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 information of the mouse in study. For example mouse ID, sex, date of birth as well as the name of the operator doing the study.</li>
	<li>Turn on the temperature-controlled EKG board by clicking the &#34;TEMP ON/OFF&#34; button. Set it to a temperature of 39 &#176;C using the up and down arrows.</li>
	<li>To start setting up the anesthesia chamber, first make sure the oxygen and isoflurane levels are appropriate in each of the delivery systems.</li>
	<li>Place the mouse in the anesthesia chamber and induce with isoflurane and oxygen until recumbent and then transfer it to face mask. Confirm that the mouse is properly anesthetized by pinching its paw and checking for lack of movement. Maintain the isoflurane at the lowest possible level that avoids a positive paw withdrawal response.</li>
	<li>Place the mouse in a supine position on the temperature-controlled EKG board and tape each paw to the correspondent transducer element on the board. Optionally, use transducer cream to improve the signal.</li>
	<li>Apply artificial tears ophthalmic lubricant ointment on the mouse&#39;s eyes to prevent dryness.</li>
	<li>Once the mouse is taped to the EKG board, observe an EKG tracing in the lower part of the monitor. Monitor the heart rate throughout the whole study to ensure best results. A heart rate of 400 beats/min is ideal. Heart rates below this range may suggest isoflurane effects.</li>
	<li>Make sure the mouse keeps receiving oxygen and isoflurane by face mask.</li>
	<li>Use an electric razor to shave the body fur from the mouse&#39;s anterior thorax and upper abdominal area. Use depilatory cream if necessary to further ensure fur removal.</li>
	<li>Put a generous amount of transducer gel in the chest of the mouse.</li>
</ol>
2. Image Acquisition
<ol>
	<li>Left Atrium measurement
	<ol>
		<li>Locate the handle beneath the EKG board. Use the handle to adjust the angle of the movable EKG board in which the mouse was placed. Begin by adjusting the EKG board to a 30 - 45&#176; angle along the X axis and 0&#176; angle along the Y axis. Authors refer to it as &#34;supine&#34; position. Use this position for all of the Left Atrium measurements acquisition.</li>
		<li>Place the 25 MHz scan head in the echocardiographic movable arm clamp in a long axis view. Make sure to select the Cardiac Measurement Filter and 2D B-Mode in the echocardiographic system.</li>
		<li>Notice that the echocardiographic set has X and Y axis adjustment torques that can be moved clockwise or counterclockwise. Rotate them to get a long axis view of the heart at the level of the aortic outflow tract and acquire a 5 s movie recording. Acquire the scan by pressing the &#34;Cine Store&#34; button on the keyboard of the echocardiographic system. Use this to calculate the Superior-Inferior measurement of the LA.</li>
		<li>After the movie acquisition, switch the echocardiographic view to M-Mode by pressing the &#34;M-Mode&#34; button on the keyboard of the echocardiographic system.</li>
		<li>Observe a sagittal view and a cursor. Place the cursor so that it encompasses the whole LA at the level of the aortic outflow tract. Acquire 3 images of this new view. Acquire the scan by pressing the &#34;Frame Store&#34; button on the keyboard of the echocardiographic system. Use this to calculate the Anterior-Posterior measurement of the LA.</li>
		<li>Return to B-Mode by pressing the &#34;B-Mode&#34; button on the keyboard of the echocardiographic system.</li>
		<li>Rotate the movable arm clamp of the set 90&#176; clockwise. This will set the scan head in a short axis view.</li>
		<li>Using the Y axis torque find the intraarticular septum. Make sure to visualize the wall of the atrium. Acquire a 5 s movie recording. Acquire the scan by pressing the &#34;Cine Store&#34; button on the keyboard of the echocardiographic system. Use this to calculate the Medio-Lateral measurement of the LA.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55214/55214fig1.jpg" /> 
Figure 1: &#34;Supine&#34; Position. Representation of a mouse in the &#34;Supine&#34; Position. Approximately 30 - 45&#176; angle along the X axis and 0&#176; angle along the Y axis. This position is used to acquire all of the Left Atrium measurements. <a href="http://cloudfront.jove.com/files/ftp_upload/55214/55214fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Great Vessels Measurement
	<ol>
		<li>Continue by exchanging the 25 MHz scan head for the 30 MHz scan head. Remove the 25 MHz scan head from the movable arm clamp and place the 30 MHz scan head instead. Make sure to select the Abdominal Measurements Filter and 2D B-Mode in the echocardiographic system.</li>
		<li>Locate the handle beneath the EKG board. Use the handle to adjust the angle of the movable EKG board in which the mouse lies. Begin by adjusting the EKG board to a 5&#176; angle along the X axis and 60&#176; angle along the Y axis. Use this podition for the acquisition of the Aorta and Pulmonary Artery measurements. Refer to this as &#34;standing-up&#34; position.</li>
		<li>Using the X and Y axis adjustment torques in the echocardiographic set, get a long axis view of the heart at the level of the aortic outflow tract and acquire a 5 s movie recording. Acquire the scan by pressing the &#34;Cine Store&#34; button on the keyboard of the echocardiographic system. Use this to calculate the Aortic Diameter.</li>
