This&#160;research was supported by NIH 1 U01 HL097973, NHLBI 5 R01 HL116511-02, UCSF-CTSI Grant Number UL1 TR000004, the Robert Wood Johnson Health and Society Scholars Program, and the Lisa and John Pritzker Family Foundation. The authors also wish to acknowledge Michelle Stephens for her assistance with scoring the ANS data as well as Vanessa Tearnan, Marialma Gonzales-Cruz, Yurivia Cervantes, Amy Engler, Stephanie Grover and Karen Jones-Mason for their assistance in collecting the ANS data, and Michael Coccia for his help with the data. We are also thankful to the families for their generous participation in this research and to the volunteers who helped us to test and refine this protocol, including the first author&#8217;s two children. We want to acknowledge our mentors who taught us about measuring ANS and why measuring ANS responsivity is meaningful in young children: W. Thomas Boyce, Gary Bernston, and Dave Lozano.

这项研究是经委员会加州大学旧金山分校的人类研究。 1.预协议建立（参见设备的完整列表材料的电子表格） <ol><li>通过打开计算机和连接ANS数据采集单元到电源安装设备。 注意：不要把采集单元上。 </li><li>在此之前开始任何数据采集，建立了ANS数据采集软件。选择设置，以符合图1。 </li></ol><img alt="图1" src="/files/ftp_upload/53652/53652fig1.jpg" /> 图1. BIOLAB软件配置设置。如在步骤1.2中描述此图显示的ANS数据收集期间将要使用的配置设置的屏幕截图。 <ol start="3"><li>现货保持电极在一个密封的包装，直至需要它们，因为电极质量下降了时间暴露于空气中。抓拍点电极到每个使用硬表面7捕捉线索。确保引线解开并牢固地连接到电极上。 </li><li>打开和提示音频和视觉应用（见下文步骤4）的片剂。设置的音量，以在片剂和全卷上的扬声器最大体积的80％。 </li><li>设置在不显眼的地方三脚架和摄像机。 </li><li>事先设置柠檬汁任务物品开始​​的协议。倒入一小纸杯一些水柠檬汁任务（约3盎司）之后冲洗。把柠檬汁极少量从分组到另一个杯和柠檬汁2滴（该量将达到的移液管的第一缩小和等于约0.1毫升）填充一次性塑料吸管。在纸巾或柠檬汁杯休息滴管，并设置出的方式。 </li></ol> 2.准备的发展挑战协议的母亲和儿童<ol><li>确保两个人，儿童评估机构（CA）和电脑操作员（CO），准备以管理协议。引进人员的母亲和孩子，让他们舒服。眼神接触，跟我们的孩子，以建立母，子，和员工之间的信任关系。让孩子一定的空间，使其恢复到CA和CO。不要关注压倒了孩子。 </li><li>问母如果孩子需要被馈送或具有电极置于孩子之前以避免以后中断他或她的尿布改变。让母亲开始之前的协议顾不上孩子的需求。 </li><li>让孩子在地板上或引进过程中一个开放的空间发挥。一旦协议开始，指导母亲让孩子在她的腿上，直到它完成。 </li><li>通过阅读剧本审查与母亲协议（见补充信息）。此外，发言的孩子直接左右了我回事访问期间发生。 </li></ol> 3.连接ANS采集设备儿童<ol><li>指示施加在电极对孩子的颈部和躯干（ 见图2）在CA，而CO分散了孩子。使用白色圆圈贴纸展示一个娃娃电极位置，如果有必要搞的孩子。快速移动和放置电极孩子的衬衫下面，以尽量减少可能性孩子将触摸或移开电极。 </li></ol><img alt="图2" src="/files/ftp_upload/53652/53652fig2.jpg" /> 图2.电极配置18个月孩子注 ：2 ECG电极在铅II配置应用。此外，对于颈背替代电极放置在此显示。 <ol start="2"><li>直接应用在电极到皮肤上。按用手指在纸带凝胶周围，以确保Ť帽子当场电极附着到皮肤上。所有电极必须从邻近的电极，以确保正确的信号传导被置于相距至少3厘米。对于那些难以确定任何距离，应用卷尺测量和通过测量每个电极的中心之间的长度验证此最小距离。 注意：将电极对儿童的下列顺序一直有效，以幼儿良好的耐受性。 <ol><li>放置连接到红色的电极（ - ）上的下部的中心引回。 </li><li>放置附着在白色电极（ - ）导致正上方的剑突，它位于xiphosternal结（胸骨底部尖端;其中所述左和右肋骨相遇）。确保该电极是红上述至少3个厘米（ - ）点电极。 </li><li>放置连接到刚刚离开的锁骨的边缘胸骨上切迹（评估的右侧）的白色（+）导线的电极。 <ol><li>使用卷尺测量，验证并记录在厘米2前电极之间的距离（（白色（+）和白 - ）），用于在阻抗得分程序以后使用。 </li></ol></li><li>将连接到棕色第一ECG电极（ - ）铅对右锁骨那里的锁骨符合肩膀骨。按下电极周围的泡沫和感觉锁骨。将电极沿纵向骨。 </li><li>放置连接至左下肋棕色（+），引线的第二ECG电极。按在电极周围的泡沫和感觉肋骨。 </li><li>放置连接到黑色的接地电极（ - ）铅对儿童的右下肋符合左下肋电极的水平面。 </li></ol></li><li>包和剪辑导致孩子的衣服下端，以尽量减少引线的运动。指示CO监控计算机。指示CA从事儿童和审查的Activiti参与与母亲的协议ES（见脚本B中的补充信息）。 </li><li>为了获得阻抗和心电图（ECG）信号转ANS数据获取单元上。按“重新检测”，开始心电图分析，然后单击一次“开始”旁边的绿灯在程序窗口的右下角。 <ol><li>等待5分钟后，在电极被放置以允许凝胶充分粘附到孩子的皮肤对所述ECG和阻抗信号的最佳电导。 5分钟后，检查电脑屏幕，查看信号，并确保他们看起来正常（CO）。转动摄像机（CO）上。 <ol><li>避免在这段时间与孩子闲聊。报价孩子举行一个标准化的“可选”的毛绒玩具，如果他或她（以协议开始计划从手工删除玩具前夕），似乎不舒服。 注：解释给孩子和母亲，这是需要等待一个双向吨至确保设备工作正常。 </li><li>显示孩子的心脏活动，母亲的计算机图像并解释每个信号代表。提醒母亲，这一活动是不用于诊断任何健康问题。向她保证，如果研究小组注意到令人关注的东西，她会收到通知。按照脚本B（见补充信息）。 </li></ol></li></ol></li></ol> 4.管理任务的发展挑战议定书<ol><li>打开片剂媒体播放器。播放第1分钟的摇篮曲。保持平板电脑出孩子够不到的地方。 </li><li>持有玩偶的盒子在孩子面前。慢慢的旋转手臂，播放音乐，直到袜子猴子弹出，使得它需要15秒才能完成一个回合。重复四次。 </li><li>开始与脚本C柠檬汁任务（见补充信息）。将2滴浓柠檬汁对孩子的舌头的中心，稍有BAC从前面K，所以果汁不顺口。 注：如果吸管在果汁杯休息，抖落之前放弃它在舌头上的吸管外额外滴。 <ol><li>静静地等待10秒，一个友好的，中性的表情。说：“什么味道？”如果孩子需要安慰。 注：柠檬汁任务必须至少需要20秒，从通过等到后滴在他们口中，然后看是否有反应讲述液体孩子开始。 </li><li>滴柠檬汁后至少10秒说，“现在是时候喝一些水”，并给孩子喝了一口水。如果孩子拒绝，等待几秒钟，说，再试试“现在你可以拿点水喝。”按F4键作为评估开始提供水（CO）。 </li></ol></li><li>玩患病婴儿使用媒体平板电脑上哭30秒记录播放器。保持平板电脑出孩子够不到的地方。 </li><li>播放使用媒体播放器平板电脑的第二个1分钟的摇篮曲。保持平板电脑出孩子够不到的地方。 </li><li>播放使用媒体播放器平板电脑的2分钟视频中性。握住平板电脑的孩子的脸大致12英寸，但不要让他们抓住不放的平板电脑。 </li></ol> 5.标记协议段在发展方面的挑战管理局（CO） <ol><li>指示CO推功能（F）键（由用户设定的先验数据评分期间确定的任务或潜在的问题），以纪念在ANS数据中的重要步骤协议（ 见表1）。按下F11键表示一个任务提前结束（键入“结束任务早”，并解释到文本框）或重大协议问题或违法行为，如电极脱落或有人在房间里走动和分散注意力的孩子（类型解释到文本框中）。 </li></ol><table><tbody><tr><td> Marker键 </td><td> 事件 </td><td> 持续时间后开始 </td></tr><tr><td> F1 </td><td>摇篮曲1日开始</td><td> 60秒</td></tr><tr><td> F2 </td><td>杰克在框中启动</td><td> 60秒</td></tr><tr><td> F3 </td><td>柠檬汁脚本启动</td><td>至少20秒</td></tr><tr><td> F4 </td><td>柠檬汁端（水喝提供） </td><td>至少有10个SES </td></tr><tr><td> F5 </td><td>婴儿哭声的开始</td><td> 30秒</td></tr><tr><td> F6 </td><td>摇篮曲2启动</td><td> 60秒</td></tr><tr><td> F7 </td><td>视频开始</td><td> 120秒</td></tr><tr><td> F10 </td><td> DCP结束（视频终端） </td><td rowspan="2"></td></tr><tr><td> F11 </td><td>严重的不规则事件</td></tr></tbody></table> 表1：分子标记键协议事件标志期间挑战的任务。如在步骤5.1中所述标记的键被显示，用于指示在ANS数据文件重要协议事件和很少发生。 6.邮政发展挑战协议<ol><li>有CA对母亲说：“那么，如何是你 （暂停听到回应） 这是有趣的，看看孩子对这些各种各样的活动如何应对？现在是时候要轻轻地从孩子的皮肤去除电极“。 </li><li>感人/取出电极之前先关闭ANS的数据采集。 注：该设备将无法进行，或当它被关闭传输电​​力。 </li><li>通过使用一个手指紧握皮肤，同时另一方面消除电极，除去从孩子电极。拉起电极，并从标签指向的方向走的标签。除去最敏感电极最后一个（前部和颈部的背面）。让母亲重新移动电极如果孩子特别不安或者如果她期望。