我们感谢曼侬Laprise培训在超声心动图和安伯兰采取在图1和图2所示的照片。这项工作是由卫生研究院授予澳门币44363加拿大学院和加拿大主席950-216684支持。

在这个协议中显示的所有程序已批准红外对抗动物保护委员会。 1。超声系统和终端准备<ol><li>启动超声成像系统，并根据制造商的指示（ 图1）连接的扫描头以及生理学控制器单元。选择心脏测量程序和相对应的30兆赫传感器的扫描头。 </li><li>填用离子交换水的扫描头的物镜转换器。避免产生气泡，因为他们与成像分辨率干涉。将扫描头与手柄侧面上的保持器附近的成像平台（ 图1A）。 </li><li>消毒成像平台和工作区。 </li><li>完全填充瓶超声波凝胶，避免泡沫的形成，并把它倒挂在其prewarming容器设置为37°C（ 图1B）。 </li><li>验证氧基的含量根和异氟醚和管道系统进行麻醉。一个实验〜8胚胎会使用约15-20 L氧气。 </li><li>将眼科唇膏，脱毛霜及附近的成像平台（ 图1B）电极凝胶的瓶子。 </li><li>确认红外线烤灯功能。在训练过程中，设置热级中，灯的位置和使灯泡的​​小鼠以维持恒定的体温和心脏速率之间的距离。 注意：适当准备的系统和材料是至关重要的。冗长的程序，可能会影响胎儿的生理和血流动力学特征，只要麻醉通过降低心脏心率，血压和血液氧合水平踩下心脏功能。的处理时间〜8胎（平均产仔数的C57BL / 6）应为大约1小时。其他小鼠品系可以提供一些胎儿较高，可能与较大的女性的大小。训练是必不可少的，以优化processi纳克时间。 </li></ol> 2。鼠标准备<ol><li>麻醉的妊娠女性中，用2％异氟烷在氧气中连续供给（200毫升/分钟）的腔室，直到它变得无响应。 </li><li>将鼠标的成像平台上仰卧位，调整管连续吸入氧气1.5％异氟醚（200毫升/分钟），并固定吸入管带（ 图2A）。足够的麻醉应在手术过程中通过鼠标的轻松的姿势，并没有对尾部和脚趾捏的任何响应的证实。 </li><li>仅在左上角和右下角电极片（右前腿和左后腿，也称为Ⅱ导联位置）心电图电极放置凝胶。 II导地位提供了更好的定义正面直立P波和QRS波群，以确定心脏速率。使用胶带（ 图2A）固定四只爪子到每个焊盘。为了防止眼睛干涩，适用1滴眼科唇膏为每只眼睛。 </li><li>从胸部剃腹部膀胱理发推子。然后申请脱毛霜2分钟，用纱布和/或棉签擦去小心删除任何剩余的头发。注意不要切断乳头。 </li><li>插入预润滑与电极凝胶进入女性的直肠监测体温的温度计（应保持在37±0.5℃下，通过调整在红外加热灯下放在上面的平台）。心脏率应该是450±50次/分（BPM）。温度和心脏速率显示在生理学控制器单元（ 图1B）上。 注：龙麻醉，脱发和超声波凝胶（尽管预热的）可以导致体温降低，这可能会影响女性的心脏速率。如果体温低于36.5℃时，停止该程序，并修改所述加热灯的位置来调节温度和心脏速率。等待一两分钟BEF矿石再次出发。 </li></ol> 3。胚胎鉴定<ol><li>轻轻按在腹部赤裸下来，找到胚胎。慢慢地，轻轻地传播出来有大部分胚胎在腹部表面下的单层。在每个子宫角，囊直线地彼此连接。尝试蔓​​延时要尊重这个秩序。 </li><li>马克每个胚胎在女性的裸体腹部与他们的前/后和背/腹方向上的永久性标记。知道方向将促进与探头定位的心脏。数在右边的胚胎和离开角从子宫颈（1，2，3 ...和1&#39;，2&#39;，3&#39;...分别; 图2B）开始。 附注：（i）避免与传播过度用力的胚胎。画出其上的一张纸的位置来跟踪它们（ 图2C）。 （二）C57BL / 6女性有每胎〜8胎。然而，胚胎0-2在每个垃圾都升ocated别人之下，使他们的成像是不可能的。从分析中排除这些胚胎，如果需要评估更多的窝。 </li></ol> 4。心脏速率的测定<ol><li>将少量的赤裸的腹部超声预热凝胶均匀涂抹均匀。避免形成气泡。上的特定区域进行图像添加较大量的凝胶（〜5ml）中。 </li><li>将探头与厚的凝胶层（10mm）的接触，并逐渐移动探头向皮肤，同时寻找跳动的心脏。一次跳动的心脏被可视屏幕上，调整探头的角度，有两个心室中的最大尺寸在成像平面（ 图2D）。 </li><li>开始采集图像。用前臂休息站上，放置在超声凝胶换能器，以获得实时图像的观察屏（ 图2D）上。保持传感器顶部的脊，然后点击扫描头倾向¼Ñ​​标记（在图像的左上角）允许指针的动作及可视区域在屏幕上的协调。 </li><li>从子宫颈开始，移动到标记（在右侧或左侧子宫角1或1&#39;，分别）以可视化的心脏跳动在屏幕上最接近的胚。由于焦点深度是固定的，轻轻移动探头向上/向下和横向不丢失与凝胶接触，以获得所需的图像平面。 </li><li>由代表聚焦区域获得更好的数据采集黄色虚线表示的区域内的屏幕中心跳动的心脏的位置。 </li><li>采集现场录音。获得侦察员形象和恢复录制（ 图3A）。一旦获得最低10秒稳定的记录的，停止录音并保存。 </li><li>继续下一个胚胎。 </li><li>一旦所有的胚胎进行了分析在键盘上，按“浏览”来查看记录列表。发挥每个记录的M型崔西纳克和执行相邻峰（时间/流循环）之间的间隔的多个测量值（至少是5,00跟踪），以获取平均心脏速率（ 图3B）。 注意：某些协议建议使用固定支架的扫描头，以避免晃动。然而，它抑制分析的角度，使得横向观察胚胎的困难。它也减慢了分析，如支架，必须调整每个胚。拿着扫描头同时解决前臂站上有效地减少震动。培训与5-10怀孕的女性，建议优化的结果。 </li></ol> 5。基因分型<ol><li>使用清洁的外科剪刀和镊子，切割纵向皮肤和腹部的肌肉层。找到囊中的每个子宫角和相匹配的数字。剖开卵黄囊揭露胚胎在上面使用的顺序。如果不是以线性排列，一块胚胎位置的草图纸将是至关重要的，一旦在皮肤上的标记是不可见。 </li><li>用于基因分型只切〜4毫米尾基因组DNA是从胚胎尾部非常有效地提取。 注：（ⅰ）所述的外科手术过程可能需要在一个单独的位置，从成像室进行。然而，它不移动鼠标并具有以保持编号的胚胎的轨道成像平台上的外科手术是非常重要的。咨询动物设施经理优化外科手术。 （二）怀孕的女性很快因失血死亡的胎儿影像被删除后，进行基因分型。后尾切，胎儿被放置3分钟，冰浴麻醉体温过低，然后断头用剪刀的建议，我们的动物保护委员会。 </li></ol>

