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>
	<li>Le, V. P., Wagenseil, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+Characterization+of+Postnatal+Development+in+Mice+with+Reduced+Arterial+Elasticity.">Echocardiographic Characterization of Postnatal Development in Mice with Reduced Arterial Elasticity.</a> Cardiovascular Engineering and Technology. 3 (4), 424-438 (2012).</li><li>Nagueh, S. F., 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=Recommendations+for+the+Evaluation+of+Left+Ventricular+Diastolic+Function+by+Echocardiography:+An+Update+from+the+American+Society+of+Echocardiography+and+the+European+Association+of+Cardiovascular+Imaging.">Recommendations for the Evaluation of Left Ventricular Diastolic Function by Echocardiography: An Update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging.</a> European Heart Journal: Cardiovascular Imaging. 17 (12), 1321-1360 (2016).</li><li>Lang, R. 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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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S., Fiolkoski, J., Berko, B., Reichek, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiographic+visualization+of+coronary+artery+anatomy+in+the+adult.">Echocardiographic visualization of coronary artery anatomy in the adult.</a> Journal of the American College of Cardiology. 11 (3), 565-571 (1988).</li><li>Lambertz, H., Lethen, H., Tries, H. 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.

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	<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>
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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 ...

Questo protocollo può essere utilizzato per studiare il ruolo di alterazioni genetiche o interventi farmacologici nella funzione cardiaca e nel flusso coronarico nelle prime età postnatali. Il principale vantaggio di questa tecnica è il riconoscimento precoce delle cardiopatologie e degli studi di follow-up longitudinale nell'emodinamica ventricolare sinistra e nei parametri del flusso coronarico in diversi modelli di topi. Gli studi ecocardiografici nei topi neonatali sono tecnicamente impegnativi.Questo protocollo è progettato per studiare l'insorgenza precoce delle malattie cardiovascolari nei topi neonatali che possono consentire ai ricercatori di progettare le misure terapeutiche appropriate. Inizia con il posizionamento del gel ECG sui cuscinetti degli elettrodi della piattaforma riscaldati. Posizionare strisce di alluminio sopra i cuscinetti degli elettrodi per estendere la gamma di elettrodi e fissarli con del nastro.Successivamente, posizionare il gel ECG sopra le strisce di alluminio. Assicurarsi che il gel sotto le strisce di alluminio non si asciughi durante la procedura. Se ciò si verifica, aggiungere più gel per mantenere la conduttività.Posizionare il cucciolo anestetizzato in posizione supina sulla piattaforma di imaging e fissare il dito guantato ritagliato intorno al naso del cucciolo. Posizionare le zampe sopra i cuscinetti di alluminio e fissarli con del nastro adesivo. Assicurarsi che il circuito elettrico sia completo e che l'ECG stia registrando.Utilizzare due rotoli di garza per mantenere il gel ad ultrasuoni in posizione. Posizionare uno spesso strato di gel ad ultrasuoni preriscaldato sulla parte superiore del corpo del cucciolo. Utilizzare una lampada riscaldante per mantenere la normale temperatura corporea del cucciolo.Eseguire l'ecocardiografia transtoracica utilizzando uno strumento ecocardiografico dotato di un trasduttore lineare a 40 megahertz per B-Mode e a 32 megahertz per Doppler seguendo protocolli di ecocardiografia di topi adulti. Posizionare il trasduttore nel supporto con il segno indice verso la spalla destra del cucciolo. Abbassare il trasduttore fino a quando non è in contatto con il gel e visualizzare il tratto di efflusso ventricolare sinistro in modalità B.Evitare di esercitare una pressione eccessiva sulla cavità toracica del cucciolo quando si posiziona l'ecotrasduttore durante l'acquisizione dell'immagine ecocardiografica. Cattura la vista dell'asse lungo parasternale del ventricolo sinistro, del tratto di efflusso ventricolare sinistro e dell'atrio sinistro. Utilizzare M-Mode sui lembi aortici per misurare il diametro massimo dell'atrio sinistro a N-sistole.Registrare i dati premendo il pulsante Cine Store. Ruotare il trasduttore di circa 90 gradi in senso orario rispetto all'asse lungo parasternale per ottenere la vista dell'asse corto parasternale. Cattura la vista dell'asse corto parasternale del ventricolo sinistro per misurare le dimensioni della camera, lo spessore della parete, il flusso aortico e il flusso polmonare.Posizionare la sonda a livello dei muscoli papillari e utilizzare M-Mode per misurare i diametri interni ventricolari sinistri e lo spessore della parete durante la sistole e la diastole. Registrare i dati premendo il pulsante Cine Store. Spostare il trasduttore verso la base del cuore e utilizzare il Color Doppler per visualizzare l'arteria polmonare.Premere