Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

Podsumowanie

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

Streszczenie

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

Wprowadzenie

An abdominal aortic aneurysm (AAA) represents a significant cardiovascular disease characterized by a permanent localized dilation of the aorta exceeding the original vessel diameter by 1.5 times¹. AAA ranks among the top 13 causes of mortality in the United States². The progression of AAA is attributed to the degeneration of the aortic wall and elastin fragmentation, ultimately leading to aortic rupture. These changes in the aortic wall may occur without a significant increase in the maximal intraluminal diameter (MILD), thus suggesting that MILD alone is not sufficient to predict the severity of the disease³. Therefore, additional factors need to be identified to detect initial changes in the aortic wall, which may guide early treatment options. The overall goal of this protocol is to provide a practical guide for assessing aortic functional properties using ultrasound imaging as characterized by measurements of pulse propagation velocity (PPV), distensibility and radial strain.

A well characterized experimental model to study AAA, first described by Daugherty and colleagues, involves subcutaneous infusion of angiotensin II (AngII) via osmotic pumps in Apoe^-/- mice⁴. Precise measurement of MILD using ultrasound imaging has been instrumental in characterizing AAA in this mouse model⁵. Although histological changes during the development of AAA have been extensively studied, changes in the functional properties of the vessel wall such as aortic stiffness have not been well characterized. This protocol emphasizes the use of high-frequency ultrasound in combination with the sophisticated analyses as powerful tools for studying the temporal progression of AAA. Specifically, these approaches allow us to assess the functional properties of the vessel wall as measured by PPV, distensibility and radial strain.

Recent clinical studies in human subjects with AAA, as well as in the murine elastase-induced AAA model, suggest a positive correlation between aortic stiffness and aortic diameter^6,7. PPV, an indicator of aortic stiffness, is accepted as an excellent measurement for quantifying changes in stiffness in vessel wall^6,8. PPV is calculated by measuring the transit time of the pulse waveform at two sites along the vasculature, thus providing a regional assessment of aortic stiffness. We have recently demonstrated that increased aortic stiffness as measured by PPV, and at the cellular level as determined using atomic force microscopy, positively correlates with aneurysm development⁹. Further, the literature suggests that aortic stiffness may precede aneurysmal dilation and thus may provide useful information about regional intrinsic properties of the vessel wall during development of AAA¹⁰. Similarly, distensibility and strain measurements are the quantification tools to measure earlier changes of arterial fitness. Healthy arteries are flexible and elastic, whereas with increased stiffness and less elasticity, distensibility and strain is decreased. Here, we provide a practical guide and step by step protocol for the use of a high-frequency ultrasound system to measure MILD, PPV, distensibility and radial strain in mice. The protocol provides technical approaches that should be used in conjunction with the basic information provided by manuals for specific ultrasound imaging instruments and the accompanying video tutorial. Importantly, in our hands the described imaging protocol provides reproducible and accurate data that appear valuable in the study of the development and progression of experimental AAA.

To further demonstrate the utility of ultrasound imaging, we provide example images and measurements taken from our own studies aimed at using pharmacological approaches for preventing experimental AAA¹¹. Specifically, notch signaling has been proposed to be involved in multiple aspects of vascular development and inflammation¹². Using gene haploinsufficiency and pharmacologic approaches, we have previously demonstrated that Notch inhibition reduces the development of AAA in mice by preventing infiltration of macrophages at the site of vascular injury^13,14,15. For the current article, using the pharmacological approach for Notch inhibition we focus on the relationship between aortic stiffness and factors relating to AAA. These studies illustrate that Notch inhibition reduces aortic stiffness, which is a measure of AAA progression¹¹.

Protokół

The protocol for handling of mice and ultrasound imaging was approved by the University of Missouri Institutional Animal Care and Use Committee (animal protocol number 8799) and was conducted according to AAALAC International.