<ol>
	<li>Hoffman, J. I., Kaplan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+incidence+of+congenital+heart+disease.">The incidence of congenital heart disease.</a> J. Am. Coll. Cardiol. 39, 1890-1900 (2002).</li><li>Miller, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+brain+development+in+newborns+with+congenital+heart+disease.">Abnormal brain development in newborns with congenital heart disease.</a> N. Engl. J. Med. 357, 1928-1938 (2007).</li><li>Bruneau, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+genetics+of+congenital+heart+disease.">The developmental genetics of congenital heart disease.</a> Nature. 451, 943-948 (2008).</li><li>Wessels, A., Sedmera, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+anatomy+of+the+heart:+a+tale+of+mice+and.">Developmental anatomy of the heart: a tale of mice and.</a> 15, 165-176 (2003).</li><li>Yu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ENU+induced+mutations+causing+congenital+cardiovascular+anomalies.">ENU induced mutations causing congenital cardiovascular anomalies.</a> Development. 131, 6211-6223 (2004).</li><li>Seidah, N. G., Prat, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+and+therapeutic+targeting+of+the+proprotein+convertases.">The biology and therapeutic targeting of the proprotein convertases.</a> Nat. Rev. Drug Discov. 11, 367-383 (2012).</li><li>Roebroek, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Failure+of+ventral+closure+and+axial+rotation+in+embryos+lacking+the+proprotein+convertase+Furin.">Failure of ventral closure and axial rotation in embryos lacking the proprotein convertase Furin.</a> Development. 125, 4863-4876 (1998).</li><li>Roebroek, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+redundancy+of+the+proprotein+convertase+furin+in+mouse+liver.">Limited redundancy of the proprotein convertase furin in mouse liver.</a> J. Biol. Chem. 279, 53442-53450 (2004).</li><li>Kisanuki, Y. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tie2-Cre+transgenic+mice:+a+new+model+for+endothelial+cell-lineage+analysis+in+vivo.">Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo.</a> Dev. Biol. 230, 230-242 (2001).</li><li>Kim, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+endothelial+furin+leads+to+cardiac+malformation+and+early+postnatal+death.">Loss of endothelial furin leads to cardiac malformation and early postnatal death.</a> Mol. Cell Biol. 32, 3382-3391 (2012).</li></ol>

