作为STMAP所表示的有效性产生的视频这一点很重要。因此，考试相结合的比例尺与实际拍摄的关键是要纠正的地图解释。 视频录制和肠和结肠段的时空映射已被应用于各种物种，包括斑马鱼26的 ，小鼠25,27 - 30，大鼠7,9,30 - 33，豚鼠5,6,8,13 - 19 24,30,32,34,35，刷尾负鼠12,36，兔2,30,37,38，鸡39，猪40,41和人类42。最广​​泛研究的物种是豚鼠​​。这并不奇怪，因为豚鼠肠神经系统H作为被最完全地表征和历史上它已经研究最多在体外对于肠43的推进运动的动物。时空映射已经主要适用于小动物肠道管段;然而，在兔和猪使用改性系统的研究表明这种方法要更大的动物中的应用。在兔子的情况下，该方法是相同的，所不同的是更大的片段和器官浴，使用30小动物。在猪中使用的方法是使用肠的形象化循环从麻醉的猪一解剖组织段的，而不是浸没到的器官浴。此外，通过互相关，而不是在大多数研究40中使用的透照法生成STmaps。用于视频录制和时空测绘的分离，vascularly循环灌注编制也被应用到较小的品种，如鼠<sup&gt; 33。最近的一项研究Kuizenga 等 。是第一个使用视频STmaps记录在人体肠道42的体外部分运动模式;虽然，STmapping方法已在体内 3,44被施加到测压（压力）记录中的分析人类。在人体组织中所记录的运动模式是类似于那些已经记录在动物模型中使用类似的技术和验证这种方法来人体组织的延伸。值得注意的是，该研究结合STmaps来源于录像与记录的力传感器肌肉收缩的测量。通过插入离体段光纤测压导管腔内压力测试也被转换成STMAP，显示STMAP的通用性，可视化多变化的管腔直径。这种组合方法相关肌肉紧张，腔内压力和室壁运动允许从视频记录所产生的STmaps的一个更深入的功能分析。 从墙的动作和变化，管腔直径（也称为DMAPS）产生STmaps研究允许的运动模式的详细描述，如推进蠕动波和局部节段性收缩。虽然这些图案是由早期的实验方法确定的，目前的方法使局部收缩运动，如波纹和新型抗蠕动收缩9,24,25,30,31,42一个更精确的定义。 STmaps和改变运动模式的分析的结构已应用于在小肠和结肠的胃肠蠕动关键问题。这些措施包括:神经源性肌和收缩的分化和定义Cajal间6,9,11,12,16,24,26,27,29质细胞的作用- 31,33,37 - 40,42，理解复杂对各种运动模式的圆形和纵向的肌肉层2,7,8,11,12,32,39,40之间的相互作用，考察腔内的营养物质10,18,19的影响，微生物菌种34，和粘度12,36，和理解各种内源性神经激素剂和外源性药剂2,4的 ​​作用- 7,9,10,13 - 17,28,35,40中的产生和运动的修饰。这种技术的未来涉及与其他测量，包括压力，电生理学和张力/收缩偶联它。最近的研究经常与视频记录和时空映射联合以提供额外的相关细节2,42并入其中的一个或多个测量结果。此外，该系统可被用于测量运动在其它管状和非管状器官。例如，已经尝试在测量使用胃动力这样的系统，但技术和软件需要改进，以更好地量化运动以这样的非管状器官45。毫无疑问的是，使用单独或与分析的更传统的方法相结合的时空映射技术将导致更深入的和未来的肠胃蠕动的全面了解。

