This video aims to demonstrate step by step a protocol for a research-grade echocardiographic examination in a large animal model such as the rabbit. This is accomplished by first anesthetizing the animal, and then carefully preparing the chest for the procedure. Next, after placing the rabbit on a thermal blanket and verifying that an ECG signal is correctly displayed, position the animal in a right recumbent position to obtain the imaging planes in both a parasternal long axis view, and a parasternal short axis view of the heart.
Then position the rabbit in a left recumbent position to obtain the different imaging planes available in an apical four chamber view, and an apical five chamber view of the heart. Movies and image stills should be acquired in B-Mode and M-Mode for analysis of cardiac systolic function as well as a combination of B-Mode, color doppler, and pulsed-wave doppler, PWD, for blood flow analysis. Or a combination of B-Mode and tissue doppler imaging, TDI, modes for analysis of the myocardial velocities in rabbit heart.
Longitudinal evaluation for cardiac function using echocardiographic enlarged animal models this serves as methodology for apical assessment of heart failure therapies. The purpose of this vidoe is to demonstrate a step-by-step echocardiographic protocol for the evaluation of cardiac function using a critical ultrasound system on a large preclinical model such as the rabbit. The protocol shown in this video is in line with current international guidelines.
Practical recommendations based on our own experiences in clinical and experimental settings are also included. Before proceeding, start by injecting a combination of Ketamine and Medetomidine to anesthetize the animal. Once the animal is anesthetized, use a hair clipper to remove the hair from the skin of the thorax.
Shave one to three centimeters of the internal face of the right forelimb as well as the mediotibial regions of both right and left hindlimbs. After placing the rabbit on a thermal blanket apply a suitable conducting gel to the electrodes and position these in the shaved regions of the limbs. Fix the electrodes with surgical tape.
Verify that a correct ECG signal is displayed on the screen of the system. To obtain a parasternal long axis view of the heart, place the animal on the right lateral recumbent position with the forelimbs outstretched away from the thorax with surgical tape. It is important to maintain the skin of the thorax as flat as possible.
For this, hold the forelimbs away from the thorax with one hand whilst using the free hand to identify any skin folds and pockets, and flatten these from top to bottom moving any skin folding away from the chest and displacing the abdominal organs towards the caudal region through gentle massaging. Apply ultrasound transmission gel to the head of the transducer. Next position the transducer closely to the skin of the right hemithorax at the level of the second to third intercostal space and about one to three centimeters away from the right parasternal line with the transducer orientation mark pointing to the right shoulder of the animal.
You should obtain two imaging planes in the parasternal short axis view of the heart. An imaging plane which sections the heart longitudinally and where all four chambers of the heart, two atria and two ventricles can be identified. Perform subtle movements of the transducer such as sweeping, rocking, and rotation relative to the intercostal space as well as the antero, posterior, and dorsoventral angle of the ultrasound beam to obtain the other imaging plane of the parasternal long axis view.
Once you obtain this imaging plane, you will also be able to identify the left ventricular outflow tract, LVOT, and the aorta. After obtaining the appropriate imaging plane, use a combination of B-mode and color doppler to assess blood flow across all valves and the integrity of the interventricular septum. With the transducer in a parasternal long axis view, perform a counterclockwise rotation of the transducer of approximately 90 degrees to obtain a right parasternal short axis view.
This time the transducer orientation mark should be pointing towards the left shoulder of the rabbit. To help maintain the transducer in the same location of the chest whilst rotating the transducer, use the left hand to perform the rotation from the cord of the transducer. In the parasternal short axis view, you should routinely obtain three imaging planes by sweeping the transducer along the axis of the heart.
The midventricular imaging plane which sections the heart at the papillary muscles. Here you typically can visualize the right ventricle at the top and the left ventricle at the bottom of the image. Using B-mode, you can evaluate radial and circumferential contraction and relaxation of the left ventricle.
Use M-mode and then place the cursor in the middle of the left ventricle between both papillary muscles. By sweeping the transducer towards the cephalic region you should obtain a mitral valve plane. Use B-mode and M-mode and place the cursor along the middle of the left ventricle to evaluate the integrity and motility of the mitral valve leaflets.
Sweeping the transducer further cranially will result in an imaging plane at the level of the high base also known as the aortic valve plane. To completely visualize the pulmonary artery and its bifurcation, a greater angulation and sometimes a cranial displacement of the transducer and intercostal space may be necessary. You should identify in this view the aortic valve and its leaflets, the right atrium, the left atrium, the right ventricular outflow tract, the pulmonary valve, and the pulmonary artery.