用湿擦拭以去除来自电极的皮肤和干纱布垫剩余如果皮肤潮湿或有凝胶在皮肤上的任何多余的残留物。 </li><li>关闭摄像机。保存ANS数据文件，退出程序，并关闭计算机。 注：ANS数据采集软件创建两个数据文件，“.mw”文件（原始ANS数据）和一个“.event”文件，它是按下的功能键的文本文件。 </li></ol> 7.呼吸性窦性心律心律失常评分<ol><li>打开心率变异性（HRV）分析方案，以清洁的心电数据和得分率心脏（HR），RSA和呼吸频率（RR）。 </li></ol><img alt="图3" src="/files/ftp_upload/53652/53652fig3.jpg" /> 图3. HRV分析主导航画面。为计分用的HRV蹒跚学步ANS数据设置alysis记分软件显示出来。得分段，拖动事件到“事件类型”框中。 <ol start="2"><li>查看数据收集任何可用的现场记录，以了解可能发生的异常情况或错误。找到并双击从弹出的窗口中攻入“.mw”文件（“选择或输入文件路径”）。 注：被砍下应该出现在白框在屏幕顶部的参与者数据的文件名。 <ol><li>将“通道标签”如下：1通道心电图，通道2 - 莫宁，通道3 DZ / DT。验证该采样频率被设置为1,000。点击“确定”，在屏幕的底部打开主导航画面（ 图3）。 注意：这些通道应与数据收集过程中设置的。 </li><li>选择设置以匹配的身影。将模式设置为“事件”。设置事件标识符为“名”查看任务的名称（ 即 “玩偶的盒子”）。将“邮事件时间（s）”，以30秒。 注意：文件长度为每个孩子的文件有所不同。 “类型”是指由CO协议期间按功能（F）键。如果模式是不是事件，而是时间，“细分时代”，可以在几秒钟内（ 即 30设置为不同的时间长度或60）。 </li><li>点击“HRV校准设置”标签。一套计算方法，“整个”。呼吸信号设置为“咗”（DZ不是/ DT）。设置窗口函数为“海明”。设置VLF频带范围为0.003至0.04赫兹（Hz），低频或LF波段范围0.04到0.15赫兹，和高频HF频带范围0.15-1.04赫兹婴幼儿。 </li><li>点击下一个标签，“R＆山顶神器设置”设置IBI和MAD MED为“on”。设置最大心率（每分钟心跳（BPM））230和最小心率40 BPM。对于R峰值检测灵敏度滑块设置为1。设置手动操作来关闭。打开基线和肌肉噪声滤波器（亮绿色）和陷波滤波器关闭（深灰色）。噪声滤波器设置为60 Hz。 </li><li>点击下一个标签，“其他设置”。点击是/输出设置部分没有框（“使用默认输出路径”）来选择默认位置来保存输出文件。勾选“显示报告”按钮（亮绿色）。 注：同步的视频可以通过点击“是/否”框下被视为一个以前编辑过的数据文件可以通过单击“使用编辑的数据”是/否盒被视为“使用默认的视频路径。” </li><li>回到“事件和模式”​​选项卡。点击“分析”按钮。 </li><li>选择“是”或“否”当记者问，“难道心电图反转？否/是“。 注：多数心电图不反转。分析窗口将打开，显示数据的30秒。该HRV分析程序，标志着淡蓝色圆点每个R峰值时有一个潜在的“异常值”或不规则图案，标志着一个黄色的星星。离群值可以被标记时两个R峰之间的距离是不同的，为了这个个体经常RR间隔。 </li><li>检查所有的异常值（黄星），以确定黄星的原因（见7.1.1.9编辑选项）。在数据记录的利息登录的变量进行任何必要的编辑之后。 注：分析窗口右下角的框显示由这一细分市场，包括HR，RR和RSA的计算值。 </li><li>点击“编辑R”原则（在控制窗口中的绿色按钮）来检查异常值。点击放大镜图标，以进一步放大]。 </li><li>使用红色的“删除”按钮来删除黄色的星星被放置不正确和白色的“插入”按钮为纪念第r峰。 注：通常是清楚其中R峰值应删除或手动标记。极杂乱部分可以使用黄色“删除数据部分”按钮被移除，左右滑动的部分中的浅蓝色X游标之后被删除。这应该只在开头或分段的端部进行;必须有数据来计算一个30秒段的有效分数的连续20秒。 </li><li>点击“确定”在屏幕底部的保存编辑，返回到先前的屏幕，显示编辑的数据。点击“写入”（顶部工具栏中的白色按钮）来输出数据打进到Excel文件。重复此过程，每30秒间隔或任务进行评分。 注：“取消”退出而不保存更改。 </li><li>点击“完成”的“控制”面板上的编辑和写入数据的全部30秒后段。 注：弹出窗口将请求的文件名，编辑DAT一个。所有编辑的数据，并打进了每个孩子的数据将被保存一个.edh文件。 </li></ol></li></ol> 8.预射血期评分注意：阻抗（IMP）分析程序允许一个清洁阻抗数据，并获得人力资源和PEP值。 <img alt="图4" src="/files/ftp_upload/53652/53652fig4.jpg" /> 图4.进出口分析阻抗校准设置屏幕为计分采用阻抗分析软件得分蹒跚学步ANS数据的设定显示。 <ol><li>打开IMP分析程序。在IMP分析程序双击，然后选择刚刚打进HRV分析同一.mw文件。选择每个相同的渠道“频道标签：”通道1心电图，通道2 - 莫宁，通道3 DZ / DT。按“OK。”关于事件和模式选项卡中，选择相同的设置作为HRV分析程序。 <ol><li>点击“ImpedanCE校准设置“选项卡（ 图4）。选择设置以匹配图。设置“电极式”为“点”。 （所测量和数据收集期间记录白色电极之间的距离）进入电极的距离。设置“合奏长度（毫秒）”，以400为幼儿。 </li><li>设置的“Q点的计算方法”，以“最小值KR间隔”。对于18个月年轻人（K值可以基于每个子基，如果需要进行调节）设置K值到25。