Name: high-frequency ultrasound echocardiography to assess zebrafish cardiac function
Uploaded: 2020-03-12T15:00:00
Description: Watch this Scientific Journal Video about High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function at JoVE.com

We thank Fred Roberts&#39; technical support and revision of the manuscript.

All procedures involving zebrafish were approved by our Institutional Animal Care and Use Committee and are in compliance with the USDA Animal Welfare Act.
1. Experimental set-up
<ol>
	<li>Setting up the platform for image acquisition
	<ol>
		<li>Using small scissors or a scalpel make an incision on a sponge at the 12 o&#39;clock position to hold the fish during scanning. Place the sponge in a glass container (Figure 2A). 
...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+tissue+Doppler+imaging+of+the+zebrafish+heart+during+its+regeneration.">High-resolution tissue Doppler imaging of the zebrafish heart during its regeneration.</a> Zebrafish. 12 (1), 48-57 (2015).</li><li>Kang, B. 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We describe a systematic method for echocardiographic imaging and assessment of cardiac function in adult zebrafish. Echocardiography is the only available non-invasive and most robust method for live adult fish cardiac imaging and functional analysis, and it is becoming increasingly popular in zebrafish cardiovascular research. The amount of time needed is short and allows for high-throughput and longitudinal studies. However, there is considerable variation in the methodology employed and data analysis. Standardization...

The zebrafish (Danio rerio) is a well-established vertebrate model for studies of developmental processes and human diseases1. Zebrafish have high genetic similarity to humans (70%), genetic tractability, high fecundity, and optical transparency during embryonic development, which allows direct visual analysis of organs and tissues, including the heart. Despite having just one atrium and one ventricle, the zebrafish heart (Figure 1) is physiologically similar to mammalian four-chambered hearts. Importantly, the zebrafish heart rate, electrocardiogram morphology, and action potential shape resemble those of humans more than murine species2. These features have made zebrafish an excellent model for cardiovascular research and have provided major insights into cardiac development3,4, regeneration5, and pathologic conditions1,3,4, including arteriosclerosis, cardiomyopathies, arrhythmias, congenital heart diseases, and amyloid light chain cardiotoxicity1,4,6. Assessment of cardiac function has been possible during the embryonic stage (1-days post fertilization) through direct video analysis using high-speed video microscopy7,8. However, zebrafish lose their transparency beyond the embryonic stage, limiting functional evaluations of normal mature hearts and late-onset heart conditions. To overcome this limitation, echocardiography has been successfully employed as a high-resolution, real-time, noninvasive imaging alternative to evaluate adult zebrafish heart function9,10,11,12,13,14,15.
In zebrafish, the heart is located ventrally in the thoracic cavity immediately posterior to the gills with the atrium located dorsal to the ventricle. The atrium collects venous blood from the sinus venosus and transfers it to the ventricle where it is further pumped to the bulbus arteriosus (Figure 1). Here, we describe a physiological, underwater, protocol to assess cardiac function in adult zebrafish by non-invasive echocardiography using a linear array ultrasound probe with a center frequency of 50 MHz for B-mode imaging at a resolution of 30 &#181;m. Since ultrasound waves can easily travel through water, keeping close proximity between the fish and the scanning probe underwater provides enough contact surface for heart detection with no need for ultrasound gel and is overall less stressful for the fish. Although alternative zebrafish echocardiography systems were reported by several authors9,12,13, here we present the general and most commonly used setup that applies to high-frequency ultrasound in animals.
The method allows high resolution imaging of the adult zebrafish heart, tracing of cardiac structures, and quantification of peak-velocities from Doppler blood flow measurements. We show reliable in vivo quantification of important systolic and diastolic parameters, such as ejection fraction (EF), fractional area change (FAC), ventricular blood inflow and outflow velocities, heart rate (HR), and cardiac output (CO). We contribute to establishing a reliable range of normal healthy adult zebrafish cardiac functional and dimensional parameters to allow a more precise evaluation of pathologic states. Overall, we provide a robust method to assess cardiac function in zebrafish, which has proven extremely useful in establishing and validating zebrafish heart disease models6,16, heart injury and recovery10,13, and regeneration11,12, and can be further used to evaluate potential drugs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Double sided tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fish net</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass container - 100 inch high</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High frequency transducer</td>
			<td>Fujifilm/VisualSonics</td>
			<td>MX700</td>
			<td>Band width 29-71 MHz, Centre transmit 50 MHz, Axial resolution 30 &#181;m</td>
		</tr>
		<tr>
			<td>Plastic teaspoon</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel or scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small fish tanks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sponge (kitchen sponge)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipets (graduated 3 mL)</td>
			<td>Samco Scientific</td>
			<td>212</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine (MS-222)</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo 3100 Imaging system and imaging station</td>
			<td>Fujifilm/VisualSonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo LAB sofware v 1.7.1</td>
			<td>Fujifilm/VisualSonics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

high-frequency ultrasound echocardiography to assess zebrafish cardiac function

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its embryonic transparency, genetic tractability, and amenity to rapid, high-throughput studies. However, the loss of transparency limits heart function analysis at the adult stage, which complicates modeling of age-related heart conditions. To overcome such limitations, high-frequency ultrasound echocardiography in zebrafish is emerging as a viable option. Here, we present a detailed protocol to assess cardiac function in adult zebrafish by non-invasive echocardiography using high-frequency ultrasound. The method allows visualization and analysis of zebrafish heart dimension and quantification of important functional parameters, including heart rate, stroke volume, cardiac output, and ejection fraction. In this method, the fish are anesthetized and kept underwater and can be recovered after the procedure. Although high-frequency ultrasound is an expensive technology, the same imaging platform can be used for different species (e.g., murine and zebrafish) by adapting different transducers. Zebrafish echocardiography is a robust method for cardiac phenotyping, useful in the validation and characterization of disease models, particularly late-onset diseases; drug screens; and studies of heart injury, recovery, and regenerative capacity.

The described protocol allows for measurement of important cardiac dimensional and functional parameters, analogous to the technique used in human and animal echocardiography. The B-Mode images allow for tracing of ventricular inner wall in systole and diastole (Figure 5) and obtaining of dimensional data, such as chamber and wall dimensions, and functional data, such as heart rate, stroke volume, and cardiac output as well as parameters of ventricular systol...

Watch this Scientific Journal Video about High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function at JoVE.com

High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function

We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities.

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its ...

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