This work was supported by R01HL124155 (CPH) and funding from the Research Institute at the University of Missouri to CPH.

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
<ol>
	<li>Equipment setup
	<ol>
		<li>Turn on the ultrasound instrument, ultrasonic gel warmer and the heating pad.</li>
		<li>Open the ultrasound program and enter the study name and descriptive information for each mouse.</li>
		<li>Select the application as General Imaging.</li>
		<li>Choose the appropriate transducer for abdominal imaging (Figure 1B,C). In this experiment, MS400 transducer is used.</li>
		<li>Ensure anesthesia isoflurane and oxygen levels are adequate for each experimental session.</li>
		<li>Clean the ultrasound animal imaging platform.</li>
	</ol>
	</li>
	<li>Mouse preparation
	<ol>
		<li>Place the mouse cage on top of a heating pad (36.5 to 38.5 &#176;C).</li>
		<li>Gently hold the mouse by its tail base and place in the oxygen-filled isoflurane chamber.</li>
		<li>Direct the isoflurane and oxygen flow to the induction chamber.</li>
		<li>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.</li>
		<li>After ~2 min, confirm the adequate depth of anesthesia by the absence of withdrawal reflexes upon pinching the foot pad of the mouse.</li>
		<li>Next, turn off the induction chamber supply branch and turn on the branch directed to the anesthesia nose cone.</li>
		<li>Transfer the mouse from the induction chamber to the ultrasound imaging stage and position the anesthesia cone over the nose of the animal.</li>
		<li>Tilt the animal imaging platform around 10&#176; to the lower right corner for optimal scanning (Figure 1B).</li>
		<li>Put one drop of sterile ophthalmic solution in both eyes of mice to prevent drying under anesthesia.</li>
		<li>Position the mouse in the supine position with its nose inserted into the anesthesia cone.</li>
		<li>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).</li>
		<li>Use clippers to shave hair at the imaging site and then apply depilatory cream to remove remaining fur. Leave for less than 1 min.</li>
		<li>Gently wipe off the cream and hair with a damp paper towel.</li>
		<li>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.</li>
		<li>Apply prewarmed ultrasonic gel (37 &#176;C) to the prepared skin site and attach the transducer to its holder and lower down until it touches the gel (Figure 1C).</li>
	</ol>
	</li>
</ol>
2. Ultrasound imaging of the abdominal aorta
<ol>
	<li>Position the transducer horizontally (i.e., perpendicular to the midline of the mouse).</li>
	<li>Smooth the ultrasonic gel and remove bubbles using the wood stick of a cotton swab.</li>
	<li>Lower the transducer and place 0.5 - 1 cm below the diaphragm after touching the gel. Now start to observe the images.</li>
	<li>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.</li>
	<li>Gently rotate the transducer 90&#176; 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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Record at least 100 frames of B-mode images on the suprarenal aorta.</li>
	<li>Press cinestore to save the B-mode images.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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., &#62; 6 mm).</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Post-imaging steps
<ol>
	<li>Gently wipe the ultrasonic gel from the abdominal area of the mouse with a paper towel moistened with warm water.</li>
	<li>Place the mouse back in its home cage on a heating pad.</li>
	<li>Turn off the isoflurane machine, clean the animal imaging platform and transducer with damp wipes.</li>
	<li>Transfer the image data collected during the ultrasound scan to the hard drive.</li>
	<li>Turn off the ultrasound instrument.</li>
	<li>After the mouse recovers from anesthesia and is alert, remove the heating pad and return the cage to the animal housing rack.</li>
</ol>
4. Analysis of abdominal aortic images
<ol>
	<li>Analysis of M-mode images to measure MILD
	<ol>
		<li>Open the ultrasound program and enter the study name and descriptive information for each mouse.</li>
		<li>Open the ultrasound data in the analysis software and open the M-mode image and pause the heartbeat.</li>
		<li>Click on Measurements.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Analysis for pulse propagation velocity (PPV)
	<ol>
		<li>Open the EKV image and pause the heartbeat.</li>
		<li>Open a new window on the analysis software (e.g., Vevo Vac) by clicking on the name icon.</li>
		<li>Click on the PPV option (arrow in Figure 3D). A small window will further appear with the image of the aorta.</li>
		<li>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&#39;s always better to manually adjust the purple lines to set the exact inflection point on the pulse waves and PPV will change accordingly.</li>
		<li>Finally, select the Accept command to save the PPV values. Export the figures and the data to the data storage drive.</li>
	</ol>
	</li>
	<li>Analysis for distensibility and radial strain
	<ol>
		<li>Open the EKV image and pause the heartbeat.</li>
		<li>Click on the software icon. The software will open a new window.</li>
		<li>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).</li>
		<li>Now Accept the trace. The software will calculate the distensibility (1/Mpa) in the selected ROI.</li>
		<li>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.</li>
		<li>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).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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...

