This work was supported by NIH grants HL111121 (JDM) and TR000954 (JDM).

<ol>
	<li>Ngo, D. T., 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=Determinants+of+occurrence+of+aortic+sclerosis+in+an+aging+population.">Determinants of occurrence of aortic sclerosis in an aging population.</a> JACC Cardiovasc Imaging. 2, 919-927 (2009).</li><li>Nkomo, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+prevention+of+valvular+heart+diseases+and+infective+endocarditis+in+Africa.">Epidemiology and prevention of valvular heart diseases and infective endocarditis in Africa.</a> Heart. 93, 1510-1519 (2007).</li><li>Amato, M. C., Moffa, P. J., Werner, K. E., Ramires, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+decision+in+asymptomatic+aortic+valve+stenosis:+role+of+exercise+testing.">Treatment decision in asymptomatic aortic valve stenosis: role of exercise testing.</a> Heart. 86, 381-386 (2001).</li><li>Bonow, R. O., 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=Focused+update+incorporated+into+the+ACC/AHA+2006+guidelines+for+the+management+of+patients+with+valvular+heart+disease:+a+report+of+the+American+College+of+Cardiology/American+Heart+Association+Task+Force+on+Practice+Guidelines+(Writing+Committee+to+Revise+the+1998+Guidelines+for+the+Management+of+Patients+With+Valvular+Heart+Disease):+endorsed+by+the+Society+of+Cardiovascular+Anesthesiologists,+Society+for+Cardiovascular+Angiography+and+Interventions,+and+Society+of+Thoracic+Surgeons.">Focused update incorporated into the ACC/AHA 2006 guidelines for the management of patients with valvular heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the 1998 Guidelines for the Management of Patients With Valvular Heart Disease): endorsed by the Society of Cardiovascular Anesthesiologists, Society for Cardiovascular Angiography and Interventions, and Society of Thoracic Surgeons.</a> Circulation. 118, e523-e661 (2008).</li><li>Yutzey, K. E., 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=Calcific+aortic+valve+disease:+a+consensus+summary+from+the+Alliance+of+Investigators+on+Calcific+Aortic+Valve+Disease.">Calcific aortic valve disease: a consensus summary from the Alliance of Investigators on Calcific Aortic Valve Disease.</a> Arterioscler Thromb Vasc Biol. 34, 2387-2393 (2014).</li><li>Rajamannan, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcific+aortic+valve+disease:+cellular+origins+of+valve+calcification.">Calcific aortic valve disease: cellular origins of valve calcification.</a> Arterioscler Thromb Vasc Biol. 31, 2777-2778 (2011).</li><li>Weiss, R. M., Miller, J. D., Heistad, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrocalcific+aortic+valve+disease:+opportunity+to+understand+disease+mechanisms+using+mouse+models.">Fibrocalcific aortic valve disease: opportunity to understand disease mechanisms using mouse models.</a> Circ Res. 113, 209-222 (2013).</li><li>Sider, K. L., Blaser, M. C., Simmons, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+calcific+aortic+valve+disease.">Animal models of calcific aortic valve disease.</a> Int J Inflam. 2011, 364310 (2011).</li><li>Miller, J. D., Weiss, R. M., Heistad, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcific+aortic+valve+stenosis:+methods,+models,+and+mechanisms.">Calcific aortic valve stenosis: methods, models, and mechanisms.</a> Circ Res. 108, 1392-1412 (2011).</li><li>Ram, R., Mickelsen, D. M., Theodoropoulos, C., Blaxall, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+approaches+in+small+animal+echocardiography:+imaging+the+sounds+of+silence.">New approaches in small animal echocardiography: imaging the sounds of silence.</a> Am J Physiol Heart Circ Physiol. 301, H1765-H1780 (2011).</li><li>Moran, A. M., Keane, J. F., Colan, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+pressure+and+volume+load+on+growth+of+aortic+annulus+and+left+ventricle+in+patients+with+critical+aortic+stenosis.">Influence of pressure and volume load on growth of aortic annulus and left ventricle in patients with critical aortic stenosis.</a> J Am Coll Cardiol. 37, 471a (2001).</li><li>Thibault, H. 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=Noninvasive+assessment+of+murine+pulmonary+arterial+pressure:+validation+and+application+to+models+of+pulmonary+hypertension.">Noninvasive assessment of murine pulmonary arterial pressure: validation and application to models of pulmonary hypertension.</a> Circ Cardiovasc Imaging. 3, 157-163 (2010).</li><li>Baumgartner, H., 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=Echocardiographic+assessment+of+valve+stenosis:+EAE/ASE+recommendations+for+clinical+practice.">Echocardiographic assessment of valve stenosis: EAE/ASE recommendations for clinical practice.</a> J Am Soc Echocardiogr. 22, 1-23 (2009).</li><li>Lang, R. 