High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function

Summary

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.

Abstract

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.

Introduction

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 development^3,4, regeneration⁵, and pathologic conditions^1,3,4, including arteriosclerosis, cardiomyopathies, arrhythmias, congenital heart diseases, and amyloid light chain cardiotoxicity^1,4,6. Assessment of cardiac function has been possible during the embryonic stage (1-days post fertilization) through direct video analysis using high-speed video microscopy^7,8. 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 function^{9,10,11,12,13,14,15}.

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 µm. 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 authors^9,12,13, 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 models^6,16, heart injury and recovery^10,13, and regeneration^11,12, and can be further used to evaluate potential drugs.

Protocol

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

1. Setting up the platform for image acquisition
   1. Using small scissors or a scalpel make an incision on a sponge at the 12 o'clock position to hold the fish during scanning. Place the sponge in a glass container (Figure 2A).
      NOTE: The position of the incision should allow enough room to move transducer and also to keep the fish bellow the water line when the platform is tilted for scanning (Figure 2). The incision can vary depending on the size of the fish; however, for a standard size and weight, the incision should be approximately 2.5 cm x 0.7 cm x 0.5 cm (length, width, and depth, respectively). The glass container should be at least 6 cm deep to avoid water leakage while imaging the fish.
   2. Affix the glass box containing the sponge on the ultrasound platform, for instance using double-sided tape. Ensure the glass box is at the center of the platform and firmly attached (Figure 2B).
   3. Tilt the platform forward about 30° using the knob on the left side of the platform holder (Figure 2B). Fill the glass square with 200-250 mL of fish system water containing 0.2 mg/mL tricaine methanesulfonate (MS222).
      NOTE: Tricaine can be prepared as a 4 mg/mL stock solution in Tris 40 mM pH 7 and further diluted to the desired concentration in fish system water; 0.2 mg/mL was found to be the best concentration¹⁶. The 4 mg/mL tricaine stock solution can be stored for a long period of time at -20 °C or at 4 °C for one month.
   4. 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 (see Figure 2B). Leave enough room (10 cm on both sides) for the now connected transducer-rail system to move along the x- and y-axes.
   5. Log in to the control software and choose Mouse (Small) Vascular. Create a new study as well as a new series for each animal included in the study. Find the new study button located on the bottom left side of the screen on the browser page (the view starts in B-mode).

2. Handling the Fish

NOTE: Zebrafish used in this study were adult, 11-month-old males of the wild-type strain AB/Tuebingen (AB/TU). Zebrafish were maintained in a stand-alone flow-through aquarium system at 28 °C in a constant light cycle set as 14 h light/10 h dark. Zebrafish were fed twice daily with brine shrimp (Artemia nauplii) and dry food flakes.

1. Using a fish net, transfer the fish into a small tank containing system water with 0.2 mg/mL tricaine. Wait until the fish is fully anesthetized (no movement and no response to touch).
2. Using a plastic teaspoon, gently and quickly transfer the fish into the glass box containing the sponge into the previously made incision with ventral side of the fish facing up.
   NOTE: Make sure the head of the fish is positioned towards the operator (same direction as the notch of the transducer) and at a slightly higher level compared to the rest of the body to achieve better heart visualization.
3. Gently lower the transducer (keeping its original position) using the handle on the rail system, placing it longitudinally and close to the ventral side of the fish with the notch of the transducer facing the operator. Leave 2-3 mm (no more than 1 cm) clearance from the fish. Adjust the platform in respect to the transducer using the micromanipulator in all 3 axes until the fish heart is visualized and then start image acquisition. The angle of the transducer should not be changed during the entire image acquisition (Figure 2C).
   NOTE: As long as there is enough proximity (up to 1 cm), the water on top of the fish will provide a contact surface via liquid surface tension that allows transmission of the ultrasound waves between the probe and the fish. Therefore, there is no need to push the transducer against the fish. Try to complete this step and finish the scan in less than 3 minutes to prevent fish death or a decrease of the heart rate during image acquisition. If needed, use a timer. The heart can be found on the upper side of the screen towards the left side of the eye, which can be easily visualized if moving the x-axis all the way to the right. If there is continued difficulty in finding the heart while in B-Mode, switch to color Doppler mode, which will allow for tracking blood flow (red indicates blood flowing towards the operator) and locating the heart.

3. Image acquisition

NOTE: See Table of Materials for imaging system and image analysis software.