没有利益冲突声明。

M型超声心动图是在小鼠胚胎的子宫内心脏率来衡量一种有效而简单的方法。市售的转换器提供足够的分辨率以可视化的小跳动的心。因此，它们允许一个高度精确的心脏速率测量时，相对于其他方法，如脉冲的测量，并且可以替换高分辨率视频显微镜。然而，目前的工具不允许所有胚胎的同时分析，这意味着一个繁琐的过程可视化每个胚胎。此外，鉴于相当窄的领域和不存在字段?...

冠心病是死亡在出生1最常见的非感染性原因。通常需要多次手术，以纠正其科目的生活质量可能会保持1受损的结构性缺陷。冠心病的孩子经常发生，即使他们没有经历手术神经系统紊乱，说明在发展2,3 宫内的后果非常重要。遗传和环境因素，如接触到病毒或化学品（酒精）怀孕期间，引起冠心病。研究遗传贡献者仍然处于早期阶段，但发展迅速。为了找出这些贡献者和了解他们在心脏发育的作用，表型分型突变小鼠用一个简单而强大的工具将是非常有益的。 鼠标确实是选择冠心病研究的动物模型，和大多数人的情况下，可以在小鼠4,5重现。因此，胎鼠心脏表型已成为INCreasingly重要的是要研究人类冠心病的病因，需要足够的工具。虽然固定的标本组织学研究是非常宝贵的，活的动物的实时成像关键的是要了解心脏的生理机能。视频显微镜提供实时成像。但是，它需要剖腹暴露的胚胎，从而危及到他们的生理和血流动力学状态。最近，超声心动图已成为在诊所以及在​​小鼠心脏评估的标准成像技术。 小鼠胎儿超声心动图是使用标准的临床超声诊断系统，以及超高频超声系统进行。后者提供30 MHz或产生二维图像，并允许早期胚胎阶段的评价较高频率换能器。这些传感器具有相对较差的穿透深度（〜13毫米）的，这一点，然而，足以获得足够的成像平面，并确定基本心脏每年rameters，如心脏速率，左，右心室内径在舒张期和收缩期和隔垫和壁厚，不进行剖腹手术。 在我们的研究中，我们使用了一个超高频超声系统，以评估在胚胎一天E18.5小鼠胚胎的心脏速率。我们选择了30 MHz的换能器，提供20毫米×20毫米，这是理想的给定的胎儿的大小，具有12.7毫米的焦距的视场。然而，较高的频率换能器可以被选择为分析早期发育阶段。所选择的M-模式允许组织在运动中由于1000帧/秒的高时间分辨率的可视化。完整的程序很简单，应该尽快进行，以避免胎儿的生理和血流动力学状态中的任何扰动。约8个胚胎的分析需要约1小时。

