Name: echocardiographic approaches and protocols for comprehensive phenotypic characterization of valvular heart disease in mice
Uploaded: 2017-02-14T12:30:00.000+00:00
Duration: 12 min 12 s
Description: Watch this Scientific Journal Video about Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice at JoVE.com

This work was supported by NIH grants HL111121 (JDM) and TR000954 (JDM).

<ol>
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M., 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+cardiac+chamber+quantification+by+echocardiography+in+adults:+an+update+from+the+American+Society+of+Echocardiography+and+the+European+Association+of+Cardiovascular+Imaging.">Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging.</a> Eur Heart J Cardiovasc Imaging. 16, 233-270 (2015).</li><li>Devereux, R. 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+determination+of+left+ventricular+mass+in+man.+Anatomic+validation+of+the+method.">Echocardiographic determination of left ventricular mass in man. Anatomic validation of the method.</a> Circulation. 55, 613-618 (1977).</li><li>Ommen, S. 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G., Lindsey, M. L., Lange, R. A., Aune, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Assessment+in+Pediatric+Mice:+Strain+Analysis+as+a+Diagnostic+Measurement.">Cardiac Assessment in Pediatric Mice: Strain Analysis as a Diagnostic Measurement.</a> Echocardiography. 31, 375-384 (2014).</li><li>Ferferieva, 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=Assessment+of+strain+and+strain+rate+by+two-dimensional+speckle+tracking+in+mice:+comparison+with+tissue+Doppler+echocardiography+and+conductance+catheter+measurements.">Assessment of strain and strain rate by two-dimensional speckle tracking in mice: comparison with tissue Doppler echocardiography and conductance catheter measurements.</a> Eur Heart J Cardiovasc Imaging. 14, 765-773 (2013).</li><li>Fine, N. 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The authors have nothing to disclose.

A indução da anestesia indução e manutenção de anestesia adequada é fundamental para a avaliação precisa das mudanças na válvula cardíaca e função cardíaca em ratos. Dada a rápida indução da anestesia induzida por isoflurano eo tempo de wash-out relativamente longo deste anestésico após anestesia profunda, não usamos uma câmara de anestesia stand-alone para a indução. Em vez disso, tal como referido em pormenor acima, os animais são guiados directamente...

O envelhecimento está associado com aumentos progressivos na calcificação cardiovascular 1. Estenose da valva aórtica hemodinamicamente significativa afeta 3% da população com idade superior a 65 2, e os pacientes com estenose da válvula aórtica, mesmo moderada (velocidade de pico de 3-4 m / s) têm uma sobrevida livre de eventos em 5 ano de menos de 40% 3. Atualmente, não existem tratamentos eficazes para retardar a progressão da calcificação da válvula aórtica e substituição da válvula aórtica cirúrgica é o único tratamento disponível para a estenose da válvula aórtica avançada 4. Estudos destinados a ganhar uma compreensão mais profunda dos mecanismos que contribuem para o início e progressão de calcificação da válvula aórtica é um primeiro passo fundamental no caminho para métodos farmacológicos e não-cirúrgicos para gerenciar estenose da valva aórtica 5, 6. Genéticocamundongos ly-alteradas têm desempenhado um papel importante no desenvolvimento de nossa compreensão dos mecanismos que contribuem para uma variedade de doenças e agora estão vindo à tona de estudos sobre os mecanismos que visam a compreensão da biologia da estenose da válvula aórtica 6, 7, 8. Ao contrário de outras doenças cardiovasculares, tais como aterosclerose e insuficiência cardíaca onde-protocolos padrão para avaliar a função ventricular e vascular são, na maior parte bem estabelecida-existem desafios únicos associados com a fenotipagem in vivo da função de válvula de coração em ratinhos. Enquanto comentários recentes têm proporcionado debates aprofundados sobre as vantagens e desvantagens de inúmeras imagens e modalidades invasivos utilizados para avaliar a função da válvula em roedores 9, 10, 11, até à data, não temos conhecimento de uma publicação que fornece uma comprehensive, passo-a-passo protocolo para a função de válvula cardíaca fenotipagem em camundongos. O objetivo deste artigo é descrever os métodos e protocolos para o fenótipo função de válvula cardíaca em camundongos. Todos os métodos e procedimentos foram aprovados pelo Comitê Animal Care Institucional e Use a Mayo Clinic. Os principais componentes deste protocolo incluem a profundidade da anestesia, a avaliação da função cardíaca, bem como a avaliação da função de válvula cardíaca. Esperamos que este relatório não só servirá para orientar os investigadores interessados ​​em prosseguir a investigação no domínio da doença da válvula do coração, mas também vai iniciar um diálogo nacional e internacional relacionada com a padronização do protocolo para garantir a reprodutibilidade dos dados e validade neste campo de rápido crescimento. Importante, uma imagem com o uso de sistemas de ultra-som de alta resolução exige um conhecimento prático dos princípios da ultra-sonografia (e terminologia comumente utilizados em ultra-sonografia), uma compreensão do princip fundamentaisles da fisiologia cardíaca e experiência significativa com a ultra-sonografia para permitir a avaliação precisa e tempo-eficiente da função cardíaca em roedores.