设置的“B点的计算方法”，以“的DZ / dt的时间+ C百分数”设置的百分比DZ / dt的峰值为“55”，而常数“4”。将“DZ / dt的来源”为“测量”。验证“Detrend DZ / dt的”灯亮了非移动采集单元。 </li><li>点击“R＆山顶神器设置”标签。验证IBI最小/最大和MAD MED检查都“在“设置最大心率（BPM），以230为幼儿和最小心率（BPM）为40设置峰值检测灵敏度1.将手动覆盖到了。打开“基线和肌肉噪声滤波器”（亮绿色）。关闭“陷波滤波器”（深绿色）。关闭60 Hz的设置。 </li><li>点击“更多设置”标签。不要选中“自动分析所有数据段”按钮。选择“是”，在“使用编辑的数据”是/否框，如果任何编辑都在HRV程序进行。双击从弹出框的编辑（.edh）文件。选择“创先争优”为输出模式。点击明亮的绿色“分析（回车）”按钮。 注：如果在得分HRV没有编辑被做了，这是没有必要（或可能）在这里使用编辑数据。 </li><li>点击“编辑合奏”看到合奏波，这是图像的全屏视图的平均值所有波形从段。 </li><li>检查Q点，这是通过在设置中输入K值确定的位置。通过所有段的滚动检查在Q位置。 注：Q点应该只是之前的红色（EKG）线开始上升到R峰值的最低点。如果有一个逐渐倾斜“预Q”区，则Q应在点被标记，其中斜率至R开始更壁立。 </li><li>返回阻抗校准设置选项卡，如果Q点盯人不正确的，当K = 25增加5 K值。再次回顾段。继续更改到K，直到在Q标志是最优的。 注：K值需要为每个孩子保持一致;没有针对不同的细分选择不同的价值观，不要用手移动任何标记。 </li><li>记录PEP值，每个段得分。 注：PEP是每一个人在相当稳定，一般由4-6毫秒整个评估的波动每个孩子。然而，PEP值可以是在整个儿童非常不同。 </li><li>为了输出的得分数据点击“写入”按钮，Excel文件。重复这个过程被打进每个部分。 </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+reactivity+to+psychological+challege:+Conceptual+and+measurement+considerations.">Cardiovascular reactivity to psychological challege: Conceptual and measurement considerations.</a> Psychosom. Med. 65, 9-21 (2003).</li><li>Fox, N. A., Schmidt, L. A., Henderson, H. A., Marshall, P. J., Cacioppo, J. T., Tassinary, L. G., Bernston, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+20,+Developmental+Psychophysiology:+Conceptual+and+Methodological+Issues.">Chapter 20, Developmental Psychophysiology: Conceptual and Methodological Issues.</a> Handbook of Psychophysiology. , 453-481 (2007).</li><li>Gilissen, R., Koolstra, C., van Ijzendoorn, M., Bakermans-Kranenburg, M., van der Veer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+reactions+of+preschoolers+to+fear-inducing+film+clips:+Effects+of+temperamental+fearfulness+and+quality+of+the+parent-child+relationship.">Physiological reactions of preschoolers to fear-inducing film clips: Effects of temperamental fearfulness and quality of the parent-child relationship.</a> Dev. Psychobiol. 49, 187-195 (2007).</li><li>Bush, N. R., Alkon, A., Stamperdahl, J., Obradovi&#263;, J., Boyce, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiating+Challenge+Reactivity+from+Psychomotor+Activity+in+Studies+of+Children’s+Psychophysiology:+Considerations+for+Theory+and+Measurement.">Differentiating Challenge Reactivity from Psychomotor Activity in Studies of Children’s Psychophysiology: Considerations for Theory and Measurement.</a> J. Exp. Child Psychol. 110, 62-79 (2011).</li></ol>

