项目编号NVKP_16-1-2016-0017（"国家心脏计划"）已在匈牙利国家研究，发展和创新基金的支持下实施，该基金由NVKP_16资助计划资助。该研究由匈牙利创新与技术部的主题卓越计划（2020-4.1.1.-TKP2020）在塞梅尔威斯大学的治疗开发和生物成像主题计划的框架内资助。

该方案遵循该机构人类研究伦理委员会的指导方针，临床病例的患者对该研究给予书面知情同意。
1. 技术要求
<ol><li>对于 3D 采集和分析，请使用适当的软件和硬件。使用超声心动图设备的心电电缆;此外，对于下面描述的完整3D采集协议，这是强制性的。</li>
	<li>对于 3D 采集，请使用 3D 超声心动图探头和兼容 3D 的超声机。对于 3D RV 体积分析，请使用专用软件。</li>
</ol>2. 收购
<ol><li>在绝大多数情况下，使用顶点视图对 RV 进行 3D 采集。与以左心室为中心的观点相反，建议采用不同的患者体位。如果通过在正确的顶点视图上切换到一个肋间空间可以实现更好的图像质量，则这种透视缩短的视图可以实现更好的3D图像质量。透视收缩可以在 3D 分析过程中进行校正。<ol><li>与建议使用左侧卧位（患者躺在左侧，左臂伸展在头部上方）的标准根尖超声心动图采集相比，患者稍微向后倾斜，以使换能器的位置更侧。</li>
		<li>选择仅包含 RV 的图像深度。 不必要的大深度可能会降低采集帧速率，同时对 RV 体积分析缺乏有益的影响。</li>
	</ol></li>
	<li>从 2D 超声心动图图像中确认正确的右心室聚焦视图。如果即使从这个角度看，RV的自由壁也很难看到，那么预期的3D图像质量将无法成为进一步分析的最佳选择。</li>
	<li>使用 4D 按钮切换到实时 3D 成像，可以对 RV 视图进行进一步校正。</li>
	<li>虽然 3D 实时 模式在美学上可能非常令人愉悦，但对 3D 视图使用 12 Slice 模式，该模式显示感兴趣区域的三平面图像以及可以自由修改的 9 个横截面平面。通过旋转和正确定位切割平面，确认整个 RV 自由壁（包括流出道和顶端段）的可见性。</li>
	<li>通过使用扇区的左倾（触摸屏上的第二页）进一步调整图像，以改善 RV 可视化。</li>
	<li>使用两种 3D 采集模式进行 RV 体积分析: 多拍 和 单拍 模式。在每位患者中使用这两种方法，但在某些情况下（例如，某些心律失常，患者的严重呼吸困难），只有后一种方法可能是可行的。</li>
	<li>使用 单拍 模式，在图像质量和帧速率之间实现权衡。选择最佳图像深度、宽度和帧速率（触摸屏的下面板），无需任何进一步操作即可获得 RV 的 3D 循环。这种方法在大多数患者中是可行的;但是，与 多拍 方法相比，它产生的图像质量和帧速率通常较低。<ol><li>在平均（60-70/分钟）心率的情况下，保持 16 帧/秒的帧速率下限，以便进行充分的 RV 分析;但是，如果存在心动过速，则建议使用更高的帧速率。</li>
	</ol></li>