<table><tbody><tr><td>High resolution ultrasound machine</td><td>VisualSonics, Fujifilm</td><td>Vevo 2100&amp;nbsp;</td><td></td></tr><tr><td>Isoflurane diffuser (capable of delivering 1 % to 1.5 % isoflurane mixed with 1 L/min 100% O&lt;sub&gt;2&lt;/sub&gt;</td><td>VisualSonics, Fujifilm</td><td>N/A</td><td></td></tr><tr><td>Transducers for small mice (550D) or larger mice (400)</td><td>MicroScan, VisualSonics, Fujifilm</td><td>MS 550D, MS 400</td><td></td></tr><tr><td>Animal platform</td><td>VisualSonics, Fujifilm</td><td>11503</td><td></td></tr><tr><td>Advanced physiological monitoring unit</td><td>VisualSonics, Fujifilm</td><td>N/A</td><td></td></tr><tr><td>Isoflurane</td><td>Terrell</td><td>NDC 66794-019-10</td><td></td></tr><tr><td>Nose cone and tubing connected to isoflurane diffuser and 100% O&lt;sub&gt;2&lt;/sub&gt;</td><td>Custom Engineered in-house</td><td>--</td><td></td></tr><tr><td>Hair razor</td><td>Andis Super AGR+ vet pack clipper</td><td>AD65340</td><td></td></tr><tr><td>Ultrasound gel</td><td>Parker Laboratories</td><td>REF 01-08</td><td></td></tr><tr><td>Electrode gel&amp;nbsp;</td><td>Parker Laboratories</td><td>REF 15-25</td><td></td></tr><tr><td>Adhesive tapes</td><td>Fisher Laboratories</td><td>1590120B</td><td></td></tr><tr><td>Paper towels</td><td></td><td></td><td></td></tr></tbody></table>

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.

Exemplos de imagens que são rotineiramente obtidos a partir de animais imagiologia ultra-sons cardíacos são incluídos neste manuscrito. Uma ilustração do posicionamento do transdutor sobre o peito do animal é fornecida para dar ao leitor uma compreensão clara de que o transdutor está posicionado para obtenção de imagens tal como descrito. Uma fotografia do laboratório de ultra-som set-up também está incluído para enfatizar a importância do equipamento adequado, especialme...

Watch this Scientific Journal Video about Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice at JoVE.com

Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in Mice

This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice.

Abordagens ecocardiograma e protocolos de Comprehensive Caracterização fenotípica de doença cardíaca valvular em ratos

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 ...

echocardiographic-approaches-protocols-for-comprehensive-phenotypic

Research

JoVE Journal

Medicine

15.9K visualizações.  Mayo Clinic. This protocol provides a detailed description of the echocardiographic approach for comprehensive phenotyping of heart and heart valve function in mice. 

Vídeo: Echocardiographic Approaches and Protocols for Comprehensive Phenotypic Characterization of Valvular Heart Disease in 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.

Ecocardiografia transtorácica em Ratos

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

Medicina

Murine Echocardiography and Ultrasound Imaging

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1 Echocardiography provides a powerful, non-invasive tool to serially assess cardiac morphometry and function in a living animal.2 However, using this technique on mice poses unique challenges owing to the small size and rapid heart rate of these animals.3 Until recently, few ultrasound systems were capable of performing quality echocardiography on mice, and those generally lacked the image resolution and frame rate necessary to obtain truly quantitative measurements. Newly released systems such as the VisualSonics Vevo2100 provide new tools for researchers to carefully and non-invasively investigate cardiac function in mice. This system generates high resolution images and provides analysis capabilities similar to those used with human patients. Although color Doppler has been available for over 30 years in humans, this valuable technology has only recently been possible in rodent ultrasound.4,5 Color Doppler has broad applications for echocardiography, including the ability to quickly assess flow directionality in vessels and through valves, and to rapidly identify valve regurgitation. Strain analysis is a critical advance that is utilized to quantitatively measure regional myocardial function.6 This technique has the potential to detect changes in pathology, or resolution of pathology, earlier than conventional techniques. Coupled with the addition of three-dimensional image reconstruction, volumetric assessment of whole-organs is possible, including visualization and assessment of cardiac and vascular structures. Murine-compatible contrast imaging can also allow for volumetric measurements and tissue perfusion assessment.