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=Recommendations+for+cardiac+chamber+quantification+by+echocardiography+in+adults:+an+update+from+the+American+Society+of+Echocardiography+and+the+European+Association+of+Cardiovascular+Imaging.">Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging.</a> Eur Heart J Cardiovasc Imaging. 16, 233-270 (2015).</li><li>Devereux, R. B., Reichek, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+determination+of+left+ventricular+mass+in+man.+Anatomic+validation+of+the+method.">Echocardiographic determination of left ventricular mass in man. Anatomic validation of the method.</a> Circulation. 55, 613-618 (1977).</li><li>Ommen, S. R., 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=Clinical+utility+of+Doppler+echocardiography+and+tissue+Doppler+imaging+in+the+estimation+of+left+ventricular+filling+pressures:+A+comparative+simultaneous+Doppler-catheterization+study.">Clinical utility of Doppler echocardiography and tissue Doppler imaging in the estimation of left ventricular filling pressures: A comparative simultaneous Doppler-catheterization study.</a> Circulation. 102, 1788-1794 (2000).</li><li>Tei, C., 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=New+index+of+combined+systolic+and+diastolic+myocardial+performance:+a+simple+and+reproducible+measure+of+cardiac+function--a+study+in+normals+and+dilated+cardiomyopathy.">New index of combined systolic and diastolic myocardial performance: a simple and reproducible measure of cardiac function--a study in normals and dilated cardiomyopathy.</a> J Cardiol. 26, 357-366 (1995).</li><li>Koshizuka, R., 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=Longitudinal+strain+impairment+as+a+marker+of+the+progression+of+heart+failure+with+preserved+ejection+fraction+in+a+rat+model.">Longitudinal strain impairment as a marker of the progression of heart failure with preserved ejection fraction in a rat model.</a> J Am Soc Echocardiogr. 26, 316-323 (2013).</li><li>Ishizu, T., 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=Left+ventricular+strain+and+transmural+distribution+of+structural+remodeling+in+hypertensive+heart+disease.">Left ventricular strain and transmural distribution of structural remodeling in hypertensive heart disease.</a> Hypertension. 63, 500-506 (2014).</li><li>Yamada, 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=Induced+pluripotent+stem+cell+intervention+rescues+ventricular+wall+motion+disparity,+achieving+biological+cardiac+resynchronization+post-infarction.">Induced pluripotent stem cell intervention rescues ventricular wall motion disparity, achieving biological cardiac resynchronization post-infarction.</a> J Physiol. 591, 4335-4349 (2013).</li><li>Andrews, T. G., Lindsey, M. L., Lange, R. A., Aune, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Assessment+in+Pediatric+Mice:+Strain+Analysis+as+a+Diagnostic+Measurement.">Cardiac Assessment in Pediatric Mice: Strain Analysis as a Diagnostic Measurement.</a> Echocardiography. 31, 375-384 (2014).</li><li>Ferferieva, V., 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=Assessment+of+strain+and+strain+rate+by+two-dimensional+speckle+tracking+in+mice:+comparison+with+tissue+Doppler+echocardiography+and+conductance+catheter+measurements.">Assessment of strain and strain rate by two-dimensional speckle tracking in mice: comparison with tissue Doppler echocardiography and conductance catheter measurements.</a> Eur Heart J Cardiovasc Imaging. 14, 765-773 (2013).</li><li>Fine, N. 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=Left+and+right+ventricular+strain+and+strain+rate+measurement+in+normal+adults+using+velocity+vector+imaging:+an+assessment+of+reference+values+and+intersystem+agreement.">Left and right ventricular strain and strain rate measurement in normal adults using velocity vector imaging: an assessment of reference values and intersystem agreement.</a> Int J Cardiovasc Imaging. 29, 571-580 (2013).</li><li>Pernot, M., Fujikura, K., Fung-Kee-Fung, S. D., Konofagou, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECG-gated,+mechanical+and+electromechanical+wave+imaging+of+cardiovascular+tissues+in+vivo.">ECG-gated, mechanical and electromechanical wave imaging of cardiovascular tissues in vivo.</a> Ultrasound Med Biol. 33, 1075-1085 (2007).</li><li>Liu, J. H., Jeng, G. S., Wu, T. K., Li, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECG+triggering+and+gating+for+ultrasonic+small+animal+imaging.">ECG triggering and gating for ultrasonic small animal imaging.</a> IEEE Trans Ultrason Ferroelectr Freq Control. 53, 1590-1596 (2006).</li><li>Monin, J. 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=Low-gradient+aortic+stenosis:+operative+risk+stratification+and+predictors+for+long-term+outcome:+a+multicenter+study+using+dobutamine+stress+hemodynamics.">Low-gradient aortic stenosis: operative risk stratification and predictors for long-term outcome: a multicenter study using dobutamine stress hemodynamics.</a> Circulation. , 319-324 (2003).</li></ol>

