The study was funded by the Ludwig Boltzmann Institute for Lung Vascular Research.

The study was performed according to national regulations for animal experimentation and EU Directive 2010/63. Prepare equipment as described previously by Brittain&#160;et al.8.
1. Mouse Preparation
<ol>
	<li>Obtain 12&#160;to 13 week-old male C57Bl6/J mice and house them with a 12 h light/dark cycle, at a constant room temperature, and with ad libitum access to standard laboratory chow and water, until the start of the experiment.</li>
	<li>Anesthetize the mouse using general anesthesia approved by the Institute and check for the lack of response to the toe pinch. Under mild anesthesia with isoflurane 0.8%&#8211;1.2%, fix the mouse on a heated platform. Apply electrode gel to its extremities for the continuous monitoring of its heart rate and temperature.</li>
	<li>Depilate the mouse&#39;s chest hair using depilation creme. To reduce pressure on its thorax, do not apply the ultrasound coupling gel directly on the thorax; rather, apply a layer of the gel to the tip of the transducer.</li>
</ol>
2. Image Acquisition
<ol>
	<li>Apical four-chamber view with a left and caudal tilt of the platform
	<ol>
		<li>After the mouse preparation, angulate the platform to the left at 10&#176;&#8211;15&#176; and then caudally at 10&#176;&#8211;15&#176;.</li>
		<li>Position the transducer above the apex with the imaging plane ~45&#176; to the coronal plane and the central axis of the ultrasound beam directed cranially, posterior, and to the left to obtain the apical four-chamber view. Press the B-Mode button to activate the B-mode/2-D image. 
		NOTE: The transducer can be held manually or fixed by a stage. The term &#34;B-mode&#34; comes from the imaging system that was used instead of the more familiar term &#34;two-dimensional&#34; (2-D) and is used throughout the protocol.</li>
		<li>Look for the appearance of the following structures in the acoustic window: the left ventricle (LV), the left atrium (LA), the RV, the right atrium (RA), the mitral valve (MV), and the tricuspid valve (TV).</li>
		<li>Manipulate the imaging plane in the coronal plane and rotate clock- and counterclockwise around the central axis until both ventricles are visualized at their longest dimension and both atria are visible. This is the four-chamber view (Figure 1).</li>
		<li>Press the Cine store button to save the recording.</li>
		<li>Press the Scan/Freeze button to pause the system.</li>
	</ol>
	</li>
	<li>Measurement of transtricuspid blood flow velocities
	<ol>
		<li>Press the Scan/Freeze button to activate the system.</li>
		<li>Press the Overlay button several times to activate the sample volume for PW (pulsed wave) mode.</li>
		<li>While keeping the obtained four-chamber view, use the trackball to position the sample volume at the opening of the tricuspid valves for the measurement of inflow velocities (E and A peak velocities).</li>
		<li>Press the PW mode button for the measurement of inflow velocities (E and A peak velocities). 
		NOTE: Because tricuspid valves are difficult to visualize in this position, performing several measurements helps to align correctly the sample volume with the blood flow. Perform the Doppler sampling with the smallest incidence angle between the Doppler beam and the blood flow direction. The obtained blood flow profile should correspond to the following criteria: 1) an inflow profile similar to an M-shape with the first peak lower than the second; 2) a respiratory modulation with an increased amplitude at inspiration; 3) a maximal amplitude of velocities in several measurements (Figure 2).</li>
		<li>Press the Cine store button to save the optimized recording.</li>
		<li>Press the Scan/Freeze button to pause the system.</li>
	</ol>
	</li>
	<li>Measurement of the tricuspid annular plane systolic excursion (TAPSE)
	<ol>
		<li>Press the Scan/Freeze button to activate the system.</li>
		<li>Switch to B-mode by pressing the B-Mode button. Some manipulations on the image might be necessary to regain the correct four-chamber view.</li>
