The authors thank Chad M. Warren, MS (University of Illinois at Chicago), for editing this manuscript. This work was supported by NIH/NHLBI K01HL155241 and AHA CDA849387 grants to PCR.

All experiments were approved by the Animal Care and Use Committee of the University of Illinois at Chicago. For the experiments, 7-day-old FVB/N mice were used. The protocol is divided into mouse preparation, echocardiography image acquisition, and post-imaging animal care.
1. Mouse preparation
<ol>
	<li>Obtain the 7-day-old mice from the breeding cage. 
	NOTE: At this early age, it is difficult to determine the sex of the animal by physical examination.</li>
	<li>Plac...

<ol>
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M., Wikstrom, J., Bergstrom, G., Wandt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+imaging+of+coronary+arteries+in+living+mice+using+high-resolution+echocardiography.">Non-invasive imaging of coronary arteries in living mice using high-resolution echocardiography.</a> Scandinavian Cardiovascular Journal. 38 (2), 121-126 (2004).</li><li>Gan, L. M., Wikstrom, J., Fritsche-Danielson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+flow+reserve+from+mouse+to+man--from+mechanistic+understanding+to+future+interventions.">Coronary flow reserve from mouse to man--from mechanistic understanding to future interventions.</a> Journal of Cardiovascular Translational Research. 6 (5), 715-728 (2013).</li><li>Krzanowski, M., Bodzon, W., Dimitrow, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+all+three+coronary+arteries+by+transthoracic+echocardiography.+An+illustrated+guide.">Imaging of all three coronary arteries by transthoracic echocardiography. An illustrated guide.</a> Cardiovascular Ultrasound. 1, 16 (2003).</li><li>Constantinides, C., Mean, R., Janssen, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+isoflurane+anesthesia+on+the+cardiovascular+function+of+the+C57BL/6+mouse.">Effects of isoflurane anesthesia on the cardiovascular function of the C57BL/6 mouse.</a> ILAR Journal. 52 (3), 21-31 (2011).</li><li>Ha, T. W., Oh, B., Kang, J. 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In the era of preventive medicine, early assessment of alterations in cardiovascular function is required to establish the onset of the disease and design appropriate interventional therapies. Mice are increasingly being used as preclinical models in cardiac research, and echocardiographic studies are typically conducted with young adult mice. However, to study the role of genetic alterations or pharmacological interventions in the early stages of cardiac diseases, echocardiographic imaging needs to be initiated earlier ...

The overall goal of this protocol is to examine cardiac morphology, function, and coronary artery flow in 7-day-old neonatal mouse pups using echocardiography. The rationale behind the development of this technique is to determine early changes in coronary flow and cardiac function in mouse models of cardiac disease1. The non-invasive nature of echocardiography is advantageous because it allows researchers to assess cardiovascular function under physiological conditions and provides researchers with a screening tool for the study of targeted therapies to treat cardiovascular diseases2,3. Traditionally, echocardiographic studies are conducted with young adult mice (4-6 weeks); however, some mice models (i.e., genetically modified models) already exhibit pathological changes and cardiac dysfunction at this age. Therefore, cardiac research using animal models has focused primarily on therapeutic agents that ameliorate or treat cardiac dysfunction. In contrast, more recently, research efforts have been redirected to focus on preventive measures and early interventions in cardiac diseases4.
Previous studies have described the use of echocardiography to measure cardiac function in models of myocardial infarction in neonatal mice5,6; however, these studies failed to measure coronary flow and, most importantly, failed to record an electrocardiogram (ECG) and heart rate (HR) data during the procedure, most likely due to the small size of the pups&#39; limbs, which could not reach the electrode pads. We overcome this problem in this protocol by attaching aluminum foil to the limbs to enable them to reach the electrode pads and create an ECG circuit. Furthermore, this protocol describes and characterizes coronary artery flow in neonatal mice.
This study obtained B-mode and M-mode images in parasternal long and short axis views to measure structural and functional parameters2,3. The morphological parameters included left atrial dimensions, left ventricular (LV) dimensions, LV wall thickness, LV mass, and relative wall thickness (RWT). The functional parameters included ejection fraction (EF), fractional shortening (FS), cardiac output (CO), and velocity of circumferential fiber shortening (Vcf). Pulse wave (PW) Doppler was used to measure aortic flow in the parasternal short-axis (PSAX) view and to measure mitral blood flow in the apical four-chamber view. The apical four-chamber view was also used to perform Tissue Doppler at the septal part of the mitral valve annulus. Coronary flow at the left anterior descending (LAD) coronary artery was also examined using a modified parasternal long-axis (PLAX) view. Coronary flow reserve (CFR) was calculated after a stress challenge induced by increased isoflurane concentration.
The present protocol demonstrates that echocardiographic studies can be performed at a very early age in neonatal mice, thus allowing early recognition of cardiac pathologies and longitudinal follow-up studies of LV hemodynamics and coronary flow parameters in different mice models. This technique can be used to study the role of genetic alterations or pharmacological interventions in cardiac function at early postnatal ages. Moreover, the protocol provides a valuable tool for determining the onset of cardiac diseases early in life, thus enabling researchers to unlock the molecular mechanisms underlying the initial stages of cardiac diseases in different mouse models.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Depilating agent</td>
			<td>Nair Hair Remover</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode gel</td>
			<td>Parker Laboratories</td>
			<td>15-60</td>
			<td></td>
		</tr>
		<tr>
			<td>High Frequency Ultrasound</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td>Vevo 2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>MedVet</td>
			<td>RXISO-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear array high frequency transducer</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td>MS550D</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice breeding pair</td>
			<td>Charles River Laboratories</td>
			<td>FVB/N</td>
			<td>Strain Code 207</td>
		</tr>
		<tr>
			<td>Ultrasound Gel</td>
			<td>Parker Laboratories</td>
			<td>11-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo Lab Software</td>
			<td>FUJIFILM VisualSonics, Inc.</td>
			<td></td>
			<td>Verison 5.5.1</td>
		</tr>
	</tbody>
</table>