The authors declare that they have no competing financial interests.

这项研究表明，18个月大的孩子，标准化的休息和挑战任务，旨在从一系列域（惊吓，感官，社交，休息）的得到响应样品中导致了一系列的ANS响应。这些休息和挑战的条件下发现，平均ANS反应是类似于谁已经测量ANS的反应与使用类似的任务8,11,27,29幼儿人报道ANS值。在我们的样本中发现的ANS措施的平均水平是那些通常在婴幼儿发现和那些在年龄较大的儿童中发现，这是符合证据之间的心?...

在孩子们的自主神经系统的个体差异（ANS）活性发挥的生理和心理健康问题12-16发展和维护具有重要作用。越来越多的证据体现出，在休息和反应性ANS措施个体差异预测儿童和外向精神病理学4,9,17-19和身体健康7,20的变化。除了​​ANS运作这些直接或主要作用，研究的积累的身体已经发现，孩子们的反应可以用上下文的危险因素，如不良事件或婚姻冲突，中度对儿童的福祉和健康的影响进行交互（见参考文献21复审）。尽管越来越多的证据，儿童的自主反应起到保健作用在整个生命过程中，在这些过程中伴随的兴趣，有是需要更多的研究三岁下检查ANS功能在儿童中的发展。 该ANS包括两个分支，副交感神经和交感神经系统。它调节内脏（如心脏速率，呼吸，消化，性兴奋）的功能，主要是通过无意识的机制，以应对日常和不良反应1。副交感神经系统（PNS）是“休息和恢复”的系统，睡觉或放松交感神经系统（SNS）时保持较低的休息心脏率和恢复状态是响应紧急情况或威胁“战斗或逃跑”系统通过加速一个人的心脏率23的情况。呼吸性窦性心律不齐（RSA）是对心脏功能的PNS影响的可靠指标，和射血前期（PEP）是对汽车在SNS影响的可靠指标DIAC工作。 RSA和PEP已经发现通过在成人24,25的样品的实验药理学阻断有效的措施。最近，这项工作已经扩展到儿童和青少年的样本，进一步确立了RSA和PEP在整个发展8,26,27 ANS活动的有效和可靠的措施。使用带和现场电极可靠心阻抗测量（PEP）已成人样本28被使用相当长的时间。最近，平行测量和分析技术也被证明是可靠的，并在儿童11,29,30的样本有效的，尽管在幼儿的PEP措施集合是罕见的。 有对PNS在婴幼儿，包括迷走神经张力和呼吸性窦性心律不齐措施发挥作用的现存的大量文献。有使用PEP的SNS的研究较少，很少同时检查这两个副交感神经和交感神经系统，这是建立自己的高度集成的功能的理解是至关重要的。幼儿PEP的一些研究中，大多数发现自己的协议不产生响应的挑战8.31的PEP显著集团层面的变化。有研究表明，有稳定搁在婴儿期开始PNS措施，他们与气质，行为和健康，13,16,26,32,33关系，但有SNS的理解有限响应和稳定性随着时间的推移，关系发展，塑造其发展轨迹的因素。 PNS响应已被主要归因于社会参与16和SNS的反应提出了体现“战斗或逃跑反应”1以及奖励灵敏度（见参考文献34复审）。在年幼的孩子，这是具有挑战性的，以模拟在laborato具体的挑战RY和他们搞由于他们的注意力持续时间短。还有谁可能感到不适与同时测量PNS与SNS需要七个点电极测量幼儿的挑战。此外，发展科学领域内，有关于如何激发“应激反应”，同时充分尊重也与儿童工作儿童的发展需求和职业道德标准很少。本文介绍了关于由设计引起从18个月大的孩子和副交感神经交感神经系统的反应休息和挑战性的环境的标准化协议的管理和评分信息。 ANS反应通常是概念化为离散相对于外部刺激的生理反应比较或休息状态，横跨生物个体差异22。的精神和肉体的生命早期病因的检查，他ALTH问题已导致科学家在理解孩子们的反应，以唤起自适应响应，或者可能被认为是紧张的情况下挑战特别感兴趣。文献的优势是指这种现象为“压力反应”，虽然在遇到挑战性的刺激具有在宽范围的域的引出应答的潜力。因此，协议中所述下方被设计为跨多个领域，包括两种形式的休息引起ANS反应（一边听着舒缓的摇篮曲，并同时观看了冷静，中立视频）和18个月的孩子3发育相适应的挑战：预测/惊吓（玩偶的箱），感官（柠檬汁），和社会情感（听一个生病的婴儿哭声的）。该协议是改编自我们现有的12个月孩子8和3-5岁儿童11协议，以使其发育挑战性，参与和容忍的幼儿（18-21个月）。 在这里，我们目前的发展挑战协议（DCP）和ANS从应力的18个月大的考察访问数据，进食和孕妇和他们的后代的早期发展（SEED）研究。健康孕妇怀孕期间参加了2013-14孕期体重增加和产前应激的研究中招募和超重，低收入，以及种族和多族裔。刚出生后并再次之前的数据收集为这里所报告的研究中，当后代是18-21个月的年龄时2014-2015的开始获得父母对她的后代的研究的知情同意。

