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>
	<li>Santoriello, C., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooked!+Modeling+human+disease+in+zebrafish.">Hooked! Modeling human disease in zebrafish.</a> Journal of Clinical Investigation. 122 (7), 2337-2343 (2012).</li><li>Verkerk, A. O., Remme, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+novel+research+tool+for+cardiac+(patho)electrophysiology+and+ion+channel+disorders.">Zebrafish: a novel research tool for cardiac (patho)electrophysiology and ion channel disorders.</a> Frontiers in Physiology. 3, 255 (2012).</li><li>Bakkers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+to+study+cardiac+development+and+human+cardiac+disease.">Zebrafish as a model to study cardiac development and human cardiac disease.</a> Cardiovascular research. 91 (2), 279-288 (2011).</li><li>Poon, K. L., Brand, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+model+system+in+cardiovascular+research:+A+tiny+fish+with+mighty+prospects.">The zebrafish model system in cardiovascular research: A tiny fish with mighty prospects.</a> Global Cardiology Science and Practise. 2013 (1), 9-28 (2013).</li><li>Jopling, C., 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=Zebrafish+heart+regeneration+occurs+by+cardiomyocyte+dedifferentiation+and+proliferation.">Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation.</a> Nature. 464 (7288), 606-609 (2010).</li><li>Mishra, S., 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=Zebrafish+model+of+amyloid+light+chain+cardiotoxicity:+regeneration+versus+degeneration.">Zebrafish model of amyloid light chain cardiotoxicity: regeneration versus degeneration.</a> American Journal of Physiology Heart Circulatory Physiology. 316 (5), H1158-H1166 (2019).</li><li>Shin, J. T., Pomerantsev, E. V., Mably, J. D., MacRae, C. A. <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+cardiovascular+function+confirms+functional+orthology+of+myocardial+contractility+pathways+in+zebrafish.">High-resolution cardiovascular function confirms functional orthology of myocardial contractility pathways in zebrafish.</a> Physiologycal Genomics. 42 (2), 300-309 (2010).</li><li>Mishra, S., 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=Human+amyloidogenic+light+chain+proteins+result+in+cardiac+dysfunction,+cell+death,+and+early+mortality+in+zebrafish.">Human amyloidogenic light chain proteins result in cardiac dysfunction, cell death, and early mortality in zebrafish.</a> American Journal of Physiology Heart Circulatory Physiology. 305 (1), H95-H103 (2013).</li><li>Ernens, I., Lumley, A. I., Devaux, Y., Wagner, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Coronary+Ultrasound+Imaging+to+Evaluate+Ventricular+Function+in+Adult+Zebrafish.">Use of Coronary Ultrasound Imaging to Evaluate Ventricular Function in Adult Zebrafish.</a> Zebrafish. 13 (6), 477-480 (2016).</li><li>Gonz&#225;lez-Rosa, J. 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=Use+of+Echocardiography+Reveals+Reestablishment+of+Ventricular+Pumping+Efficiency+and+Partial+Ventricular+Wall+Motion+Recovery+upon+Ventricular+Cryoinjury+in+the+Zebrafish.">Use of Echocardiography Reveals Reestablishment of Ventricular Pumping Efficiency and Partial Ventricular Wall Motion Recovery upon Ventricular Cryoinjury in the Zebrafish.</a> PLoS One. 9 (12), (2014).</li><li>Huang, C. C., Su, T. H., Shih, C. 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. J., 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=High-frequency+dual+mode+pulsed+wave+Doppler+imaging+for+monitoring+the+functional+regeneration+of+adult+zebrafish+hearts.">High-frequency dual mode pulsed wave Doppler imaging for monitoring the functional regeneration of adult zebrafish hearts.</a> Journal of the Royal Society Interface. 12 (103), (2015).</li><li>Lee, J., 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=Hemodynamics+and+ventricular+function+in+a+zebrafish+model+of+injury+and+repair.">Hemodynamics and ventricular function in a zebrafish model of injury and repair.</a> Zebrafish. 11 (5), 447-454 (2014).</li><li>Sun, L., Lien, C. L., Xu, X., Shung, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Cardiac+Imaging+of+Adult+Zebrafish+Using+High+Frequency+Ultrasound+(45-75+MHz).">In Vivo Cardiac Imaging of Adult Zebrafish Using High Frequency Ultrasound (45-75 MHz).</a> Ultrasound in Medicine and Biology. 34 (1), 31-39 (2008).</li><li>Wang, L. W., Kesteven, S. H., Huttner, I. G., Feneley, M. P., Fatkin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Frequency+Echocardiography-+Transformative+Clinical+and+Research+Applications+in+Humans,+Mice,+and+Zebrafish.">High-Frequency Echocardiography- Transformative Clinical and Research Applications in Humans, Mice, and Zebrafish.</a> Circulation Journal. 82 (3), 620-628 (2018).</li><li>Wang, L. W., 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=Standardized+echocardiographic+assessment+of+cardiac+function+in+normal+adult+zebrafish+and+heart+disease+models.">Standardized echocardiographic assessment of cardiac function in normal adult zebrafish and heart disease models.</a> Disease Models &amp; Mechanisms. 10 (1), 63 (2017).</li><li>Lee, L., 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=Functional+Assessment+of+Cardiac+Responses+of+Adult+Zebrafish+(Danio+rerio)+to+Acute+and+Chronic+Temperature+Change+Using+High-Resolution+Echocardiography.">Functional Assessment of Cardiac Responses of Adult Zebrafish (Danio rerio) to Acute and Chronic Temperature Change Using High-Resolution Echocardiography.</a> PLOS ONE. 11 (1), e0145163 (2016).</li><li>Genge, C. E., et al., Nilius, B., 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=.">.</a> Reviews of Physiology, Biochemistry and Pharmacology. 171, 99-136 (2016).</li></ol>

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 ...