	<li>使用 多拍 模式，从可在触摸屏上选择的给定数量的心跳周期重建获取的 3D 循环（可以使用 2、3、4 和 6 拍模式）。与单拍采集相比，通常期望更好的图像质量和帧速率;然而，由于强制性屏气动作，它需要相对恒定的心动周期长度以及患者的依从性。该操作对于避免所谓的拼接伪影至关重要:当将获取的3D体积缝合在一起时，不相等的心动周期长度和/或由于呼吸引起的运动可能会导致这种现象。<ol><li>正确定位探头并设置机器后（类似于"单拍"模式），要求患者深呼吸并屏住呼吸。在这种情况下，扩张的肺通常覆盖整个图像。</li>
		<li>要求患者在指导下缓慢呼气。在肺部放气的同时，RV 再次变得可见。</li>
		<li>当整个RV（游离壁和隔膜）再次出现时，要求患者在这种状态下屏住呼吸。</li>
		<li>通过在屏幕上单击 多拍 ，开始采集，3D循环在给定的心动周期内建立。</li>
		<li>当采集准备就绪（整个房车可视化）时，要求患者再次自由呼吸。</li>
		<li>检查获得的循环以确保没有拼接或丢失伪影。</li>
	</ol></li>
</ol>3. 4D 房车分析
<ol><li>使用专用软件对RV进行3D体积分析。从患者库中选择以 RV 为中心的 3D 循环后，从"卷"文件夹中的"测量"窗口中打开软件。</li>
	<li>打开软件后，将 RV 定向到四个预定义的切割平面上。<ol><li>将两个标记（电视中心）放在左上和左下长轴平面的三尖瓣中心。使用旋转工具将图像的长轴调整为 RV 的实际长轴。右上角的参考图像显示了正确方向的显示方式。</li>
		<li>在右上和右下面板上，通过旋转将短轴图像对齐到正确的位置。与上一步类似，参考图像也有助于此过程。</li>
	</ol></li>
	<li>完成后，单击 设置地标 以执行下一步分析。在两个图像中设置地标。<ol><li>在左侧，在自由壁（电视自由壁）和隔膜（电视隔）处标记三尖瓣环，并在先前定向的顶端四室视图上标记 RV 顶点。</li>
		<li>在右侧，设置右心室后部（左心室/右心室后部）和前插入点（左心室/右心室前部）和右心室游离壁（右心室游离壁）。与上一个窗口类似，右上角的参考图像有助于正确设置。设置所有地标后，软件会自动跳转到下一个窗口（查看）。</li>
	</ol></li>
	<li>在此窗口（查看）中，如有必要，查看并手动纠正整个心动周期中的自动心内膜边界检测。默认情况下，可以看到 9 个面板:左侧是 3 个移动环（1 个长轴和 2 个短轴），中间是相同图像的舒张末期帧，右侧是收缩末期帧。<ol><li>在错误跟踪的情况下，通过单击它们自由纠正心内膜边界（绿线），跟踪边界。使用短轴上的旋转工具，查看沿 RV 整个圆周跟踪的图像。 通过选择右侧面板上的笔大小来调整校正幅度。如果认为跟踪正确，请单击同一面板中的结果。</li>
	</ol></li>
	<li>在最后一部分中，查看右上角的最终 3D 体积数据和其他计算参数（"工作表 "面板）。除了 RV 体积和射出分数外，该软件还显示 2D 参数，例如线性（中轴、基轴和长轴）直径，以及从预定义的顶端四腔室视图得出的 FAC 和 TAPSE 值。该软件还显示了RV的长轴和短轴（左侧），RV的3D实时模型（中上）和腔室的体积-时间曲线（右下）。<ol><li>如果需要进一步调整跟踪，可以通过单击右侧面板上的每个先前步骤进行更正。如果跟踪和 3D 参数被视为有效，请单击同一面板上的"批准并退出"来保存结果。</li>
	</ol></li>
</ol>

