This work was supported by the Huffington Center on Aging at Baylor College of Medicine, Geriatrics, CVS DeBakey Heart Center at Houston Methodist Hospital and BCM, NIH RO-1 HL 13870 to ML Entman, as well as a Career Development Award # IK2BX002410 from the United States Department of Veterans Affairs (LMP), Biomedical Laboratory Research and Development Program.
We are immensely grateful to Mark Entman M.D. for sharing his wisdom and supporting this work.

All animals were cared for in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals at Baylor College of Medicine.
1. Set-up
<ol>
	<li>Turn on the echocardiographic system. Start the software by selecting &#34;Cardiac Measurements&#34; from the drop down menu displayed and click &#34;Initialize&#34;.</li>
	<li>To begin a new study click on the &#34;New&#34; button on the upper part of the screen and enter the demographic informat...

<ol>
	<li>Upadhya, B., Taffet, G. E., Ping, C., Kitzman, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+failure+with+preserved+ejection+fraction+in+the+elderly:+scope+of+the+problem.">Heart failure with preserved ejection fraction in the elderly: scope of the problem.</a> Journal of Molecular and Cellular Cardiology. 83, 73-87 (2015).</li><li>Mclaughlin, V. V., Archer, S. 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=ACCF+/+AHA+Expert+Consensus+Document+Expert+Consensus+Document+ACCF+/+AHA+2009+Expert+Consensus+Document+on+Pulmonary+Hypertension+A+Report+of+the+American+College+of+Cardiology+Foundation+Task+Force+on+Expert+Consensus+Documents+and+the+American+Heart+Association.">ACCF / AHA Expert Consensus Document Expert Consensus Document ACCF / AHA 2009 Expert Consensus Document on Pulmonary Hypertension A Report of the American College of Cardiology Foundation Task Force on Expert Consensus Documents and the American Heart Association.</a> Circulation. , 2250-2294 (2009).</li><li>Cieslik, K. A., Taffet, G. E., Carlson, S., Hermosillo, J., Trial, J., Entman, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune-inflammatory+dysregulation+modulates+the+incidence+of+progressive+fibrosis+and+diastolic+stiffness+in+the+aging+heart.">Immune-inflammatory dysregulation modulates the incidence of progressive fibrosis and diastolic stiffness in the aging heart.</a> Journal of Molecular and Cellular Cardiology. 50 (1), 248-256 (2011).</li><li>Tate, C. A., Taffet, G. E., Hudson, E. K., Blaylock, S. L., McBride, R. P., Michael, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+calcium+uptake+of+cardiac+sarcoplasmic+reticulum+in+exercise-trained+old+rats.">Enhanced calcium uptake of cardiac sarcoplasmic reticulum in exercise-trained old rats.</a> Am J Physiol Heart Circ Physiol. 258 (2), H431-H435 (1990).</li><li>Kitzman, D. W., Daniel, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diastolic+Heart+Failure+in+the+Elderly.">Diastolic Heart Failure in the Elderly.</a> Heart Failure Clinics. 3 (4), 437-453 (2007).</li><li>Sanderson, J. E., Rusconi, 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=How+to+diagnose+diastolic+heart+failure+a+consensus+statement+on+the+diagnosis+of+heart+failure+with+normal+left+ventricular+ejection+fraction+by+the+Heart+Failure+and+Echocardiography+Associations+of+the+European+Society+of+Cardiology.">How to diagnose diastolic heart failure a consensus statement on the diagnosis of heart failure with normal left ventricular ejection fraction by the Heart Failure and Echocardiography Associations of the European Society of Cardiology.</a> Eur Heart J. , 2539-2550 (2007).</li><li>Medrano, G., Hermosillo-Rodriguez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+Atrial+Volume+and+Pulmonary+Artery+Diameter+Are+Noninvasive+Measures+of+Age-Related+Diastolic+Dysfunction+in+Mice.">Left Atrial Volume and Pulmonary Artery Diameter Are Noninvasive Measures of Age-Related Diastolic Dysfunction in Mice.</a> The Journals of Gerontology Series A: Biological Sciences and Medical Sciences. (16), 1-10 (2015).</li><li>Franco, F., Thomas, G. D., 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=Magnetic+Resonance+Imaging+and+Invasive+Evaluation+of+Development+of+Heart+Failure+in+Transgenic+Mice+With+Myocardial+Expression+of+Tumor+Necrosis+Factor-+.">Magnetic Resonance Imaging and Invasive Evaluation of Development of Heart Failure in Transgenic Mice With Myocardial Expression of Tumor Necrosis Factor- .</a> Circulation. 99, 448-454 (1999).</li><li>Jenkins, C., Bricknell, K., Marwick, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+real-time+three-dimensional+echocardiography+to+measure+left+atrial+volume:+Comparison+with+other+echocardiographic+techniques.">Use of real-time three-dimensional echocardiography to measure left atrial volume: Comparison with other echocardiographic techniques.</a> Journal of the American Society of Echocardiography. 18 (9), 991-997 (2005).</li><li>Ujino, K., Barnes, M. 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=Two-dimensional+echocardiographic+methods+for+assessment+of+left+atrial+volume.">Two-dimensional echocardiographic methods for assessment of left atrial volume.</a> The American journal of cardiology. 98 (9), 1185-1188 (2006).</li><li>Scalia, G. M., Scalia, I. G., 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=ePLAR+-+The+echocardiographic+Pulmonary+to+Left+Atrial+Ratio+-+A+novel+non-invasive+parameter+to+differentiate+pre-capillary+and+post-capillary+pulmonary+hypertension.">ePLAR - The echocardiographic Pulmonary to Left Atrial Ratio - A novel non-invasive parameter to differentiate pre-capillary and post-capillary pulmonary hypertension.</a> International Journal of Cardiology. 212, 379-386 (2016).</li></ol>