echocardiographic characterization of left ventricular structure, function, and coronary flow in neonate mice

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular disease and is used to assess the impact of potential treatments in preclinical studies. Echocardiographic studies are usually conducted in young adult mice (i.e., 4-6 weeks of age). The evaluation of early neonatal cardiovascular function is not usually performed because of the small size of the mouse pups and the associated technical difficulties. One of the most important challenges is that the short length of the pups&#39; limbs prevents them from reaching the electrodes in the echocardiography platform. Body temperature is the other challenge, as pups are very susceptible to changes in temperature. Therefore, it is important to establish a practical guide for performing echocardiographic studies in small mouse pups to help researchers detect early pathological changes and study the progression of cardiovascular disease over time. The current work describes a protocol for performing echocardiography in mouse pups at the early age of 7 days old. The echocardiographic characterization of cardiac morphology, function, and coronary flow in neonatal mice is also described.

This study used 7-day-old mouse pups to characterize cardiac morphology, function, and coronary artery flow. Mouse handling needs to be done with care, and the mouse platform must be adapted for the small size of the pups, as described in Figure 1. A representative image of the PLAX view is shown in Figure 2A and Supplementary Video 1. In this view, M-mode was used to measure the left atrium (LA) diameter (Figure 2B...

Watch this Scientific Journal Video about Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice at JoVE.com

Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice

The present protocol describes the echocardiographic assessment of left ventricular morphology, function, and coronary blood flow in 7-day old neonate mice.

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular ...

