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

1. Prepare the Materials and Equipment (Table 1 and Figure 1)
<ol>
	<li>Turn on the ultrasound machine. Enter the animal ID, date, and time (for serial imaging experiments) and other relevant information.</li>
	<li>Use a high-frequency ultrasound transducer, 40 MHz for imaging mice less than ~20 g or 30 MHz for mice greater than ~20 g.</li>
	<li>Connect the platform to the electrocardiogram (ECG) monitor for ECG gating of imaging for certain modalities. 
	NOTE: Critically, this also allows for the instantaneous ca...

<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.

Induction of anesthesia
Proper induction and maintenance of anesthesia is critical for the accurate assessment of changes in heart valve and cardiac function in mice. Given the rapid induction of anesthesia elicited by isoflurane and the relatively long wash-out time of this anesthetic following deep anesthesia, we do not use a stand-alone anesthesia chamber for induction. Instead, as noted in detail above, animals are guided directly to the anesthesia cone, which allows for rapi...

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 border="0" cellpadding="0" cellspacing="0" width="1906">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">High resolution ultrasound machine</td>
			<td style="width:453px;">VisualSonics, Fujifilm</td>
			<td style="width:293px;">Vevo 2100&#160;</td>
			<td style="width:467px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:693px;">Isoflurane diffuser (capable of delivering 1 % to 1.5 % isoflurane mixed with 1 L/min 100% O2</td>
			<td>VisualSonics, Fujifilm</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;width:693px;">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 height="34">
			<td height="34" style="height:34px;width:693px;">Animal platform</td>
			<td>VisualSonics, Fujifilm</td>
			<td>11503</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">Advanced physiological monitoring unit</td>
			<td>VisualSonics, Fujifilm</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">Isoflurane</td>
			<td>Terrell</td>
			<td>NDC 66794-019-10</td>
			<td></td>
		</tr>
		<tr height="74">
			<td height="74" style="height:74px;width:693px;">Nose cone and tubing connected to isoflurane diffuser and 100% O2</td>
			<td>Custom Engineered in-house</td>
			<td>--</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">Hair razor</td>
			<td>Andis Super AGR+ vet pack clipper</td>
			<td>AD65340</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">Ultrasound gel</td>
			<td>Parker Laboratories</td>
			<td>REF 01-08</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">Electrode gel&#160;</td>
			<td>Parker Laboratories</td>
			<td>REF 15-25</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">Adhesive tapes</td>
			<td>Fisher Laboratories</td>
			<td>1590120B</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:693px;">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 metho...

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 ...