记录和分析肠道蠕动的各种方法已经发展了近150年1。这些已经从最初的不等体内威廉博蒙特并测量与肌肉紧张，腔内压力，和/或膜电位的多站点记录的解释的更近的方法沃尔特炮的观察和描述（ 即，交界电位）2 - 6。后面的这些方法提供了整体运动模式的快照，而是由记录的位点的数目和数据的内插的区域，以在记录点之间的有效性是有限的。 视频录制和时空映射（STMAP）技术的最新发展使人们可以观察和分析体外全结肠和小肠段复杂的运动模式。最初的办法，先为INTES描述tinal段在20世纪90年代后期7,8，取决于调查设计的软件来分析录像;现在已经创建或修改的软件用于此目的2,8几组- 12。虽然有许多组织已经产生自己的软件包或插件，他们都分析直径组织段以及各种直径转换为灰度表示。市售的记录和分析系统，称为胃肠蠕动监测系统（GIMM）提供了交钥匙的方式，允许双方通过粪便颗粒速度的决心推进运动豚鼠远端结肠13，以及对推进和混合动力模式的分析分析与流体刺激在完整的肠段4,5,14 - 19。这后一种方法取决于STmaps的产生和分析，并在本文中描述。该方法的目标是增加吨他能力定性和定量分析存在于肠道内的各种运动的模式。而其它基团已通过自己的软件中使用的STMAP的运动分析，这是如何使用GIMM来分析运动型态通过产生STmaps的第一次描述。在本文中，我们提供了详细的一步一步的说明，:肠道组织的录像，录像参数进行合适的设置的准备，最大限度的检测能力改变组织的直径，创造STmaps的，还有解释和使用GIMM系统和ImageJ的软件STmaps的分析。 这里介绍的方法是特定的流体或含有影响肠道蠕动型态化合物半固体的腔灌注的分析。一种粪便颗粒推进的分析方法的论文Mawe和他的同事13进行说明。此处描述的通用方法可以是应用到其他管状平滑肌器官如:小肠，血管，尿道，输尿管， 等等。虽然自身这个方法不提供对压力变化或肌肉张力数据，它可以再加上使用的压力换能器，力传感器或电生理学测量，以提供运动模式作为一些其它组的一个更完整的画面显示2,15,20,21。

<table>
	<tbody>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Fisher</td>
			<td>BP358</td>
			<td>For Krebs buffer.</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher</td>
			<td>BP366</td>
			<td>For Krebs buffer.</td>
		</tr>
		<tr>
			<td>Potassium Phosphate (KH2PO4)</td>
			<td>Fisher</td>
			<td>P285</td>
			<td>For Krebs buffer.</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (MgSO4)</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td>For Krebs buffer.</td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td>For Krebs buffer.</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Fisher</td>
			<td>BP328</td>
			<td>For Krebs buffer.</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td>For Krebs buffer.</td>
		</tr>
		<tr>
			<td>Carboxygen (95%O2/5%CO2)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting pins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting trays/dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dunkin Hartley Guinea Pigs</td>
			<td>Charles River</td>
			<td>Strain 051</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td>http://imagej.nih.gov/ij/</td>
			<td>Freely available online.</td>
		</tr>
		<tr>
			<td>GastroIntestinal Motility Monitor (GIMM)</td>
			<td>Catamount Inc., St. Albans, Vermont</td>
			<td></td>
			<td>Includes parts listed below.</td>
		</tr>
		<tr>
			<td>Peristaltic Pumps</td>
			<td></td>
			<td></td>
			<td>Included with GIMM.</td>
		</tr>
		<tr>
			<td>Bath Cameras</td>
			<td></td>
			<td></td>
			<td>Included with GIMM.</td>
		</tr>
		<tr>
			<td>Bath TransIllumination Backlights</td>
			<td></td>
			<td></td>
			<td>Included with GIMM.</td>
		</tr>
		<tr>
			<td>Organ Baths</td>
			<td></td>
			<td></td>
			<td>Included with GIMM.</td>
		</tr>
		<tr>
			<td>Backlight Intensity Controls</td>
			<td></td>
			<td></td>
			<td>Included with GIMM.</td>
		</tr>
		<tr>
			<td>GIMM Processor ImageJ Plugin</td>
			<td></td>
			<td></td>
			<td>Included with GIMM.</td>
		</tr>
		<tr>
			<td>Polyethylene Tubing</td>
			<td></td>
			<td></td>
			<td>Included with GIMM.</td>
		</tr>
		<tr>
			<td>Tubing Connectors</td>
			<td></td>
			<td></td>
			<td>Included with GIMM.</td>
		</tr>
		<tr>
			<td>Masterflex tubing for Peristaltic Pumps</td>
			<td></td>
			<td></td>
			<td>Included with GIMM.</td>
		</tr>
		<tr>
			<td>Heating Bath/Water Circulator</td>
			<td></td>
			<td></td>
			<td>Included with GIMM.</td>
		</tr>
	</tbody>
</table>

