Ultrasound imaging and functional analysis are very powerful techniques to determine the growth and stability of abdominal aortic aneurysms, beyond the traditional methods of aortic size and diameter. This non-invasive technique measures the pulse wave velocity, distance ability, and in distance of the aorta to monitor subtle changes in the aortic functions along with the aortic diameter. Aortic stiffness is an indicator of abdominal aortic aneurysm progression and the stability can be useful in assessing the effects of therapeutic agents of interest in preclinical trials.
Also, velocity and strain measurements can also be correlated to the vascular changes that occur during the development of various cardiovascular diseases, such as hypertension, diabetes, obesity, and atherosclerosis. Demonstrating the procedure will be Dr.Sun, a research assistant professor from the Imaging Core facility. To prepare the mouse for the imaging procedure, after confirming a lack of response to pedal reflex, apply opthalmic solution to the anesthetized mouse's eyes and place the mouse in the supine position on the animal imaging platform tilted 10 degrees to the lower right.
Apply electrode gel to all four paws and tape the paws to the copper electrocardiogram leads on the imaging platform. Use clippers to shave the hair at the imaging site, and apply depilatory cream to the exposed skin. After less then a minute, use a damp paper towel to remove the cream and hair, and apply 37 degrees Celsius ultrasonic gel to the prepared site.
Then use the wooden end of a cotton swab to smooth the gel and to remove any bubbles. For abdominal aorta imaging, attach the transducer to its holder and place the transducer perpendicular to the midline of the mouse. Lower the transducer until it touches the gel and position the tip 0.5 to one centimeter below the diaphragm.
When the transducer is in place, visualize the abdominal aorta in the short-axis view. Next, gently rotate the transducer 90 degrees clockwise and slowly adjust the X-axis micro manipulator knob to visualize the aorta in the long-axis view. Use the focus zone and focus depth toggles to set the focus'zone and depth at the region of the aorta.
Manually adjust the time gain compensation slider to darken the lumen of the aorta to achieve an optimal contrast of the aorta wall. Adjust the Y-axis manipulator to visualize the branching points of the superior mesenteric and the right renal arteries. The right renal artery can be used as a landmark to capture an image of the super renal aorta.
After recording at least 100 frames of B-mode images on the super renal aorta, save the images, and select the end mode. Roll the cursor ball to bring the yellow indicator line to a normal aorta section with a clear vessel wall image or to the sections where the maximal diameter of aneurysm is observed. Click the SV gate toggle and adjust the cursor ball to ensure that the vessel walls are included in the measurement bracket.
Then press update to record the M-mode measurements and send to store to capture the measurements. To obtain EKV images press the B-mode button to go back to B-mode recording and press the Physio Settings button to allow respiration gating selection. Manually adjust the gating delay and window to record the data only during the flatter parts of the respiration wave.
The recording sections will be shown as colored blocks on the tracing of the respiration wave. Press the EKV button to enable the EKV mode. In the appropriate menu set the Acquisition Type to Standard resolution, the Frame Rate to 3, 000 or higher and click Scan to record the EKV images.
Then press send to store to save the images and use the EKV mode image to obtain measurements of the PPV, distensibility and radial strain. To measure the mild, open the ultrasound program and enter the study name and descriptive information for each mouse. Open the ultrasound data and the M-mode image and pause the heartbeat.
Click measurements and select the Vascular Package and Depth from the drop-down options. Then draw a line across the aortic lumen extending from inner wall to wall. For PPV analysis open the EKV image and pause the heartbeat.
Click the icon to open a new window on the analysis software and select the PPV option. A small window will appear with the image of the aorta. Click on the upper vessel wall and drag the pointer to cover both walls of the super renal aorta with an approximately four millimeter box, then click Accept to save the PPV values.
For distensibility analysis in the opened EKV image, click the software icon and select Trace New Region of Interest to draw a rectangular box on both walls of the vessel. The software will automatically trace the upper and lower walls of the vessel. Click on the green points to adjust the trace align if necessary and click Accept.
The software will calculate the distensibility within the selected region of interest. To obtain the radial strain measurement select the appropriate strain option from the menu and click Strain. The images for radial strain and tangential strain will open.
Then move the cursor to the peak of the curve to obtain the value for the radial strain. For optimal reproducibility and detection of certain stages the images should be analyzed by the same person at the same anatomical location to ensure consistency within the data. Here, representative M-mode images of normal and aneurysmal mouse abdominal aortas are shown.
The super renal abdominal aorta can be identified by its location next to the right branch of the renal artery and the superior mesenteric artery. As illustrated in these images, mild can be calculated using three different heartbeats of the systolic cardiac cycle in both normal and aneurysmal aortas. In the case of an aortic aneurysm, the luminal diameter can be determined by drawing a perpendicular yellow line between the two inner edges of the lumen at the area of maximal dilation.
In this representative analysis, trans-abdominal ultrasound imaging revealed a progressive increase in mild and PPV and a decrease in the distensibility and radial strain in response to angiotensin II at day 28. Angiotensin II infusion marginally increased mild from day 28 to 56, while treatment with a notch inhibitor did not significantly affect mild compared to angiostatin II alone. It is important to note that PPV correlated strongly with mild at day 28, whereas at day 56, the correlation was relatively weak.
Histologically, angiotensin II infusion increased collagen degradation and proteolytic activity in the media layer of the aorta. Notch inhibitor treatment, however, minimized these changes in the extra cellular matrix degradation. Really setting up the animal and the probe and identifying the abdominal aorta are the most critical steps.
Beginners I advise to use the micromanipulator knob to make fine adjustments. Using this method we can also measure the block flow in the aorta or the renal artery, evaluate cardiac function, and monitor for peripheral vascular disease. After reaching certain size, abdominal aortic aneurysms do not grow excessively.
Under these circumstances certain structure changes are rapidly detected by these techniques in response to pharmacological agents or other manipulations. While administering the anesthesia be sure to use the charcoal and solvent for isofluorent, and the exhaust wear. Also make sure there is no leakage from the nose hole.
