Name: echocardiographic characterization of left ventricular structure, function, and coronary flow in neonate mice
Uploaded: 2022-04-07T13:00:00
Description: Watch this Scientific Journal Video about Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice at JoVE.com

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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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=Guidelines+for+the+echocardiographic+assessment+of+the+right+heart+in+adults:+a+report+from+the+American+Society+of+Echocardiography+endorsed+by+the+European+Association+of+Echocardiography,+a+registered+branch+of+the+European+Society+of+Cardiology,+and+the+Canadian+Society+of+Echocardiography.">Guidelines for the echocardiographic assessment of the right heart in adults: a report from the American Society of Echocardiography endorsed by the European Association of Echocardiography, a registered branch of the European Society of Cardiology, and the Canadian Society of Echocardiography.</a> Journal of the American Society of Echocardiography. 23 (7), 685-713 (2010).</li><li>Wilson, J. 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P., Kersting, S. <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+assessment+of+coronary+flow+reserve+-+valuable+functional+information+in+cardiac+workflow.">Non-invasive assessment of coronary flow reserve - valuable functional information in cardiac workflow.</a> Ultraschall in der Medizin. 25 (1), 25-33 (2004).</li><li>Lenzarini, F., Di Lascio, N., Stea, F., Kusmic, C., Faita, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+isoflurane-induced+vasodilation:+A+Doppler+ultrasound+study+of+the+left+coronary+artery+in+mice.">Time course of isoflurane-induced vasodilation: A Doppler ultrasound study of the left coronary artery in mice.</a> Ultrasound in Medicine and Biology. 42 (4), 999-1009 (2016).</li><li>Gan, L. 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. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrocardiogram+recordings+in+anesthetized+mice+using+lead+II.">Electrocardiogram recordings in anesthetized mice using lead II.</a> Journal of Visualized Experiments. (160), e61583 (2020).</li><li>Chu, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+non-invasively+recording+electrocardiograms+in+conscious+mice.">Method for non-invasively recording electrocardiograms in conscious mice.</a> BMC Physiology. 1, 6 (2001).</li><li>Hartley, C. 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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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Research

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Medicine

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

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, ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

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 ...

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 ...

Technique of Minimally Invasive Transverse Aortic Constriction in Mice for Induction of Left Ventricular Hypertrophy

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure overload-induced left ventricular hypertrophy (LVH) and its progression to heart failure. In the majority of the reported investigations, this procedure is performed with intubation and ventilation of the animal which renders it demanding and time-consuming and adds to the surgical burden to the animal. The aim of this protocol is to describe a simplified technique of minimally invasive TAC without intubation and ventilation of mice. Critical steps of the technique are emphasized in order to achieve low mortality and high efficiency in inducing LVH.
Male C57BL/6 mice (10-week-old, 25-30 g, n=60) were anesthetized with a single intraperitoneal injection of a mixture of ketamine and xylazine. In a spontaneously breathing animal following a 3-4 mm upper partial sternotomy, a segment of 6/0 silk suture threaded through the eye of a ligation aid was passed under the aortic arch and tied over a blunted 27-gauge needle. Sham-operated animals underwent the same surgical preparation but without aortic constriction. The efficacy of the procedure in inducing LVH is attested by a significant increase in the heart/body weight ratio. This ratio is obtained at days 3, 7, 14 and 28 after surgery (n = 6 - 10 in each group and each time point). Using our technique, LVH is observed in TAC compared to sham animals from day 7 through day 28. Operative and late (over 28 days) mortalities are both very low at 1.7%.
In conclusion, our cost-effective technique of minimally invasive TAC in mice carries very low operative and post-operative mortalities and is highly efficient in inducing LVH. It simplifies the operative procedure and reduces the strain put on the animal. It can be easily performed by following the critical steps described in this protocol.

The aim of this protocol is to describe step-by-step the technique of minimally invasive transverse aortic constriction (TAC) in mice. By elimination of intubation and ventilation which are mandatory for the commonly used standard procedure, minimally invasive TAC simplifies the operative procedure and reduces the strain put on the animal.

Transverse aortic constriction (TAC) in mice is one of the most commonly used surgical techniques for experimental investigation of pressure ...

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.

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

Studying Left Ventricular Reverse Remodeling by Aortic Debanding in Rodents

To better understand the left ventricular (LV) reverse remodeling (RR), we describe a rodent model wherein, after aortic banding-induced LV remodeling, mice undergo RR upon removal of the aortic constriction. In this paper, we describe a step-by-step procedure to perform a minimally invasive surgical aortic debanding in mice. Echocardiography was subsequently used to assess the degree of cardiac hypertrophy and dysfunction during LV remodeling and RR and to determine the best timing for aortic debanding. At the end of the protocol, terminal hemodynamic evaluation of the cardiac function was conducted, and samples were collected for histological studies. We showed that debanding is associated with surgical survival rates of 70-80%. Moreover, two weeks after debanding, the significant reduction of ventricular afterload triggers the regression of ventricular hypertrophy (~20%) and fibrosis (~26%), recovery of diastolic dysfunction as assessed by the normalization of left ventricular filling and end-diastolic pressures (E/e&#39;&#160;and LVEDP). Aortic debanding is a useful experimental model to study LV RR in rodents. The extent of myocardial recovery is variable between subjects, therefore, mimicking the diversity of RR that occurs in the clinical context, such as aortic valve replacement. We conclude that the aortic banding/debanding model represents a valuable tool to unravel novel insights into the mechanisms of RR, namely the regression of cardiac hypertrophy and the recovery of diastolic dysfunction.

Here we describe a step-by-step protocol of surgical aorta debanding in the well-established mice model of aortic-constriction. This procedure not only allows studying the mechanisms underlying the left ventricular reverse remodeling and regression of hypertrophy but also to test novel therapeutic options that might accelerate myocardial recovery.

To better understand the left ventricular (LV) reverse remodeling (RR), we describe a rodent model wherein, after aortic banding-induced LV remodeling, ...