This video demonstrates use of a rail-mounted high-frequency ultrasound probe to perform echocardiography on an anesthetized mouse. The methods describe both conventional two-dimensional and M-mode measurements of cardiac function in addition to newer, more powerful tools such as color Doppler, strain analysis, as well as general and targeted contrast imaging.

Ecocardiografia murino e ultra-sonografia

Rodent models of cardiac pathophysiology represent a valuable research tool to investigate mechanism of disease as well as test new therapeutics.1  ...

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.

Avaliação ecocardiográfica do coração direito em Ratos

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

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.

Um Pipeline para Caracterizar Defeitos Cardíacos Estruturais em Rato Fetal

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 ...

Biologia do Desenvolvimento

Assessment of Cardiac Morphological and Functional Changes in Mouse Model of Transverse Aortic Constriction by Echocardiographic Imaging

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable noninvasive method is essential to assess real-time cardiac morphological and functional changes in animal models of heart disease. Transthoracic echocardiography represents an important tool for noninvasive assessment of cardiac structure and function2. Here we used a high-resolution ultrasound imaging system to monitor myocardial remodeling and heart failure progression over time in a mouse model of TAC. B-mode, M-mode, and Doppler imaging were used to precisely assess cardiac hypertrophy, ventricular dilatation, and functional deterioration in mice following TAC. Color and pulse wave (PW) Doppler imaging was used to noninvasively measure pressure gradient across the aortic constriction created by TAC and to assess transmitral blood flow in mice. Thus transthoracic echocardiographic imaging provides comprehensive noninvasive measurements of cardiac dimensions and function in mouse models of heart disease.

The goal of this protocol is to noninvasively assess cardiac structural and functional changes in a mouse model of heart disease created by transverse aortic constriction, using B- and M-mode echocardiography and color/pulse wave Doppler imaging.

Avaliação da morfológicas e funcionais cardíacas Mudanças no Modelo rato de Transverse aórtica constrição por ecocardiográfica de imagem

Transverse aortic constriction (TAC) in mice has been used as a valuable model to study mechanisms of cardiac hypertrophy and heart failure1. A reliable ...

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.

Murino Ecocardiografia de Esquerda Atrium, Aorta, ea artéria pulmonar

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.

Exame ecocardiográfico e histológico da morfologia cardíaca no Mouse

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.

Mensuração ecocardiográfica dos parâmetros diastólicos do ventrículo direito no mouse

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

Revealing Subtle Changes in Cardiac Function using Transthoracic Dobutamine Stress Echocardiography in Mice

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic dobutamine stress echocardiography in humans, a reductionist investigation approach to unmask subtle changes in cardiac function has become possible. Here, we provide a protocol for using this technique in mice to facilitate expanded analysis of LV architecture and function in physiology and pathology enabling the observation of alterations in models of cardiac disease hidden in unstressed hearts. This investigation can be performed in one and the same animal and allows both, basal and pharmacologically stress-induced measurements. We outline detailed criteria for appropriate anesthesia, imaging-based LV analysis, consideration of intra- and interobserver variability, and obtaining positive inotrope response that can be attained in mice after intraperitoneal injection of dobutamine under near physiological conditions. To recapitulate the characteristics of human physiology and disease in small animal models, we highlight critical pitfalls in evaluation, e.g., a pronounced Bowditch effect in mice. To further meet translational objectives, we compare stress-induced effects in humans and mice. When used in translational studies, attention must be paid to physiological differences between mice and human. Experimental rigor dictates that some parameters assessed in patients can only be used with caution due to restrictions in spatial and temporal resolution in mouse models.

<meta charset="utf8"></head><body>Revelando alterações sutis na função cardíaca usando ecocardiografia transtorácica de estresse com dobutamina em camundongos

Left ventricular dysfunction constitutes the final common pathway for a host of cardiac disorders. We here provide a detailed protocol of transthoracic dobutamine stress echocardiography approach for comprehensive evaluation of the left ventricular function of mouse models of cardiac disease as well as cardiac phenotyping.