Project no. NVKP_16-1&#8211;2016-0017 (&#8217;National Heart Program&#8217;) has been implemented with the support provided from the National Research, Development and Innovation Fund of Hungary, financed under the NVKP_16 funding scheme. The research was financed by the Thematic Excellence Programme (2020-4.1.1.-TKP2020) of the Ministry for Innovation and Technology in Hungary, within the framework of the Therapeutic Development and Bioimaging thematic programmes of the Semmelweis University.

The protocol follows the guidelines of the institution&#39;s human research ethics committee and the patients of the clinical cases gave their written informed consent to the study.
1. Technical requirements
<ol>
	<li>For 3D acquisition and analysis, use appropriate software and hardware. Use ECG cables of the echocardiography device; moreover, it is mandatory for the complete 3D acquisition protocol described below.</li>
	<li>For 3D acquisition, use a 3D echocardiographic probe and 3D-compatible ultrasound machine. For 3D RV volumetric analysis, use dedicated software.</li>
</ol>
2. Acquisition
<ol>
	<li>In the vast majority of cases, perform 3D acquisition of the RV using apical views. In opposed to the LV-focused views, a different patient positioning is recommended. If substantially better image quality can be achieved by switching to one intercostal space over the correct apical view, this foreshortened view may enable better 3D image quality. The foreshortening can be corrected during 3D analysis.
	<ol>
		<li>Compared to the standard apical echocardiographic acquisition, where the left lateral decubitus position (patient lying on the left side with the left arm stretched above the head) is recommended, have the patient lean back slightly more to enable a more lateral position of the transducer.</li>
		<li>Choose an image depth that only includes the RV. Unnecessarily large depth may lower the acquisition frame rate with the lack of beneficial effects regarding RV volumetric analysis.</li>
	</ol>
	</li>
	<li>Confirm the correct RV-focused view from 2D echocardiography images. If the free wall of the RV is poorly visualized even from this view, the expected 3D image quality will not be optimal for further analysis.</li>
	<li>Switch to the live 3D imaging using the 4D button, where further correction of the RV view can be performed.</li>
	<li>While the 3D live mode may be quite pleasing aesthetically, use the 12 Slice mode for the 3D view, which shows a triplane image of the region of interest as well as 9 cross-sectional planes that can be freely modified. By rotation and correct positioning of the cut planes, confirm the visibility of the entire RV free wall (including the outflow tract and apical segments).</li>
	<li>Further adjust the image by using the left tilting of the sector (second page on the touch screen) to improve RV visualization.</li>
	<li>Use two 3D acquisition modes for RV volumetric analysis: the multi beat and the single beat mode. Use both of these approaches in every patient however, in some cases (e.g., certain arrhythmias, severe dyspnea of the patient), only the latter one may be feasible.</li>
	<li>Using single beat mode, achieve a tradeoff between image quality and frame rate. Choose an optimal image depth, width, and frame rate (lower panel of the touch screen) and obtain 3D loops of the RV without any further action. This method is feasible in the majority of patients; however, it yields generally lower image quality and frame rate compared to the multi beat approach.
	<ol>
		<li>In case of an average (60-70/min) heart rate, keep a lower frame rate limit of 16 frames/s for adequate RV analysis; however, if tachycardia is present even higher frame rates are recommended.</li>
	</ol>
	</li>
	<li>Using the multi beat mode, reconstruct the acquired 3D loop from a given number of heart cycles that can be selected on the touch screen (2,3,4 and 6 beat modes can be used). In contrast with the single beat acquisition, generally better image quality and frame rate are expected; however, it requires relatively constant heart cycle lengths and also patient compliance due to the mandatory breath-hold maneuver. The maneuver is essential to avoid the so-called stitching artefacts: when the acquired 3D volume is stitched together, unequal cardiac cycle lengths and/or motion due to breathing may result in this phenomenon.
	<ol>
		<li>After correct positioning of the probe and setting of the machine (similarly to &#34;single beat&#34; mode), ask the patient to take a deep breath and hold it. In this case, the expanding lungs usually cover the entire image.</li>
		<li>Ask the patient to exhale slowly, strictly with guidance. In parallel with the deflation of the lungs, the RV becomes visible again.</li>
		<li>When the entire RV (free wall and septum) reappears, ask the patient to hold breath in this state.</li>
		<li>By clicking to multi beat on the screen, begin the acquisition, and the 3D loop builds up during the given amount of heart cycles.</li>
		<li>When the acquisition is ready (the entire RV is visualized), ask the patient to breathe freely again.</li>
		<li>Check the obtained loop to ensure if there are no stitching or drop-out artifacts.</li>
	</ol>
	</li>
</ol>
3. 4D RV analysis
<ol>
	<li>Using dedicated software, perform 3D volumetric analysis of the RV. After choosing the RV-focused 3D loop from the patient library, open the software from the Measurement window found in the Volume folder.</li>
	<li>After opening the software, orient the RV on four predefined cut planes.
	<ol>
		<li>Put two markers (TV Center) to the center of the tricuspid valve in the upper and lower left long-axis planes. Adjust the long-axis of the image to the actual long axis of the RV by using the rotation tool. Reference images on the upper right edges show how the correct orientation should appear.</li>
		<li>On the upper and lower right panels, align the short-axis images into the correct position by rotation. Similarly to the previous step, reference images help in this process as well.</li>
	</ol>
	</li>
	<li>After finishing, click Set landmarks to the next step of the analysis. Set landmarks in two images.
	<ol>
		<li>On the left side, mark the tricuspid annulus at the free wall (TV free wall) and septum (TV septum) and the RV apex on the previously oriented apical four-chamber view.</li>
		<li>On the right side, set the RV posterior (LV/RV posterior) and anterior insertion points (LV/RV anterior) and the RV free wall (RV free wall). Similarly to the previous window, reference images in the upper right corner help regarding the correct setup. After setting all landmarks, the software automatically jumps to the next window (Review).</li>
	</ol>
	</li>
	<li>In this window (Review), review and manually correct the automatic endocardial border detection throughout the entire cardiac cycle, if necessary. By default, 9 panels can be seen: on the left side, 3 moving loops (1 long-axis and 2 short-axis), on the middle the end-diastolic frames of the same images, and on the right side the end-systolic ones.
	<ol>
		<li>In the case of false tracking, freely correct the endocardial borders (green lines), the tracked border by clicking on them. Using the rotation tool on the short axis, review images tracking along the entire circumference of the RV. Adjust the magnitude of correction by choosing Pen size on the right side panel. If the tracking is considered to be correct, click on the Results in the same panel.</li>
	</ol>
	</li>
	<li>In the last section, review the final 3D volumetric data and other calculated parameters on the upper right side (Worksheet panel). Beyond RV volumes and ejection fraction, the software also displays 2D parameters, such as linear (mid, basal, and long-axis) diameters, as well as FAC and TAPSE values derived from the predefined apical four-chamber view. The software also shows a long- and short axis of the RV (left side), a 3D live model of the RV (upper middle), and a volume-time curve of the chamber (lower right).
	<ol>
		<li>In case of the need for further adjustments in the tracking, every previous steps are available for correction by clicking on them on the right panel. If the tracking and the 3D parameters are considered to be valid, save the results by clicking on &#34;Approve and exit&#34; on the same panel.</li>
	</ol>
	</li>
</ol>

