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>Place ECG gel (see Table of Materials) on the warmed platform electrode pads. Place aluminum foil strips (~1.5 in x 0.25 in) on top of the electrode pads to extend the electrode range and secure with tape (Figure 1A). Subsequently, place the ECG gel on top of the aluminum foil strips. 
	NOTE: 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.</li>
	<li>Cut out a finger from a nitrile glove and fit it to cover both the isoflurane/oxygen nose cone on one side and the mouse nose on the other side (Figure 1B).</li>
	<li>Place the mouse pup into the isoflurane induction chamber and start isoflurane delivery at 2.5% concentration driven by 100% oxygen (Figure 1C).</li>
	<li>Place the anesthetized pup in a supine position on the imaging platform with the paws on top of the aluminum foil pads and secure with tape. Ensure that the electrical circuit is complete and that the ECG is recording.</li>
	<li>Decrease the isoflurane delivery to 1.5%&#160;driven by 100% oxygen. Secure the cut-out finger from the glove around the pup&#39;s nose with tape. Confirm the depth of anesthesia by pinching the pup&#39;s paws.</li>
	<li>Place a thick layer of prewarmed ultrasound gel on top of the pup&#39;s upper body. Use two gauze rolls to keep the ultrasound gel in place (Figure 1D).</li>
	<li>Use a heating lamp to maintain the pup&#39;s normal body temperature (Figure 1E). 
	&#8203;NOTE: A rectal probe was not used to monitor body temperature in the current study due to the small size of the pup.</li>
</ol>
2. Echocardiographic image acquisition and analyses
<ol>
	<li>Perform transthoracic echocardiography using an echocardiography instrument equipped with a linear array transducer at 40 MHz for B-mode and at 32 MHz for Doppler (frame rate 233) (see Table of Materials), following adult mice echocardiography protocols7,8,9.</li>
	<li>Avoid placing excessive pressure on the pup&#39;s chest cavity when placing the echo transducer during echocardiographic image acquisition. 
	NOTE: Due to the pup&#39;s small size, the weight of the transducer itself may result in altered cardiac function or death.</li>
	<li>Capture the PLAX view of the left ventricular outflow tract and the left atrium.
	<ol>
		<li>Place the transducer in the holder, with the index mark toward the right shoulder of the pup.</li>
		<li>Lower the transducer until it is in contact with the gel and visualize the left ventricular outflow tract in B-mode (Figure 2A).</li>
		<li>Use M-mode at the aortic leaflets to measure the left atrium (LA) maximum diameter at end-systole (Figure 2B, Table 1). Press the Cine Store button to record the data.</li>
	</ol>
	</li>
	<li>Capture the PSAX view of the left ventricle to measure the chamber dimensions, wall thickness, aortic flow, and pulmonary flow.
	<ol>
		<li>Rotate the transducer ~90&#176; clockwise of the PLAX to obtain the PSAX view.</li>
		<li>Place the probe at the level of the papillary muscles and use M-mode to measure the left ventricular internal diameters (LVID), interventricular septum thickness (IVS), and PW during systole and diastole (Figure 3A, Table 1). Press the Cine Store button to record the data.</li>
		<li>Calculate the RWT, an index of hypertrophy, using diastolic chamber dimensions as follows3,10: 
		(PW + IVS at end-diastole) / (LVID at end-diastole)</li>
		<li>Move the transducer toward the base of the heart and use the color Doppler to visualize the pulmonary artery. Press PW Doppler to quantify the pulmonary peak flow velocity, pulmonary flow profiles, pulmonary ejection time (PET), and pulmonary acceleration time (PAT)11,12 (Figure 3B). Press the Cine Store button to record the data.</li>
		<li>Move the transducer further toward the base and use color Doppler to visualize the aortic flow (Figure 3C). Use PW Doppler to visualize the blood flow and measure the aortic ejection time (AET). Press the Cine Store button to record the data.</li>
		<li>Calculate the Vcf (circ/sec)13,14, an indicator of myocardial performance, using LVID end-diastole (LVIDd), LVID end-systole (LVIDs), and AET as follows (Table 1): 
		(LVIDd - LVIDs) / (LVIDd x AET)</li>
	</ol>
	</li>
	<li>Capture the apical four-chamber view.
	<ol>
		<li>Place the platform in the Trendelenburg position, tilt it to the left, and adjust the probe to visualize the four chambers (Figure 4A).</li>
		<li>Use color Doppler to visualize the blood flow and PW Doppler at the tip of the mitral valve leaflets in the center of the mitral valve orifice to record the mitral flow. Press the Cine Store to record the data.</li>
		<li>In this view, calculate the following parameters2,3,10 (Figure 4B and Table 1):
		<ol>
			<li>Calculate E/A ratio, which is the maximal velocity of blood flow in the early phase of diastole (E) over the maximal velocity of blood flow in the late phase of diastole (A).</li>
			<li>Determine E wave deceleration time (DT), which is the time from peak E to the end of the early diastole.</li>
			<li>Calculate LV isovolumic relaxation time (IVRT), which is the time from aortic valve closure to mitral valve opening.</li>
			<li>Calculate LV isovolumic contraction time (IVCT), which is the time from mitral valve closure to aortic valve opening.</li>
		</ol>
		</li>
		<li>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 (e&#39;) and late diastolic filling (a&#39;), as well as the peak systolic myocardial contraction velocity (s&#39;) (Figure 4C and Table 1). Press the Cine Store button to record the data.</li>
	</ol>
	</li>
	<li>Capture the modified PLAX view to examine the left anterior descending coronary artery.
	<ol>
		<li>Use a modified PLAX view15, moving the transducer laterally and tilting the beam toward the anterior (Figure 5A).</li>
		<li>Move the probe and use color Doppler to visualize the origin of the left main coronary artery (LCA) that generates from the aorta. Identify the LAD artery that generates from the LCA and runs between the left ventricular anterior wall and the right ventricular outflow tract16,17. In this position, apply PW Doppler to measure the LAD flow (Figure 5B). Press the Cine Store button to record the data.</li>
		<li>Calculate the following LAD coronary artery flow parameters (Figure 5C and Table 2): peak coronary flow velocity (CFV), mean CFV, and velocity-time integral (VTI). 
		NOTE: All these parameters are measured at a basal isoflurane concentration of 1.5% (baseline).</li>
		<li>Increase the isoflurane concentration to 2.5% and wait for 5 min to achieve maximal flow (Figure 5C). Press the Cine Store button to record the data. Calculate CFR as the ratio of diastolic peak CFV at maximal flow to diastolic peak CFV at baseline18,19,20 (Table 2): 
		CFR = diastolic peak CFV (2.5%) / diastolic peak CFV (1.5%)</li>
	</ol>
	</li>
</ol>
3. Post-imaging animal monitoring and care
<ol>
	<li>After completing the echocardiographic imaging, carefully clean the pup and allow it to recover from the anesthesia for approximately 2 min.</li>
	<li>Before returning the pup to its cage, smear the pup with the cage mother&#39;s bedding to prevent rejection or cannibalization.</li>
	<li>Observe the mother&#39;s behavior for about 30 min after the procedure. If aggressive behavior is observed, euthanize the pup following the animal procedure guidelines.</li>
</ol>