		<li>Press the Overlay button several times to activate the sample volume of the M-mode. Using the trackball, align the sample volume with the lateral part of the tricuspid annulus. By pulling the edges of the sample volume using the trackball, align the length of the sample volume to cover the entire amplitude of the cardiac movement during the cardiac cycle.</li>
		<li>Press the M-Mode button to activate M-mode. Tricuspid annulus&#39; movements should appear as a wave (Figure 2).</li>
		<li>Press the Cine store button to save the recording.</li>
		<li>Press the Scan/Freeze button to pause the system.</li>
	</ol>
	</li>
	<li>Measurement of tissue Doppler parameters
	<ol>
		<li>Press the Scan/Freeze button to activate the system.</li>
		<li>Press the B-Mode button to activate B-mode. 
		NOTE: Some manipulations by angulation in the coronal plane and rotation clock- and counterclockwise around the central axis of the image might be necessary to regain the correct four-chamber view.</li>
		<li>Press the Overlay button several times to activate the sample volume for TDI (tissue Doppler imaging). Using the trackball, align the sample volume with the lateral part of the tricuspid annulus, where the RV free wall creates an angle with the tricuspid valve. By pulling the edges of the sample volume using the trackball, adjust the sample volume to include both the systolic and the diastolic extreme positions of the annulus.</li>
		<li>Press the Tissue button to activate TDI mode. 
		NOTE: Yellow tracing of the TDI recording appears corresponding to the following criteria:1) a recording similar to an inverted M-shape; 2) clearly distinguishable E&#39; and A&#39; peaks during diastole and S&#39; peak during systole; 3) a maximal amplitude of velocities in several measurements (Figure 2).</li>
		<li>Press the Cine store button to record an optimized image.</li>
		<li>Press the Scan/Freeze button to pause the system.</li>
	</ol>
	</li>
	<li>Apical four-chamber view with right and cranial tilt of the platform
	<ol>
		<li>Angulate the platform to the right at 10&#176;&#8211;15&#176; and then cranial at 10&#176;&#8211;15&#176;. Perform the measurements as described in the previous sections for the LeCa steps (steps 2.1, 2.2, 2.3, and 2.4). 
		NOTE: During the investigation, isoflurane should be titrated between 0.&#8211;1.2 to keep the mouse&#39;s heart rate at 400&#8211;440 bpm. In this range, separate peaks of transtricuspid blood flow and tissue Doppler (DTI) velocities are measurable. To avoid the effects of the heat loss on hemodynamics, the data are recorded, and the analysis is performed off-line. Only signals obtained at the end-expiration are used for analysis. Measurements of 3 - 5 heartbeats are averaged.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Egemnazarov, B., Crnkovic, S., Nagy, B. M., Olschewski, H., Kwapiszewska, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Right+ventricular+fibrosis+and+dysfunction:+Actual+concepts+and+common+misconceptions.">Right ventricular fibrosis and dysfunction: Actual concepts and common misconceptions.</a> Matrix Biology: Journal of the International Society for Matrix Biology. 68-69, 507-521 (2018).</li><li>Rain, S., 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=Right+ventricular+diastolic+impairment+in+patients+with+pulmonary+arterial+hypertension.">Right ventricular diastolic impairment in patients with pulmonary arterial hypertension.</a> Circulation. 128, 1-10 (2013).</li><li>Egemnazarov, B., 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=Pressure+overload+creates+right+ventricular+diastolic+dysfunction+in+a+mouse+model:+assessment+by+echocardiography.">Pressure overload creates right ventricular diastolic dysfunction in a mouse model: assessment by echocardiography.</a> Journal of the American Society of Echocardiography. 28, 828-843 (2015).</li><li>Crnkovic, S., 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=Functional+and+molecular+factors+associated+with+TAPSE+in+hypoxic+pulmonary+hypertension.">Functional and molecular factors associated with TAPSE in hypoxic pulmonary hypertension.</a> American Journal of Physiology. Lung Cellular and Molecular Physiology. 311, 59-73 (2016).</li><li>Shi, L., 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=miR-223-IGF-IR+signalling+in+hypoxia-+and+load-induced+right-ventricular+failure:+a+novel+therapeutic+approach.">miR-223-IGF-IR signalling in hypoxia- and load-induced right-ventricular failure: a novel therapeutic approach.