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 diameter6,7. PPV, an indicator of aortic stiffness, is accepted as an excellent measurement for quantifying changes in stiffness in vessel wall6,8. 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 injury13,14,15. 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.

<table><tbody><tr><td>Angiotensin II</td><td>Sigma</td><td>A9525</td><td></td></tr><tr><td>Apoe-/- mice</td><td>The Jackon lab</td><td>&lt;img src=&quot;/files/ftp_upload/60515/60515mateq.jpg&quot;/&gt;</td><td></td></tr><tr><td>Clippers</td><td>WAHL</td><td>1854</td><td></td></tr><tr><td>Cotton swab</td><td>Q-tips</td><td></td><td></td></tr><tr><td>DAPT</td><td>Sigma</td><td>D5942</td><td></td></tr><tr><td>Depilatory cream</td><td>Nair</td><td>LL9038</td><td></td></tr><tr><td>Electrode cream</td><td>Sigma</td><td>17-05</td><td></td></tr><tr><td>Gel warmer</td><td>Thermasonic (Parker)</td><td>82-03 (LED)</td><td></td></tr><tr><td>Heating pad</td><td>Stryker</td><td>T/pump professional</td><td></td></tr><tr><td>Isoflurane</td><td>VetOne</td><td>Fluriso TM</td><td></td></tr><tr><td>Isoflurane vaporizer</td><td>Visualsonics</td><td>VS4244</td><td></td></tr><tr><td>Lubricating ophthalmic ointment</td><td>Lacri-lube</td><td></td><td></td></tr><tr><td>Osmotic pumps</td><td>Alzet</td><td>Model 2004</td><td></td></tr><tr><td>Oxygen tank</td><td>Air gas</td><td></td><td></td></tr><tr><td>Tranducer</td><td>Visualsonics</td><td>MS-400 or MS550D</td><td></td></tr><tr><td>Ultrasonic gel</td><td>Parker</td><td>Aquasonic clear</td><td></td></tr><tr><td>Ultrasound Imaging System</td><td>Visualsonics</td><td>Vevo 2100</td><td></td></tr><tr><td>Vevo Vasc Software</td><td>Visualsonics</td><td></td><td></td></tr></tbody></table>

measurement of pulse propagation velocity, distensibility and strain in an abdominal aortic aneurysm mouse model

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.

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...

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

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.

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 ...

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6.1K Views.  University of Missouri. 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.

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

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

Medicine

Ultrasound-based Pulse Wave Velocity Evaluation in Mice

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This parameter is being increasingly investigated in small rodent models in which it is used for assessing alterations in vascular function related to particular genotypes/treatments or for characterizing cardiovascular disease progression. This protocol describes an image processing algorithm which leads to non-invasive arterial PWV measurement in mice using ultrasound (US) images only. The proposed technique has been used to assess abdominal aorta PWV in mice and evaluate its age-associated changes.
Abdominal aorta US scans are obtained from mice under gaseous anesthesia using a specific US device equipped with high-frequency US probes. B-mode and Pulse-Wave Doppler (PW-Doppler) images are analyzed in order to obtain diameter and mean velocity instantaneous values, respectively. For this purpose, edge detection and contour tracking techniques are employed. The single-beat mean diameter and velocity waveforms are time aligned and combined in order to achieve the diameter-velocity (lnD-V) loop. PWV values are obtained from the slope of the linear part of the loop, which corresponds to the early systolic phase.
With the present approach, anatomical and functional information about the mouse abdominal aorta can be non-invasively achieved. Requiring the processing of US images only, it may represent a useful tool for the non-invasive characterization of different arterial sites in the mouse in terms of elastic properties. The application of the present technique can be easily extended to other vascular districts, such as the carotid artery, thus providing the possibility to obtain a multi-site arterial stiffness assessment.