<ol>
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RV 的 3D 分析是日常心脏病学实践中的重要一步。随着人们对以前被忽视的心腔的形态和功能的兴趣日益浓厚，这些新颖的解决方案提供了有关心脏右侧的临床有意义的信息。虽然3D采集有几个方面与2D超声心动图成像明显不同，但通过特别注意关键点并使用全面的协议，3D RV分析可以从科学工具发展成为超声心动图检查的重要步骤。凭借最佳的图像质量和适当的专业知识，使用超声心动图进行RV体?...

超声心动图从 1950 年代的首次临床应用开始了漫长的道路1.第一个一维超声探头旨在提供腔室壁和腔的简单线性直径;然而，它们无疑代表了心血管成像的一个里程碑。二维（2D）超声成像的发展是另一个重要步骤，它提供了更精确的形态和功能量化，并且仍然被认为是日常临床实践中的标准方法。然而，基于2D超声心动图的评估仍然存在该技术的主要局限性:从几个断层扫描平面对给定腔室的成像不能充分表征三维（3D）结构的形态和功能。这个问题在右心室 （RV） 的情况下更为明显:与相对简单的子弹形左心室 （LV） 相比，右心室具有复杂的几何形状2 ，无法使用线性直径或区域3 充分量化。尽管有这些广为人知的事实，但RV的形态和功能通常在临床实践中通过这些简单的参数来测量。
几十年来，与左翼对应物相比，RV 被认为在流通中的作用要小得多。几篇具有里程碑意义的论文推翻了这一观点，表明RV几何形状和功能在多种疾病中的强烈预后作用4，5，6，7。大量研究表明，即使使用相对简单的常规参数，RV测量的增量价值也突出了对具有潜在有意义的临床价值的腔室进行更精确定量的重要性和需求。
3D超声心动图克服了心腔2D评估的几个局限性。虽然在LV的情况下，没有几何假设的体积和功能参数的测量也可能引起高度关注，但在RV8的评估中可能变得特别重要。3D 衍生的右心室体积和射血分数 （EF） 在各种心血管疾病中具有显着的预后价值9，10。
如今，一些供应商提供用于 3D RV 评估的半自动解决方案，并根据金标准心脏磁共振 （MR） 测量结果验证结果11，12。3D评估的技术要求是当今最先进的心血管成像部门的重要组成部分，预计它很快就会成为每个超声心动图实验室通用设备的一部分。凭借在3D采集和后处理方面的适当专业知识，3D RV分析可以很容易地实施到标准检查方案中。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3V-D/4V-D/4Vc-D</td>
			<td>General Electric</td>
			<td>n.a.</td>
			<td>ultrasound probe</td>
		</tr>
		<tr>
			<td>4D Auto RVQ</td>
			<td>General Electric</td>
			<td>n.a.</td>
			<td>software for analysis</td>
		</tr>
		<tr>
			<td>E9/E95</td>
			<td>General Electric</td>
			<td>n.a.</td>
			<td>ultrasound machine</td>
		</tr>
		<tr>
			<td>EchoPac v203</td>
			<td>General Electric</td>
			<td>n.a.</td>
			<td>software for analysis</td>
		</tr>
	</tbody>
</table>

morphological and functional assessment of the right ventricle using 3d echocardiography

传统上，人们认为心脏的右侧在循环中起着次要的作用;然而，越来越多的数据表明，右心室（RV）功能对各种心血管疾病具有很强的诊断和预后能力。由于其复杂的形态和功能，通过传统的二维超声心动图评估RV是有限的:日常临床实践通常依赖于简单的线性尺寸和功能测量。三维 （3D） 超声心动图克服了这些限制，提供了不受几何假设影响的 RV 体积定量。在这里，我们提供了一个分步指南，使用领先的商用软件获取和分析RV的3D超声心动图数据。我们将量化 3D RV 体积和射出分数。几个技术方面也可能有助于提高RV采集和分析的质量，我们以实用的方式介绍。我们回顾了该方法的当前机会和限制因素，并强调了3D RV评估在当前临床实践中的潜在应用。

RV 的 3D 分析在各种心血管疾病中是可行的。病例1是一名健康的志愿者，心室容量和功能正常（图1）。病例 2 是一名二尖瓣修复术后患者，他是传统 2D 评估结果相互矛盾的典型示例:虽然 TAPSE 明显降低，但患者未表现出任何右心室功能障碍的迹象，并且 3D RV EF 正常证实右心室整体收缩功能维持（图 2）。两位患者都有出色的超?...

Watch this Scientific Journal Video about Morphological and Functional Assessment of the Right Ventricle Using 3D Echocardiography at JoVE.com

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

在这里，我们为右心室的3D体积评估提供了一步一步的采集和分析方案，主要侧重于最大化该技术可行性的实际方面。

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 ...

morphological-functional-assessment-right-ventricle-using-3d

Research

JoVE Journal

Medicine

2.7K 次观看.  Semmelweis University Heart and Vascular Center. 在这里，我们为右心室的3D体积评估提供了一步一步的采集和分析方案，主要侧重于最大化该技术可行性的实际方面。

视频: Morphological and Functional Assessment of the Right Ventricle Using 3D 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

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

右心小鼠的超声心动图评价

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

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, ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

心肌梗死和功能预后评估的猪

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

慢性血栓栓塞性肺动脉高压和右心室功能的仔猪评估

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

在横向主动脉缩窄的小鼠模型和心脏形态学评估功能改变通过超声心动图成像

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

Transthoracic Speckle Tracking Echocardiography for the Quantitative Assessment of Left Ventricular Myocardial Deformation

