The overall goal of this procedure is to use echocardiography to assess right ventricular size and function, as well as the presence and severity of pulmonary vascular disease in mice. This is accomplished by first positioning the mouse and the echocardiography equipment for optimal imaging. In the second step, the size of the right ventricle is determined.
Next, the blood flow through the pulmonary artery is measured to assess right ventricular function. In the final step, the pulmonary artery size and pulmonary acceleration, time is measured to evaluate the extent of the pulmonary vascular disease. Ultimately, the presence and severity of the right ventricular dysfunction and pulmonary vascular disease is determined by echocardiography.
The main advantage of this technique over existing methods like right heart catheterization, is that echocardiography is noninvasive and can be performed Serially demonstrating this technique will be Nikki Pinner, a research assistant from our laboratory. Before beginning the procedure, examine the ultrasound transducer for defects. If an air bubble is observed, remove the screw located on the right side of the transducer head and add sterile water through the hole with a 26 gauge needle.
Next, open the software and choose the cardiac package from the dropdown menu along with the appropriate transducer. Click initialize to initialize the probe, then select the operator animal, and date on the demographic screen. Click start to begin The program am now apply a small amount of lubricating ointment to the mouse's eyes after anesthesia to avoid damaging the corneas, use depilatory cream to remove the hair from the animal's chest.
Apply a skin moisturizer following the hair removal. Then place the mouse in a ventral dorsal position on a heated table set to 37 degrees Celsius and gently tape down all four paws. Apply a dime size amount of transduction gel to the chest to image the animal in the parasternal long axis view.
First, block the ultrasound transducer into place inside the mount on the rail system. Then rotate the instrument 10 degrees counterclockwise so that the metal probe of the transducer is positioned directly over the heart. Next, manipulate the x and y axis located on the rail system until the correct view is obtained.
Then select the B mode to project A 2D live image or view the following anatomic structures on the monitor. The normal right ventricle, the normal interventricular septum, the apex, the normal left ventricle and the posterior wall of the left ventricle. Then use the mouse to pull back through the video loop at the bottom of the image until the left ventricle is in systole and the aorta is at its greatest diameter.
Click the scan button to freeze the image and obtain one diametric measurement of the aorta. Save the measurement by clicking the frame store button. Finally, create a video loop by clicking C store to image the animal in the para sternal short axis view.
Reposition the transducer to the transverse three and nine o'clock positions. Then angle the transducer mount slightly coddly with the metal probe positioned horizontally and directly over the sternum to achieve the best view of the aorta and the left ventricle lumen. Manipulate the x and Y AEs on the rail system until the left ventricle, lumen, and anterolateral and postal medial papillary muscles can be seen.
Then further manipulate the Y axis cran from the papillary muscle view until the semilunar valve of the aorta comes into focus. Obtain measurements of the aorta just above the valve at the greatest diameter, and then manipulate the x and y axis again until the main pulmonary artery bifurcates. Now freeze the image and obtain the pulmonary artery measurement in systole.
Next, select PW mode to produce a Doppler reading of blood flow through the aorta. Then place the sample volume just above the level of the aortic valve. Once a triangular doppler envelope with dense white borders and a black interior are achieved, freeze the image and trace the border of the doppler envelope.
To calculate the velocity time integral measurements, rotate the angle knob located on the system console clockwise until the segmented yellow line seen in the image on the upper right hand side of the monitor is at zero degrees. Then place the sample volume proximal to the level of the pulmonary valve in the center of the right ventricular outflow tract and repeat the velocity time integral measurements as just demonstrated. Resume the B mode and reposition the transducer to obtain the reference.
View a cross sectional view of the left ventricle at the level of the papillary muscles. Then select the M mode to produce a continuous video feed and freeze the image. Pull back through the video loop if needed to a point where respiration is not occurring, and then obtain the following measurements using the diagonal line icon.
Three measurements of left ventricle and diastolic dimension, and three measurements of left ventricle and systolic dimension. Then click the heart icon and measure the heart rate from systolic peak to systolic peak of the left ventricle posterior wall. Then in beam mode, click the sin store button to record a video loop of the short para sternal axis view.
Finally, go to file and select browse study to recap the measurements. Then click end session followed by commit session data and export the data as a CSV file for subsequent analysis. In this first figure, representative echocardiographic images of Marian anatomy are shown here.
The parasternal long axis view of a mouse heart with a normal anatomy can be observed. While in this image, the anatomy is viewed along the per sternal short axis. Note that in this second image, the right ventricle is enlarged.
The principle goals of this protocol are to quantify the right ventricle size and function and to understand the degree to which the pulmonary vasculature is diseased. The right ventricle size is best assessed in the per sternal long axis view and is measured as the distance from the free wall to the interventricular septum using M mode as just demonstrated, this image shows a normal size right ventricle in a control mouse. While here, severe right ventricular enlargement in a mouse that underwent pulmonary artery banding can be observed in mice.
It is not possible to accurately measure the usual metrics of right ventricle function as in humans. However, using pulse wave Doppler to measure the velocity time integral at the level of the right ventricular outflow tract and the diameter of the pulmonary artery, it is possible to estimate the right ventricle stroke volume. The right ventricle velocity time integral can be further interrogated to estimate the pulmonary artery pressure by measuring the pulmonary artery acceleration time in humans, the pulmonary artery acceleration time is used to dichotomize the pulmonary artery pressure as high or low, and may be used to estimate the pulmonary artery pressure when a tricuspid regurgitant jet is not present.
Once mastered, this technique can be performed in 15 minutes.
