This method can help answer key questions in the regenerative field, such as how cardiac regeneration progresses following myocardial injury. The main advantage of this technique is that it provides high resolution, noninvasive, and repeatable evaluation of cardiac function in the axolotl. Additionally, this method can also be applied to other amphibian model organisms, such as newts and xenopus.
Begin with positioning an anesthetized axolotl supine in a lip-shaped animal bed. When the medium gets added, the animal will float, so it must be secured with loose rubber bands. Next, fill the bed with medium containing anesthetics so that the thorax is three to five millimeters deep.
If only using B-mode, color-Doppler mode, and pulse-wave Doppler mode data, then the axolotl can be alert for this procedure. In this situation, position the animal prone in a hammock, and let it recover from the stress for 30 to 60 minutes before proceeding. For white or albino animals, have a cold light source ready to help place the transducer.
For a transducer, use either a 40-or 50-hertz model depending on the animal's mass. Then, prepare the transducer ultrasound gel, and proceed with collecting data. Begin with positioning the ultrasound transducer over the midline of the animal in the thoracic region, and parallel to its long axis.
A small portion of the ventricle, positioned to the right in the thoracic cavity, should appear in-frame at the ventricular diastole. A large portion of both atria should also be visible, as should the sinus venosus. These structures should be identifiable in the diastole or systole view.
Next, translate the transducer one to three millimeters to the right, to obtain the ventricular long-axis view. Ultimately, the correct position is attained when the cross-sectional area of the end-systole ventricle is at its maximum. Correct 2D ultrasound measurements are highly dependent on correct positioning of the transducer.
Practice the transducer placement meticulously and perform inter-operator analysis to minimize subjectivity. Now, in B-mode, acquire at least three cardiac cycles at a minimum of 50 frames per second. Choose between using the general imaging mode or the cardiology mode.
Next, translate the transducer along the long axis of the animal until the center of the ventricle is in the middle of the screen. Then, rotate the transducer 90 degrees clockwise to obtain the midventricular short-axis view. From this position, evaluate the circular shape of the ventricle by translating the transducer along the long axis of the heart.
Then, return the transducer to the long axis plane, and translate it back towards the midline to obtain an atrial long-axis two-chamber view. The correct position is achieved when the cross-sectional areas of the end-systole atria are at their maxima, and the two atria combined assume the outline of the number eight when tilted about 45 degrees to the left. Then, collect B-mode images.
To proceed, translate the transducer to the right until the outflow tract appears. The ventricular end-systole view is correct when the diameter of the outflow is at its largest, and when, during mid-injection, two of the semiluminar valves, at the entrance of the outflow, are visible. From this view, velocity and flow measurements can be made using Doppler imaging.
Apply the color-Doppler mode to map blood flow velocities in the outflow tract during cardiac injection. Next, apply color-Doppler imaging and power-Doppler imaging to visualize the blood flow in the ventricular view, and do the same in the atrial view. Next, use color-Doppler mode directed at the location of maximum blood velocity in the outflow tract running directly towards the transducer.
When the outflow is not completely perpendicular to the transducer, apply a beam angle and angular correction to correct the image by up to 45 degrees. Next, collect velocity-time data in the pulse-wave Doppler mode. During no phase of the cardiac cycle should the spiral valve overlap the view of the outflow.
Collect data from at least three cardiac cycles. Then, without moving the transducer, acquire data in B-mode from at least three cardiac cycles. Next, for anesthetized animals only, rotate the animal 90 degrees so that the right part of the animal is facing upwards, and move the transducer to the oblique para-gill view, just parallel and posterior to the protruding gills.
This view offers an alternative measurement of the blood flow velocity in the outflow tract. The outflow tract should be running downward at about 45 degrees, and the atria should appear under the outflow tract during injection. Finally, switch to pulse-wave Doppler mode and position the transducer to view the maximum blood velocity running away from the transducer in the outflow tract.
As needed, use up to 45 degrees of beam angle and angular correction to make the outflow perpendicular to the transducer. Then, collect data in pulse-wave Doppler mode and in B-mode from the same position as before. 3D acquisition takes a while, so the axolotl must be anesthetized.
Place it supine in the lip-shaped animal bed. Secure it with rubber bands, and submerge its thoracic surface in three to five millimeters of ultrasound medium containing an anesthetic. Movements during 3D acquisition are detrimental to subsequent reconstruction.
If the animal moves during 3D ultrasound acquisition, the procedure must be repeated from the beginning. Next, position the transducer over the midline in the thoracic region, and place it parallel to the long axis for a sagittal 3D recording, or orthogonal to the long axis for transversal 3D recording. Then, translate the transducer to insure that the entire cardiac region gets covered in the subsequent 3D capture.
Move it in both the in-plane dimension and in the out-of-plane dimension. For the imaging mode, if the animal's heart rate is between 20 and 60 beats per minute, select the general imaging mode. Otherwise, choose the cardiology mode.
Turn off the light. Now, in the raw B-mode image, adjust the 2D gain to a level where the anatomical structures are barely recognizable. This will increase signal-to-noise ratio in the final reconstructions.
Then, decide the size of the Z-step or slice thickness. Now, translate the transducer one Z-step at a time, taking a recording containing 1, 000 frames at each Z-step until the entire cardiac region has been covered. A 2D echocardiographic analysis of a 10-gram, 10-centimeter axolotl was performed using the described technique.
The long-axis view provided a good starting point. The midline plane showed the sinus venosus, atria, and parts of the ventricle. Viewing the ventricular plane showed the ventricle to be spherical and highly trabeculated.
In the atrial plane, the atria appeared more irregular and barely trabeculated. The center of the outflow tract is close to the center of the ventricle. Measurements of the cardiac cycle using pulse-wave Doppler from the long-axis and from the oblique para-gill plane had some background noise.
This noise was surmountable when making measurements of the velocity time integral. Color-Doppler and power-Doppler imaging showed the flow pattern through the heart chambers. Views were possible from the ventricle, from the atria, and from the outflow tract.
3D echocardiography was also performed. This multiplanar view of the heart has many uses, such as surface and volume reconstructions, or segmentation and generation of 3D models. After watching this video, you should have a good understanding of how to perform 2D and 3D echocardiography in the axolotl.
Once mastered, the 2D ultrasound technique can be done in less than five minutes per animal, if it is performed properly, whereas the 3D acquisition can take up to an hour per animal. Following this procedure, other methods like heart extraction and histology can be performed in order to answer additional questions related to cardiac structure and anatomy.