The authors have nothing to disclose.

Aging is associated with progressive increases in cardiovascular calcification1. Hemodynamically significant aortic valve stenosis affects 3% of the population over the age of 652, and patients with even moderate aortic valve stenosis (peak velocity of 3-4 m/s) have a 5 year event-free survival of less than 40%3. Presently, there are no effective treatments to slow the progression of aortic valve calcification, and surgical aortic valve replacement is the only available treatment for advanced aortic valve stenosis4.
Studies aimed at gaining a deeper understanding of the mechanisms that contribute to the initiation and progression of aortic valve calcification are a key first step in moving towards pharmacological and non-surgical methods to manage aortic valve stenosis5,6. Genetically-altered mice have played a major role in developing our understanding of the mechanisms that contribute to a variety of diseases and are now coming to the forefront of mechanistic studies aimed at understanding the biology of aortic valve stenosis6,7,8. Unlike other cardiovascular diseases such as atherosclerosis and heart failure-where standard protocols for evaluating vascular and ventricular function are for the most part well-established-there are unique challenges associated with in vivo phenotyping of heart valve function in mice. While recent reviews have provided thorough discussions regarding the advantages and disadvantages to numerous imaging and invasive modalities used to assess valve function in rodents9,10,11, to date, we are not aware of a publication that provides a comprehensive, step-by-step protocol for phenotyping heart valve function in mice.
The purpose of this manuscript is to describe the methods and protocols to phenotype heart valve function in mice. All methods and procedures have been approved by the Mayo Clinic Institutional Animal Care and Use Committee. Key components of this protocol include the depth of anesthesia, the evaluation of cardiac function, and the evaluation of heart valve function. We hope this report will not only serve to guide investigators interested in pursuing research in the field of heart valve disease, but will also start a national and international dialogue related to protocol standardization to ensure data reproducibility and validity in this rapidly-growing field. Importantly, successful imaging using high-resolution ultrasound systems requires a working knowledge of the principles of sonography (and terminology commonly used in sonography), an understanding of the fundamental principles of cardiac physiology, and significant experience with sonography to allow for accurate and time-efficient assessment of cardiac function in rodents.