		<li>After the movie acquisition, rotate the movable arm clamp of the set 90&#176; clockwise. This will set the scan head in a short axis view.</li>
		<li>Using the Y axis torque locate the Pulmonary Artery at the level of its bifurcation and acquire a 5 s movie recording. Acquire the scan by pressing the &#34;Cine Store&#34; button on the keyboard of the echocardiographic system. Use this to calculate the Pulmonary Artery Diameter. 
		NOTE: The researcher may use Doppler imaging as a tool to ensure correct identification of the Pulmonary Artery. If desired, once completing step 2.2.5 press the &#34;PW&#34; button on the keyboard of the echocardiographic system to obtain Doppler imaging. If characteristic pulmonary wave tracings are seen, the researcher can be reassured that the correct location was identified.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/55214/55214fig2.jpg" /> 
Figure 2: &#34;Standing-up&#34; Position. Representation of a mouse in the &#34;Standing-up&#34; Position. Approximately 5&#176; angle along the X axis and a 60&#176; angle along the Y axis. This position is used to acquire the Aorta and Pulmonary Artery diameters. <a href="http://cloudfront.jove.com/files/ftp_upload/55214/55214fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55214/55214fig3.jpg" /> 
Figure 3: Pulmonary Artery Doppler. Characteristic Pulmonary Artery Doppler imaging. The researcher may use Doppler imaging as a tool to ensure correct identification of the Pulmonary Artery. <a href="http://cloudfront.jove.com/files/ftp_upload/55214/55214fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Post Procedure Animal Recovery
<ol>
	<li>To safely remove the mouse from the EKG board, remove the gel from the chest of mouse and remove the tape from the extremities.</li>
	<li>Turn off the anesthesia and oxygen delivery systems. Return the mouse to its cage and do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
</ol>
4. Image Analysis
<ol>
	<li>Image Transferring
	<ol>
		<li>Use a USB flash drive to transfer the images collected in the echocardiographic system into a computer. Insert the USB flash drive into a USB port in the echocardiographic system.</li>
		<li>Select the study or studies to transfer by clicking on the study name(s).</li>
		<li>Click on the &#34;Copy To&#34; button located on the upper part of the screen. A new window showing files and directories will appear. Select the directory corresponding to the USB flash drive just inserted and click &#34;OK&#34;.</li>
		<li>Remove the USB flash drive from the echocardiographic system and insert it into a USB port in the computer to be used for analysis.</li>
		<li>Launch the echocardiographic software. Click on the &#34;Copy From&#34; button located on the upper part of the screen. Observe a new window showing files and directories. Select the studies to be copied from the directory corresponding to the USB flash drive just inserted and click &#34;OK&#34;.</li>
		<li>After copying the files, observe them in the study list. To begin analyzing a study, double click on its name and select the name of the operator analyzing.</li>
	</ol>
	</li>
	<li>Left Atrium Analysis 
	NOTE: For all the following atrial measurements use the EKG as a guide to select the P-wave as the point where the researcher wants to measure the LA. This corresponds to the pre-atrial contraction time when the LA is at its largest capacity.
	<ol>
		<li>Select the movie clip corresponding to the Superior-Inferior dimension of the Left Atrium (Step 2.1.3). Determine the pre-atrial contraction time using the EKG as a guide. This way the cavity is measured at its largest capacity.</li>
		<li>From the tool bar on the top of the screen, click on &#34;Tools&#34; and then &#34;Measurements&#34;. A new window will appear. Click on the &#34;Linear distance Measurement Tool&#34; icon (it corresponds to the first icon on the left upper corner of the window). Measure the cavity at its largest diameter.</li>
		<li>Select the image corresponding to the Anterior-Posterior dimension of the Left Atrium (Step 2.1.5). Determine the pre-atrial contraction time using the EKG as a guide. This way the cavity is measured at its largest capacity. To measure it repeat step 4.2.2.</li>
		<li>Select the image corresponding to the Medio-Lateral dimension of the Left Atrium (Step 2.1.8). Determine the pre-atrial contraction time using the EKG as a guide. This way the cavity is measured at its largest capacity. Measure the cavity from the interatrial septum to the opposing wall in a diagonal fashion. To measure it repeat step 4.2.2. 
		NOTE: Repeat steps 4.2.1 thru 4.2.4 at least 3 times and acquire the average values to minimize error.</li>
	</ol>
	</li>
	<li>Great Vessels Analysis 
	NOTE: For all the following great vessels measurements use the EKG as a guide to select a time after the QRS complex as the point where the researcher wants to measure. This corresponds to the point where the great vessels are at their largest capacity.
	<ol>
		<li>Select the movie clip corresponding to the Aorta (Step 2.2.3). Determine a point after the QRS using the EKG. Measure the diameter at a 90&#176; angle using the vessel wall as a reference. To measure it repeat step 4.2.2</li>
		<li>Select the movie clip corresponding to the Pulmonary Artery (Step 2.2.5). Determine a point after the QRS using the EKG. Measure the diameter at a 90&#176; angle using the vessel wall as a reference. Select a plane above the pulmonary artery bifurcation. To measure it repeat step 4.2.2.</li>
	</ol>
	</li>
</ol>