Arterial stiffness represents a key factor in cardiovascular disease and pulse wave velocity (PWV) can be considered as a surrogate index for arterial stiffness. This protocol describes an image processing algorithm for calculating PWV in mice based on ultrasound image processing that is applicable at different arterial sites.

Arterial stiffness can be evaluated by calculating pulse wave velocity (PWV), i.e., the speed with which the pulse wave travels in a conduit vessel. This ...

Creation of a Rodent Model of Abdominal Aortic Aneurysm by Blocking Adventitial Vasa Vasorum Perfusion

The adventitial vasa vasorum (VV) provides oxygen and nourishment to the aortic wall. Hypoxia in the aortic wall can cause enlarged abdominal aortic aneurysms (AAAs). This article introduces and describes a standard protocol used to induce AAAs through adventitial VV hypoperfusion created with a combination of polyurethane catheter insertion into the aortic lumen and suture ligation of the infrarenal abdominal aorta.
The protocol involves the use of male rats weighing 300-400 g, which are provided food and water ad libitum. After laparotomy with a ventral midline abdominal incision, exfoliation of the aorta is performed, which blocks blood flow from the perivascular tissue. Aortotomy involving a small incision adjacent to the renal artery branches is performed, and a polyurethane catheter is inserted using an 18-gauge indwelling needle. After repairing the incision, tight ligation of the aorta over the catheter blocks VV blood flow from the proximal direction through the aortic wall without disturbing the aortic blood flow. This technique can induce an AAA with progressive aortic dilatation.
The greatest benefit of this model is that VV hypoperfusion causes tissue hypoxia and the development of an infrarenal AAA, which has morphological and pathological characteristics similar to those of a human AAA.

Polyurethane catheter insertion into the aortic lumen and suture ligation of the aorta induce chronic hypoxia due to hypoperfusion of the adventitial vasa vasorum. This article describes a novel animal model of abdominal aortic aneurysm (AAA) with characteristics similar to those of AAA in humans.

The adventitial vasa vasorum (VV) provides oxygen and nourishment to the aortic wall. Hypoxia in the aortic wall can cause enlarged abdominal aortic ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...

Ultrasound Imaging of the Thoracic and Abdominal Aorta in Mice to Determine Aneurysm Dimensions

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been widely used to measure aortic dimensions in mouse models of aortic aneurysms. Aortic aneurysms are defined as permanent dilations of the aorta, which occur most frequently in the ascending and abdominal regions. Sequential measurements of aortic dimensions by ultrasound are the principal approach for assessing the development and progression of aortic aneurysms in vivo. Although many reported studies used ultrasound imaging to measure aortic diameters as a primary endpoint, there are confounding factors, such as probe position and cardiac cycle, that may impact the accuracy of data acquisition, analysis, and interpretation. The purpose of this protocol is to provide a practical guide on the use of ultrasound to measure the aortic diameter in a reliable and reproducible manner. This protocol introduces the preparation of mice and instruments, the acquisition of appropriate ultrasound images, and data analysis.

Ultrasound imaging has become a common modality to determine the luminal dimensions of thoracic and abdominal aortic aneurysms in mice. This protocol describes the procedure to acquire reliable and reproducible two-dimensional ultrasound images of the ascending and abdominal aorta in mice.

Contemporary high-resolution ultrasound instruments have sufficient resolution to facilitate the measurement of mouse aortas. These instruments have been ...

Drug Treatment by Central Venous Catheter in a Mouse Model of Angiotensin II Induced Abdominal Aortic Aneurysm and Monitoring by 3D Ultrasound

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse models, are applied to advance the understanding of the disease pathogenesis and to identify potential therapeutic targets. Testing novel drug candidates to block AAA growth in these models generally requires repeated drug administration during the time course of the experiment. Here, we describe a compiled protocol for AAA induction, insertion of an intravenous catheter to facilitate prolonged therapy, and serial AAA monitoring by 3D ultrasound. Aneurysms are induced in apolipoprotein E (ApoE) deficient mice by angiotensin II release over 28 days from osmotic mini-pumps implanted subcutaneously into the mouse back. Subsequently, the surgical procedure for external jugular vein catheterization is conducted to allow for daily intravenous drug treatment or repeated blood sampling via a subcutaneous vascular access button. Despite the two dorsal implants, the monitoring of AAA development is readily facilitated by sequential semi-automated 3D ultrasound analysis, which yields comprehensive information on the expansion of aortic diameter and volume and on aneurysm morphology, as illustrated by experimental examples.