<table><tbody><tr><td>High resolution ultrasound machine</td><td>VisualSonics, Fujifilm</td><td>Vevo 2100&amp;nbsp;</td><td></td></tr><tr><td>Isoflurane diffuser (capable of delivering 1 % to 1.5 % isoflurane mixed with 1 L/min 100% O&lt;sub&gt;2&lt;/sub&gt;</td><td>VisualSonics, Fujifilm</td><td>N/A</td><td></td></tr><tr><td>Transducers for small mice (550D) or larger mice (400)</td><td>MicroScan, VisualSonics, Fujifilm</td><td>MS 550D, MS 400</td><td></td></tr><tr><td>Animal platform</td><td>VisualSonics, Fujifilm</td><td>11503</td><td></td></tr><tr><td>Advanced physiological monitoring unit</td><td>VisualSonics, Fujifilm</td><td>N/A</td><td></td></tr><tr><td>Isoflurane</td><td>Terrell</td><td>NDC 66794-019-10</td><td></td></tr><tr><td>Nose cone and tubing connected to isoflurane diffuser and 100% O&lt;sub&gt;2&lt;/sub&gt;</td><td>Custom Engineered in-house</td><td>--</td><td></td></tr><tr><td>Hair razor</td><td>Andis Super AGR+ vet pack clipper</td><td>AD65340</td><td></td></tr><tr><td>Ultrasound gel</td><td>Parker Laboratories</td><td>REF 01-08</td><td></td></tr><tr><td>Electrode gel&amp;nbsp;</td><td>Parker Laboratories</td><td>REF 15-25</td><td></td></tr><tr><td>Adhesive tapes</td><td>Fisher Laboratories</td><td>1590120B</td><td></td></tr><tr><td>Paper towels</td><td></td><td></td><td></td></tr></tbody></table>

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.