Use B-mode then use M-mode and place the cursor along the aortic valve and left atrium. Finally use color doppler and pulsed wave doppler modes to record the velocity of blood flow at the pulmonary valve level by placing the sample volume just below the opening of the pulmonary valve leaflets. To obtain an apical four chambers view, place the rabbit in the left lateral recumbent position maintain the skin of the thorax flat in a similar way as described before.
Apply ultrasound gel to the transducer then position it closely to the skin of the left hemithroax at the level of the fourth and fifth intercostal spaces with the midclavicular line with the transducer orientation mark pointing towards the back of the rabbit in the direction of the left scapula. From this position if necessary move the transducer upward one intercostal space at a time, a maneuver often called window shopping. Once you reach the appropriate intercostal space you should observe an image of the heart from the apex to the base of the heart.
The typical heart shape where all four chambers can be seen with the left and right ventricles at the top and both atria at the bottom of the image. Use B-mode to check for regional wall motion abnormalities and have a global view of the left ventricular function. Use color doppler to evaluate flow across the atrioventricular valves and use pulsed wave doppler and position the sample volume at the level of the mitral valve leaflet tips to obtain images of the mitral valve inflow spectrum.
Use tissue doppler imaging mode and place the sample volume at the septal and lateral sides of the mitral valve anulus. Starting with the transducer in the apical four chambers view, perform a gentle tilting cordially until the left ventricular outflow tract and the aortic valve come into view. This is the apical five chambers view of the heart.
Use B-mode to evaluate the left ventricular outflow tract, the movement of the aortic valve leaflets, as well as the left ventricular cavity size and function. Use color doppler mode for evaluation of outflow across the aortic valve and use pulsed wave doppler to asses flow velocity across this valve by positioning the sample volume just behind the aortic valve. Here we show an imaging plane of the right parasternal long axis view where the four chambers of the heart are clearly distinguishable.
You can identify in this view the right ventricle, left ventricle, the right atrium, the left atrium, interventricular septum, the free wall of the left ventricle, as well as the mitral valve. In this view, it is possible to estimate accurately the left ventricular volume using the biplane method of discs. This is the Modified Simpsons Rule as shown in this image in diastole and in systole.
Here the other imaging plane of the right parasternal long axis is shown. The location for placement of the calipers for accurate measurement of the left ventricular outflow tract is shown in this image. In this image, a right parasternal short axis view of the heart at the level of the papillary muscles plane is shown.
You can identify in this view the right ventricle, left ventricle, interventricular septum, the left ventricular free wall, as well as the anterolateral and posteromedial papillary muscles. An example of an M-mode trace in the parasternal short axis view at the papillary muscles level is shown in this image where the placement of calipers for the measurements of the right ventricle, left ventricle, interventricular septum, and left ventricular free wall is also demonstrated. The measurements corresponding to the left ventricular end diastolic diameter, LVDd, and left ventricular end systolic diameter, LVDs, are also shown.
This is an example of an M-mode trace at the level of the mitral valve plane in the parasternal short axis view. The location of the calipers for the measurement of the E-point Septal separation, EPSS, of the mitral valve is also shown. An example of the pulmonary valve outflow analysis using both color doppler and pulsed wave doppler is shown in this image.
An example of mitral valve inflow using color doppler in an apical four chambers view is shown here. In this same view and using pulsed wave doppler, the mitral valve inflow spectrum can be assessed as shown in this image where the Early, E, and Late, A, filling waves during diastole are easily differentiated. This image shows an example of myocardial tissue velocities using tissue doppler imaging with the sample volume located at the septal wall of the mitral valve anulus.
The systolic component is denoted by the S-wave whilst the E prime and A prime waves correspond with the myocardial movement of the mitral valve anulus during early filling, E prime, and late filling, A prime, components of diastole. This image shows an example of color doppler positioned at the left ventricular outflow tract in an apical five chambers view. Here we show an example of how to quantitate the aortic valve outflow using pulsed wave doppler signal to evaluate the volume-time integral, VTI, systolic ejection time, ET, as well as the pre-ejection period, PEP, of the aortic valve.
The development of a standardized protocol and correct technique are essential to minimize the influence of the operator and to generate reliable quantitative data. After watching this video, you should have a good understanding of the essential steps of how to perform a transthoracic and cardiographic examination in the rabbit model. If you go and practice under supervision of a mentor will help to find and develop your echocardiographic skills.
In experienced hands this examination can be completed within 15 minutes after inducing anesthesia.
