Name: Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse (Video) | JoVE Journal
Uploaded: 2019-04-27T12:30:00.000+00:00
Duration: 6 min 4 s
Description: Learn echocardiographic measurement of right ventricular diastolic parameters in mice using apical four-chamber imaging with dual tilting positions. This technique improves visualization rates and enables reliable cardiac function assessment.

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5 minutes

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/pubmed?term=((Right+ventricular+fibrosis+and+dysfunction%3A+Actual+concepts+and+common+misconceptions%5BTitle%5D)+AND+%22Matrix+Biology%3A+Journal+of+the+International+Society+for+Matrix+Biology%22%5BJournal%5D)">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/pubmed?term=((Right+ventricular+diastolic+impairment+in+patients+with+pulmonary+arterial+hypertension%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">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/pubmed?term=((Pressure+overload+creates+right+ventricular+diastolic+dysfunction+in+a+mouse+model%3A+assessment+by+echocardiography%5BTitle%5D)+AND+%22Journal+of+the+American+Society+of+Echocardiography%22%5BJournal%5D)">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/pubmed?term=((Functional+and+molecular+factors+associated+with+TAPSE+in+hypoxic+pulmonary+hypertension%5BTitle%5D)+AND+%22American+Journal+of+Physiology.+Lung+Cellular+and+Molecular+Physiology%22%5BJournal%5D)">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/pubmed?term=((miR-223-IGF-IR+signalling+in+hypoxia-+and+load-induced+right-ventricular+failure%3A+a+novel+therapeutic+approach%5BTitle%5D)+AND+%22Cardiovascular+Research%22%5BJournal%5D)">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/pubmed?term=((Tyrosine+kinase+inhibitor+BIBF1000+does+not+hamper+right+ventricular+pressure+adaptation+in+rats%5BTitle%5D)+AND+%22American+Journal+of+Physiology+-+Heart+and+Circulatory+Physiology%22%5BJournal%5D)">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/pubmed?term=((Comprehensive+transthoracic+cardiac+imaging+in+mice+using+ultrasound+biomicroscopy+with+anatomical+confirmation+by+magnetic+resonance+imaging%5BTitle%5D)+AND+%22Physiological+Genomics%22%5BJournal%5D)">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/pubmed?term=((Echocardiographic+assessment+of+the+right+heart+in+mice%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((Nonparametric+vs+parametric+tests+of+location+in+biomedical+research%5BTitle%5D)+AND+%22American+Journal+of+Ophthalmology%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/29209453">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/pubmed?term=((Non-invasive+indices+of+right+ventricular+function+are+markers+of+ventricular-arterial+coupling+rather+than+ventricular+contractility%3A+insights+from+a+porcine+model+of+chronic+pressure+overload%5BTitle%5D)+AND+%22European+Heart+Journal+Cardiovascular+Imaging%22%5BJournal%5D)">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/pubmed?term=((Strain+Imaging%3A+From+Physiology+to+Practical+Applications+in+Daily+Practice%5BTitle%5D)+AND+%22Cardiology+in+Review%22%5BJournal%5D)">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/pubmed?term=((Use+of+myocardial+strain+imaging+by+echocardiography+for+the+early+detection+of+cardiotoxicity+in+patients+during+and+after+cancer+chemotherapy%3A+a+systematic+review%5BTitle%5D)+AND+%22Journal+of+the+American+College+of+Cardiology%22%5BJournal%5D)">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/pubmed?term=((Global+Longitudinal+Strain+Is+a+Superior+Predictor+of+All-Cause+Mortality+in+Heart+Failure+With+Reduced+Ejection+Fraction%5BTitle%5D)+AND+%22JACC%3A+Cardiovascular+Imaging%22%5BJournal%5D)">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/pubmed?term=((Physiological+Mechanisms+Mediating+the+Coupling+between+Heart+Period+and+Arterial+Pressure+in+Response+to+Postural+Changes+in+Humans%5BTitle%5D)+AND+%22Frontiers+in+Physiology%22%5BJournal%5D)">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>
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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 ...

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

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 (<strong class...

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

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Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

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