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><tbody><tr><td>RMV-707B scan head 30 MHz</td><td>Visual Sonics</td><td>P/N 11459</td><td>mouse scan head</td></tr><tr><td>VisualSonics Vevo 770&amp;reg; High-Resolution Imaging System</td><td>Visual Sonics</td><td>770-230</td><td>ultrasound machine</td></tr><tr><td>Veet depilation creme for sensitive skin</td><td>Veet</td><td>07768307&amp;nbsp;</td><td></td></tr><tr><td>Surgical tape Durapore 3M</td><td>3M Deutschland GmbH</td><td>1538-1</td><td>for fixation</td></tr><tr><td>Askina Brauncel cellulose swabs</td><td>B.Braun</td><td>9051015</td><td></td></tr><tr><td>Aquasonic ultrasound gel</td><td>Parker Laboratories Inc.</td><td>BT025-0037L</td><td></td></tr><tr><td>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><td>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

7.5K 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

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 ...

Right Ventricular Systolic Pressure Measurements in Combination with Harvest of Lung and Immune Tissue Samples in Mice

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is a common modifier of heart and lung function, by elaborating cellular infiltration, production of cytokines and growth factors, and by initiating remodeling processes 1.
Compared to the left ventricle, the right ventricle is a low-pressure pump that operates in a relatively narrow zone of pressure changes. Increased pulmonary artery pressures are associated with increased pressure in the lung vascular bed and pulmonary hypertension 2. Pulmonary hypertension is often associated with inflammatory lung diseases, for example chronic obstructive pulmonary disease, or autoimmune diseases 3. Because pulmonary hypertension confers a bad prognosis for quality of life and life expectancy, much research is directed towards understanding the mechanisms that might be targets for pharmaceutical intervention 4. The main challenge for the development of effective management tools for pulmonary hypertension remains the complexity of the simultaneous understanding of molecular and cellular changes in the right heart, the lungs and the immune system.
Here, we present a procedural workflow for the rapid and precise measurement of pressure changes in the right heart of mice and the simultaneous harvest of samples from heart, lungs and immune tissues. The method is based on the direct catheterization of the right ventricle via the jugular vein in close-chested mice, first developed in the late 1990s as surrogate measure of pressures in the pulmonary artery5-13. The organized team-approach facilitates a very rapid right heart catheterization technique. This makes it possible to perform the measurements in mice that spontaneously breathe room air. The organization of the work-flow in distinct work-areas reduces time delay and opens the possibility to simultaneously perform physiology experiments and harvest immune, heart and lung tissues.
The procedural workflow outlined here can be adapted for a wide variety of laboratory settings and study designs, from small, targeted experiments, to large drug screening assays. The simultaneous acquisition of cardiac physiology data that can be expanded to include echocardiography5,14-17 and harvest of heart, lung and immune tissues reduces the number of animals needed to obtain data that move the scientific knowledge basis forward. The procedural workflow presented here also provides an ideal basis for gaining knowledge of the networks that link immune, lung and heart function. The same principles outlined here can be adapted to study other or additional organs as needed.

A specific and rapid protocol to simultaneously investigate right heart function, lung inflammation, and the immune response is described as a learning tool. Video and figures describe physiology and microdissection techniques in an organized team-approach that is adaptable to be used for small to large sized studies.

The function of the right heart is to pump blood through the lungs, thus linking right heart physiology and pulmonary vascular physiology. Inflammation is ...

Immunology and Infection

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, ...

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in rodents, with detailed descriptions of the appropriate methods for anesthesia, the echocardiographic windows used, the imaging planes and probe orientations for image acquisition, the methods for data analysis, and the limitations of emerging technologies for the evaluation of cardiac and valvular function. Importantly, we also highlight several future areas of research in cardiac and heart valve imaging that may be leveraged to gain insights into the pathogenesis of valve disease in preclinical animal models. We propose that using a systematic approach to evaluating cardiac and heart valve function in mice can result in more robust and reproducible data, as well as facilitate the discovery of previously underappreciated phenotypes in genetically-altered and/or physiologically-stressed mice.

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

The aim of this manuscript and accompanying video is to provide an overview of the methods and approaches used for imaging heart valve function in ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

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 ...

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 Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular disease and is used to assess the impact of potential treatments in preclinical studies. Echocardiographic studies are usually conducted in young adult mice (i.e., 4-6 weeks of age). The evaluation of early neonatal cardiovascular function is not usually performed because of the small size of the mouse pups and the associated technical difficulties. One of the most important challenges is that the short length of the pups&#39; limbs prevents them from reaching the electrodes in the echocardiography platform. Body temperature is the other challenge, as pups are very susceptible to changes in temperature. Therefore, it is important to establish a practical guide for performing echocardiographic studies in small mouse pups to help researchers detect early pathological changes and study the progression of cardiovascular disease over time. The current work describes a protocol for performing echocardiography in mouse pups at the early age of 7 days old. The echocardiographic characterization of cardiac morphology, function, and coronary flow in neonatal mice is also described.

The present protocol describes the echocardiographic assessment of left ventricular morphology, function, and coronary blood flow in 7-day old neonate mice.

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular ...

Induction of Right Ventricular Failure by Pulmonary Artery Constriction and Evaluation of Right Ventricular Function in Mice

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of RVF. Previous rat models imitating RVF progression have been described. Compared with rats, mice are more accessible, economical, and widely used in animal experiments. We developed a pulmonary artery constriction (PAC) approach which is comprised of banding the pulmonary trunk in mice to induce right ventricular (RV) hypertrophy. A special surgical latch needle was designed that allows for easier separation of the aorta and the pulmonary trunk. In our experiments, the use of this fabricated latch needle reduced the risk of arteriorrhexis and improved the surgical success rate to 90%. We used different padding needle diameters to precisely create quantitative constriction, which can induce different degrees of RV hypertrophy. We quantified the degree of constriction by evaluating the blood flow velocity of the PA, which was measured by noninvasive transthoracic echocardiography. RV function was precisely evaluated by right heart catheterization at 8 weeks after surgery. The surgical instruments made inhouse were composed of common materials using a simple process that is easy to master. Therefore, the PAC approach described here is easy to imitate using instruments made in the lab and can be widely used in other labs. This study presents a modified PAC approach that has a higher success rate than other models and an 8-week postsurgery survival rate of 97.8%. This PAC approach provides a useful technique for studying the mechanism of RVF and will enable an increased understanding of RVF.

Here, we provide a useful approach for studying the mechanism of right ventricular failure. A more convenient and efficient approach to pulmonary artery constriction is established using surgical instruments made inhouse. In addition, methods to evaluate the quality of this approach by echocardiography and catheterization are provided.

The mechanism of right ventricular failure (RVF) requires clarification due to the uniqueness, high morbidity, high mortality, and refractory nature of ...