The overall goal of this procedure is to establish a non-invasive method to evaluate the structure and function of the right ventricle in a mouse model of pulmonary artery constriction. This is accomplished by first obtaining by ultrasound, the parasternal long axis B and M mode views to obtain right ventricle chamber dimension fractional shortening, and right ventricle wall thickness. The second step is to obtain a para sternal short axis view at the mid papillary level to calculate fractional area change.
Next, the para sternal short axis view at the aortic valve level is obtained to attain RV wall thickness and pulmonary artery peak velocity. The final step is to obtain a modified parasternal long axis view of RV and PA To calculate pulmonary artery peak velocity PA calculation can be done from the short and long axis. Ultimately, based on the above images, step five, data calculation and analysis can be performed offline.
The main advantage of this technique of existing methods like RV characterization, is that echocardiography is a non-invasive method. This method can help answer key questions in assessment of RV structure and function. To begin this protocol, first, obtain eight-week old male C 57 black six mice and allow them to acclimate for one week prior to any experimental procedures.
Then fully anesthetize animals and perform pulmonary artery occlusion in accordance with A VMA guidelines and your institution's approved iacuc protocols. Note that during imaging the proper choice of anesthesia, such as a short duration of inhaled isoflurane is crucial in the maintenance of heartbeat at normal physiological rates. Images as seen here are collected using the vivo 2100 system.
Similar images may be obtained using ultrasound imaging systems from other manufacturers. It is recommended that all images should be obtained and analyzed in a blinded fashion whenever possible. Begin by using B mode to obtain a full left ventricle parasternal long axis view with the animal in a supine position.
On the platform. Position the 40 megahertz ultrasound probe, also known as the MS five 50 D on the animal with about a 30 degree angle counterclockwise to the left para sternal line with the notch pointing in the coddle direction. Then adjust the probe angle by tilting slightly along the Y axis as seen here to obtain a full left ventricle chamber view in the center of the screen.
Once the proper landmarks are clearly visualized switch to M mode, an indicator line will show up on the screen. In the M mode setting, the line should be positioned to go through the widest portion of the right ventricular chamber using the aorta as a landmark, as illustrated here in both a pulmonary artery occlusion model and a sham mouse. In this view, the right ventricle wall and intraventricular septum should be clearly visible, ensure that the focus depth lies in the center of the right ventricle chamber.
Record the data in the highest possible frame rate for measurement of the right ventricle chamber dimension fractional shortening, and right ventricle wall thickness offline. Next to obtain fractional area changes, switch to B mode. Then find the long axis view as before, and then turn the probe 90 degrees clockwise To obtain the short axis view, tip the probe slightly along the X axis of the probe to prevent the obstructive view of the sternum.
Then move slightly up and down along the Y axis of the probe. To obtain the mid papillary view, look for the view with the largest chamber dimension in this view, the papillary muscles are typically located at the two and five o'clock positions as seen here now from the para sternal short axis view, move the probe at the Y axis toward cranium until the aortic valve cross section can be seen in the middle of the window. The right ventricular outflow tract should be visible on the top as a crescent shaped structure with the tricuspid valve separating the right ventricle from the right atrium.
As illustrated here, record the data using ctor for the measurement of the right ventricle. Wall thickness offline remain at the same position. Switch to color doppler mode and position the yellow PW dashed line parallel to the direction of flow in the vessel.
Note that blue and red colors indicate flow away from and toward the probe respectively. Then place the pulse wave cursor on the stenosed area. It is acceptable to have a probe angle less than 20 for accurate measurement record data using ctor for measurement of PA peak velocity offline.
Next, to obtain a modified parasternal long axis view, continue on B mode setting and position the probe to the right para sternal line and slowly tilt the probe to about a 30 to 45 degree angle on the Y axis of the probe toward the chest to clearly visualize the pulmonary artery crossing over the aorta. Then switch to color doppler mode and position the yellow PW dashed line parallel to the direction of flow in the vessel. Doppler angle should be as close to zero degrees as possible while maintaining directionality parallel to PA flow.
Place the PW cursor at the tip of the pulmonary valve leaflets and record data using ctor and measure PA peak velocity. Offline right ventricle wall thickness can be calculated from the B mode data obtained from the right ventricle parasternal short axis view at the aortic level. Begin by selecting the 2D area tracing tool to trace the area of the right ventricle wall at diastole as seen here in pink.
Then use the distance tracing tool to trace the inner and outer circumferences of the wall of the right ventricle outflow track. Take the average of the inner and outer circumferences right ventricle wall thickness can be calculated using the equation seen here. Consistent measurements of right ventricle wall thickness area, or dimensions can be made using multiple acoustic windows in both the long and short axis.
The choice of some of these windows will depend on operator experience and may account for some variability as seen here. Systolic function of the right ventricle can be measured in plaques, view as percent fractional shortening or in mid papillary muscle view as percent fractional area change respectively. The RV dilation can be measured in the long and short axis as right ventricle chamber dimension and right ventricle area.
In diastole right ventricle free wall thickness as a marker of RV hypertrophy can be determined accurately either using M mode or the area trace method. Similarly, the pulmonary artery peak velocity can also be obtained with either the para sternal long axis or short axis mode. Reliable measurements of PA peak velocity and thus peak pressure gradient within the PA can be obtained using color doppler in both short and long axis acoustic windows.
Lastly, in this study, we show that non-invasive echocardiography is a viable alternative to the terminal right heart catheterization method currently used as the gold standard for RVSP measurement for five animals. Catheterization was performed for comparison to RVSP. Measurement and calculations of pressure were highly comparable.
Once measured, this technique can be done in 15 to 20 minutes if it is performed properly. While attempting this procedure, it's important to remember to keep the heart rate of the mice at physiological levels. Following this procedure, esta like rib ventricular case catheterization can be performed in order to obtain rib ventricular systolic pressure.
After watching this video, you should have a good understanding of how to measure the structural and functional changes in the right ventricle using echocardiography in mice after pulmonary artery constriction.