<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>

The authors have nothing to disclose.

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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		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Vevo 770 high-resolution in vivo micro-imaging system</td>
			<td style="width:85px;">Visual Sonics</td>
			<td style="width:119px;">Vevo 770-120</td>
			<td style="width:267px;">Echocardiographic Equipment</td>
		</tr>
		<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>
		</tr>
		<tr height="84">
			<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>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:203px;">Vevo integrated rail system including physiological monitoring unit&#160;</td>
			<td style="width:85px;">Visual Sonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Inhalation Anesthesia System</td>
			<td style="width:85px;">VetEquip</td>
			<td style="width:119px;">VE2627</td>
			<td>Anesthesia System</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:203px;">Isofluorane</td>
			<td style="width:85px;">Henry Schein</td>
			<td style="width:119px;">50033</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Electric razor&#160;</td>
			<td style="width:85px;">Wahl</td>
			<td style="width:119px;">General supply</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Hair removal cream</td>
			<td style="width:85px;">Nair</td>
			<td style="width:119px;">General supply</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Transductor cream</td>
			<td style="width:85px;">Parker</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Transductor gel</td>
			<td style="width:85px;">Parker</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Standard Gauze pads&#160;&#160;</td>
			<td style="width:85px;">McKeeson</td>
			<td style="width:119px;">General supply</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Tape</td>
			<td style="width:85px;">Durapore</td>
			<td style="width:119px;">General supply</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Nose cone&#160;</td>
			<td style="width:85px;"></td>
			<td style="width:119px;"></td>
			<td>For anesthesia delivery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Water&#160;</td>
			<td style="width:85px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Vet eye ointment&#160;</td>
			<td style="width:85px;">Puralube</td>
			<td style="width:119px;">General supply&#160;</td>
			<td>To prevent dryness&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">Cotton tipped applicators&#160;</td>
			<td style="width:85px;"></td>
			<td style="width:119px;">General supply</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:203px;">USB Flash Drive</td>
			<td style="width:85px;"></td>
			<td style="width:119px;">General supply</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

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Research

JoVE Journal

Medicine

14.2K Views.  Baylor College of 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.

Video: Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

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  ...

Murine Fetal Echocardiography

Transgenic mice displaying abnormalities in cardiac development and function represent a powerful tool for the understanding the molecular mechanisms underlying both normal cardiovascular function and the pathophysiological basis of human cardiovascular disease. Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development 1-3. In order to study the role of genetic or pharmacologic alterations in the early development of cardiac function, ultrasound imaging of the live fetus has become an important tool for early recognition of abnormalities and longitudinal follow-up. Noninvasive ultrasound imaging is an ideal method for detecting and studying congenital malformations and the impact on cardiac function prior to death 4. It allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies 5,6. Until recently, imaging of fetal mouse hearts frequently involved invasive methods. The fetus had to be sacrificed to perform magnetic resonance microscopy and electron microscopy or surgically delivered for transillumination microscopy. An application of high-frequency probes with conventional 2-D and pulsed-wave Doppler imaging has been shown to provide measurements of cardiac contraction and heart rates during embryonic development with databases of normal developmental changes now available 6-10. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. High-frequency ultrasound imaging of the fetus has improved 2-D resolution and can provide excellent information on the early development of cardiac structures 11.

Fetal and perinatal death is a common feature when studying genetic alterations affecting cardiac development. High-frequency ultrasound imaging has improved 2-D resolution and can provide excellent information on early cardiac development and is an ideal method to detect the impact on cardiac structure and function prior to death.