Examples of images that are routinely obtained from animal cardiac ultrasound imaging are included in this manuscript. An illustration of transducer placement on the animal&#39;s chest is provided to give the reader a clear understanding of where the transducer is positioned to obtain the images as described. A photograph of the ultrasound laboratory set-up is also included to emphasize the importance of the proper equipment, particularly the ultrasound transducer to be used and the method of anesthesia. The 2D/B-mode, M-mode, and color and Doppler displays of the normal and abnormal valves, right and left ventricles, and aortic root are properly labeled. Although strain-rate imaging is not routinely performed, an example is also included.
Mitral regurgitation is characterized by a high, usually non-laminar blood flow velocity (mosaic coloring) across the valve during systole (Figure 5). The presence of such a mosaic-color Doppler flow pattern from the left ventricle to the left atrium across the MV, occurring after the QRS complex in the ECG tracing, allows for an unequivocal diagnosis of MR. When this occurs in the absence of aortic valve regurgitation and/or left ventricular dysfunction, this can be characterized as isolated mitral valve prolapse. If there is significant dilation of the left ventricle (due to experimentally-induced heart failure or excessive depth of anesthesia), this can be characterized as ischemic mitral regurgitation (or regurgitation secondary to cardiac dysfunction). A pulsed-wave spectral Doppler display can be used to confirm the presence and timing of a regurgitant jet of blood flow.
A normal aortic valve has three thin, pliable cusps that open and close adequately during each cardiac cycle. Aortic valve cusp separation is measured in 2D-guided M-mode of the aortic valve in the long-axis view. Electronic calipers are used to measure from the leading edge of the right aortic cusp to the leading edge of the left aortic cusp (Figure 3). Aortic valve cusp-separation distance in normal mice is 0.9 to 1.3 mm. Color Doppler shows a laminar flow across the valve and into the aortic root during systole. Turbulent flow can be appreciated in conditions of increased flow, such as in aortic valve regurgitation, or increased pressure, as in aortic valve stenosis. This is demonstrated as mosaic coloring in the outflow tract. Even small amounts of aortic valve regurgitation can result in significant increases in peak transvalvular velocity due to hyperdynamic cardiac function and increased left ventricular preload. Peak aortic velocity in normal mice ranges from 0.90 m/s to 1.50 m/s. Peak aortic valve velocity of &#62; 5 m/s has been recorded in mice with severe aortic valve stenosis.
The pulsed-wave spectral Doppler tracings can also be used to provide an index of pulmonary artery hemodynamics12 (Figure 8). Pulmonary artery acceleration time is the time interval from the onset of systolic pulmonary arterial flow to the peak flow velocity. Right ventricular ejection time is the interval between the onset of right ventricular ejection to the point at which there is cessation of systolic pulmonary artery systolic flow. The combination of a shortened pulmonary artery acceleration time with a decrease in the ratio of pulmonary artery acceleration time to right ventricular ejection time suggests the presence of pulmonary arterial hypertension (which can be confirmed using invasive or direct measures of pulmonary arterial or right ventricular pressure).
<img alt="Figure 1" src="/files/ftp_upload/54110/54110fig1.jpg" /> 
Figure 1: Animal Cardiac Ultrasound Laboratory. The laboratory is equipped with the small-animal-dedicated ultrasound machine with high frequency (30 MHz and 40 MHz) transducers (MS 400 and MS 550D), isoflurane diffuser, animal platform, temperature and heart rate monitor, 1% to 1.5% isoflurane mixed with 1 L/min 100% O2, nose cone and tubing connected to isoflurane diffuser and 100% O2, hair razor, ultrasound gel, electrode gel, adhesive tapes, and paper towels. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54110/54110fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54110/54110fig2.jpg" /> 
Figure 2: Basic Transducer Positions. (A) Parasternal window. The transducer head is positioned in the left parasternal border, with the image index marker of the transducer directed caudally. From this position, the long-axis view of the left ventricle, aortic valve, and aortic root and the short-axis view of the pulmonic valve can be obtained. (B) From the parasternal window, the transducer head is rotated counterclockwise, with the notch directed posteriorly. From this position, the short-axis view of the left ventricle and aortic valve and the long-axis view of the pulmonic valve can be obtained. (C) Apical window. The transducer head is positioned at the apex of the heart. From this position, the long-axis view of the right and left ventricles and mitral and tricuspid valves can be obtained. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54110/54110fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54110/54110fig3.jpg" /> 
Figure 3: Assessment of Aortic Valve Function in a Normal Mouse versus Aortic Valve Function in a Mouse with Calcific Aortic Valve Disease. (A) 2D image of a normal aortic valve in the long-axis view. Note that the aortic valve opens well during systole. (B) M-mode image depicting normal aortic valve function (box-like appearance). Note that the cusp-separation distance is measured at 1.12 mm. (C) Spectral Doppler display of peak velocity across the normal aortic valve was meaured at 1.3 m/s. (D) 2D image of a calcified aortic valve in the long-axis view from a low-density lipoprotein receptor deficient (ldlr-/-) and apolipoprotein B100-only (apoB100/100) mouse fed with western diet. The cusps are thickened and have increased echogenicity, which results in restricted opening during systole. (E) An M-mode image depicting the same stenotic aortic valve shows a cusp-separation distance measurement of 0.7 mm. (F) The spectral Doppler display of peak velocity across the stenotic aortic valve was meaured at 4.6 m/s. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54110/54110fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54110/54110fig4.jpg" /> 