This protocol describes the consecutive implantation of an osmotic pump to induce abdominal aortic aneurysm by angiotensin II release in apolipoprotein E (ApoE) deficient mice and of a vascular access port with a jugular vein catheter for repeated drug treatment. Monitoring of aneurysm development by 3D ultrasound is effectively conducted despite dorsal implants.

Since pharmaceutical treatment options are lacking in the clinical management of abdominal aortic aneurysm (AAA), animal models, in particular mouse ...

Investigating the Molecular Mechanisms Underlying Murine Abdominal Aortic Aneurysm

A Mouse Abdominal Aortic Aneurysm Model by Periadventitial Calcium Chloride and Elastase Infiltration

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the abdominal aorta with at least a 50% increase in arterial diameter. Various animal models of AAA have been introduced to mimic the pathophysiological changes and study the underlying mechanisms of AAA. Among these models, the calcium chloride (CaCl2)- and elastase-induced AAA models are commonly used in mice. However, these methods have certain limitations. Traditional intraluminal porcine pancreatic elastase (PPE) perfusion is associated with high technical difficulty and a high rupture rate, while periadventitial administration of PPE yields inconsistent results. In addition, the CaCl2-induced AAA model lacks human AAA features, such as atherothrombosis and aneurysm rupture. Therefore, the combined application of CaCl2 and PPE has been proposed as an approach to enhance success rates and induce greater diameter increases in AAA animal models. This manuscript presents a comprehensive protocol for establishing a mouse AAA model through periaortic infiltration of PPE and CaCl2 in the infrarenal segment of the abdominal aorta. By following this protocol, we can achieve an AAA formation rate of approximately 90% with technical simplicity and reproducibility. Further ultrasound and histological experiments confirm that this model effectively replicates the morphological and pathological changes observed in human AAA.

Author Spotlight: Development and Characterization of a Mouse Model for Abdominal Aortic Aneurysm

This manuscript presents a protocol for establishing a mouse abdominal aortic aneurysm model using calcium chloride and elastase, combining the advantages of previous modeling methods. This model can be utilized to investigate the pathophysiological mechanisms underlying abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) is a life-threatening disease associated with high mortality rates. It is characterized by the permanent dilation of the ...

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. Currently, there are several distinct experimental models of AAA, each emphasizing a different aspect in the pathogenesis of AAA. The elastase-induced AAA model is the second most used rodent AAA model. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. Due to technical challenges, most elastase-induced AAA model nowadays is performed with the external application rather than an intraluminal infusion of PPE. The infiltration of elastase will cause degradation of elastic lamellae in the medial layers, resulting in the loss of aortic wall integrity and subsequent dilation of the abdominal aorta. However, one disadvantage of the elastase-induced AAA model is the inevitable variation of how the surgery is performed. Specifically, the surgical technique of isolating the infrarenal segment of the aorta, the material used for aorta wrapping and PPE incubation, the enzymatic activity of PPE, and the time duration of PPE application can all be important determinants that affect the eventual AAA formation rate and aneurysm diameter. Notably, the difference in these factors from different studies on AAA can lead to reproducibility issues. This article describes a detailed surgical process of the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse. Following this procedure, a stable AAA formation rate of around 80% in male and female mice is achievable. The consistency and reproducibility of AAA studies using an elastase-induced AAA model can be significantly enhanced by establishing a standard surgical procedure.

The present protocol describes a standardized surgical method for the elastase-induced AAA model through the direct application of elastase to the adventitia of infrarenal abdominal aorta in mice.

Abdominal aortic aneurysm (AAA), although primarily asymptomatic, is potentially life-threatening as the rupture of AAA usually has a devastating outcome. ...

