The overall goal of this procedure is to acquire and analyze echocardiographic images for the determination of the left atrial volume and the aorta, and pulmonary artery diameters in mice. This technique can discriminate between increases in the pulmonary artery resistance and diameter with no changes in the left atrial volume from models in which the PA and the LA are both enlarged. The main advantage of this technique, is that it is a non-terminal, non invasive approach that allows for serial assessment of the same animal.
This technique has the potential to further our ability to monitor interventions and to initiate other studies in the field of cardiopulmonary science. Demonstration of this technique is critical as the proper probe and mouse positioning are necessary for optimal image acquisitioning. Before beginning the procedure, turn on the echocardiographic system and select cardiac measurements from the drop down menu.
Click initialize to start the software. Then, click the new button to begin a new study, and enter the demographic information of the experimental animal. Click temp on and off to turn on the temperature controlled EKG board, and use the up and down arrows to set the temperature to 39 degrees Celsius.
Next, turn on the oxygen delivery system and anesthesia chamber and confirm that the mouse is properly anesthetized by lack of response to paw pinch. Place the animal in the supine position on the temperature controlled EKG board. Apply lubricant ointment to the animal's eyes, and then tape each paw to the correspondent EKG electrode on the board.
Observe the EKG tracing in the lower part of the monitor. Then, use an electric razor to shave the fur from the anterior thorax and upper abdominal area, and cover the chest with a generous amount of transducer gel. To measure the left atrium, use the handle beneath the EKG board to adjust the angle of the board to 30 to 45 degrees along the X axis, and 0 degrees along the Y axis.
Next, place the 25 MHz scan head in the echocardiographic movable arm clamp in the long axis view, and select the cardiac measurement filter, and the 2-D B-Mode in the echocardiographic system. Rotate the X and Y adjustment torques to obtain a long axis view of the heart at the level of the aortic outflow tract and press the Cine Store button to acquire a five second movie recording. Now, switch the echocardiographic view to M-Mode, a sagittal view of the heart and a cursor will appear.
Move the cursor so that it encompasses the whole left atrium at the level of the aortic outflow tract. And press the Frame Store button on the keyboard to acquire three images on this view. In B-Mode, rotate the movable arm clamp 90 degrees clockwise to set the scan head in the short axis view.
Then, use the Y axis torque to find the intra-articular septum, and acquire a five second recording that includes the wall of the atrium. We're measuring the left atrium diameters using EKG tracing as a timing guide to select the image corresponding to the P wave time point. This corresponds to the pre-atrial contraction time, when the left atrium is at its largest capacity.
To measure the great vessels, replace the 25 MHz scan head with a 30 MHz scan head, and select the abdominal measurements filter, and the 2-D B-Mode. Use the handle under the EKG board to adjust the angle of the board to a 5 degree angle along the X axis and 60 degrees along the Y axis. Use the X and Y axis adjustment torques to obtain a long axis view of the heart at the level of the aortic outflow tract, and acquire a five second movie recording.
Rotate the movable arm clamp of the set 90 degrees clockwise to set the scan head in the short axis view. Then, use the Y axis torque to locate the pulmonary artery at the level of its bifurcation, and acquire a five second movie recording. To ensure the correct location of the pulmonary artery, you can also acquire the pulmonary artery doppler tracing by pressing the PW button.
Observe the normal respiratory variation in the tracing. For the great vessels measurements, use the EKG tracing as a timing guide to select a time point after a QRS complex. This corresponds to the point where the great vessels are at the largest capacity.
Echocardiographic assessment of the left atrium volume and the pulmonary artery diameter permits the discrimination between models, where an increased pulmonary artery resistance results in an increased pulmonary artery diameter with no change in the left atrium volume from instances when the pulmonary artery and left atrial enlargement are both a result of elevated filling pressures on the left side of the heart when the mitral valve is normal. By comparison, older animals develop an increase in both their pulmonary artery diameter, and left atrium volume, suggesting that the backward propagation of pressure drives the increase in the pulmonary artery diameter observed in this group. Using the demonstrated technique to measure the left atrium and great vessels allows the acquisition of both reproducible and consistent data between the experimental animals, as evidenced by these measurements obtained from a group of 19-month-old mice that underwent two subsequent echocardiographic assessments.
Further, these results are easily reproduced between researchers, as apparent in the similarities observed between these four images obtained for the measurement of the left atrium super inferior diameter, and the pulmonary artery diameter acquired by two different researchers. Once this technique has been mastered, the images can be acquired in 15 minutes if it is performed properly. While attempting this procedure, it's important to monitor the heart rate throughout the whole study.
A heart rate of 400 beats per minute is ideal. Following this procedure, other methods like dopplers can be performed to answer additional questions about systolic and diastolic cardiac functions. We hope that this technique will pave the way for researchers in the field of cardiopulmonary science to explore age-associated left ventricular diastolic dysfunction and pulmonary hypertension in mice, as well as therapeutic interventions for these and other pathologies.