spatiotemporal mapping of motility in ex vivo preparations of the intestines

多种方法已经被用于记录和评估胃肠蠕动，包括:在肌肉张力的变化记录，腔内的压力，和膜电位。所有这些方法依赖于活性测定在沿着肠道的一个或多个位置同时其然后解释为提供整体运动模式的感觉。近日，视频录制和时空映射（STMAP）技术的发展使人们可以观察和分析体外全结肠和小肠段复杂的图案。一旦记录和数字化，视频​​记录可以被转换为在该管腔直径被转换为灰度或STmaps颜色[称为直径的地图（DMAPS）]。 STmaps可以提供运动方向（ 即静止的，蠕动，抗蠕动），速度，时间，频率和收缩运动模式的强度数据。这种方法的优点包括:analysi相互作用或在同一段的不同区域不同运动模式同步发展秒，蠕动图案的可视化随时间改变，以及如何活动的分析在一个区域影响活性在另一个区域。视频录音可以重放具有不同的时间表和分析参数，以便独立STmaps和蠕动模式可以进行更详细的分析。该协议具体细节腔内液体扩张和腔内刺激影响运动产生的影响。使用管腔受体激动剂和拮抗剂提供特定的模式如何发起和如何一个图案可以被转换成另一种模式的机械信息。该技术是通过仅测量运动引起的变化的腔直径，而不提供关于腔内压力的变化或肌肉张力数据，并通过基于实验装置的工件的产生的能力的限制;虽然，analysi小号的方法可以考虑到这些问题。相较于以往的技术录像和STMAP方法提供胃肠道功能比较全面的了解。