</a> Cardiovascular Research. 111, 184-193 (2016).</li><li>de Raaf, M. A., 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=Tyrosine+kinase+inhibitor+BIBF1000+does+not+hamper+right+ventricular+pressure+adaptation+in+rats.">Tyrosine kinase inhibitor BIBF1000 does not hamper right ventricular pressure adaptation in rats.</a> American Journal of Physiology - Heart and Circulatory Physiology. 311, 604-612 (2016).</li><li>Zhou, Y. 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=Comprehensive+transthoracic+cardiac+imaging+in+mice+using+ultrasound+biomicroscopy+with+anatomical+confirmation+by+magnetic+resonance+imaging.">Comprehensive transthoracic cardiac imaging in mice using ultrasound biomicroscopy with anatomical confirmation by magnetic resonance imaging.</a> Physiological Genomics. 18, 232-244 (2004).</li><li>Brittain, E., Penner, N. L., West, J., Hemnes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+assessment+of+the+right+heart+in+mice.">Echocardiographic assessment of the right heart in mice.</a> Journal of Visualized Experiments. (81), e50912 (2013).</li><li>Kitchen, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonparametric+vs+parametric+tests+of+location+in+biomedical+research.">Nonparametric vs parametric tests of location in biomedical research.</a> American Journal of Ophthalmology. 147, 571-572 (2009).</li><li>Yan, F., Robert, M., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statistical+methods+and+common+problems+in+medical+or+biomedical+science+research.">Statistical methods and common problems in medical or biomedical science research.</a> International Journal of Physiology, Pathophysiology and Pharmacology. 9, 157-163 (2017).</li><li>Guihaire, 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=Non-invasive+indices+of+right+ventricular+function+are+markers+of+ventricular-arterial+coupling+rather+than+ventricular+contractility:+insights+from+a+porcine+model+of+chronic+pressure+overload.">Non-invasive indices of right ventricular function are markers of ventricular-arterial coupling rather than ventricular contractility: insights from a porcine model of chronic pressure overload.</a> European Heart Journal Cardiovascular Imaging. 14, 1140-1149 (2013).</li><li>Sareen, N., Ananthasubramaniam, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+Imaging:+From+Physiology+to+Practical+Applications+in+Daily+Practice.">Strain Imaging: From Physiology to Practical Applications in Daily Practice.</a> Cardiology in Review. 24, 56-69 (2016).</li><li>Thavendiranathan, 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=Use+of+myocardial+strain+imaging+by+echocardiography+for+the+early+detection+of+cardiotoxicity+in+patients+during+and+after+cancer+chemotherapy:+a+systematic+review.">Use of myocardial strain imaging by echocardiography for the early detection of cardiotoxicity in patients during and after cancer chemotherapy: a systematic review.</a> Journal of the American College of Cardiology. 63, 2751-2768 (2014).</li><li>Sengelov, M., 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=Global+Longitudinal+Strain+Is+a+Superior+Predictor+of+All-Cause+Mortality+in+Heart+Failure+With+Reduced+Ejection+Fraction.">Global Longitudinal Strain Is a Superior Predictor of All-Cause Mortality in Heart Failure With Reduced Ejection Fraction.</a> JACC: Cardiovascular Imaging. 8, 1351-1359 (2015).</li><li>Silvani, A., 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=Physiological+Mechanisms+Mediating+the+Coupling+between+Heart+Period+and+Arterial+Pressure+in+Response+to+Postural+Changes+in+Humans.">Physiological Mechanisms Mediating the Coupling between Heart Period and Arterial Pressure in Response to Postural Changes in Humans.</a> Frontiers in Physiology. 8, 163 (2017).</li><li>Mohan, M., Anandh, B., Thombre, D. P., Surange, S. G., Chakrabarty, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+posture+on+heart+rate+and+cardiac+axis+of+mice.">Effect of posture on heart rate and cardiac axis of mice.</a> Indian Journal of Physiology and Pharmacology. 31, 211-217 (1987).</li></ol>

