Approcci ecocardiografici e protocolli per completa caratterizzazione fenotipica di malattia valvolare cardiaca nei topi

The overall goal of this echo protocol is to show methods and approaches used to image heart valve function in rodents. The methods we'll be showing you today can answer key questions in the field of heart disease, with a specific emphasis on evaluating the heart as integrated unit under conditions of heart valve disease. The implications of this approach are quiet profound for the field of heart valve diseases because the comprehensive echocardiographic evaluation of the heart will not only allow for detection of valvular dysfunction in multiple heart valves, but will also allow investigators to understand the complex interplay between cardiac function and various measures of heart valve function.

Though this method can be leveraged to characterize animal models in which heart valve disease is our primary defect, it can also be applied to other systems where heart valve dysfunction may appear secondary to other insults such as heart failure, or a thoracic aortic aneurysm. Thus, it is our recommendation that comprehensive cardiac and heart valve phenotyping should be routinely performed in most research studies. We first had the idea to film our approach to comprehensive cardiac and heart valve phenotyping after attending a number of meetings where there was a profound variability in the methods and protocols used to assess heart valve function.

It is our hope that this manuscript and the accompanying video spurs discussion in the field regarding the best methodologies and standards to follow, which ultimately aligns with recent initiatives from the National Institutes of Health to improve rigor and reproducibility of research studies. The main advantage of echocardiography is that it is able to noninvasively assess morphology and function of the heart in real time. It is easy to perform, and can be performed serially.

Thus, it is most ideal in serial follow up studies. Generally, individuals new to cardiac and heart valve imaging will struggle due to logistical challenges associated with administration and titration of anesthesia, as well as the technical challenges associated with the very small anatomic windows that can be used for echocardiography and the need to precisely identify cardiac structures and probe orientations while imaging. We hope that visual demonstration of our approach accelerates the rate at which investigators can learn to conduct echocardiography in mice, and will ultimately serve as a platform to discuss best practices for small animal imaging.

To begin, turn on the ultrasound machine equipped with a high frequency ultrasound transducer. Enter the ID of the animal to be measured, along with date, time, and any other relevant information. Preheat the platform to 37 degrees celsius, and then gently pick up the mouse by it's tail, and firmly hold the animal at the nape of it's neck.

Guide the nose of the animal into the nose cone and initiate anesthesia as described in the accompanying text protocol. Once sedated, quickly and accurately lay the animal on the platform in a supine position, making sure that the forefeet and hindfeet lie on the ECG sensors of the platform. Use adhesive tape to secure the animal on all four limbs, to stabilize the head in the nose cone apparatus, and to stabilize the tail.

Next, check the heart rate using the ECG sensors in the platform, or using an external device. Ensure that the baseline heart rate is between 600 to 700 beats per minute. Monitor the heart rate and ensure that it does not fall below 450 beats per minute under any circumstances.

In addition, monitor the body temperature using a rectal thermometer and keep the temperature between 36.5 degrees celsius and 38 degrees celsius. As long as the vitals are stable, use an electric clipper designed for use with fine hair to shave off the hair from the mouse's chest. Then, take a damp paper towel and wipe the chest to remove any remaining hairs.

With the animal securely fastened on the platform, and the head facing away, tilt the table 15 to 20 degrees to the left to bring the heart closer to the chest wall. Then, apply a generous amount of ultrasound gel on the transducer or directly on the animal's chest. Position the transducer parasternally about 90 degrees perpendicular with the long axis of the heart so that the image index marker of the transducer is pointing posteriorly.

While in two DB mode slide the transducer's defilade until the aortic valve comes into view along the short axis. Next, rotate the transducer clockwise until the image index marker points cadad. This is the parasternal long axis view.

Observe the aortic route, aortic valve, left ventricular outflow tract, mitral valve, left atrium, and part of the right ventricular outflow tract on the image display. Measure the largest atrial posterior dimension of the aorta using the electronic caliper associated with the measurement tool that is embedded in the machine. Next, reduce the image width so that only the aortic valve is on the image display by adjusting the image width button in the control panel.