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The authors have nothing to disclose.

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 in life. Problematically, echocardiographic studies in neonate mice are technically challenging. In this study, we have established a protocol for performing echocardiographic measurements in mice as young as 7 days old. This is especially important for transgenic mouse models, in which the deletion or overexpression of a gene is believed to cause cardiovascular dysfunction. Early recognition of the cardiovascular abnormalities in these animal models allows researchers to design pharmacological treatments that prevent disease progression.
Due to the small size of the 7-day-old mice, some technical considerations in this protocol included maintaining their normal body temperatures and minimizing the length of the echocardiographic procedure. A heated platform, a heating lamp, and prewarmed acoustic gel were used to prevent hypothermia. Ideally, the animal&#39;s temperature should be monitored using a rectal probe; however, given the small size of the pups in this study, we were unable to use a rectal probe during the procedure. Moreover, hyperthermia is also a concern, and care needs to be taken to avoid the pups being in close proximity to the heating lamp. The duration of the echo procedure needs to be kept to less than 1 h to minimize major temperature variations and avoid the physiological effects of prolonged anesthesia22. Additionally, since the size of the echocardiographic probe is designed to image adult mice, using a thicker layer of acoustic gel is recommended to adjust the focal distance. It is also important to mention that the imaging system used in this study calculates respiration rate and heart rate from the ECG signal detected by the platform electrode pads (Table 2). As the ECG pads were extended to reach the pup&#39;s limbs using aluminum foil, the signal detected may have been distorted. Another problem encountered was that, by the end of the procedure, we noticed that the gel underneath the aluminum foil strips had dried out, which may have affected the conductivity and the ECG signal. Ideally, a platform with electrode pads that match the animal&#39;s size or needle electrodes that contact the pup&#39;s limbs should be used to obtain a more reliable ECG signal23,24.
The limitations of the current study include the higher isoflurane concentrations needed for the anesthesia of neonatal mice. This protocol used 1.5% isoflurane to perform echocardiographic analyses, including coronary flow dynamics. The isoflurane concentration was increased from 1.5% to 2.5% to induce hyperemia and evaluate CFR. In adult mice, resting coronary flow velocity assessment is performed at 1% isoflurane, and the hyperemic response is made at 2.5%18,25,26. However, in neonatal mice, 1% isoflurane is not sufficient to maintain an adequate level of anesthesia. Nevertheless, the shift from 1.5% to 2.5% isoflurane in neonatal mice increased peak CFV, mean CFV, and VTI (Figure 5C and Table 2), thus verifying isoflurane-induced coronary artery vasodilation. It is also important to mention that, in this protocol, a modified PLAX view was used to visualize and examine the LAD coronary flow parameters15,16,21; however, LAD can also be visualized using a modified PSAX16,19,21 or a modified apical four-chamber view16,21. In the present study, the modified PLAX gave us more consistent results in the correct visualization and assessment of LAD coronary flow and CFR in neonatal mice.
This article provides a practical guide for imaging and assessing cardiovascular function in neonatal mice. It must be considered that cardiac function parameters vary according to mice strain and age. In this study, we used FVB/N mice, and these results may be used as reference values for future studies with the same strain (Table 1 and Table 2).