The authors have nothing to disclose.

The echocardiographic RV function and dimension assessment from parasternal positions have been well described. In contrast, the apical position in mouse echocardiography has been neglected partly due to technical difficulties. Using a horizontal platform position, it is difficult to obtain a sufficient acoustic window for four-chamber view imaging. To facilitate the imaging of this position, the platform can be tilted to the left, a manipulation similar to the left-sided positioning of patients. This should result in a leftward and more superior positioning of the heart, thereby improving the acoustic window. Therefore, LeCa is our standardized position for apical visualization. However, in approximately 30%&#8211;35% of mice, the image quality in this position can be insufficient. Here, imaging in the RiCr position can be helpful.
From these positions, transtricuspid blood flow velocities (E and A) and tissue Doppler velocities (E&#39; and A&#39;) can be measured, providing information about the RV diastolic function. We observed a good correlation between TDI parameters obtained from the two positions. Less satisfactory was the correlation of E. In general, the visualization of the transtricuspidal blood flow profile was the most challenging part of the protocol presented here and exhibited the highest variability. The measurement of TAPSE and S&#39; by tissue Doppler provided an estimate of the RV systolic function. However, in the light of recent findings, the physiological meaning of TAPSE is not clear11. We do not routinely measure the RV fractional area of contraction from the apical position because, in the conditions of pressure overload, the lateral part of the enlarged RV is partly covered by the sternum and not completely visible from this position3. Thus, the visualization of the apical position in mice enables the measurement of the parameters routinely used in the clinic and, thereby, delivers more information, which allows a more complete functional characterization.
Strain, strain rate analysis, and speckle tracking echocardiography are novel modalities of cardiac ultrasound12. Its high sensitivity can detect cardiac dysfunction at initial stages13 and has the power to predict mortality14; therefore, its application is also warranted in experimental studies. Unfortunately, in mice, the RV free wall is partly hidden behind the sternum&#39;s shadow, which might hinder the analysis of strain. Furthermore, stain analysis requires good image quality and visualization of the entire free wall.
The cardiovascular system responds quickly to changes in the posture by activating baroreceptor mechanisms15. Therefore, it could be expected that the cranial tilt of the platform would cause reflectory changes in the measured cardiac parameters. Indeed, both the head-up and the head-down tilt position caused a transient change in the heart rate and cardiac electric axis in mice16. While a 90&#176; head-up tilt causes an increased heart rate, a 90&#176; head-down tilt caused transient and statistically insignificant bradycardia. In contrast, we recommend tilting the mouse only by 10&#176;&#8211;15&#176; in either direction. These mild changes in posture did not cause any measurable hemodynamic perturbances.
LV diastolic function in mice is another understudied area. Although not tested in this study, the protocol presented here should be able to be used for the quantification of the LV diastolic function.
Theoretical and practical limitations of the small animal EchoCG have been described in detail elsewhere8. In this protocol, measurements are performed at heart rates of 400&#8211;440 bpm. At this range of heart rate, measurements of the E and A velocity peaks, as well as of TDI indexes, are feasible. At higher heart rates, peaks merge, making quantification impossible. Since the physiological heart rate for mice is 500&#8211;600 bpm, the heart rate used in this protocol is rather low. Nevertheless, the measurements at this heart rate range appear reliable and enable distinguishing between physiologic and dysfunctional phenotypes3.
We described a protocol for two positions facilitating the assessment of RV functional parameters from four-chamber views in mice. The positions provide comparable results and can be used interchangeably.

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling1 and is associated with pressure-overload conditions2. Echocardiography (EchoCG) can be used for the characterization of RV diastolic dysfunction3,4. Despite recent developments in small animal echocardiography, measurements of diastolic parameters are rarely reported. In contrast, measurements of the systolic function are widely used for the characterization of transgenic mice5, as well as for the evaluation of a treatment response6.
This can be partly explained by the difficulties in the measurement of diastolic parameters from the apical four-chamber view. Visualization of the heart in this position can be facilitated by tilting the fixation platform LeCa or RiCr. Even if these manipulations are used, echocardiographers do not report them in their manuscripts4,7. Therefore, it remains unclear whether these manipulations provide comparable results. Moreover, this also precludes a development of standardized nomenclature of this position for mice.
The aim of this study was to describe two positions for apical four-chamber view visualization and compare their results. To determine the differences between the two positions, we have utilized the mouse pulmonary artery banding (PAB) model, in which a tantalum clip leads to a partial occlusion of the pulmonary artery. This occlusion results in right ventricle remodeling and dysfunction. Full details of the PAB operation can be found in previously published work3. Sham-operated mice, where the clip was placed next to the pulmonary artery, were used for comparison. EchoCG investigations were performed three weeks post-operation using the imaging system with a 30 MHz scan head (see Table of Materials for both). Nomenclature for the description of the positions and orientations between the mouse and the ultrasound beam is used as described by Zhou et al.7.