The protocol allows noninvasive and high-resolution representation and analysis of adult zebrafish heart function. This method can be used for validation of adult heart disease models and potential drug screens. This technique allow rapid positioning of the fish for correct imaging providing accurate and reproducible results.It is noninvasive and performed underwater which allows the rapid recovery of the fish. To set up the platform for image acquisition, use small scissors or a scalpel to make an incision on a sponge at the 12 o'clock position. Place the sponge in a glass container.Using double-sided tape, affix the glass container containing the sponge on the ultrasound platform. Ensure the glass container is at the center of the platform and firmly attached. Tilt the platform forward about 30 degrees using the knob on the left side of the platform holder.Fill the glass container with approximately 200 milliliters of fish system water containing 0.2 milligrams per milliliter tricaine methanesulfonate. Insert the transducer within the micromanipulator holder on the working rail station turning the notch of the transducer towards the operator. Keep the array parallel to the ground with the working side longitudinal with respect to the stage.Leave enough room about 10 centimeters on both sides for the now connected transducer rail system to move along the x and y-axes. Log in to the control software and choose mouse small vascular. Create a new study as well as a new series for each animal induced in the study.Press the new study button located on the bottom left side of the screen on the browser page. The view starts in B-mode. Using a fish net, transfer the fish into a small tank containing system water with 0.2 milligrams per milliliter of tricaine.Wait until the fish is fully anesthetized with no movement or no response to touch. Gently and quickly transfer the fish into the glass container with the sponge into the previously made incision with the ventral side of the fish facing up. Gently lower the transducer using the handle on the rail system placing it longitudinally and close the ventral side of the fish with the notch of the transducer facing the operator.Leave two to three millimeters of clearance from the fish. Adjust the platform in respect to the transducer using the micromanipulator in all three axes until the fish heart is visualized. Immediately after localizing the heart, select or stay in B-mode and reduce the field by zooming in to achieve a closer look at the heart.If needed, enhance the quality or contrast of the image by setting the dynamic range to 45 to 50 decibels. Go to the B-mode controls in the more controls option and subsequently save the change to mode presets. Tap mode presets to select the optimized image acquisition setting every time before starting to image a new series.Take as many images as desired in the long axis plane. Next, switch to color Doppler for blood flow detection and acquisition. Using the touchscreen, position the quadrant on top of the atrioventricular valve and localize the inflow which is distinguished by the red color signal.Reduce the quadrant area as much as possible to increase the frame rate. Activate the pulse wave Doppler move to sample ventricular blood inflow velocity. Position the sample volume gate at the center of the atrioventricular valve to detect the maximum flow velocity.The red color signal becomes more yellowish. Adjust the pulse wave angle on the screen using fingers so it aligns with the direction of the blood inflow. Press start or update to start sampling the velocity of blood flowing into the ventricle.Determine the outflow velocity by placing the color Doppler quadrant at the junction between the ventricle and the bulbus and localize the flow. This is distinguished by a blue color signal. Position the sample volume gate right before the ventricle bulbus junction and adjust the angle correction line to match the direction of the blood flow.Adjust the baseline raising it in the flow velocity panel in order to completely detect and trace the signal peaks. As soon as the image acquisition is complete, use a teaspoon and transfer the fish into regular system aerated water free of tricaine and let the fish recover. To help recovery, squirt water repeatedly over the gills using a transfer pipette to promote aeration of the water and oxygen transfer.It usually takes 30 seconds to two minutes to resume gill movement and swimming. Open the image analysis software. Select an image and click on the image processing icon.Adjust brightness and contrast of the image to allow clear visualization of ventricular walls or blood flow pattern. Using the B-mode image, open the dropdown list from the parasternal long axis option on the cardiac package measurements. Select LV trace and trace the ventricular inner wall at systole and diastole to obtain the ventricular area in systole and diastole and diastolic volume and end systolic volume.In this study, the B-mode images allowed for tracing of ventricular inner wall in systole and diastole and obtaining of dimensional data such as chamber and wall dimensions. Functional data was also obtained such as heart rate, stroke volume, and cardiac output as well as parameters of ventricular systolic function including fractional area change and ejection fraction. Measurements at the level of the atrioventricular valve using color Doppler mode images also provided ventricular inflow and outflow blood velocities.Based on the pulse wave Doppler image, a heart rate value was generated by tracing three consecutive aortic flow peaks. The most important thing is to position the ultrasound platform and the fish correctly for accurate imaging and to minimize the fish exposure to tricaine. Additional methods to support this procedure include fish electrocardiogram for rapid detection of heart dysfunction like arrhythmias or histology methods to evaluate heart tissue structure or cell death.These techniques allow evaluation of zebrafish heart function and establish a cardiac phenotype in zebrafish heart disease models. It also allows to test new drugs, study heart injury and regenerative capacity. Tricaine MS-222 is a skin, eye, and respiratory irritant.When preparing tricaine solution, protective equipment should include a dust mask, eye shield and gloves.

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