<table border="0" cellpadding="0" cellspacing="0" width="591">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="44">
			<td height="44" style="height:44px;width:164px;">Isoflurane</td>
			<td style="width:160px;">Pharmaceutical Partners of Canada Inc.</td>
			<td style="width:104px;">CP0406v2</td>
			<td style="width:163px;">1-chloro-2,2,2-trifluoroethyl difluoromethyl ether</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Ultrasound gel</td>
			<td>Parker Laboratories Inc.</td>
			<td>Aquasonic Clear</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Electrode gel</td>
			<td>Parker Laboratories Inc.</td>
			<td>Spectra 360</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Ophthalmic gel</td>
			<td>Novartis</td>
			<td>Tear-Gel</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Depilatory cream</td>
			<td>Church &#38; Dwight Co., Inc.</td>
			<td>Nair</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Hair clipper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Gauze/cotton swap</td>
			<td>Q-tips</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Permanent marker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:164px;">High-Resolution In vivo Ultrasound Imaging System</td>
			<td style="width:160px;">Visual Sonics</td>
			<td style="width:104px;">Vevo770</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">30 MHz Transducer</td>
			<td>Visual Sonics</td>
			<td>RMV707B</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:164px;">Imaging platform and physiology controller unit</td>
			<td>Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Anesthetic System</td>
			<td>Cyprane North America Inc.</td>
			<td>312462</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Infrared heating lamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in utero measurement of heart rate in mouse by noninvasive m-mode echocardiography

先天性心脏疾病（CHD）死亡是出生时最常见的非感染性原因。冠心病的发病率介乎4至50/1，000胞胎（疾病和损伤区域的估计，世界卫生组织，2004年）。手术，往往危及生命的质量需要纠正心脏缺陷，提醒发现冠心病的原因的重要性。突变小鼠模型和实时成像技术已成为必不可少的工具来研究这种疾病的病因。虽然先进的方法让心中异常胚胎的实时成像，后者的生理和血流动力学状态往往是由于手术和/或冗长的程序损害。非侵入性超声成像，但是，可以使用，不用手术暴露的胚胎，从而保持它们的生理机能。在此，我们用简单的M型超声在子宫内评估在E18.5胚胎的心脏速率。心脏异常率的检测的确是一个不错的籼对心脏功能障碍的器，从而构成，可能导致心脏衰竭发育缺陷的识别的第一步。

上述方法被用于评估在子宫内对小鼠胚胎的心脏速率存在或不存在的丝氨酸蛋白酶弗林蛋白酶的内皮细胞在E18.5的影响。弗林属于前蛋白转化酶（个人电脑）的切割蛋白前体的碱性氨基酸残基后的家庭。弗林蛋白酶和它的底物是分泌蛋白和裂解可以发生在高尔基体，内体或在细胞表面。弗林蛋白酶的关键底物包括TGFB，以及TGFB样因子，如骨形态发生蛋白（BMPs）中发挥心脏发育中起重要作用?...

Watch this Scientific Journal Video about In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography at JoVE.com

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

超高频超声是一个功能强大的实时成像工具，研究在小动物心脏异常。其无创允许维持胚胎的生理状态。在这里，我们将展示采用M型超声测量胚胎的心脏率E18.5 在子宫内 。

心率在小鼠中通过无创M型超声心动图测量子宫内

Congenital heart disease (CHD) is the most frequent noninfectious cause of death at birth. The incidence of CHD ranges from 4 to 50/1,000 births (Disease ...