<table border="0" cellpadding="0" cellspacing="0" style="width:839px;" width="838">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">RMV-707B scan head 30 MHz</td>
			<td style="width:189px;">Visual Sonics</td>
			<td style="width:239px;">P/N 11459</td>
			<td style="width:167px;">mouse scan head</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">VisualSonics Vevo 770&#174; High-Resolution Imaging System</td>
			<td>Visual Sonics</td>
			<td>770-230</td>
			<td>ultrasound machine</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Veet depilation creme for sensitive skin</td>
			<td>Veet</td>
			<td>07768307&#160;</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Surgical tape Durapore 3M</td>
			<td>3M Deutschland GmbH</td>
			<td>1538-1</td>
			<td>for fixation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Askina Brauncel cellulose swabs</td>
			<td>B.Braun</td>
			<td>9051015</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Aquasonic ultrasound gel</td>
			<td>Parker Laboratories Inc.</td>
			<td>BT025-0037L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrode Gel</td>
			<td>GE medical systems information technologies Inc.</td>
			<td>2034731-002</td>
			<td>apply to extremities for countinous ECG and heart rate monitoring</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Thermasonic gel warmer</td>
			<td>Parker Laboratories Inc.</td>
			<td>82-04-20</td>
			<td>to reduce heat loss warm up the ultrasound gel before use</td>
		</tr>
	</tbody>
</table>

echocardiographic measurement of right ventricular diastolic parameters in mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

The apical four-chamber view is difficult to obtain in mice. Therefore, manipulations of the platform position can help to visualize the heart by changing its position in the thorax. The tilting of the platform to the left and to the right improved the acoustic window and provided images of comparable quality in B-mode (Figure 1). After obtaining the correct positions, measurements in PW-, M-, and TDI-modes provided images of comparable quality (Figure 2). The measurement of diastolic parameters was performed on sham- and PAB-operated mice (Table 1). Both positions (RiCr and LeCa) gave similar results in the diastolic parameters (Table 2). Furthermore, the EchoCG investigations in both positions revealed similar differences between the sham and PAB groups (Table 2, Dunnet&#39;s test). Correlation analysis revealed a good agreement between values obtained from these two facilitated positions (Figure 3). As small groups of animals were used for this study, non-parametric tests have been applied9,10. Intra-observer variability for some analyzed parameters has been published previously3.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58021/58021fig1.jpg" /> 
Figure 1: Representative images of the apical four-chamber view. The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). LA = left atrium; LV = left ventricle; RA = right atrium; RV = right ventricle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58021/58021fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58021/58021fig2.jpg" /> 
Figure 2: Representative images of the TAPSE, TDI, and transtricuspid flow measurements obtained from two facilitated apical four-chamber view positions. TAPSE = tricuspid annulus plane systolic excursion; E&#39; = early peak of right ventricular relaxation velocity; A&#39; = late peak of right ventricular relaxation velocity; S&#39; = velocity of the right ventricular contraction; E = early peak of diastolic tricuspid inflow; A = late peak of diastolic tricuspid inflow. Note the change in the transtricuspid blood flow profile at the inspiration (Insp). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58021/58021fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58021/58021fig3.jpg" /> 
Figure 3: Correlation analysis of data obtained from two facilitated apical positions. Correlation analysis was performed using non-parametric Spearman&#39;s test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58021/58021fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/58021/58021table1.jpg" /> 
Table 1: Characterization of the operated groups three weeks after the operation. RVFW = right ventricular free wall thickness; VTI = velocity-time interval.
<img alt="Table 2" src="/files/ftp_upload/58021/58021table2.jpg" /> 
Table 2: Comparison of the results obtained from the apical four-chamber view facilitated by the left caudal or right cranial platform tilt. EchoCG-derived RV functional parameters are shown. As every mouse was investigated in both positions, the signed rank Wilcoxon test was used for intra-group comparisons. &#167; p &#62; 0.05 between RiCr and LeCa. The Kruskal-Wallis test, followed by Dunnet&#39;s post hoc test, was used for multiple group comparisons. The results of two selected intergroup comparisons are presented in the table. * p &#60; 0.05, ** p &#60; 0.01. PAB = pulmonary artery banding; LeCa = left caudal tilt; RiCR = right cranial tilt; E = early peak of diastolic tricuspid inflow; A = late peak of diastolic tricuspid inflow; TAPSE = tricuspid annulus plane systolic excursion; e&#39; = early peak of right ventricular relaxation velocity; a&#39; = late peak of right ventricular relaxation velocity; S&#39; = velocity of the right ventricular contraction; HR = heart rate; bpm = beats per minute.