了解时空地图（STmaps）为直径地图（DMAPS） 而通过这种技术产生的地图是时空，改变在所述组织的腔直径在距离和时间具体地可视化的参数。该STMAP描绘沿着x轴（单位mm）的组织段在y轴线（在秒）与起始时间在顶部和结束时间在底部水平距离和时间。在右上角是一个说明，其中显示的最小和最大管腔直径以及缩放对于x和y轴（ 图1-4）。因此，灰度级图像内的不同的像素的色调对应于不同的管腔直径。较暗的像素对应于更宽的直径和打火机像素对应于更小的直径。因此，环形肌收缩波将显示为亮像素化的地区，由于管腔直径减小（ <strong&gt;的图1黑色箭头）。与此相反，管腔腹胀由于圆形肌肉松弛或流体的大丸药将导致增加管腔直径和较暗的像素中的STMAP / DMAP（ 图1C白色箭头）。 STmaps的形成和意义的进一步讨论可以在纸张由拉默斯组 11中找到。 管腔腹胀引起的传播收缩 在图1中，豚鼠近端结肠而膨胀0.5，1.0，1.5和2.0毫升Krebs缓冲液在每个卷5分钟。传播收缩是由所有卷引起≥1.0毫升，并出现薄白乐队在STMAP（ 图1）。肠腔的腹胀引起传播收缩的开始。 如图1的具有较大体积的腔内的管腔的直径增加，并在STMAP中的像素对应于直径变得更暗创造一个整体的深色​​背景。在膨胀了某种程度的蠕动反射被激活（1.0毫升， 图1），从而启动收缩在口头端推进的波浪减小内腔的直径并走向段的肛门端（示出为白色带在图 1和4）。白带代表收缩往往是前面有一个暗带，它代表未来的蠕动波（ 图1C白色箭头）的反口松弛。这些白色和暗像素对应的上升收缩和蠕动反射，分别22,23降序松弛部件。在STMAP 在图1中图A中，有0推进波浪在0.0毫升，0推进波以0.5ml腹胀，3推进波以1.0ml腹胀，6推进波以1.5ml腹胀，和5推进波的牛逼2.0毫升腹胀。 营养诱导固定/混合收缩 传播收缩波是不是可以通过STmaps可视化的唯一的运动模式。蠕动混合型态如分割也可以看到在STmaps和对应的图像（图2A）。这种模式比传播收缩不同。在混合过程中的图案许多小的，固定的收缩发生在在同一时间不同的区域（可视化为多个小白色方块在同一水平线上，但不互相接触）。虽然每个节段的收缩是固定的，并且不在口腔移动到所述用于传播收缩肛门方式，STmaps来说明复杂收缩型态在长时间内的能力允许收缩的缓慢进展的可视肛门随时间（图2A黑色箭头）。的持续时间每个单独的收缩，也可以通过使用ImageJ和相关的插件（图2A）的白色收缩正方形绘制垂直线来确定。在从短链脂肪酸帧内灌注结肠产生这个特定STMAP，平均静止收缩持续时间为〜2秒（范围:1.9至2.1秒）。而剩下的肠系膜上的组织段可导致在分析工件，可用于通过肠系膜（图1B，2B）产生的垂直线的工件，以确定纵向肌肉运动。在图1B和 2B中的黑色垂直线的水平移动是由于收缩和纵肌松弛。纵肌这个横向运动可以被可视上STmaps如通过肠系膜工件产生的垂直带的水平移动（图1B，2B）。 ImageJ的和PlugiñSTmaps分析两者传播波（图3）以及收缩的持续时间（图2）的速度可以通过使用ImageJ和GIMMProcessor插件来确定。在图3中，既orthograde和逆行波的传播速度为〜0.25毫米/秒（范围:0.21到0.35毫米/秒）。如可以将ST地图上方的视频图像中所看到的，分析线（红线形成所述视频图像上的框）分别设置精确组织周围段的一个边缘，而不是整个段周围。这是在软件的准则的重要用途。这些准则精确的设置是非常重要的，在讨论部分为进一步分析视频的正确分析。这允许可视生肌波纹收缩24的STMAP的生成。这些收缩是肌源性的起源和不改变管腔直径很大。此外，不同的目录传播ections（orthograde或逆行），这是常见的波纹，可以使用这种技术（图3）进行分析。 如图2收缩的持续时间可以在组织段的不同区域而变化。 STmaps正确分析 与创建STmaps之一潜在的问题是可能的伪影的产生是由于实验方法。例如，肠系膜留在组织的外部将增加直径读数的组织的该段建立一个垂直黑线的STMAP（ 图1，图2B）。肠系膜的存在还人为地扩大通过增加最宽管腔测量，这导致规模的对比度测量结果的钝化灰度地图。由于这个原因，最好是从段尽可能完全地除去肠系膜未经穿孔组织。一个<html>其他可能的伪影STMAP是一种白色垂直线由于管腔液 ​​内的气泡，不能对比为黑色（ 图4B）。这些气泡可能使管腔直径显得更小到分析软件或覆盖白/光区域上的STMAP的运动模式。因此，设置在分析前的视频记录的组织段和适当的反衬的是绝对至关重要的构建STmaps（图4）的成功。正确和不正确的设置的对比示于图4中 。