<table>
	<tbody>
		<tr>
			<td>ANS equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laptop and power cord&#160;</td>
			<td>Dell</td>
			<td>60-0107-3.0</td>
			<td>Mindware BioLab software requires a PC with 2 USB ports</td>
		</tr>
		<tr>
			<td>ANS data collection software</td>
			<td>Mindware</td>
			<td>contact company</td>
			<td>BioLab acquisition program</td>
		</tr>
		<tr>
			<td>ANS data acquisition unit and cords</td>
			<td>Mindware</td>
			<td>50-3711-08</td>
			<td>BioNex 8-slot Chassis</td>
		</tr>
		<tr>
			<td>Snap leads</td>
			<td>Mindware</td>
			<td>93-0498-00</td>
			<td>2 white, 2 red, 2 brown, 1 black</td>
		</tr>
		<tr>
			<td>Spot electrodes</td>
			<td>Kendall</td>
			<td>31112496</td>
			<td>130 model; 7 for each assessment + extras-to be sealed in a ziplock bag at all times)</td>
		</tr>
		<tr>
			<td>ANS scoring/editing software</td>
			<td>Mindware</td>
			<td>contact company</td>
			<td>HRV Analysis, version 3.1.0F and IMP Analysis, version 3.1.0I</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Protocol equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tablet computer</td>
			<td>Apple</td>
			<td></td>
			<td>iPad; for playing audio and video clips</td>
		</tr>
		<tr>
			<td>portable speaker</td>
			<td>Polariod</td>
			<td>PBT510</td>
			<td></td>
		</tr>
		<tr>
			<td>Jack in the Box</td>
			<td>Schylling</td>
			<td>SMJB</td>
			<td>Sock Monkey theme</td>
		</tr>
		<tr>
			<td>Lemon juice</td>
			<td>ReaLemon</td>
			<td>98077</td>
			<td>0.14oz shelf-stable packets</td>
		</tr>
		<tr>
			<td>Pasture pipettes</td>
			<td>Grainger</td>
			<td>6FAV9</td>
			<td>1 ml size, disposable</td>
		</tr>
		<tr>
			<td>Portion cups</td>
			<td>Dart Solo Con</td>
			<td>250PC</td>
			<td>2.5 oz, disposable, plastic</td>
		</tr>
		<tr>
			<td>audio files of baby cry</td>
			<td>Alkon lab</td>
			<td></td>
			<td>30 second clip recorded in a neonatal intensive care unit</td>
		</tr>
		<tr>
			<td>audio file of baseline lullaby</td>
			<td>Rockabye Baby Music</td>
			<td></td>
			<td>60 seconds of &#34;Across the Universe&#34; on &#34;Rockabye Baby Lullaby Renditions of the Beatles&#34; CD</td>
		</tr>
		<tr>
			<td>audio file of final lullaby</td>
			<td>Rockabye Baby Music</td>
			<td></td>
			<td>60 seconds of &#34;Here Comes the Sun&#34; on &#34;Rockabye Baby Lullaby Renditions of the Beatles&#34; CD</td>
		</tr>
		<tr>
			<td>video file of neutral video</td>
			<td></td>
			<td></td>
			<td>2 minutes of Baby Einstein video</td>
		</tr>
		<tr>
			<td>Retractable measuring tape</td>
			<td>Rollfix</td>
			<td>TM04</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Miscellaneous materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surge protector</td>
			<td></td>
			<td></td>
			<td>to plug in all electronics</td>
		</tr>
		<tr>
			<td>2-prong adapter for surge protector</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td>Sony</td>
			<td></td>
			<td>Handycam HDR-CX440</td>
		</tr>
		<tr>
			<td>Video Encoder, power and input cords</td>
			<td>Mindware</td>
			<td>50-8614-01</td>
			<td>optional; to sync video and ANS data files</td>
		</tr>
		<tr>
			<td>Baby wipes and gauze</td>
			<td></td>
			<td></td>
			<td>for cleaning and drying oily skin</td>
		</tr>
		<tr>
			<td>Toys</td>
			<td></td>
			<td></td>
			<td>to distract children during assessment</td>
		</tr>
		<tr>
			<td>Visual aids</td>
			<td></td>
			<td></td>
			<td>pictures of child with electrodes on, description of protocol to show participants</td>
		</tr>
		<tr>
			<td>Script of protocol for research assistants</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Charging cords for tablet and speaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Doll and reinforcer</td>
			<td></td>
			<td></td>
			<td>for demonstration if necessary</td>
		</tr>
	</tbody>
</table>

measuring cardiac autonomic nervous system (ans) activity in toddlers - resting and developmental challenges

自主神经系统（ANS）包括两个分支，副交感神经和交感神经系统，并控制内部器官（如心脏速率，呼吸，消化）的功能，并响应日常和不良事件1。已发现儿童ANS措施涉及到的行为问题，情绪调节和健康2-7。因此，幼儿期了解影响ANS发展的因素是很重要的。在ANS的两个分支影响青少年儿童对刺激心血管反应，并已测量无创性，通过外部监测设备，采用生理变化8-11有效和可靠的措施。不过，也有非常年幼的儿童与副交感神经和交感神经系统的同步措施，限制了两个系统的集成功能的了解一些研究。此外，Majori的现有幼儿的研究对婴幼儿休息ANS措施或者其反应报告TY常用的母子互动范例，而较少有人知道ANS反应到其他具有挑战性的条件。我们提出了一个研究设计和标准化的协议，用于心脏自主控制在18个月大的孩子一个非侵入性和快速评估。我们家庭或实验室访问期间描述了连续监测ANS的休息和挑战条件下，副交感神经和交感神经分支的方法，从我们的使用验证的设备和软件得分140种族多元化的幼儿提供样本描述调查结果。结果表明，该协议可能会产生一系列既休息和发育困难的条件下的生理反应，在心脏的变动和副交感神经和交感神经活动指数所示。个人休息的水平，respons证明变异ES挑战，挑战和反应性，它提供了额外的证据，该协议旨在为幼儿ANS个体差异考试有用。

种子研究纳入162母子二人组合（37％非裔美国人，30％的拉丁裔; 16％是白人，17％为其他种族或）。对于18月的访问中，我们完成了140名儿童（已登记的样本87％）的人的评价; 6名参加搬走了，剩下的参与者无法访问或这次访问不可用。为参观ANS数据收集组件的拒绝率是可供此访的母亲牌3/140（2％）。三次访问分别为21个月（超过3个月以后在目标年龄）后进行的，导致在135儿童在目标时间内收集的AN...