Transgenic mice displaying abnormalities in  cardiac development and function represent a powerful tool for the understanding  the molecular mechanisms ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Transthoracic Speckle Tracking Echocardiography for the Quantitative Assessment of Left Ventricular Myocardial Deformation

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the examiners&#39; expertise. Speckle tracking Echocardiography (STE) is a promising but technically challenging method that can be used to quantitatively assess regional and global systolic and diastolic myocardial performance. Myocardial strain and strain rate can be measured in all three dimensions&#160;&#8212; radial, circumferential, longitudinal &#8212; of myocardial deformation. Standard cross-sectional two-dimensional B-mode images are recorded and subsequently postprocessed by automated continuous frame-by-frame tracking and motion analysis of speckles within the myocardium. Images are recorded as digital loops and synchronized to a 3-lead EKG for timing purposes. Longitudinal deformation is assessed in the apical 4-, 3-, and 2-chamber views. Circumferential and radial deformation are measured in the parasternal short axis plane.
Optimal image quality and accurate tissue tracking are paramount for the correct determination of myocardial performance parameters. Utilizing transthoracic STE in a healthy volunteer, the present article is a detailed outline of the essential steps and potential pitfalls of quantitative echocardiographic myocardial deformation analysis.

Speckle tracking echocardiography is an emerging diagnostic imaging technique for the quantitative assessment of global and regional myocardial performance. Standard view echocardiographic motion images are recorded and deformation parameters are subsequently measured by automated continuous frame-by-frame tracking and motion analysis of speckles within the B-mode images of the myocardium.

The value of conventional echocardiography is limited by differences in inter-individual image interpretation and therefore largely dependent on the ...

Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by reduced blood supply to the heart, resulting in the loss of oxygen to and the ensuing necrosis of the heart muscle. The MI model has gained popularity for its use as a short-term ischemia-reperfusion model and a long-term permanent ligation model. Below, we describe a reliable method for the permanent ligation of the LAD. With mouse genetic engineering technology becoming more advanced, and with an increasing availability of quality murine surgical instruments, the mouse has become a popular model for MI surgeries. Our surgical model incorporates the use of an easily reversible anesthetic for the rapid recovery of the mouse; a minimally invasive endotracheal intubation without involving a tracheotomy; and a thoracentesis through the original thoracotomy site without creating an additional incision in the chest, as is done in some other methods, to effectively remove excess blood and air from the chest cavity. This method is comparatively less invasive than other methods, which dramatically reduces surgical and post-surgical complications and mortality and improves reproducibility.

Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by ...

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery (ALCAPA)

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ischemia and infarction in children. If left untreated, it results in a 90% mortality rate in the first year of life. In patients who survive to the adulthood, the coronary steal phenomenon and retrograde left-sided coronary flow provide a substrate for chronic subendocardial ischemia, which may lead to left ventricular dysfunction, ischemic mitral regurgitation, malignant ventricular arrhythmias, and sudden cardiac death. The average age of life-threatening presentation is 33 years and of sudden cardiac death 31 years. Therefore, surgical correction is highly recommended as soon as the diagnosis is made, regardless of age. In adult-type ALCAPA originating from the right-facing sinus of the pulmonary artery, direct re-implantation of the ALCAPA into the aorta is the more physiologically sound repair technique to re-establish the dual-coronary perfusion system and is recommended. This protocol describes the technique of direct re-implantation of adult-type ALCAPA into the aorta.

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery ALCAPA

Surgical correction of ALCAPA is highly recommended, regardless of age or the degree of intercoronary collateralization. This protocol presents a technique for the direct re-implantation of adult-type ALCAPA into the aorta to re-establish the dual-coronary perfusion. Whenever feasible, direct re-implantation is preferred to other surgical correction techniques.

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ...

Left Atrial Stenosis Induced Pulmonary Venous Arterialization and Group 2 Pulmonary Hypertension in Rat

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of group 2 PH, due to mitral stenosis (MS), to facilitate the investigation of disease mechanisms and potential therapeutic strategies. We present a novel rat model of pulmonary venous congestion-induced pulmonary venous arterialization and group 2 PH caused by left atrial stenosis (LAS). LAS is achieved by constricting the left atrium using a half-closed titanium clip. After the LAS surgery, a rat model with a transmitral inflow velocity greater than or equal to 2.0 m/s on echocardiography gradually develops pulmonary venous arterialization and group 2 PH over an 8- to 10-week period. In this protocol, we provide the step-by-step procedure of how to perform the LAS surgery. The presented LAS rat model mimics MS in humans and is useful for studying the underlying molecular mechanism of pulmonary venous arterialization and for the preclinical evaluation of therapies for group 2 PH.

Left atrial stenosis (LAS) is a novel surgical technique used for studying group 2 pulmonary hypertension (PH) and mechanisms underlying pulmonary venous arterialization. Here, we present a protocol to constrict the left atrium using a titanium clip to cause pulmonary venous arterialization and moderate PH in a rat.

The mechanism of mitral stenosis-induced pulmonary venous arterialization and group 2 pulmonary hypertension (PH) is unclear. There is no rodent model of ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...