Position the M mode line of interrogation where it intersects the tips of the aortic valve to accurately assess aortic valve cusp separation. In the M mode display of the aortic valve, measure the cusp separation distance using the electronic caliper. While still in the parasternal long axis view of the aortic valve, press the color doppler control key in the control panel to apply color doppler to the region of the aortic valve.

Next, press the post wave doppler control key. Using the track ball, place the post wave sample volume in the proximal ascending aorta just above the aortic valve, making sure that the angle between the ultrasound beam and the blood flow is less than 60 degrees by tilting the platform, and or the transducer. Then, measure the peak velocity from the spectral display using the electronic calipers.

Start by placing the transducer in the apical position in B mode and positioning the transducer so that it is angled towards the head of the mouse. Observe the right ventricle, left ventricle, right atrium and left atrium, on the image display. From the apical four chamber view bring the mitral valve in focus by reducing the image width.

Then, place the M mode cursor across the mitral valve to assess the thickness of the leaflets. Using the apical four chamber view, apply color doppler to image the flow from the left atrium through the mitral valve during diastole. Observe the flow across the mitral valve which is encoded in red.

From the apical lung axis view, tilt or point the transducer tip using a rocking motion so that the right ventricle is at the center of the image display. Reduce the image width so that only the right ventricle is visible in the image display. Then, apply color doppler in the region of the tricuspid valve.

Next, move the transducer to a modified parasternal long axis position at the level of the aortic valve. Then, tilt the transducer slightly upward to obtain a short axis view of the pulmonic valve. In this view, apply M mode imaging to evaluate the separation distance of the pulmonic valve.

Then, apply color doppler in the region of the pulmonic valve to assess for valvular regurgitation and stenosis. In two DB mode, place the transducer in the parasternal short axis position at the level of the papillary muscles to obtain a short axis view of the left ventricle. From this view, press the M mode button located in the control panel.

Using the track ball, position the M mode cursor at the center of the left ventricular cavity at the level of the papillary muscles, and obtain M mode images. Then, measure the left ventricular cavity dimension at end diastole where the distance between the interior wall and posterior wall is the largest, and an end systole where the inward motion of both interior and posterior walls is maximal. Next, move the transducer to the apical window, apply color on the mitral valve, and place the sample volume at the tips of the mitral valve leaflets.

Measure the peak mitral inflow velocity from the spectral display of pulse waved doppler velocities across the mitral valve. With the sample volume positioned between left ventricle inflow and outflow not the mitral and aortic valve closing and opening signals. Measure the isovolumic relaxation time, isovolumic contraction time, and left ventricular ejection time.

Then, perform tissue doppler imaging of the mitral annulus in the apical long axis view. Press the TGI control key and place the sample volume at the medial aspect of the mitral annulus. When finished, remove any excess ultrasound gel from the mouse.

Gently remove the tape securing the animal, and turn off the anesthesia. Place the animal on an absorbent paper towel and observe the animal until sternal recumbency is attained. Shown here is an assessment of aortic valve function in a normal mouse versus the aortic valve function in a mouse with calcific aortic valve disease.

In the mouse with calcified valves, the cusps are thickened and have increased echogenicity which results in restricted opening during systole. From the apical window a long axis view of the mitral valve is presented. The mitral leaflet thickness can be measured using the M mode line of interrogation.

Measurement of the thickness of the mitral valve can be extremely challenging given the thin, poorly echogenic, and rapidly moving leaflets of the normal mitral valve. The color doppler shown in this modified long axis view of the mitral valve shows a mosaic color jet at the mitral valve during systole. The blue color is evidence of mitral valve regurgitation.

From the parasternal window, both short and long axis views of the pulmonic valve are obtained. In this image, the M mode line of interrogation is applied across the pulmonic valve, and the valve cusp separation distance can be measured from this view. Once mastered, this technique can be done in 15 to 20 minutes if it is performed properly.

While attempting this procedure, it's important to have a basic knowledge of cardiac anatomy and physiology, a working knowledge of the principles and terminology of sonography, and experience in cardiac ultrasound to allow for accurate and time efficient assessment of cardiac function in rodents. After watching this video you should have a good understanding of how to perform cardiac ultrasound in rodents by obtaining images from various imaging planes, and measuring echo doppler parameters that adequately describe cardiac and heart valve function.