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Medicine

In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

11.3K 次观看.  Institut de Recherches Cliniques de Montréal. 超高频超声是一个功能强大的实时成像工具，研究在小动物心脏异常。其无创允许维持胚胎的生理状态。在这里，我们将展示采用M型超声测量胚胎的心脏率E18.5 在子宫内 。 

视频: In utero Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

经胸超声心动图在小鼠

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

科研

医学

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

右心室结构和功能的肺动脉缩窄经胸超声小鼠模型的评估

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

心脏异常的实验性自身免疫性心肌炎的无创性评估磁共振成像显微镜在鼠标

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

Noninvasive Determination of Vortex Formation Time Using Transesophageal Echocardiography During Cardiac Surgery

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular (LV) filling compared with a continuous linear jet. Vortex ring development is most often quantified with vortex formation time (VFT), a dimensionless parameter based on fluid ejection from a rigid tube. Our group is interested in factors that affect LV filling efficiency during cardiac surgery. In this report, we describe how to use standard two-dimensional (2D) and Doppler transesophageal echocardiography (TEE) to noninvasively derive the variables needed to calculate VFT. We calculate atrial filling fraction (&#946;) from velocity-time integrals of trans-mitral early LV filling and atrial systole blood flow velocity waveforms measured in the mid-esophageal four-chamber TEE view. Stroke volume (SV) is calculated as the product of the diameter of the LV outflow track measured in the mid-esophageal long axis TEE view and the velocity-time integral of blood flow through the outflow track determined in the deep transgastric view using pulse-wave Doppler. Finally, mitral valve diameter (D) is determined as the average of major and minor axis lengths measured in orthogonal mid-esophageal bicommissural and long axis imaging planes, respectively. VFT is then calculated as 4 &#215; (1-&#946;) &#215; SV/(&#960;D3). We have used this technique to analyze VFT in several groups of patients with differing cardiac abnormalities. We discuss our application of this technique and its potential limitations and also review our results to date. Noninvasive measurement of VFT using TEE is straightforward in anesthetized patients undergoing cardiac surgery. The technique may allow cardiac anesthesiologists and surgeons to assess the impact of pathological conditions and surgical interventions on LV filling efficiency in real time.

We describe a protocol to measure vortex formation time, an index of left ventricular filling efficiency, using standard transesophageal echocardiography techniques in patients undergoing cardiac surgery. We apply this technique to analyze vortex formation time in several groups of patients with differing cardiac pathologies.

经食管超声心动图在心脏手术中无创地测定涡旋形成时间

Trans-mitral blood flow produces a three-dimensional rotational body of fluid, known as a vortex ring, that enhances the efficiency of left ventricular ...

Transdermal Measurement of Glomerular Filtration Rate in Mice

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of acute kidney injury and chronic kidney disease. The measurement system consists of a miniaturized fluorescence detector that is directly attached to the skin on the back of conscious, freely moving animals, and measures the excretion kinetics of the exogenous GFR tracer, fluorescein-isothiocyanate (FITC) conjugated sinistrin (an inulin analog). This system has been described in detail in rats. However, because of their smaller size, measurement of transcutaneous GFR in mice presents additional technical challenges. In this paper we therefore provide the first detailed practical guide to the use of transdermal GFR monitors in mice based on the combined experience of three different investigators who have been performing this assay in mice over a number of years.

Here we describe a protocol to measure glomerular filtration rate (GFR) in conscious, freely moving mice using a transdermal GFR monitor.

小鼠肾小球滤过率的透皮测定

Transdermal analysis of glomerular filtration rate (GFR) is an established technique that is used to assess renal function in mouse and rat models of ...