Watch this Scientific Journal Video about Measuring Cardiac Autonomic Nervous System ANS Activity in Toddlers - Resting and Developmental Challenges at JoVE.com

Measuring Cardiac Autonomic Nervous System ANS Activity in Toddlers - Resting and Developmental Challenges

我们描述了与18个月大的孩子休息和挑战的条件下连续监测的自主神经系统的方法。结果表明，该协议能产生有意义的生理反应的自主神经系统的两个分支和响应模式引起显著的个体差异。

测量心脏自主神经系统（ANS）活性的幼儿 - 安静和发展挑战

The autonomic nervous system (ANS) consists of two branches, the parasympathetic and sympathetic nervous systems, and controls the function of internal ...

measuring-cardiac-autonomic-nervous-system-ans-activity-toddlers

Research

Education

JoVE Journal

JoVE Core

Introduction to Psychology

Anatomy and Physiology

Medicine

Unit 1

Measuring Cardiac Autonomic Nervous System (ANS) Activity in Toddlers - Resting and Developmental Challenges

Biopsychology

The Autonomic Nervous System

SCIENTISTS IN ACTION

15.2K 次观看.  University of California, San Francisco. 我们描述了与18个月大的孩子休息和挑战的条件下连续监测的自主神经系统的方法。结果表明，该协议能产生有意义的生理反应的自主神经系统的两个分支和响应模式引起显著的个体差异。 

视频: Measuring Cardiac Autonomic Nervous System ANS Activity in Toddlers - Resting and Developmental Challenges

Quantitative Autonomic Testing

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be invaluable tool for evaluation of these disorders, both in clinic and research. There are number of autonomic tests, however, only few were validated clinically or are quantitative. Here, fully quantitative and clinically validated protocol for testing of autonomic functions is presented. As a bare minimum the clinical autonomic laboratory should have a tilt table, ECG monitor, continuous noninvasive blood pressure monitor, respiratory monitor and a mean for evaluation of sudomotor domain. The software for recording and evaluation of autonomic tests is critical for correct evaluation of data. The presented protocol evaluates 3 major autonomic domains: cardiovagal, adrenergic and sudomotor. The tests include deep breathing, Valsalva maneuver, head-up tilt, and quantitative sudomotor axon test (QSART). The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores (CASS). Detailed protocol is provided highlighting essential aspects of testing with emphasis on proper data acquisition, obtaining the relevant parameters and unbiased evaluation of autonomic signals. The normative data and CASS algorithm for interpretation of results are provided as well.

Standardized, comprehensive and fully quantitative testing of autonomic functions is described. The autonomic tests consist of evaluation of all three major autonomic domains including cardiovagal, adrenergic and sudomotor. The severity and distribution of dysautonomia is quantitated using Composite Autonomic Severity Scores.

定量自主测试

Disorders associated with dysfunction of autonomic nervous system are quite common yet frequently unrecognized. Quantitative autonomic testing can be ...

科研

医学

Measuring Cardiac Autonomic Nervous System (ANS) Activity in Children

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory rate, salivation, perspiration and renal function. The ANS has two main branches: the sympathetic nervous system, preparing the human body for action in times of danger and stress, and the parasympathetic nervous system, which regulates the resting state of the body.
ANS activity can be measured invasively, for instance by radiotracer techniques or microelectrode recording from superficial nerves, or it can be measured non-invasively by using changes in an organ's response as a proxy for changes in ANS activity, for instance of the sweat glands or the heart. Invasive measurements have the highest validity but are very poorly feasible in large scale samples where non-invasive measures are the preferred approach. Autonomic effects on the heart can be reliably quantified by the recording of the electrocardiogram (ECG) in combination with the impedance cardiogram (ICG), which reflects the changes in thorax impedance in response to respiration and the ejection of blood from the ventricle into the aorta. From the respiration and ECG signals, respiratory sinus arrhythmia can be extracted as a measure of cardiac parasympathetic control. From the ECG and the left ventricular ejection signals, the preejection period can be extracted as a measure of cardiac sympathetic control. ECG and ICG recording is mostly done in laboratory settings. However, having the subjects report to a laboratory greatly reduces ecological validity, is not always doable in large scale epidemiological studies, and can be intimidating for young children. An ambulatory device for ECG and ICG simultaneously resolves these three problems.
Here, we present a study design for a minimally invasive and rapid assessment of cardiac autonomic control in children, using a validated ambulatory device 1-5, the VU University Ambulatory Monitoring System (VU-AMS, Amsterdam, the Netherlands,<a href="http://www.vu-ams.nl" target="_blank"> www.vu-ams.nl</a>).

Measuring Cardiac Autonomic Nervous System ANS Activity in Children

Measurement of autonomic nervous system activity usually confines the researcher and participant to the laboratory, which may provide an intimidating environment to children. The VU University Ambulatory Monitoring System (VU-AMS) device can record cardiac autonomic control in any setting. The VU-AMS proved very amenable to testing in children.