Watch this Scientific Journal Video about Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse at JoVE.com

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

echocardiographic-measurement-right-ventricular-diastolic-parameters

Nanyang Technological University

Research

JoVE Journal

Medicine

7.4K Views.  Ludwig Boltzmann Institute for Lung Vascular Research. Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

Video: Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

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, ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

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 Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

A Pulmonary Trunk Banding Model of Pressure Overload Induced Right Ventricular Hypertrophy and Failure

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary disorders. Reliable and reproducible animal models of RV failure are therefore warranted in order to investigate disease mechanisms and effects of potential therapeutic strategies. Banding of the pulmonary trunk is a common method to induce isolated RV hypertrophy but in general, previously described models have not succeeded in creating a stable model of RV hypertrophy and failure.
We present a rat model of pressure overload induced RV hypertrophy caused by pulmonary trunk banding (PTB) that enables different phenotypes of RV hypertrophy with and without RV failure. We use a modified ligating clip applier to compress a titanium clip around the pulmonary trunk to a pre-set inner diameter. We use different clip diameters to induce different stages of disease progression from mild RV hypertrophy to decompensated RV failure.
RV hypertrophy develops consistently in rats subjected to the PTB procedure and depending on the diameter of the applied banding clip, we can accurately reproduce different disease severities ranging from compensated hypertrophy to severe decompensated RV failure with extra-cardiac manifestations.
The presented PTB model is a valid and robust model of pressure overload induced RV hypertrophy and failure that has several advantages to other banding models including high reproducibility and the possibility of inducing severe and decompensated RV failure.

We present a surgical method to induce right ventricular hypertrophy and failure in rats.

Right ventricular (RV) failure induced by sustained pressure overload is a major contributor to morbidity and mortality in several cardiopulmonary ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

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. ...

Echocardiographic Assessment of Cardiac Anatomy and Function in Adult Rats

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional imaging to study the cardiac anatomy of the left ventricle and M-mode echo to assess its dimensions. However, this could limit a comprehensive study. Herein, we describe a protocol that allows an assessment of the heart chamber size, left ventricular function (systolic and diastolic) and valvular function. A conventional medical ultrasound machine was used in this protocol and different echo views were obtained through left parasternal, apical and suprasternal windows. In the left parasternal window, the long and short axis were acquired to analyze left chamber dimensions, right ventricle and pulmonary artery dimensions, and mitral, pulmonary and aortic valve function. The apical window allows the measurement of heart chamber dimensions and evaluation of systolic and diastolic parameters. It also allows Doppler assessment with detection and quantification of heart valve disturbances (regurgitation or stenosis). Different segments and walls of the left ventricle are visualized throughout all views. Finally, the ascending aorta, aortic arch, and descending aorta can be imaged through the suprasternal window. A combination of ultrasound imaging, Doppler flow and tissue Doppler assessment have been obtained to study cardiac morphology and function. This represents an important contribution to improve the assessment of cardiac function in adult rats with impact for research using these animal models.

A non-invasive protocol for transthoracic echocardiography assessment of cardiac anatomy and function for adult rats is presented in the current study. The heart valves, all four cardiac chambers and the ascending aorta, aortic arch and descending aorta are studied in detail.

The use of experimental animal models has become crucial in cardiovascular science. Most studies using rodent models are focused on two-dimensional ...