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3D analysis of the RV represents an important step in everyday cardiology practice. In parallel with the growing interest of the morphology and function of the previously neglected cardiac chamber, these novel solutions provide clinically meaningful information about the right side of the heart. While 3D acquisition has several aspects that markedly differ from 2D echocardiographic imaging, by keeping special attention to the critical points and by using a thorough protocol, 3D RV analysis may progress from a scientific ...

Echocardiography came a long way from its first clinical applications in the 1950s1. The first one-dimensional ultrasound probes were designed to provide simple linear diameters of the chamber walls and lumens; however, they undoubtedly represent a milestone in cardiovascular imaging. The development of two-dimensional (2D) ultrasound imaging was another major step by providing much more precise quantification of morphology and function and is still considered to be the standard method in everyday clinical practice. Nevertheless, 2D echocardiography-based assessment still carries a major limitation of the technique: imaging of a given chamber from a few tomographic planes does not adequately characterize the morphology and function of a three-dimensional (3D) structure. This problem is even more pronounced in the case of the right ventricle (RV): compared to the relatively simple bullet-shaped left ventricle (LV), the RV has a complex geometry2 that cannot be adequately quantified using linear diameters or areas3. Despite these widely known facts, RV morphology and function are usually measured by such simple parameters in the clinical practice.
For many decades, the RV was considered to have a much less important role in circulation compared to its left counterpart. Several landmark papers defeated this standpoint showing the strong prognostic role of RV geometry and function in a wide variety of diseases4,5,6,7. Numerous studies demonstrated the incremental value of RV measurement even by using relatively simple conventional parameters, which highlights the importance and need for more precise quantification of the chamber with potentially meaningful clinical value.
3D echocardiography overcomes several limitations of the 2D assessment of the cardiac chambers. While the measurement of volumes and also functional parameters free of geometrical assumptions may be of high interest in the case of the LV as well, it may gain particular importance in the assessment of the RV8. 3D-derived RV volumes and ejection fraction (EF) are shown to have significant prognostic value in various cardiovascular conditions9,10.
Nowadays, several vendors provide semi-automated solutions for 3D RV assessment with validated results against gold standard cardiac magnetic resonance (MR) measurements11,12. The technical requirements of 3D assessment are essential parts of a state-of-the-art cardiovascular imaging department nowadays, and it is expected that it will soon be part of the general equipment in every echocardiography lab. With proper expertise in 3D acquisition and post-processing, 3D RV analysis can be easily implemented into the standard examination protocol.

<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

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.

3D analysis of the RV is feasible in a wide variety of cardiovascular diseases. Case 1 is a healthy volunteer with normal ventricular volumes and function (Figure 1). Case 2 is a post-mitral valve repair patient who is a typical example for the conflicting results of conventional 2D assessment: while TAPSE is markedly reduced, the patient does not show any signs of RV dysfunction and a maintained RV global systolic function was confirmed by normal 3D RV EF (<...

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

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.

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

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2.7K Views.  Semmelweis University Heart and Vascular Center. 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.

Video: 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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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