要注意的是视频调整同时在预实验摄像机标定和后实验分析窗口是至关重要的适当STMAP的生成和分析是很重要的。不正确的图像校准的可导致STmaps与不使用的数据（ 图4A）。 700"/&gt; 图中的近端结肠1.传播的波。 （A）腹胀响应曲线（豚鼠近端结肠执行）来确定合适的腔内积液量开始在这一领域（白色横带）传播的波。黑色箭头示出了全长传播波的例子。白色箭头表示该行神器造成的不完整的肠系膜切除。（B）传播通过STMAP代从类似于A组的实验取得了收缩的近距离观察，但持续时间较短的视频。这是比较容易地注意到，收缩在口腔到肛门方向前进，并确定表示为暗区域在前方的白色带的相比面板A.本图还提供了肠系膜工件的另一视图前述反口松弛。红色框标识此STMAP的扩大面板C.（三）区域此面板显示了一个更近距离观察Øf显示相同的视频和地图中标记为"流体腹胀的角度来暗的像素是由于通过流体反口管腔扩张的传播波面板B.白色箭头。这些是圆形的肌肉松弛发生反口圆形肌肉收缩区域。需要注意的是传播收缩看起来像在图A完全水平的线，由于在地图上的长时期。在板B和C的时间尺度是逐渐变短，这样的收缩波具有可衡量和报告为波浪运动速度的斜率。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53263/53263fig1highres.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/53263/53263fig2highres.jpg" width="700" /> 图2.测定收缩持续时间由STMAP。 （一）豚鼠近端结肠SHOWS响应短链脂肪酸（丁酸）的腔内灌注混合/节段的运动模式。垂直红线已通过紧缩的时期被吸引使用Image J与GIMMProcessor插件来确定收缩持续时间。黑色箭头示出了静止的收缩的传播方向反口。（B）的小鼠回肠收缩传播常表现出持续收缩的区域。垂直箭头绘制在这张地图上显示更多的口头地区仍然承包了一个持续时间较长。在此制备中，制剂的肛门端被封闭以防止流体在组织收缩而使封闭系统。在面板的顶部的图像显示时的组织的整个口腔端收缩（白上STMAP），而整个肛门端部（上STMAP黑色）鼓起由流体中的时间。在图B STMAP的中间的垂直黑色的水平/横向移动显示的移动纵肌层。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53263/53263fig2highres.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/53263/53263fig3highres.jpg" width="400" /> 图3.双向运动性模式。传播波可以在两个orthograde（口服肛门）和逆行（肛门口服）的方向移动。纯黑色箭头显示正常orthograde传播和黑色虚线箭头表示逆行传播。既orthograde的传播和逆行收缩在该STMAP的速度通过​​ImageJ的分析测定和为〜0.25毫米/秒。图像分析系（红色线形成上面的STMAP的视频图像上的框）设定在接近所述组织的一个边缘形象化生肌脉动收缩这是低幅度，浅收缩往往取向orthograde河etrograde，或两个方向。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53263/53263fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53263/53263fig4highres.jpg" width="700" /> 图4.重要性的STMAP生成正确的图像对比度。 （A）示出一个不正确的对比图像和STMAP，而面板B显示一个适当的对比图像和STMAP。在（A）中从适当的对比图像 （B）产生的水平白色条纹不一样明显，有多个工件（白色阴影）由于以灰度初始图像之中。在从正常对比图像 （B）所产生的STMAP灰度的白色阴影伪影消失，传播波更加明显。此外，黑色阴影代表的放松啊传播的收缩波的EAD可以看出，在与每个波收缩的STMAP的肛门端。在面板B中的白色箭头示出了由不能被正确地对比的图像的区域中产生一个STMAP工件。这种类型的神器主要是由于偶尔发生在实验方案的过程中准备腔内的气泡。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53263/53263fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines at JoVE.com