Pulse Wave Doppler per quantificare la velocità del flusso di picco polmonare, i profili di flusso polmonare, il tempo di eiezione polmonare e il tempo di accelerazione polmonare. Registrare i dati premendo il pulsante Cine Store. Spostare ulteriormente il trasduttore verso la base e utilizzare Color Doppler per visualizzare il flusso aortico.Utilizzare Pulse Wave Doppler per visualizzare il flusso sanguigno e misurare il tempo di eiezione aortica. Registrare i dati premendo il pulsante Cine Store. Per catturare la vista apicale a quattro camere, posizionare la piattaforma nella posizione di Trendelenburg, inclinarla verso sinistra e regolare la sonda per visualizzare le quattro camere.Utilizzare Color Doppler per visualizzare il flusso sanguigno e Pulse Wave Doppler sulla punta dei lembi della valvola mitrale al centro dell'orifizio della valvola mitrale per registrare il flusso mitrale. Registrare i dati premendo il pulsante Cine Store. Utilizzare Tissue Doppler sul lato settale dell'anulus della valvola mitrale in una vista a quattro camere per misurare la velocità di rilassamento miocardico di picco nel riempimento diastolico precoce e nel riempimento diastolico tardivo, nonché la velocità di contrazione miocardica sistolica di picco.Registrare i dati premendo il pulsante Cine Store. Cattura la vista dell'asse lungo parasternale modificata per esaminare l'arteria coronaria discendente anteriore sinistra. Utilizzare una vista dell'asse lungo parasternale modificata, spostando il trasduttore lateralmente.Spostare la sonda e utilizzare Color Doppler per visualizzare l'origine dell'arteria coronaria principale sinistra che genera dall'aorta. Identificare l'arteria discendente anteriore sinistra che genera dall'arteria coronaria principale sinistra e corre tra la parete anteriore ventricolare sinistra e il tratto di efflusso ventricolare destro. In questa posizione, misurare il flusso coronarico discendente anteriore sinistro premendo Pulse Wave Doppler.Registrare i dati premendo il pulsante Cine Store. Aumentare la concentrazione di isoflurano al 2,5% e attendere cinque minuti per ottenere il flusso massimo. Registrare i dati premendo il pulsante Cine Store.Nella vista dell'asse lungo parasternale, M-Mode è stato utilizzato per misurare il diametro dell'atrio sinistro. La vista dell'asse corto parasternale è stata utilizzata per misurare le dimensioni della camera ventricolare sinistra e lo spessore della parete, il flusso polmonare e il flusso aortico. La vista apicale a quattro camere è stata utilizzata per esaminare le velocità del flusso sanguigno attraverso la valvola mitrale, nonché le velocità di rilassamento e contrazione miocardica all'anulus della valvola mitrale.La vista dell'asse lungo parasternale modificata è stata utilizzata per esaminare i parametri di flusso dell'arteria coronaria discendente anteriore sinistra. L'aumento dei valori della velocità di picco del flusso coronarico, della velocità media del flusso coronarico e dell'integrale velocità/tempo cinque minuti dopo l'incremento dell'isoflurano ha confermato la risposta attesa all'iperemia nei topi neonatali. A causa delle piccole dimensioni dei cuccioli, è importante mantenere la loro normale temperatura corporea e attaccare strisce di alluminio agli arti per raggiungere gli elettropad e creare un circuito ECG.

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Medicine

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

Riduzione del ventricolo sinistro stress di parete e di miglioramento della funzione in mancanza di Cuori con Algisyl-LVR

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

Ricerca

Medicina

Echocardiographic Assessment of the Right Heart in Mice

La valutazione ecocardiografica del cuore destro in topi

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

Valutazione del ventricolo destro Struttura e funzione in modello murino di polmonare Arteria Costrizione da ecocardiografia transtoracica

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

Legatura permanente dell&#39;arteria discendente anteriore sinistra coronarica nei topi: un modello di post-infarto rimodellante miocardico e insufficienza cardiaca

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

Valutazione Ecografia base di coronaropatia Flow e flusso coronarico Reserve Utilizzando la pressione Sovraccarico modello nei topi

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

Approcci ecocardiografici e protocolli per completa caratterizzazione fenotipica di malattia valvolare cardiaca nei topi

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 ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Nerve-sparing Mid-ostruzione uretrale (Nemo) nel femminile piccoli roditori

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

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

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

Diretto Re-impianto di sinistra coronarica nell&#39;aorta in Adulti con Anomala origine della coronaria sinistra dall&#39;arteria polmonare (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 ...