Figure 4: M-mode of a Normal Mitral Valve. From the apical window, a long-axis view of the mitral valve is obtained. The M-mode line of interrogation is applied across the mitral valve leaflet. While mitral leaflet thickness can theoretically be measured using electronic calipers, this can be extremely challenging given the thin, poorly echogenic, and rapidly moving leaflets of the normal mitral valve. The arrows point to the M-mode of the mitral valve leaflet in systole. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54110/54110fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54110/54110fig5.jpg" /> 
Figure 5: Evidence of a Mitral Valve Regurgitant Jet using Color Doppler Imaging. From the parasternal window, a modified long-axis view of the mitral valve is obtained. Color Doppler interrogation shows a mosaic-color jet at the mitral valve during systole (highlighted by an arrow). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54110/54110fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54110/54110fig6.jpg" /> 
Figure 6: Long-axis View of the Main Pulmonary Artery and its Major Branches. The long-axis view of the main pulmonary artery (MPA) and right (RPA) and left (LPA) branches can be obtained from the parasternal window. The right ventricular outflow tract (RVOT), pulmonic valve (PV), and aorta (AO) are partly seen. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54110/54110fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54110/54110fig7.jpg" /> 
Figure 7: M-mode Image Depicting a Normal Pulmonic Valve. From the parasternal window, both short- and long-axis views of the pulmonic valve can be obtained. The M-mode line of interrogation is applied across the pulmonic valve. The pulmonic valve cusp-separation (arrows) distance can be measured from this view. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54110/54110fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/54110/54110fig8.jpg" /> 
Figure 8: Pulsed-wave Doppler Interrogation of Flow Across the Pulmonic Valve. The pulmonary artery acceleration time (PAAT) is the time interval from the onset of systolic pulmonary arterial flow to the peak flow velocity. Right ventricular ejection time (RVET) is the interval between the onset of right ventricular ejection to the point at which there is cessation of flow. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54110/54110fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/54110/54110fig9.jpg" /> 
Figure 9: M-mode Image Depicting a Short-axis View of the Left Ventricle. From the parasternal window, the short-axis view of the left ventricle is obtained by rotating the transducer head counterclockwise so that the image index marker points posteriorly or dorsally. The M-mode line of interrogation is applied across the left ventricle at the level of the papillary muscles. Left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), and anterior wall (AW) and posterior wall (PW) thicknesses can be readily measured. Be careful not to include the papillary muscle (*) in any measurements. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54110/54110fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" src="/files/ftp_upload/54110/54110fig10.jpg" />
Figure 10: Color Doppler Evaluation and Pulsed-wave Doppler Spectral Display of Mitral Valve Inflow. (A) Image showing a color Doppler evaluation of mitral valve inflow in the apical long-axis view. Note that the 2D color Doppler image can be a critical tool for guiding the appropriate sample volume position for the acquisition of pulsed-wave Doppler tracings (depicted in panel B). (B) Spectral display of mitral valve inflow using pulsed-wave Doppler. The pulsed-wave Doppler assessment of blood flow across the mitral valve (in the apical long-axis view) is performed to assess left ventricular diastolic function. The sample volume is placed at the tips of the mitral valve leaflets. The isovolumic relaxation time (IVRT), isovolumic contraction time (IVCT), left ventricular ejection time (LVET), and peak mitral inflow velocity (E) can all be derived from the spectral display of pulsed-wave Doppler velocities across the mitral valve. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54110/54110fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" src="/files/ftp_upload/54110/54110fig11.jpg" /> 
Figure 11: Tissue Doppler Imaging of the Septal Mitral Annulus. From the apical window, a long-axis view of the mitral valve is obtained. The tissue Doppler sample volume is positioned at the septal region of the mitral annulus. The ratio between the peak mitral inflow velocity (variable E in Figure 10B) and the peak mitral annulus tissue velocity (e&#39;, denoted by white arrows) is used to assess left ventricular diastolic function (commonly referred to as E/e&#39;). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54110/54110fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" src="/files/ftp_upload/54110/54110fig12.jpg" /> 
Figure 12: Assessment of the Strain and Strain Rate of the Left Ventricular Myocardium. There are specialized analysis software packages available commercially, and the strain and strain rate variables can be obtained as measures of early or sub-clinical changes in intrinsic myocardial contractile properties. The examples shown above depict radial strain and strain rate in commonly-acquired imaging planes in mice. Note that these imaging planes (and the subsequent shape of the strain tracings) can differ from images in humans, which are frequently acquired in the apical long-axis or 4-chamber view. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54110/54110fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice at JoVE.com