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, ...

spatiotemporal-mapping-motility-ex-vivo-preparations

Nanyang Technological University

Research

JoVE Journal

Biology

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

10.3K 次观看.  Virginia Commonwealth University. 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.

视频: Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

体外机械装载肌腱

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

科研

生物学

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

有针对性的表达绿色荧光蛋白在头发上使用体外病毒转导的卵泡

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

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 ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

"体外骨骼肌微血管制备离体血管反应性的调查

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An ex vivo technique for imaging mouse airway epithelia for quantitative analysis of motile cilia function important for insight into mucociliary clearance function has been established. Freshly harvested mouse trachea is cut longitudinally through the trachealis muscle and mounted in a shallow walled chamber on a glass-bottomed dish. The trachea sample is positioned along its long axis to take advantage of the trachealis muscle to curl longitudinally. This allows imaging of ciliary motion in the profile view along the entire tracheal length. Videos at 200 frames/sec are obtained using differential interference contrast microscopy and a high speed digital camera to allow quantitative analysis of cilia beat frequency and ciliary waveform. With the addition of fluorescent beads during imaging, cilia generated fluid flow also can be determined. The protocol time spans approximately 30 min, with 5 min for chamber preparation, 5-10 min for sample mounting, and 10-15 min for videomicroscopy.

Ex vivo Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

An easy and reliable technique for visualizing and quantifying airway cilia motility and cilia generated flow using mouse trachea is described. This technique can be modified to determine how a wide range of factors influence cilia motility, including pharmacological agents, genetic factors, environmental exposures, and/or mechanical factors such as mucus load.

在啮齿类动物呼吸道上皮纤毛运动功能的高分辨率成像的体外方法

An ex vivo technique for imaging mouse airway epithelia for quantitative  analysis of motile cilia function important for insight into mucociliary  ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

药物与DNA相互作用的细胞全基因组映射与COSMIC（小分子交联，染色质隔离）

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

在c-fos蛋白免疫组织学检测:一个有用​​的工具，中央通路在特定的生理反应所涉及的标记<em&gt;在体内</em&gt;和<em&gt;离体</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Ex Vivo Method for Assessing the Mouse Reproductive Tract Spontaneous Motility and a MATLAB-based Uterus Motion Tracking Algorithm for Data Analysis

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. Here, we present an easy and inexpensive method that would be instrumental for testing new drugs decreasing uterine contractility. This method utilizes the unique ability of the entire mouse reproductive tract to exhibit spontaneous motility when maintained ex vivo in a Petri dish containing oxygenated Krebs buffer. This spontaneous motility resembles the wave-like myometrial activity of the human uterus, referred to as endometrial waves. To demonstrate the effectiveness of the method, we employed a well-known uterine relaxant drug, epinephrine. We demonstrate that the spontaneous motility of the entire mouse reproductive tract can be quickly and reversibly inhibited by 1 &#181;M epinephrine in this Petri dish model. Documenting the changes of uterine motility can be easily done using an ordinary smart phone or a sophisticated digital camera. We developed a MATLAB-based algorithm allowing motion tracking to quantify spontaneous uterine motility changes by measuring the rate of uterine horn movements. A major advantage of this ex vivo approach is that the reproductive tract remains intact throughout the entire experiment, preserving all intrinsic intrauterine cellular interactions. The major limitation of this approach is that up to 10-20% of uteri may exhibit no spontaneous motility. Thus far, this is the first quantitative ex vivo method for assessing spontaneous uterine motility in a Petri dish model.

Uterine contractions are important for the well-being of females. However, pathologically increased contractility may result in dysmenorrhea, especially in younger females. Here, we describe a simple ex vivo preparation allowing quick assessment of the efficacy of smooth muscle relaxants that may be used for treating dysmenorrhea.

用于评估小鼠生殖道自发性的方法和用于数据分析的基于 MATLAB 的 Uterus 运动跟踪算法

Dysmenorrhea, or painful cramping, is the most common symptom associated with menses in females and its severity can hinder women&#39;s everyday lives. ...