There are three critical steps to successfully measure the LAV, Aorta and PA diameters. During the Set-up it is important to completely remove the fur on the chest; failure to do so will result in interference with image quality. During the Image Acquisition steps it is important to obtain each image with their corresponding probe and filter as the frames per second vary from probe to probe: that is 25 MHz and Cardiac Filter for the assessment of the LAV and 30 MHz and Abdominal Filter for assessment of the Aorta and PA ...

The present methodology teaches the investigator how to measure and use the LAV, Aorta diameter and PA diameter with 2D echocardiography in mice under isoflurane. Heart Failure with preserved Ejection Fraction (HFpEF) in elderly people affects up to 10% in those 80 years and older1 and results in significant morbidity and mortality. Significant mortality also occur from Pulmonary Hypertension (PH), an insidious disease process presenting with similar symptoms as HFpEF, in which elevated pulmonary artery pressures lead to exertional dyspnea, progressive right heart failure, and often death.2 The increasing prevalence of both HFpEF and PH signify the need for developing a method that allows for accurate evaluation and monitoring of interventions in murine models in a non-invasive, non-terminal approach.
Aging leads to a deterioration of diastolic function via alterations in ventricular-arterial stiffening, vascular dysfunction, inflammation3, impaired calcium regulation4, decreased &#946;-adrenergic responsiveness, and physical deconditioning producing slowed active relaxation and increased passive stiffness. Over time this leads to increased LV filling pressure and compensatory enlargement of the LA.5
Though other etiologies such as valvular dysfunction (mitral regurgitation or stenosis) and infiltrative processes cause elevations in pressure and volume in the LA1, the European Society of Cardiology supports the addition of LA size as a noninvasive reflection of LV function.6
Correlations between LA volume and invasive measures of diastolic function have been studied in mice. LA volume correlated with differences in function determined invasively within age groups for both 14- and 31-months-old mice. In the 14-months-old mice, LA volume correlated with three standard invasive measures of diastolic function &#8722;dP/dtmin (r2 = .5, p &#60;0.05), Tau (time constant of relaxation, (r2 = .6, p &#60;0.05), and left ventricular end diastolic pressure (r2 = .25, p &#60;0.05). For the 31 months-old mice, the correlations between LA volume and -dP/ dtmin (r2 = .92, p &#60;0.05) and LVEDP (r2 = .61, p &#60;0.05) were apparent though the relationship with Tau was less clear. Therefore, LA volume increased with diastolic impairment not only across groups but within age groups.7
Studies of cardiac function in murine models using catheter techniques to evaluate cardiac performance, although rigorous and reliable, are limited due to their incompatibility with repeated assessments.8 Alternatives to invasive measures such as MRI and 3D echocardiography may also be more accurate than 2D echocardiographic techniques, but they are more expensive; 2D echocardiography is considered adequate for LA volume evaluation. 9,10
Assessment of the LA volume and PA diameter with echocardiography allows the discrimination between models that produce primary increases in pulmonary artery resistance resulting in an increase in PA diameter with no change in LA pressure or LAV from those where the Pulmonary Artery and Left Atrium both enlarge as a result of elevated filling pressures on the left side of the heart. This approach was taken by Scalia et al. who showed that in people, the echocardiographic Pulmonary Artery to Left Atrial Ratio (ePLAR) is a parameter to accurately differentiate between patients with pre-pulmonary capillary hypertension and post-capillaryhypertension.11