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

echocardiographic-approaches-protocols-for-comprehensive-phenotypic

Nanyang Technological University

Research

JoVE Journal

Medicine

15.8K Views.  Mayo Clinic. This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice. 

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

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 ...

Murine Echocardiography and Ultrasound Imaging

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1 Echocardiography provides a powerful, non-invasive tool to serially assess cardiac morphometry and function in a living animal.2 However, using this technique on mice poses unique challenges owing to the small size and rapid heart rate of these animals.3 Until recently, few ultrasound systems were capable of performing quality echocardiography on mice, and those generally lacked the image resolution and frame rate necessary to obtain truly quantitative measurements. Newly released systems such as the VisualSonics Vevo2100 provide new tools for researchers to carefully and non-invasively investigate cardiac function in mice. This system generates high resolution images and provides analysis capabilities similar to those used with human patients. Although color Doppler has been available for over 30 years in humans, this valuable technology has only recently been possible in rodent ultrasound.4,5 Color Doppler has broad applications for echocardiography, including the ability to quickly assess flow directionality in vessels and through valves, and to rapidly identify valve regurgitation. Strain analysis is a critical advance that is utilized to quantitatively measure regional myocardial function.6 This technique has the potential to detect changes in pathology, or resolution of pathology, earlier than conventional techniques. Coupled with the addition of three-dimensional image reconstruction, volumetric assessment of whole-organs is possible, including visualization and assessment of cardiac and vascular structures. Murine-compatible contrast imaging can also allow for volumetric measurements and tissue perfusion assessment.

This video demonstrates use of a rail-mounted high-frequency ultrasound probe to perform echocardiography on an anesthetized mouse. The methods describe both conventional two-dimensional and M-mode measurements of cardiac function in addition to newer, more powerful tools such as color Doppler, strain analysis, as well as general and targeted contrast imaging.

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1  ...

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 ...

A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe CHD died before adulthood, with the maximum mortality during the first week of life. Remarkable advances in surgical techniques, diagnostic approaches, and medical management have led to marked improvements in outcomes. To address the critical research needs of understanding congenital heart defects, murine models have provided an ideal research platform, as they have very similar heart anatomy to humans and short gestation rates. The combination of genetic engineering with high-throughput phenotyping tools has allowed for the replication and diagnosis of structural heart defects to further elucidate the molecular pathways behind CHDs. The use of noninvasive fetal echocardiography to screen the cardiac phenotypes in mouse models coupled with the high fidelity of Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) histopathology with three-dimensional (3D) reconstructions enables a detailed view into the anatomy of various congenital heart defects. This protocol outlines a complete workflow of these methods to obtain an accurate diagnosis of murine congenital heart defects. Applying this phenotyping protocol to model organisms will allow for accurate CHD diagnosis, yielding insights into the mechanisms of CHD. Identifying the underlying mechanisms of CHD provide opportunities for potential therapies and interventions.

This article details murine congenital heart disease (CHD) diagnostic methods using fetal echocardiography, necropsy, and Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) followed by three-dimensional (3D) reconstruction.

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe ...

Developmental Biology

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

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

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

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

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 ...

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.

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 ...

Revealing Subtle Changes in Cardiac Function using Transthoracic Dobutamine Stress Echocardiography in Mice

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic dobutamine stress echocardiography in humans, a reductionist investigation approach to unmask subtle changes in cardiac function has become possible. Here, we provide a protocol for using this technique in mice to facilitate expanded analysis of LV architecture and function in physiology and pathology enabling the observation of alterations in models of cardiac disease hidden in unstressed hearts. This investigation can be performed in one and the same animal and allows both, basal and pharmacologically stress-induced measurements. We outline detailed criteria for appropriate anesthesia, imaging-based LV analysis, consideration of intra- and interobserver variability, and obtaining positive inotrope response that can be attained in mice after intraperitoneal injection of dobutamine under near physiological conditions. To recapitulate the characteristics of human physiology and disease in small animal models, we highlight critical pitfalls in evaluation, e.g., a pronounced Bowditch effect in mice. To further meet translational objectives, we compare stress-induced effects in humans and mice. When used in translational studies, attention must be paid to physiological differences between mice and human. Experimental rigor dictates that some parameters assessed in patients can only be used with caution due to restrictions in spatial and temporal resolution in mouse models.

Left ventricular dysfunction constitutes the final common pathway for a host of cardiac disorders. We here provide a detailed protocol of transthoracic dobutamine stress echocardiography approach for comprehensive evaluation of the left ventricular function of mouse models of cardiac disease as well as cardiac phenotyping.

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic ...

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 ...