Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice
Summary
The present protocol describes the echocardiographic assessment of left ventricular morphology, function, and coronary blood flow in 7-day old neonate mice.
Abstract
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' 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.
Introduction
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' 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.
Protocol
Representative Results
Discussion
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 …
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Depilating agent | Nair Hair Remover | ||
| Electrode gel | Parker Laboratories | 15-60 | |
| High Frequency Ultrasound | FUJIFILM VisualSonics, Inc. | Vevo 2100 | |
| Isoflurane | MedVet | RXISO-250 | |
| Linear array high frequency transducer | FUJIFILM VisualSonics, Inc. | MS550D | |
| Mice breeding pair | Charles River Laboratories | FVB/N | Strain Code 207 |
| Ultrasound Gel | Parker Laboratories | 11-08 | |
| Vevo Lab Software | FUJIFILM VisualSonics, Inc. | Verison 5.5.1 |
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