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			<td height="42" style="height:42px;width:203px;">Vevo 770 high-resolution in vivo micro-imaging system</td>
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			<td style="width:119px;">Vevo 770-120</td>
			<td style="width:267px;">Echocardiographic Equipment</td>
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		<tr height="84">
			<td height="84" style="height:84px;width:203px;">707B RMV (Real time MicroVisualization) 30 MHz Scanhead with encapsulated transducer&#160;</td>
			<td style="width:85px;">Visual Sonics</td>
			<td style="width:119px;">707B-256</td>
			<td>Real Time Microvisualization Scanhead</td>
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			<td height="84" style="height:84px;width:203px;">710B RMV (Real time MicroVisualization) 25 MHz Scanhead with encapsulated transducer&#160;</td>
			<td style="width:85px;">Visual Sonics</td>
			<td style="width:119px;">710B-159</td>
			<td>Real Time Microvisualization Scanhead</td>
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			<td height="63" style="height:63px;width:203px;">Vevo integrated rail system including physiological monitoring unit&#160;</td>
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			<td>Anesthesia System</td>
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			<td height="21" style="height:21px;width:203px;">Standard Gauze pads&#160;&#160;</td>
			<td style="width:85px;">McKeeson</td>
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			<td height="21" style="height:21px;width:203px;">Tape</td>
			<td style="width:85px;">Durapore</td>
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			<td height="21" style="height:21px;width:203px;">Nose cone&#160;</td>
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			<td>For anesthesia delivery</td>
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			<td height="21" style="height:21px;width:203px;">Water&#160;</td>
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			<td height="21" style="height:21px;width:203px;">Vet eye ointment&#160;</td>
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			<td>To prevent dryness&#160;</td>
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			<td height="21" style="height:21px;width:203px;">Cotton tipped applicators&#160;</td>
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			<td height="21" style="height:21px;width:203px;">USB Flash Drive</td>
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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.

Assessment of the LA volume and PA diameter with echocardiography allows the discrimination between models where increased pulmonary artery resistance results in increased PA diameter with no change in LAV from those where the Pulmonary Artery and Left Atrial enlargement are both a result of elevated filling pressures on the left side of the heart. Images displaying the differences in the two pathophysiologies are shown in Figure 4. Images of the LA and PA are from young,...

Watch this Scientific Journal Video about Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery at JoVE.com

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

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 ...

