High resolution ultrasound measurements have been used widely to measure aortic dimensions in mice. This protocol provides a practical guide to the application of this modality. This protocol facilitates the acquisition of reproducible and accurate two dimensional measurements of the diameter of the thoracic and abdominal aortas in mice.
Demonstrating the procedure will be Dr.Hasashi Sawada, a post doctoral scholar, and Jessica Morrleghen a research scientist, both at the University of Kentucky. Before beginning the procedure, use a cotton swab to apply dilatory cream to the imaging area of the anesthetized mouse. After one minute, gently remove all of the cream and hair, and rinse the exposed skin with warm water.
After wiping skin dry to completely remove the cream, place a dot of ultrasound gel on to each of the four copper leads on the platform. Tape each paw pad onto each lead to obtain electrocardiogram readings. And verify that the heart rate is between 450 to 450 beats per minute.
Apply pre-warmed ultrasonic gel to the imaging site and attach the probe to the holder. Then rotate the platform for optimal scanning, and lower the probe until it is in contact with the ultrasonic gel. To image the thoracic aorta, tilt the platform down toward the left side of the mouse and place the probe on the right edge of the mouse's sternum.
Orienting the reference marker on the probe caudally. Switch to the color doppler on the thoracic aorta to confirm the blood flow. And adjust the stage and probe angle to visualize the aorta clearly.
Using the knobs for image depth and width, crop the ultrasound image to increase the frame rate and change the focal depth on the dorsal side of the ascending aorta, using the knob for focal depth. Verify the ultrasound parameters as indicated in the table and use the X and Y axis stage knob to gently move the probe, to capture the longitudinal aortic image with the largest possible diameter. To image the abdominal aorta, place the probe transversely just below the sternum and xyphoid process with the reference marker on the probe facing the mouse's right side.
The abdominal aorta should be located next to the inferior vena cava, and or portal vein. Visualize the abdominal aorta with color doper to confirm the pulsatile flow and crop the ultrasound image to increase the frame rate. Change the focal depth to the posterior wall of the abdominal aorta and move the probe caudally to visualize the branch points of the celiac and superior mesenteric arteries.
Then use the right renal artery as a landmark and capture a sine loop of the region of interest, showing the maximum dilation in the abdominal aorta. After all of the images have been captured, removed the ultrasonic gel, rinse the imaging area with warm water, and gently wipe the mouse dry. Return the mouse to it's cage on the heating pad, and clean the ultrasound machine, probe, and platform with a soft cloth and disinfectant wipes.
Then download all the files collected during the scan and turn off the ultrasound machine. In this representative ultrasound image of a non-aneurysmal proximal thoracic aorta, the ascending aorta can be observed located next to the pulmonary artery, and forms a curved tube with three branches in the arch region. The innominate artery, the left common carotid artery, and the left subclavian artery.
In this representative ultrasound image of non-aneurysmal abdominal aorta, the abdominal aorta is detected dorsally to the inferior vena cava. Here, representative images of thoracic and abdominal aortic aneurysms are shown. For measurement of the aortic sinus and ascending aortic diameters, the green line is used for standardizing the aortic sinus and the ascending aortic diameter, respectively.
Lines can then be drawn perpendicularly to the green line between the two inner edges of the lumen at the aortic sinus and the maximal ascending aortic diameter. For measurement of the abdominal aorta, draw a line across the largest luminal diameter, from the inner edge to the inner edge of the vessel lumen, and trace the inner edge of the aortic lumen for the luminal area. The luminal diameters of the thoracic and abdominal aortas are different between systole and diastole.
The cardiac cycle should also be considered for accurate measurements. In this representative pilot study, there was no major difference in the measured diameters of the ascending aorta between the ultrasound and the ex vivo images. This ultrasound technique allows the acquisition of reproducible and accurate results as no inter or intra observer variabilityies were observed in this experiment between the aortic measurements made by two independent observers and measurement sessions.
Adjustment of the probe and stage settings are important steps for reliable and reproducible aortic imaging. Data analysis steps are also important for accurate aortic measurement.