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><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). The PSAX view (Supplementary Video 2) was used to measure the left ventricular chamber dimensions (Figure 3A), pulmonary flow (Figure 3B), and aortic flow (Figure 3C). The apical four-chamber view (Supplementary Video 3 and Figure 4A) was used to examine the blood flow velocities across the mitral valve (Figure 4B), as well as the myocardial relaxation and contraction velocities at the mitral valve annulus (Figure 4C).
The modified PLAX view was used to examine the LAD coronary artery flow parameters (Figure 5A,B and Supplementary Video 4), as previously described15,16,21. In Figure 5C, representative results of the diastolic peak CFV, mean CFV, and VTI are shown at a resting flow state (1.5% isoflurane) and 5 min after increasing isoflurane to 2.5% to induce maximal vasodilation. The increased values of these parameters (i.e., peak CFV, mean CFV, and VTI) 5 min after isoflurane increment confirm the expected response to hyperemia in the neonatal mice18. CFR was calculated as the ratio of diastolic peak CFV during maximal vasodilation induced by 2.5% isoflurane to diastolic peak CFV at a baseline of 1.5% isoflurane concentration18. All measurements and calculations were averaged across 3 consecutive cycles, and the representative results are shown in Table 1 and Table 2.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63539/63539fig01.jpg" /> 
Figure 1: Echocardiographic platform setup and 7-day-old mouse pup preparation. (A) Aluminum foil strips are placed on the platform electrode pads and secured with tape. (B) The glove finger is cut and adapted to fit the isoflurane/oxygen nose cone. (C) The pup is placed in the isoflurane induction chamber, and the isoflurane delivery starts at 2.5% concentration. (D) The pup is placed in a supine position with paws touching the aluminum foil strips and secured with tape. Two rolls of gauze are used to keep the acoustic gel in place. (E) A heating lamp is placed close to the pup to maintain its body temperature. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63539/63539fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63539/63539fig02.jpg" /> 
Figure 2: Parasternal long-axis (PLAX) view of the left ventricle. (A) B-mode images of the left ventricular chamber (LV), left atrium (LA), and the aorta. (B) M-mode is used to measure the LA diameter. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63539/63539fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63539/63539fig03.jpg" /> 
Figure 3: Parasternal short-axis (PSAX) view of the left ventricle. (A) B-mode images of the left ventricular chamber. (B) M-mode sample of the interventricular septum at diastole (IVSd), left ventricular internal diameter at diastole (LVIDd), and posterior wall thickness at diastole (PWd). (C) Representative images of the pulmonary peak flow velocity, pulmonary ejection time (PET), and pulmonary acceleration time (PAT). (D) Representative images of the aortic ejection time (AET). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63539/63539fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63539/63539fig04.jpg" /> 
Figure 4: Apical four-chamber view. (A) B-mode image of the left ventricle (LV), right ventricle (RV), left atrium (LA), and right atrium (RA). (B) Representative images of the maximal blood inflow velocity in the early phase of diastole (E), maximal blood inflow velocity in the late phase of diastole (A), deceleration time (DT), isovolumetric contraction time (IVCT), and isovolumetric relaxation time (IVRT). (C) Tissue Doppler sample images of the peak myocardial relaxation velocity in the early diastolic filling (e&#39;), late diastolic filling (a&#39;), and peak systolic myocardial velocity (s&#39;). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63539/63539fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63539/63539fig05.jpg" /> 
Figure 5: Modified parasternal long-axis view. (A) Platform and transducer position in modified parasternal long-axis view. (B) Visualization and recording of left anterior descending (LAD) coronary artery flow. LVOT = left ventricular outflow tract. (C) Peak coronary flow velocity (CFV), mean CFV, and velocity-time integral (VTI) in diastole are measured at 1.5% isoflurane (baseline) and 5 min after increasing isoflurane concentration to 2.5%; 7-day old mice, N = 7; data presented as mean &#177; SD. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63539/63539fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2" rowspan="2">Echocardiographic parameters</td>
