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.</l...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signalling:+a+simple+pathway+becomes+complex.">Notch signalling: a simple pathway becomes complex.</a> Nature Reviews Molecular and Cell Biology. 7, 678-689 (2006).</li><li>Hans, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Notch1+signaling+reduces+abdominal+aortic+aneurysm+in+mice+by+attenuating+macrophage-mediated+inflammation.">Inhibition of Notch1 signaling reduces abdominal aortic aneurysm in mice by attenuating macrophage-mediated inflammation.</a> Arteriosclerosis, Thrombosis and Vascular Biology. 32, 3012-3023 (2012).</li><li>Cheng, J., Koenig, S. N., Kuivaniemi, H. S., Garg, V., Hans, C. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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><img src="/files/ftp_upload/60515/60515mateq.jpg"/></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...

Watch this Scientific Journal Video about Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model at JoVE.com

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 ...

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.

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6.0K visualizações.  University of Missouri. A imagem de ultrassom e a análise funcional são técnicas muito poderosas para determinar o crescimento e estabilidade dos aneurismas abdominais, além dos métodos tradicionais de tamanho aórtico e diâmetro. Esta técnica não invasiva mede a velocidade da onda de pulso, a capacidade de distância e a distância da aorta para monitorar mudanças sutis nas funções aórticas, juntamente com o diâmetro aórtico. A rigidez aórtica é um indicador da progressão do aneurisma de aoriedade ...