This protocol can be used to study the role of genetic alterations or pharmacological interventions in cardiac function and coronary flow at early postnatal ages. The main advantage of this technique is the early recognition of cardiopathologies and longitudinal follow-up studies in left ventricular hemodynamics and coronary flow parameters in different mice models. Echocardiographic studies in neonatal mice are technically challenging.This protocol is designed to study the early onset of cardiovascular disease in neonatal mice that may allow researchers to design the appropriate therapeutic measures. Start with placing the ECG gel on the warmed platform electrode pads. Place aluminum foil strips on top of the electrode pads to extend the electrode range and secure them with tape.Subsequently, place the ECG gel on top of the aluminum strips. Ensure that the gel underneath the aluminum foil strips does not dry out during the procedure. If that occurs, add more gel to maintain the conductivity.Place the anesthetized pup in a supine position on the imaging platform and secure the cut-out glove finger around the pup's nose. Place the paws on top of the aluminum foil pads and secure them with tape. Ensure that the electrical circuit is complete and that the ECG is recording.Use two gauze rolls to keep the ultrasound gel in place. Place a thick layer of pre-warmed ultrasound gel on top of the pup's upper body. Use a heating lamp to maintain the pup's normal body temperature.Perform transthoracic echocardiography using an echocardiography instrument equipped with a linear array transducer at 40 megahertz for B-Mode and at 32 megahertz for Doppler following adult mice echocardiography protocols. Place the transducer in the holder with the index mark toward the right shoulder of the pup. Lower the transducer until it is in contact with the gel and visualize the left ventricular outflow tract in B-Mode.Avoid placing excessive pressure on the pup's chest cavity when placing the echotransducer during echocardiographic image acquisition. Capture the parasternal long axis view of the left ventricle, left ventricular outflow tract, and the left atrium. Use M-Mode at the aortic leaflets to measure the left atrium maximum diameter at N-systole.Record the data by pressing the Cine Store button. Rotate the transducer around 90 degrees clockwise of the parasternal long axis to obtain the parasternal short axis view. Capture the parasternal short axis view of the left ventricle to measure the chamber dimensions, wall thickness, aortic flow, and pulmonary flow.Place the probe at the level of the papillary muscles and use M-Mode to measure the left ventricular internal diameters and wall thickness during systole and diastole. Record the data by pressing the Cine Store button. Move the transducer toward the base of the heart and use the Color Doppler to visualize the pulmonary artery.Press Pulse Wave Doppler to quantify the pulmonary peak flow velocity, pulmonary flow profiles, pulmonary ejection time, and pulmonary acceleration time. Record the data by pressing the Cine Store button. Move the transducer further toward the base and use Color Doppler to visualize the aortic flow.Use Pulse Wave Doppler to visualize the blood flow and measure the aortic ejection time. Record the data by pressing the Cine Store button. To capture the apical four-chamber view, place the platform in the Trendelenburg position, tilt it to the left, and adjust the probe to visualize the four chambers.Use Color Doppler to visualize the blood flow and Pulse Wave Doppler at the tip of the mitral valve leaflets in the center of the mitral valve orifice to record the mitral flow. Record the data by pressing the Cine Store button. Use Tissue Doppler at the septal side of the mitral valve annulus in a four-chamber view to measure the peak myocardial relaxation velocity in the early diastolic filling and late diastolic filling, as well as the peak systolic myocardial contraction velocity.Record the data by pressing the Cine Store button. Capture the modified parasternal long axis view to examine the left anterior descending coronary artery. Use a modified parasternal long axis view, moving the transducer laterally.Move the probe and use Color Doppler to visualize the origin of the left main coronary artery that generates from the aorta. Identify the left anterior descending artery that generates from the left main coronary artery and runs between the left ventricular anterior wall and the right ventricular outflow tract. In this position, measure the left anterior descending coronary artery flow by pressing Pulse Wave Doppler.Record the data by pressing the Cine Store button. Increase the isoflurane concentration to 2.5%and wait for five minutes to achieve maximal flow. Record the data by pressing the Cine Store button.In the parasternal long axis view, M-Mode was used to measure the left atrium diameter. The parasternal short axis view was used to measure the left ventricular chamber dimensions and wall thickness, pulmonary flow, and aortic flow. The apical four-chamber view was used to examine the blood flow velocities across the mitral valve as well as the myocardial relaxation and contraction velocities at the mitral valve annulus.The modified parasternal long axis view was used to examine the left anterior descending coronary artery flow parameters. The increased values of peak coronary flow velocity, mean coronary flow velocity, and velocity/time integral five minutes after isoflurane increment confirmed the expected response to hyperemia in the neonatal mice. Due to the small size of the pups, it is important to maintain their normal body temperature and to attach aluminum foil strips to their limbs to reach the electropads and to create an ECG circuit.

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2.5K Views.  University of Illinois at Chicago. This protocol can be used to study the role of genetic alterations or pharmacological interventions in cardiac function and coronary flow at early postnatal ages. The main advantage of this technique is the early recognition of cardiopathologies and longitudinal follow-up studies in left ventricular hemodynamics and coronary flow parameters in different mice models. Echocardiographic studies in neonatal mice are technically challenging.This protocol is designed to study the early onset...