			<td>WT (n = 7)</td>
		</tr>
		<tr>
			<td>Mean &#177; SD</td>
		</tr>
		<tr>
			<td rowspan="5">Morphology</td>
			<td>LA (mm)</td>
			<td>1.25 &#177; 0.11</td>
		</tr>
		<tr>
			<td>PWd (mm)</td>
			<td>0.40 &#177; 0.06</td>
		</tr>
		<tr>
			<td>LVIDd (mm)</td>
			<td>1.98 &#177; 0.34</td>
		</tr>
		<tr>
			<td>LV Mass (g)</td>
			<td>10.92 &#177; 3.53</td>
		</tr>
		<tr>
			<td>RWT&#160;</td>
			<td>0.39 &#177; 0.09</td>
		</tr>
		<tr>
			<td rowspan="6">Systolic Function</td>
			<td>HR (bpm)</td>
			<td>500.69 &#177; 40.04</td>
		</tr>
		<tr>
			<td>EF(%)</td>
			<td>81.97 &#177; 10.76</td>
		</tr>
		<tr>
			<td>SV (ml)</td>
			<td>10.16 &#177; 3.44</td>
		</tr>
		<tr>
			<td>CO (ml/min)</td>
			<td>5.04 &#177; 1.53</td>
		</tr>
		<tr>
			<td>s&#8217; (cm/s)</td>
			<td>16.16 &#177; 3.56</td>
		</tr>
		<tr>
			<td>Vcf (circ/s)</td>
			<td>10.50 &#177; 3.12</td>
		</tr>
		<tr>
			<td rowspan="4">Diastolic Function</td>
			<td>E/A</td>
			<td>1.25 &#177; 0.11</td>
		</tr>
		<tr>
			<td>E/e&#8217;</td>
			<td>45.58 &#177; 11.44</td>
		</tr>
		<tr>
			<td>DT (s)</td>
			<td>23.97 &#177; 2.63</td>
		</tr>
		<tr>
			<td>IVRT (s)</td>
			<td>16.27 &#177; 2.11</td>
		</tr>
	</tbody>
</table>
Table 1: Echocardiographic assessment of left ventricular morphology and function in 7-day old mouse pups.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2" rowspan="2">Coronary Flow Parameters</td>
			<td>Baseline</td>
			<td>5 min</td>
			<td>CFR</td>
		</tr>
		<tr>
			<td>Isoflurane 1.5%</td>
			<td>Isoflurane 2.5%</td>
			<td>5 min/baseline</td>
		</tr>
		<tr>
			<td rowspan="3">Diastole</td>
			<td>Peak velocity (mm/s)</td>
			<td>516.58 &#177; 113.04</td>
			<td>599.43 &#177; 101.34</td>
			<td>1.18 &#177; 0.18</td>
		</tr>
		<tr>
			<td>Mean velocity (mm/s)</td>
			<td>308.50 &#177; 63.44</td>
			<td>351.50 &#177; 53.98</td>
			<td></td>
		</tr>
		<tr>
			<td>VTI (mm)</td>
			<td>25.23 &#177; 5.86</td>
			<td>30.65 &#177; 7.75</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">Sytole</td>
			<td>Peak velocity (mm/s)</td>
			<td>121.81 &#177; 40.52</td>
			<td>163.13 &#177; 32.59*</td>
			<td></td>
		</tr>
		<tr>
			<td>Mean velocity (mm/s)</td>
			<td>84.82 &#177; 27.16</td>
			<td>114.70 &#177; 21.84*</td>
			<td></td>
		</tr>
		<tr>
			<td>VTI (mm)</td>
			<td>5.21 &#177; 1.84</td>
			<td>7.76 &#177; 2.08*</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Heart rate (bpm)</td>
			<td>536.20 &#177; 128.90</td>
			<td>540.80 &#177; 233.15</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Respiratory rate (rpm)</td>
			<td>69.60 &#177; 15.89</td>
			<td>38.80 &#177; 24.18</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Echocardiographic evaluation of coronary artery flow in 7-day old mouse pups. Seven-day old mice, N = 7; data presented as mean &#177; SD; the Student&#39;s t-test was used to analyze the data; *p &#60; 0.05; CFR = coronary flow reserve; VTI = velocity time integral.
Supplementary Video 1: The parasternal long-axis view of the left ventricular outflow and left atrium. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63539/Supplementary Video 1. Paraesternal long-axis view (PLAX).mp4" target="_blank">Please click here to download this Video.</a>
Supplementary Video 2: The parasternal short-axis view of the left ventricular chamber. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63539/Supplementary Video 2. Paraesternal short-axis view (PSAX).mp4" target="_blank">Please click here to download this Video.</a>
Supplementary Video 3: The apical four-chamber view. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63539/Supplementary Video 3. Apical four-chamber view.mp4" target="_blank">Please click here to download this Video.</a>
Supplementary Video 4: The modified parasternal long-axis view of left anterior descending coronary artery flow. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63539/Supplementary Video 4. Coronary artery flow.mp4" target="_blank">Please click here to download this Video.</a>