测量心脏自主神经系统（ANS）儿童活动

The autonomic nervous system (ANS) controls mainly automatic bodily functions that are engaged in homeostasis, like heart rate, digestion, respiratory ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

颈动脉输液的紫杉烷类化合物在小鼠体内的药代动力学和药效学分析

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Electrophysiological Measurement of Noxious-evoked Brain Activity in Neonates Using a Flat-tip Probe Coupled to Electroencephalography

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. Those tools are subjective or determine secondary physiological indicators which also have certain limitations particularly when exploring the effectiveness of analgesia. As cortical processing is essential for pain perception, brain activity measures may provide a useful approach to assess pain in infants. Here we present a method to assess nociception with electrophysiological brain activity recordings optimized for the use in newborn infants. To produce highly standardized and reproducible noxious stimuli we applied mechanical stimulation with a flat-tip probe, e.g., PinPrick, which is not skin-breaking and does not cause behavioral distress. The noxious-evoked potential allows the objective measurement of nociception in non-verbal patients. This method can be used in newborn infants as early as 34 weeks of gestational age. Moreover, it could be applied in different situations such as measuring the efficacy of analgesic or anesthetic drugs.

Measuring pain in non-verbal patients is a challenge. In this study we combine EEG recording with stimulation using a flat-tip probe to detect noxious-evoked brain activity in an objective manner.

应用平板探针结合脑电图对新生儿有害诱发脑活动的电生理测量

Pain is an unpleasant sensory and emotional experience. In non-verbal patients, it is very difficult to measure pain, even with pain assessment tools. ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum of physiological, biochemical, morphological, and pharmacological parameters in centrally denervated hearts. Here, we describe a setup for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cyclic adenosine monophosphate (cAMP) dynamics. The intracardiac autonomic nervous system is modulated by the mechanical dissection of atrial fat pads-in which murine ganglia are located mainly&#8212;or by the usage of global as well as targeted pharmacological interventions. An octapolar electrophysiological catheter is introduced into the right atrium and the right ventricle, and epicardial-placed multi-electrode arrays (MEA) for high-resolution mapping are used to determine cardiac electrophysiology and arrhythmogenesis. F&#246;rster resonance energy transfer (FRET) imaging is performed for the real-time monitoring of cAMP levels in different cardiac regions. Neuromorphology is studied by means of antibody-based staining of whole hearts using neuronal markers to guide the identification and modulation of specific targets of the intracardiac autonomic nervous system in the performed studies. The ex vivo Langendorff setup allows for a high number of reproducible experiments in a short time. Nevertheless, the partly open nature of the setup (e.g., during MEA measurements) makes constant temperature control difficult and should be kept to a minimum. This described method makes it possible to analyze and modulate the intracardiac autonomic nervous system in decentralized hearts.

Impact of Intracardiac Neurons on Cardiac Electrophysiology and Arrhythmogenesis in an Ex Vivo Langendorff System

Here, we present a protocol for the modulation of the intracardiac autonomic nervous system and the assessment of its influence on basic electrophysiology, arrhythmogenesis, and cAMP dynamics using an ex vivo Langendorff setup.

心内神经元对体外 Langendorff 系统心脏电生理和心脏交感神经的影响

Since its invention in the late 19th century, the Langendorff ex vivo heart perfusion system continues to be a relevant tool for studying a broad spectrum ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

流式细胞仪测定缺氧/复氧后心肌细胞线粒体膜电位的实验研究

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

诱导大鼠心肌梗死的半微创方法及独立工作心脏系统对心脏功能的评估

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

使用GCaMP3报告器测量心脏特异性钙通量，对心脏重编程进行体外评估

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...

评估富含 EV 的血浆的血液高凝

Determination of the Procoagulant Activity of Extracellular Vesicle (EV) Using EV-Activated Clotting Time (EV-ACT)

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. However, there is an urgent need for a bedside test to assess the procoagulant activity of EV in clinical settings. This study proposes the use of thrombin activation time of EV-rich plasma as a measure of EV's procoagulant activity. Standardized procedures were employed to obtain sodium-citrated whole blood, followed by differential centrifugation to obtain EV-rich plasma. The EV-rich plasma and calcium chloride were added to the test cup, and the changes in viscoelasticity were monitored in real-time using an analyzer. The natural coagulation time of EV-rich plasma, referred to as EV-ACT, was determined. The results revealed a significant increase in EV-ACT when EV was removed from plasma obtained from healthy volunteers, while it significantly decreased when EV was enriched. Furthermore, EV-ACT was considerably shortened in human samples from preeclampsia, hip fracture, and lung cancer, indicating elevated levels of plasma EV and promotion of blood hypercoagulation. With its simple and rapid procedure, EV-ACT shows promise as a bedside test for evaluating coagulation function in patients with high plasma EV levels.

作者聚焦：破译创伤性脑损伤患者的凝血障碍 

This protocol investigates the use of extracellular vesicle (EV)-rich plasma as an indicator of the coagulative ability of EV. EV-rich plasma is obtained through a process of differential centrifugation and subsequent recalcification.

使用 EV 激活凝血时间 （EV-ACT） 测定细胞外囊泡 （EV） 的促凝血活性

The role of extracellular vesicles (EV) in various diseases is gaining increased attention, particularly due to their potent procoagulant activity. ...