1. Longitudinal View B-Mode
   1. After localizing the heart, select or stay in B-Mode (found at the bottom left side of the touchscreen after having initiated a new series) and reduce the field in order to zoom in and have a closer look at the heart for easier tracing during analysis.
   2. In order to have a closer and clearer view of the heart in B-Mode image acquisition, reduce the field by zooming in. Use the touchscreen to manually narrow the field on both the x- and y-axes.
   3. If needed, enhance the quality/contrast of the image by setting the dynamic range to 45-50 dB. 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.
   4. Take as many images as desired in the long axis plane by selecting Save Image.
      NOTE: More detailed information and training resources on image acquisition can be found at https://www.visualsonics.com/product/software/vevo-lab and https://www.visualsonics.com/Learning-hub-online-video-training-our-users
2. Longitudinal View Pulse Wave
   1. Switch to Color Doppler for blood flow detection (select Color button) and acquisition (found at the bottom left side of the touchscreen after having initiated a new series).
   2. Using the touch screen position the quadrant on top of the atrioventricular valve and localize the inflow, which will be distinguished by the red color signal (Figure 3A). Reduce the quadrant area as much as possible to increase the frame rate.
      NOTE: Lower the Color pulse-repetition-frequency (Color PRF) (velocity range) to ensure yellow color can be seen in the velocity profile of the Color Doppler image. This will increase the range of velocities that can be seen and will help to create a mosaic of color that will allow to visualize more clearly the peak velocities.
   3. Activate pulse wave (select PW) Doppler Mode to sample ventricular blood inflow velocity. Position the sample volume gate at the center of the atrioventricular valve (where the red color signal becomes more yellowish) to detect the maximum flow velocity. Adjust the PW angle on the screen using your 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.
      NOTE: Make sure the angle correct line is parallel to the blood flow in order to provide consistent and reproducible results. Placing the angle correct line so it matches the direction of blood flow will ensure that velocities are accurately captured.
   4. Repeat step 3.2.3 to determine the outflow velocity by placing the Color Doppler quadrant at the junction between the ventricle and the bulbus (bulbuventricular valve) and localize the flow, which will be distinguished by a blue color signal (Figure 3B). 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.
      NOTE: As mentioned before, to achieve accurate velocity values, make sure the PW angle is aligned with the blood flow.
   5. Adjust the baseline (bar), lowering or raising it in the flow velocity panel, in order to detect and trace completely the signal peaks (Figure 3C,D). Identify the inflow peaks in the upper/positive quadrant (signal going towards the probe) and the outflow peaks in the lower/negative quadrant (signal going away from the probe).

4. Fish recovery

1. As soon as image acquisition is complete, using a teaspoon, transfer the fish into regular system aerated water free of tricaine and let the fish recover (usually takes 30 s to 2 min to resume gill movement and swimming).
2. To help recovery, squirt water repeatedly over the gills using a transfer pipette to promote aeration of the water and oxygen transfer.

5. Image analysis

1. Open the image analysis software.
2. Select an image and click on the image processing icon (Figure 4). Using the available scale (Figure 4), adjust brightness and contrast of the image to allow clear visualization of ventricular walls or blood flow pattern.
3. Using the B-mode image, open the drop-down list from the PSLAX (parasternal long axis) option on the cardiac package/measurements (Figure 4). Select LV trace and trace the ventricular inner wall at systole and diastole to obtain the ventricular area (VA) in systole (VAs) and diastole (VAd), end diastolic volume (EDV), and end systolic volume (ESV) (Figure 5A,B).
   NOTE: Volume values are extrapolated from 2D image tracings and might deviate from the 3D entity. For all measurements, average at least 3 representative cardiac cycles per animal.
4. Note the stroke volume and ejection fraction that will be automatically calculated and displayed by the software.
   NOTE: Stroke volume, and ejection fraction can also be manually calculated using the formulas
   SV = EDV-ESV
   EF = (EDV-ESV)/EDV
   where SV is stroke volume, EDV is end diastolic volume, ESV is end systolic volume, and EF is ejection fraction
5. Calculate fractional area change using the formula
   FAC = (VAd - VAs)/ VAd
   where FAC is fractional area change, VAd is ventricular area in diastole, and VAs is ventricular area in systole.
6. Calculate the cardiac output using the formula
   CO = HR x SV
   where CO is cardiac output, HR is heart rate, and SV is stroke volume
7. Using the Pulsed Wave Doppler Mode image, measure the inflow blood velocity by selecting the MV Flow option under the cardiac package (Figure 4). Select E or A for early diastole and late diastole, respectively, and determine the peak-velocities on the graph (Figure 3C).
8. Measure the outflow blood velocity by selecting AoV Flow and determine the peaks on the tracing (Figure 3D).
9. Measure the heart rate using 2 different methodologies for a more reliable assessment:
   1. When the heart is visualized on the screen during image acquisition, count the beats within 10 s and multiply it by 6.
   2. Using the Pulse Wave Doppler image on the Vevo LAB software, choose the heart rate button and trace intervals between 3 consecutive aortic flow peaks (Figure 4 and Figure 6).
   3. To export data to a spreadsheet after having traced the LV and the peaks of the blood flow, click on report | export | save as | excel.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Double sided tape
Fish net
Glass container - 100 inch high
High frequency transducer	Fujifilm/VisualSonics	MX700	Band width 29-71 MHz, Centre transmit 50 MHz, Axial resolution 30 µm
Plastic teaspoon
Scalpel or scissors
Small fish tanks
Sponge (kitchen sponge)
Transfer pipets (graduated 3 mL)	Samco Scientific	212
Tricaine (MS-222)	Sigma-Aldrich	A5040
Vevo 3100 Imaging system and imaging station	Fujifilm/VisualSonics
Vevo LAB sofware v 1.7.1	Fujifilm/VisualSonics