1. Equipment setup and preparation of mice

1. Equipment setup
   1. Turn on the ultrasound instrument, ultrasonic gel warmer and the heating pad.
   2. Open the ultrasound program and enter the study name and descriptive information for each mouse.
   3. Select the application as General Imaging.
   4. Choose the appropriate transducer for abdominal imaging (Figure 1B,C). In this experiment, MS400 transducer is used.
   5. Ensure anesthesia isoflurane and oxygen levels are adequate for each experimental session.
   6. Clean the ultrasound animal imaging platform.
2. Mouse preparation
   1. Place the mouse cage on top of a heating pad (36.5 to 38.5 °C).
   2. Gently hold the mouse by its tail base and place in the oxygen-filled isoflurane chamber.
   3. Direct the isoflurane and oxygen flow to the induction chamber.
   4. Turn on the isoflurane vaporizer and set the isoflurane level to 1-2% vol/vol. Turn on the oxygen tank pressure to 1-2 L/min.
   5. After ~2 min, confirm the adequate depth of anesthesia by the absence of withdrawal reflexes upon pinching the foot pad of the mouse.
   6. Next, turn off the induction chamber supply branch and turn on the branch directed to the anesthesia nose cone.
   7. Transfer the mouse from the induction chamber to the ultrasound imaging stage and position the anesthesia cone over the nose of the animal.
   8. Tilt the animal imaging platform around 10° to the lower right corner for optimal scanning (Figure 1B).
   9. Put one drop of sterile ophthalmic solution in both eyes of mice to prevent drying under anesthesia.
   10. Position the mouse in the supine position with its nose inserted into the anesthesia cone.
   11. Apply the electrode gel to all four paws using a cotton swab and tape the paws to the copper leads on the animal imaging platform for electrocardiogram readings (Figure 1C).
   12. Use clippers to shave hair at the imaging site and then apply depilatory cream to remove remaining fur. Leave for less than 1 min.
   13. Gently wipe off the cream and hair with a damp paper towel.
   14. Monitor the breathing and ensure that heart rate is maintained between 450-550 beats/min. If below this level, reduce the isoflurane flow and wait until the heart rate recovers.
   15. Apply prewarmed ultrasonic gel (37 °C) to the prepared skin site and attach the transducer to its holder and lower down until it touches the gel (Figure 1C).

2. Ultrasound imaging of the abdominal aorta

1. Position the transducer horizontally (i.e., perpendicular to the midline of the mouse).
2. Smooth the ultrasonic gel and remove bubbles using the wood stick of a cotton swab.
3. Lower the transducer and place 0.5 - 1 cm below the diaphragm after touching the gel. Now start to observe the images.
4. Visualize the abdominal aorta in the short axis view (Figure 1C).
   NOTE: B-mode is the default and most effective mode to anatomically locate the aorta and position the transducer. The abdominal aorta is identified by the presence of pulsatile flow using color Doppler and power Doppler modes in the short axis (i.e., the circumferential cross-section of the aorta). Adjust the micromanipulators on the animal stage and the transducer to bring the cross section of the aorta to the center of the image.
5. Gently rotate the transducer 90° clockwise, and slowly adjust the x-axis micromanipulator knob to visualize the aorta in long axis view (longitudinal section of the aorta).
   NOTE: In many cases, gastro-intestinal gases may interfere with the image, or the aorta may not be at the optimal angle to allow a clear long axis view. Adjust the angle of the transducer slowly and horizontally until an acceptable long axis view is obtained. If problems persist, elevate the transducer, check for air bubbles under the transducer, slightly adjust the tilting angle of the animal stage, reapply gels, and repeat all the steps again.
6. Set the focus zone and depth at the region of the aorta using the Focus Zone and Focus Depth toggles, respectively. Adjust the time gain compensation slider manually to darken the lumen of the aorta to achieve an optimal contrast of the aorta wall.
7. Adjust the y-axis manipulator to visualize the branching points of the superior mesenteric and the right renal arteries. Use the right renal artery as a landmark to capture image of the suprarenal aorta (Figure 2A).
8. Record at least 100 frames of B-mode images on the suprarenal aorta.
9. Press cinestore to save the B-mode images.
10. Press M-mode button on the instrument keyboard to enable M-mode recording. Roll the cursor ball to bring the yellow indicator line to a normal aorta sections with clear vessel wall image, or to the sections where maximal diameter of aneurysm is observed.
11. Press the SV/gate toggle and adjust the cursor ball to ensure that vessel walls are included in the measurement bracket. Press update to record M-mode measurements and press cinestore to capture (Figure 2A,B).
    NOTE: Maximal diameter of the aneurysm may not be in the same imaging plane as the optimal long axis view of the aorta. Adjust the x-axis manipulator knob slightly for each M-mode measurement to ensure that the MILD of each section is captured.
12. To obtain ECG-gated Kilohertz Visualization (EKV) images, press the B-mode button to go back to B-mode recording.
    NOTE: If the images are not sharp, adjust the x-axis manipulator to achieve the sharpest image of upper wall of the lumen over a section length (i.e., > 6 mm).
13. Press Physio Settings button on the keyboard and select Respiration Gating. Adjust the gating Delay and Window manually 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.
    NOTE: Without the adjustment of the respiration gating, the EKV images will be blurred due to the normal movement of animal during breathing.
14. Press EKV button to enable the EKV mode. In the appropriate menu, select Standard Resolution and frame rate 3000 or higher. Select proceed to record EKV images. Press cinestore to save the images. Use EKV mode image to obtain measurements of pulse propagation velocity (PPV), distensibility and radial strain.
    NOTE: EKV recording may fail if there are abnormal fluctuations in respiration, animal is respiring too rapidly, or frame rates settings are too high. In those case, set the frame rate lower and wait for the animal respiration to stabilize. Setting the frame rate at 3000 is usually appropriate for both mice and rats.