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the examiners&#39; expertise. Speckle tracking Echocardiography (STE) is a promising but technically challenging method that can be used to quantitatively assess regional and global systolic and diastolic myocardial performance. Myocardial strain and strain rate can be measured in all three dimensions&#160;&#8212; radial, circumferential, longitudinal &#8212; of myocardial deformation. Standard cross-sectional two-dimensional B-mode images are recorded and subsequently postprocessed by automated continuous frame-by-frame tracking and motion analysis of speckles within the myocardium. Images are recorded as digital loops and synchronized to a 3-lead EKG for timing purposes. Longitudinal deformation is assessed in the apical 4-, 3-, and 2-chamber views. Circumferential and radial deformation are measured in the parasternal short axis plane.
Optimal image quality and accurate tissue tracking are paramount for the correct determination of myocardial performance parameters. Utilizing transthoracic STE in a healthy volunteer, the present article is a detailed outline of the essential steps and potential pitfalls of quantitative echocardiographic myocardial deformation analysis.

Speckle tracking echocardiography is an emerging diagnostic imaging technique for the quantitative assessment of global and regional myocardial performance. Standard view echocardiographic motion images are recorded and deformation parameters are subsequently measured by automated continuous frame-by-frame tracking and motion analysis of speckles within the B-mode images of the myocardium.

经胸斑点跟踪超声心动图左室心肌变形的定量评价

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the ...

Assessment of the Anticoagulant and Anti-inflammatory Properties of Endothelial Cells Using 3D Cell Culture and Non-anticoagulated Whole Blood

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm2 of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm2/mL. This increased ratio is sufficient to ensure the &#34;natural&#34; anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.

We present an in vitro model which allows the study and analysis of coagulation in whole, non-anticoagulated blood. Anticoagulation in the system depends on the natural anticoagulation effect of healthy endothelial cells and endothelial cell activation will result in clotting.

3D 细胞培养和非抗凝全血对内皮细胞抗凝及抗炎作用的评价

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood ...

2D and 3D Echocardiography in the Axolotl (Ambystoma Mexicanum)

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model species such as zebrafish and salamanders that are capable of intrinsic heart regeneration hold promise for future regenerative therapies for human patients. To evaluate the outcome of cardioregenerative experiments it is imperative that heart function can be monitored. The axolotl salamander (A. mexicanum) represents a well-established model species in regenerative biology attaining sizes that allows for evaluation of cardiac function. The purpose of this protocol is to establish methods to reproducibly measure cardiac function in the axolotl using echocardiography. The application of different anesthetics (benzocaine, MS-222, and propofol) is demonstrated, and the acquisition of two-dimensional (2D) echocardiographic data in both anesthetized and unanesthetized axolotls is described. 2D echocardiography of the three-dimensional (3D) heart can suffer from imprecision and subjectivity of measurements, and to alleviate this phenomenon a solid method, namely intra/inter-operator/observer analysis, to measure and minimize this bias is demonstrated. Finally, a method to acquire 3D echocardiographic data of the beating axolotl heart at a very high spatiotemporal resolution and with pronounced blood-to-tissue contrast is described. Overall, this protocol should provide the necessary methods to evaluate cardiac function and model anatomy, and flow dynamics in the axolotl using ultrasound imaging with applications in both regenerative biology and general physiological experiments.

2D and 3D Echocardiography in the Axolotl Ambystoma Mexicanum

Here we present echocardiography protocols for two-dimensional and three-dimensional image acquisition of the beating heart of the axolotl salamander (Ambystoma mexicanum), a model species in heart regeneration. These methods allow for longitudinal evaluation of cardiac function at a high spatiotemporal resolution.

axolotl 中的二维和三维超声心动图

Cardiac malfunction as a result of ischemic heart disease is a major challenge, and regenerative therapies to the heart are in high demand. A few model ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. However, the evaluation of right ventricular pressure (RVP) and pulmonary artery pressure (PAP) remains technically challenging in mice. RVP and PAP are more difficult to measure than left ventricular pressure because of the anatomical differences between the left and right heart systems. In this paper, we describe a stable right heart hemodynamic measurement method and its validation using healthy and PAH mice. This method is based on open-chest surgery and mechanical ventilation support. It is a complicated procedure compared to closed chest procedures. While a well-trained surgeon is required for this surgery, the advantage of this procedure is that it can generate both RVP and PAP parameters at the same time, so it is a preferable procedure for the evaluation of PAH models.

Here, we present a protocol to perform an invasive hemodynamic assessment of the right ventricle and pulmonary artery in mice using an open-chest surgery approach.

小鼠右心室系统及缺氧诱发肺动脉高血压的侵入性血液动力学评估

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...