Biology

Abdominal Aortic Aneurysm Development in Mice Using the Elastase and BAPN Method

Advanced Abdominal Aortic Aneurysm Modeling in Mice by Combination of Topical Elastase and Oral &#223;-aminopropionitrile

The topical elastase murine model of abdominal aortic aneurysm (AAA) is enhanced when combined with &#223;-aminopropionitrile (BAPN)-supplemented drinking water to reliably produce true infrarenal aneurysms with behaviors that mimic human AAAs. Topically applying elastase to the adventitia of the infrarenal aorta causes structural damage to the elastic layers of the aortic wall and initiates aneurysmal dilation. Co-administering BAPN, a lysyl oxidase inhibitor, promotes sustained wall degeneration by reducing collagen and elastin crosslinking. This combination results in large AAAs that progressively expand, form intraluminal thrombus, and are capable of rupture. Refining surgical techniques, such as circumferentially isolating the entire infrarenal aortic segment, can help standardize the procedure for a consistent and thorough application of porcine pancreatic elastase despite different operators and anatomic variations between mice. Therefore, the elastase/BAPN model is a refined approach to surgically inducing AAA in mice, which may better recapitulate human aneurysms and provide additional opportunities to study aneurysm growth and rupture risk.

Author Spotlight: Enhanced Murine AAA Model Using Elastase to Mimic Human Aneurysms

This protocol describes a methodical surgical approach to modeling advanced abdominal aortic aneurysms in mice by a combination of directly applying elastase to the infrarenal aorta and administering &#223;-aminopropionitrile through drinking water.

The topical elastase murine model of abdominal aortic aneurysm (AAA) is enhanced when combined with &#223;-aminopropionitrile (BAPN)-supplemented drinking ...

High-frequency Ultrasound Imaging of the Abdominal Aorta

Source: Amelia R. Adelsperger, Evan H. Phillips, and <a href="https://www.jove.com/author/Craig+J._Goergen">Craig J. Goergen</a>, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana
High-frequency ultrasound systems are used to acquire high resolution images. Here, the use of a state-of-the-art system will be demonstrated to image the morphology and hemodynamics of small pulsatile arteries and veins found in mice and rats. Ultrasound is a relatively inexpensive, portable, and versatile method for the noninvasive assessment of vessels in humans as well as large and small animals. These are several key advantages that ultraound offers compared to other techniques, such as computed tomography (CT), magnetic resonance imaging (MRI), and near-infrared fluorescence tomography (NIRF). CT requires ionizing radiation and MRI can be prohibitively expensive and even impractical in some scenarios. NIRF, on the other hand, is limited by the penetration depth of light required to excite the fluorescent contrast agents.
Ultrasound has limitations in terms of imaging depth; however, this may be overcome by sacrificing resolution and using a lower frequency transducer. Abdominal gas and excess body weight can severely diminish image quality. In the first case, the propagation of sound waves is limited, while in the latter case, they are attenuated by overlying tissues, such as fat and connective tissue. As a result, no contrast or faint contrast may be observed. Finally, ultrasound is a highly user-dependent technique, requiring the sonographer to be familiar with anatomy and to be able to work around issues, such as the appearance of imaging artifacts or acoustic interference.

High-frequency Ultrasound Imaging and Image Acquisition

Source: Amelia R. Adelsperger, Evan H. Phillips, and Craig J. Goergen, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, 
...

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

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Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

Ultrasound-based Pulse Wave Velocity Evaluation in Mice

Creation of a Rodent Model of Abdominal Aortic Aneurysm by Blocking Adventitial Vasa Vasorum Perfusion

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of β-aminopropionitrile-induced Aortic Aneurysm and Dissection

Ultrasound Imaging of the Thoracic and Abdominal Aorta in Mice to Determine Aneurysm Dimensions

Drug Treatment by Central Venous Catheter in a Mouse Model of Angiotensin II Induced Abdominal Aortic Aneurysm and Monitoring by 3D Ultrasound

A Mouse Abdominal Aortic Aneurysm Model by Periadventitial Calcium Chloride and Elastase Infiltration

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

Advanced Abdominal Aortic Aneurysm Modeling in Mice by Combination of Topical Elastase and Oral ß-aminopropionitrile

High-frequency Ultrasound Imaging of the Abdominal Aorta