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 ...

echocardiographic-characterization-left-ventricular-structure

Universidad San Sebastian

Research

JoVE Journal

Medicine

2.7K Views.  University of Illinois at Chicago. The present protocol describes the echocardiographic assessment of left ventricular morphology, function, and coronary blood flow in 7-day old neonate mice. 

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

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable around embryonic day (E) 8.51. Systolic left ventricular (LV) pressure is 2 mmHg at E9.5 and 11 mmHg at E14.52. At these mid-embryonic stages, the LV is clearly visible through the chest wall for invasive pressure measurements because the ribs and skin are not fully developed. Between E14.5 and birth (approximately E21) imaging methods must be used to view the LV. After birth, mean arterial pressure increases from 30 - 70 mmHg from postnatal day (P) 2 - 353. Beyond P20, arterial pressure can be measured with solid-state catheters (i.e. Millar or Scisense). Before P20, these catheters are too big for developing mouse arteries and arterial pressure must be measured with custom pulled plastic catheters attached to fluid-filled pressure transducers3 or glass micropipettes attached to servo null pressure transducers4.
Our recent work has shown that the greatest increase in blood pressure occurs during the late embryonic to early postnatal period in mice5-7. This large increase in blood pressure may influence smooth muscle cell (SMC) phenotype in developing arteries and trigger important mechanotransduction events. In human disease, where the mechanical properties of developing arteries are compromised by defects in extracellular matrix proteins (i.e. Marfan's Syndrome8 and Supravalvular Aortic Stenosis9) the rapid changes in blood pressure during this period may contribute to disease phenotype and severity through alterations in mechanotransduction signals. Therefore, it is important to be able to measure blood pressure changes during late embryonic and neonatal periods in mouse models of human disease.
We describe a method for measuring LV pressure in late embryonic (E18) and early postnatal (P1 - 20) mice. A needle attached to a fluid-filled pressure transducer is inserted into the LV under ultrasound guidance. Care is taken to maintain normal cardiac function during the experimental protocol, especially for the embryonic mice. Representative data are presented and limitations of the protocol are discussed.

Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable ...

Bioengineering

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 ...

Echocardiographic Assessment of the Right Heart in Mice

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential therapies. Given the expense and time involved in creating animal models of disease, it is critical that researchers have tools to accurately assess phenotypic expression of disease. Right ventricular dysfunction is the major manifestation of pulmonary hypertension. Echocardiography is the mainstay of the noninvasive assessment of right ventricular function in rodent models and has the advantage of clear translation to humans in whom the same tool is used. Published echocardiography protocols in murine models of PAH are lacking.
In this article, we describe a protocol for assessing RV and pulmonary vascular function in a mouse model of PAH with a dominant negative BMPRII mutation; however, this protocol is applicable to any diseases affecting the pulmonary vasculature or right heart. We provide a detailed description of animal preparation, image acquisition and hemodynamic calculation of stroke volume, cardiac output and an estimate of pulmonary artery pressure.

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice. Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular disease.

Transgenic and toxic models of pulmonary arterial hypertension (PAH) are widely used to study the pathophysiology of PAH and to investigate potential ...

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 Pipeline to Characterize Structural Heart Defects in the Fetal Mouse

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

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

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

Developmental Biology

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.

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 ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.