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 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.