The overall goal of this procedure is to acquire and analyze echocardiographic images for the determination of the left atrial volume and the aorta, and pulmonary artery diameters in mice. This technique can discriminate between increases in the pulmonary artery resistance and diameter with no changes in the left atrial volume from models in which the PA and the LA are both enlarged. The main advantage of this technique, is that it is a non-terminal, non invasive approach that allows for serial assessment of the same animal.This technique has the potential to further our ability to monitor interventions and to initiate other studies in the field of cardiopulmonary science. Demonstration of this technique is critical as the proper probe and mouse positioning are necessary for optimal image acquisitioning. Before beginning the procedure, turn on the echocardiographic system and select cardiac measurements from the drop down menu.Click initialize to start the software. Then, click the new button to begin a new study, and enter the demographic information of the experimental animal. Click temp on and off to turn on the temperature controlled EKG board, and use the up and down arrows to set the temperature to 39 degrees Celsius.Next, turn on the oxygen delivery system and anesthesia chamber and confirm that the mouse is properly anesthetized by lack of response to paw pinch. Place the animal in the supine position on the temperature controlled EKG board. Apply lubricant ointment to the animal's eyes, and then tape each paw to the correspondent EKG electrode on the board.Observe the EKG tracing in the lower part of the monitor. Then, use an electric razor to shave the fur from the anterior thorax and upper abdominal area, and cover the chest with a generous amount of transducer gel. To measure the left atrium, use the handle beneath the EKG board to adjust the angle of the board to 30 to 45 degrees along the X axis, and 0 degrees along the Y axis.Next, place the 25 MHz scan head in the echocardiographic movable arm clamp in the long axis view, and select the cardiac measurement filter, and the 2-D B-Mode in the echocardiographic system. Rotate the X and Y adjustment torques to obtain a long axis view of the heart at the level of the aortic outflow tract and press the Cine Store button to acquire a five second movie recording. Now, switch the echocardiographic view to M-Mode, a sagittal view of the heart and a cursor will appear.Move the cursor so that it encompasses the whole left atrium at the level of the aortic outflow tract. And press the Frame Store button on the keyboard to acquire three images on this view. In B-Mode, rotate the movable arm clamp 90 degrees clockwise to set the scan head in the short axis view.Then, use the Y axis torque to find the intra-articular septum, and acquire a five second recording that includes the wall of the atrium. We're measuring the left atrium diameters using EKG tracing as a timing guide to select the image corresponding to the P wave time point. This corresponds to the pre-atrial contraction time, when the left atrium is at its largest capacity.To measure the great vessels, replace the 25 MHz scan head with a 30 MHz scan head, and select the abdominal measurements filter, and the 2-D B-Mode. Use the handle under the EKG board to adjust the angle of the board to a 5 degree angle along the X axis and 60 degrees along the Y axis. Use the X and Y axis adjustment torques to obtain a long axis view of the heart at the level of the aortic outflow tract, and acquire a five second movie recording.Rotate the movable arm clamp of the set 90 degrees clockwise to set the scan head in the short axis view. Then, use the Y axis torque to locate the pulmonary artery at the level of its bifurcation, and acquire a five second movie recording. To ensure the correct location of the pulmonary artery, you can also acquire the pulmonary artery doppler tracing by pressing the PW button.Observe the normal respiratory variation in the tracing. For the great vessels measurements, use the EKG tracing as a timing guide to select a time point after a QRS complex. This corresponds to the point where the great vessels are at the largest capacity.Echocardiographic assessment of the left atrium volume and the pulmonary artery diameter permits the discrimination between models, where an increased pulmonary artery resistance results in an increased pulmonary artery diameter with no change in the left atrium volume from instances when the pulmonary artery and left atrial enlargement are both a result of elevated filling pressures on the left side of the heart when the mitral valve is normal. By comparison, older animals develop an increase in both their pulmonary artery diameter, and left atrium volume, suggesting that the backward propagation of pressure drives the increase in the pulmonary artery diameter observed in this group. Using the demonstrated technique to measure the left atrium and great vessels allows the acquisition of both reproducible and consistent data between the experimental animals, as evidenced by these measurements obtained from a group of 19-month-old mice that underwent two subsequent echocardiographic assessments.Further, these results are easily reproduced between researchers, as apparent in the similarities observed between these four images obtained for the measurement of the left atrium super inferior diameter, and the pulmonary artery diameter acquired by two different researchers. Once this technique has been mastered, the images can be acquired in 15 minutes if it is performed properly. While attempting this procedure, it's important to monitor the heart rate throughout the whole study.A heart rate of 400 beats per minute is ideal. Following this procedure, other methods like dopplers can be performed to answer additional questions about systolic and diastolic cardiac functions. We hope that this technique will pave the way for researchers in the field of cardiopulmonary science to explore age-associated left ventricular diastolic dysfunction and pulmonary hypertension in mice, as well as therapeutic interventions for these and other pathologies.

murine-echocardiography-of-left-atrium-aorta-and-pulmonary-artery

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14.1K visualizações.  Baylor College of Medicine. The overall goal of this procedure is to acquire and analyze echocardiographic images for the determination of the left atrial volume and the aorta, and pulmonary artery diameters in mice. This technique can discriminate between increases in the pulmonary artery resistance and diameter with no changes in the left atrial volume from models in which the PA and the LA are both enlarged. The main advantage of this technique, is that it is a non-terminal, non invasive approach that allows for seri...