The overall goal of this echo protocol is to show methods and approaches used to image heart valve function in rodents. The methods we'll be showing you today can answer key questions in the field of heart disease, with a specific emphasis on evaluating the heart as integrated unit under conditions of heart valve disease. The implications of this approach are quiet profound for the field of heart valve diseases because the comprehensive echocardiographic evaluation of the heart will not only allow for detection of valvular dysfunction in multiple heart valves, but will also allow investigators to understand the complex interplay between cardiac function and various measures of heart valve function.Though this method can be leveraged to characterize animal models in which heart valve disease is our primary defect, it can also be applied to other systems where heart valve dysfunction may appear secondary to other insults such as heart failure, or a thoracic aortic aneurysm. Thus, it is our recommendation that comprehensive cardiac and heart valve phenotyping should be routinely performed in most research studies. We first had the idea to film our approach to comprehensive cardiac and heart valve phenotyping after attending a number of meetings where there was a profound variability in the methods and protocols used to assess heart valve function.It is our hope that this manuscript and the accompanying video spurs discussion in the field regarding the best methodologies and standards to follow, which ultimately aligns with recent initiatives from the National Institutes of Health to improve rigor and reproducibility of research studies. The main advantage of echocardiography is that it is able to noninvasively assess morphology and function of the heart in real time. It is easy to perform, and can be performed serially.Thus, it is most ideal in serial follow up studies. Generally, individuals new to cardiac and heart valve imaging will struggle due to logistical challenges associated with administration and titration of anesthesia, as well as the technical challenges associated with the very small anatomic windows that can be used for echocardiography and the need to precisely identify cardiac structures and probe orientations while imaging. We hope that visual demonstration of our approach accelerates the rate at which investigators can learn to conduct echocardiography in mice, and will ultimately serve as a platform to discuss best practices for small animal imaging.To begin, turn on the ultrasound machine equipped with a high frequency ultrasound transducer. Enter the ID of the animal to be measured, along with date, time, and any other relevant information. Preheat the platform to 37 degrees celsius, and then gently pick up the mouse by it's tail, and firmly hold the animal at the nape of it's neck.Guide the nose of the animal into the nose cone and initiate anesthesia as described in the accompanying text protocol. Once sedated, quickly and accurately lay the animal on the platform in a supine position, making sure that the forefeet and hindfeet lie on the ECG sensors of the platform. Use adhesive tape to secure the animal on all four limbs, to stabilize the head in the nose cone apparatus, and to stabilize the tail.Next, check the heart rate using the ECG sensors in the platform, or using an external device. Ensure that the baseline heart rate is between 600 to 700 beats per minute. Monitor the heart rate and ensure that it does not fall below 450 beats per minute under any circumstances.In addition, monitor the body temperature using a rectal thermometer and keep the temperature between 36.5 degrees celsius and 38 degrees celsius. As long as the vitals are stable, use an electric clipper designed for use with fine hair to shave off the hair from the mouse's chest. Then, take a damp paper towel and wipe the chest to remove any remaining hairs.With the animal securely fastened on the platform, and the head facing away, tilt the table 15 to 20 degrees to the left to bring the heart closer to the chest wall. Then, apply a generous amount of ultrasound gel on the transducer or directly on the animal's chest. Position the transducer parasternally about 90 degrees perpendicular with the long axis of the heart so that the image index marker of the transducer is pointing posteriorly.While in two DB mode slide the transducer's defilade until the aortic valve comes into view along the short axis. Next, rotate the transducer clockwise until the image index marker points cadad. This is the parasternal long axis view.Observe the aortic route, aortic valve, left ventricular outflow tract, mitral valve, left atrium, and part of the right ventricular outflow tract on the image display. Measure the largest atrial posterior dimension of the aorta using the electronic caliper associated with the measurement tool that is embedded in the machine. Next, reduce the image width so that only the aortic valve is on the image display by adjusting the image width button in the control panel.Position the M mode line of interrogation where it intersects the tips of the aortic valve to accurately assess aortic valve cusp separation. In the M mode display of the aortic valve, measure the cusp separation distance using the electronic caliper. While still in the parasternal long axis view of the aortic valve, press the color doppler control key in the control panel to apply color doppler to the region of the aortic valve.Next, press the post wave doppler control key. Using the track ball, place the post wave sample volume in the proximal ascending aorta just above the aortic valve, making sure that the angle between the ultrasound beam and the blood flow is less than 60 degrees by tilting the platform, and or the transducer. Then, measure the peak velocity from the spectral display using the electronic calipers.Start by placing the transducer in the apical position in B mode and positioning the transducer so that it is angled towards the head of the mouse. Observe the right ventricle, left ventricle, right atrium and left atrium, on the image display. From the apical four chamber view bring the mitral valve in focus by reducing the image width.Then, place the M mode cursor across the mitral valve to assess the thickness of the leaflets. Using the apical four chamber view, apply color doppler to image the flow from the left atrium through the mitral valve during diastole. Observe the flow across the mitral valve which is encoded in red.From the apical lung axis view, tilt or point the transducer tip using a rocking motion so that the right ventricle is at the center of the image display. Reduce the image width so that only the right ventricle is visible in the image display. Then, apply color doppler in the region of the tricuspid valve.Next, move the transducer to a modified parasternal long axis position at the level of the aortic valve. Then, tilt the transducer slightly upward to obtain a short axis view of the pulmonic valve. In this view, apply M mode imaging to evaluate the separation distance of the pulmonic valve.Then, apply color doppler in the region of the pulmonic valve to assess for valvular regurgitation and stenosis. In two DB mode, place the transducer in the parasternal short axis position at the level of the papillary muscles to obtain a short axis view of the left ventricle. From this view, press the M mode button located in the control panel.Using the track ball, position the M mode cursor at the center of the left ventricular cavity at the level of the papillary muscles, and obtain M mode images. Then, measure the left ventricular cavity dimension at end diastole where the distance between the interior wall and posterior wall is the largest, and an end systole where the inward motion of both interior and posterior walls is maximal. Next, move the transducer to the apical window, apply color on the mitral valve, and place the sample volume at the tips of the mitral valve leaflets.Measure the peak mitral inflow velocity from the spectral display of pulse waved doppler velocities across the mitral valve. With the sample volume positioned between left ventricle inflow and outflow not the mitral and aortic valve closing and opening signals. Measure the isovolumic relaxation time, isovolumic contraction time, and left ventricular ejection time.Then, perform tissue doppler imaging of the mitral annulus in the apical long axis view. Press the TGI control key and place the sample volume at the medial aspect of the mitral annulus. When finished, remove any excess ultrasound gel from the mouse.Gently remove the tape securing the animal, and turn off the anesthesia. Place the animal on an absorbent paper towel and observe the animal until sternal recumbency is attained. Shown here is an assessment of aortic valve function in a normal mouse versus the aortic valve function in a mouse with calcific aortic valve disease.In the mouse with calcified valves, the cusps are thickened and have increased echogenicity which results in restricted opening during systole. From the apical window a long axis view of the mitral valve is presented. The mitral leaflet thickness can be measured using the M mode line of interrogation.Measurement of the thickness of the mitral valve can be extremely challenging given the thin, poorly echogenic, and rapidly moving leaflets of the normal mitral valve. The color doppler shown in this modified long axis view of the mitral valve shows a mosaic color jet at the mitral valve during systole. The blue color is evidence of mitral valve regurgitation.From the parasternal window, both short and long axis views of the pulmonic valve are obtained. In this image, the M mode line of interrogation is applied across the pulmonic valve, and the valve cusp separation distance can be measured from this view. Once mastered, this technique can be done in 15 to 20 minutes if it is performed properly.While attempting this procedure, it's important to have a basic knowledge of cardiac anatomy and physiology, a working knowledge of the principles and terminology of sonography, and experience in cardiac ultrasound to allow for accurate and time efficient assessment of cardiac function in rodents. After watching this video you should have a good understanding of how to perform cardiac ultrasound in rodents by obtaining images from various imaging planes, and measuring echo doppler parameters that adequately describe cardiac and heart valve function.

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15.7K vistas.  Mayo Clinic. The overall goal of this echo protocol is to show methods and approaches used to image heart valve function in rodents. The methods we'll be showing you today can answer key questions in the field of heart disease, with a specific emphasis on evaluating the heart as integrated unit under conditions of heart valve disease. The implications of this approach are quiet profound for the field of heart valve diseases because the comprehensive echocardiographic evaluation of the heart will not only ...