3. Post-imaging steps

1. Gently wipe the ultrasonic gel from the abdominal area of the mouse with a paper towel moistened with warm water.
2. Place the mouse back in its home cage on a heating pad.
3. Turn off the isoflurane machine, clean the animal imaging platform and transducer with damp wipes.
4. Transfer the image data collected during the ultrasound scan to the hard drive.
5. Turn off the ultrasound instrument.
6. After the mouse recovers from anesthesia and is alert, remove the heating pad and return the cage to the animal housing rack.

4. Analysis of abdominal aortic images

1. Analysis of M-mode images to measure MILD
   1. Open the ultrasound program and enter the study name and descriptive information for each mouse.
   2. Open the ultrasound data in the analysis software and open the M-mode image and pause the heartbeat.
   3. Click on Measurements.
   4. Select the vascular package from the drop-down options. Click on Depth and draw a line across the aortic lumen extending from inner wall to wall (Figure 2C,D).
      NOTE: For consistency, the measurements should be taken at the systolic phase of the cardiac cycle when the aorta is maximally expanded. Draw three lines across three different heartbeats to obtain accurate and average measurements of MILD. In AAA, the measurements are taken at the maximal dilatation of the aorta. It is also advisable to fast the animals 4-6 h prior to collecting images to avoid interference from bowel motility and ensure image clarity.
2. Analysis for pulse propagation velocity (PPV)
   1. Open the EKV image and pause the heartbeat.
   2. Open a new window on the analysis software (e.g., Vevo Vac) by clicking on the name icon.
   3. Click on the PPV option (arrow in Figure 3D). A small window will further appear with the image of the aorta.
   4. Draw a rectangular box by clicking on the upper vessel wall and dragging the pointer for about 4 mm covering both the walls of the suprarenal aorta.
      NOTE: Keep the length of the box consistent (~4 mm) for all the images. The user can adjust the rectangular box by rotating to align the box and selecting the line then dragging to a new position on the vessel being analyzed to obtain the most appropriate and clear inflection of the pulse wave. The vertical lines of data from the rectangle will be displayed and identified as the Left (top image) and Right (bottom image) on the ROI. For a better visualization of the inflection of the pulse wave, it is sometimes useful to the draw box only on the upper wall as shown in Figure 3. The software will automatically calculate the PPV (m/s). However, it's always better to manually adjust the purple lines to set the exact inflection point on the pulse waves and PPV will change accordingly.
   5. Finally, select the Accept command to save the PPV values. Export the figures and the data to the data storage drive.
3. Analysis for distensibility and radial strain
   1. Open the EKV image and pause the heartbeat.
   2. Click on the software icon. The software will open a new window.
   3. Click on the trace new ROI and draw a rectangular box on the both walls of the vessel. The software will automatically trace the upper and lower walls of the vessel. The user can adjust the trace to align on the wall by clicking on green points (Figure 4A,B).
   4. Now Accept the trace. The software will calculate the distensibility (1/Mpa) in the selected ROI.
   5. For the radial strain measurement, select the appropriate strain option from the menu bars on the top left. The images for radial strain and tangential strain will open.
   6. Obtain the value for radial strain (%) by moving the cursor on the peak of the curve. Export the data as images or in video format (Figure 4A,B).

Wyniki

Representative M-mode images of the normal and aneurysmal abdominal aorta from mice are shown in Figure 2A and Figure 2B, respectively. The suprarenal abdominal aorta is identified by its location next to right renal artery and the superior mesenteric artery (Figure 2A). Representative images used for the calculation of MILD, at three different heartbeats of the systolic cardiac cyc...

Dyskusje

Ultrasound imaging provides a powerful technique for determining functional properties of the aorta through measurements of PPV, distensibility and radial strain. These measurements are particularly instructive for studying mouse models of AAA and the in vivo approach allows for collection of longitudinal data that is potentially important to understanding temporal development of the aortic pathology. Specifically, measurements of in vivo aortic stiffness are determined locally in the abdominal aorta by PPV, distensibili...

Materiały

Name	Company	Catalog Number	Comments
Angiotensin II	Sigma	A9525
Apoe-/- mice	The Jackon lab
Clippers	WAHL	1854
Cotton swab	Q-tips
DAPT	Sigma	D5942
Depilatory cream	Nair	LL9038
Electrode cream	Sigma	17-05
Gel warmer	Thermasonic (Parker)	82-03 (LED)
Heating pad	Stryker	T/pump professional
Isoflurane	VetOne	Fluriso TM
Isoflurane vaporizer	Visualsonics	VS4244
Lubricating ophthalmic ointment	Lacri-lube
Osmotic pumps	Alzet	Model 2004
Oxygen tank	Air gas
Tranducer	Visualsonics	MS-400 or MS550D
Ultrasonic gel	Parker	Aquasonic clear
Ultrasound Imaging System	Visualsonics	Vevo 2100
Vevo Vasc Software	Visualsonics