Noninvasive Electrocardiography in the Perinatal Mouse

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both human and animal models of disease. Individual heart rate, rhythm, and regularity, combined with quantitative parameters collected from ECG, serve to assess the integrity of the cardiac conduction system as well as the integrated physiology of the cardiac cycle. This article provides a comprehensive description of the methods and techniques used to perform a noninvasive ECG on perinatal and neonatal mouse pups as early as the first postnatal day, without requiring the use of anesthetics. This protocol was designed to directly address a need for a standardized and repeatable method for obtaining ECG in newborn mice. From a translational perspective, this protocol proves to be entirely effective for characterization of congenital cardiopulmonary defects generated using transgenic mouse lines, and particularly for analysis of defects causing lethality at or during the first postnatal days. This protocol also aims to directly address a gap in the scientific literature to characterize and provide normative data associated with maturation of the early postnatal cardiac conduction system. This method is not limited to a specific postnatal timepoint, but rather allows for ECG data collection in neonatal mouse pups from birth to postnatal day 10 (P10), a window that is of critical importance for modeling human diseases in vivo, with particular emphasis on congenital heart disease (CHD).

Here, we present a noninvasive electrocardiography (ECG) protocol, optimized for early postnatal mice, that does not require the use of anesthetics.

围产期小鼠中的非侵入性心电图

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both ...

Morphological and Functional Assessment of the Right Ventricle Using 3D Echocardiography

Traditionally, it was believed that the right side of the heart has a minor role in circulation; however, more and more data suggest that right ventricular (RV) function has strong diagnostic and prognostic power in various cardiovascular disorders. Due to its complex morphology and function, assessment of the RV by conventional two-dimensional echocardiography is limited: the everyday clinical practice usually relies on simple linear dimensions and functional measures. Three-dimensional (3D) echocardiography overcame these limitations by providing volumetric quantification of the RV free of geometrical assumptions. Here, we offer a step-by-step guide to obtain and analyze 3D echocardiographic data of the RV using the leading commercially available software. We will quantify 3D RV volumes and ejection fraction. Several technical aspects may help to improve the quality of RV acquisition and analysis as well, which we present in a practical manner. We review the current opportunities and the limiting factors of this method and also highlight the potential applications of 3D RV assessment in current clinical practice.

Here, we provide a step-by-step acquisition and analysis protocol for the 3D volumetric assessment of the right ventricle, mainly focusing on the practical aspects that maximize the feasibility of this technique.

使用3D超声心动图对右心室进行形态学和功能评估

Traditionally, it was believed that the right side of the heart has a minor role in circulation; however, more and more data suggest that right ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

鼠标心脏中毛细血管、动脉和腹膜的动态测量和成像

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

Measurement of Heart Contractility in Isolated Adult Human Primary Cardiomyocytes

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This paper describes a protocol for assessing changes in contractility in adult human primary ventricular cardiomyocytes utilizing the MyoBLAZER, a non-invasive optical method that preserves the normal physiology and pharmacology of the cells. This optical recording method continuously measures contractility transients from multiple cells in parallel, providing both medium-throughput and valuable information for each individual cell in the field of view, enabling the real-time tracking of drug effects. The cardiomyocyte contractions are induced by paced electrical field stimulation, and the acquired bright field images are fed to an image-processing software that measures the sarcomere shortening across multiple cardiomyocytes. This method rapidly generates different endpoints related to the kinetics of contraction and relaxation phases, and the resulting data can then be interpreted in relation to different concentrations of a test article. This method is also employed in the late stages of preclinical development to perform follow-up mechanistic studies to support ongoing clinical studies. Thus, the adult human primary cardiomyocyte-based model combined with the optical system for continuous contractility monitoring has the potential to contribute to a new era of in vitro cardiac data translatability in preclinical medical therapy development.

This protocol describes how to measure contractility in adult human primary cardiomyocytes from donor hearts with the MyoBLAZER system, a reliable platform for assessing drug-induced changes in contractility during preclinical development.

分离的成人原代心肌细胞心脏收缩力的测量

The evaluation of changes in heart contractility is essential during preclinical development for new cardiac- and non-cardiac-targeted compounds. This ...