Name: quantitative micro-ct analysis of aortopathy in a mouse model of &#946;-aminopropionitrile-induced aortic aneurysm and dissection
Uploaded: 2018-07-16T13:00:00
Description: Watch this Scientific Journal Video about Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of Î²-aminopropionitrile-induced Aortic Aneurysm and Dissection at JoVE.com

We would like to thank Mark Smith for his assistance with radiographic imaging. This work is supported by the NIH T32 Grant for Interdisciplinary Research in Cardiovascular Disease (BOA), the American Heart Association (SMC), and the NIH R35 Grant (DKS).

The protocols for animal handling were approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore (animal protocol number 0116024) and conducted according to AAALAC International standards.
1. Preparation of Reagents
<ol>
	<li>Heparin
	<ol>
		<li>Dilute 250 &#181;L of 1000 U/mL heparin sulfate in 50 mL of phosphate buffered saline to make a final concentration of 5 U/mL.</li>
		<li>Warm the heparinized (5 U/mL) phosphate buffered sal...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+beta-aminopropionitrile-induced+aortic+dissection+model+in+rats+by+different+administration+and+dosage.">Comparison of beta-aminopropionitrile-induced aortic dissection model in rats by different administration and dosage.</a> Vascular. 21 (5), 287-292 (2013).</li><li>Huffman, M. D., Curci, J. A., Moore, G., Kerns, D. B., Starcher, B. C., Thompson, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+importance+of+connective+tissue+repair+during+the+development+of+experimental+abdominal+aortic+aneurysms.">Functional importance of connective tissue repair during the development of experimental abdominal aortic aneurysms.</a> Surgery. 128 (3), 429-438 (2000).</li><li>Wu, D., Shen, Y. H., Russel, L., Coselii, J. S., LeMaire, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+thoracic+aortic+dissection.">Molecular mechanisms of thoracic aortic dissection.</a> Journal of Surgical Research. 184 (2), 907-924 (2013).</li><li>Bruel, A., Ortoft, G., Oxlund, H. <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+cross-links+in+collagen+is+associated+with+reduced+stiffness+of+the+aorta+in+young+rats.">Inhibition of cross-links in collagen is associated with reduced stiffness of the aorta in young rats.</a> Atherosclerosis. 140 (1), 135-145 (1998).</li><li>Martinez-Revelles, S., 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=Lysyl+oxidase+induces+vascular+oxidative+stress+and+contributes+to+arterial+stiffness+and+abnormal+elastin+structure+in+hypertension:+Role+of+p38MAPK.">Lysyl oxidase induces vascular oxidative stress and contributes to arterial stiffness and abnormal elastin structure in hypertension: Role of p38MAPK.</a> Antioxidants &amp; Redox Signaling. 27 (7), 379-397 (2017).</li><li>Kumar, D., Trent, M. B., Boor, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allylamine+and+beta-aminopropionitrile+induced+aortic+medial+necrosis:+Mechanisms+of+synergism.">Allylamine and beta-aminopropionitrile induced aortic medial necrosis: Mechanisms of synergism.</a> Toxicology. 125 (2-3), 107-115 (1998).</li><li>Ren, W., 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=&#946;-Aminopropionitrile+monofumarate+induces+thoracic+aortic+dissection+in+C57BL/6+mice.">&#946;-Aminopropionitrile monofumarate induces thoracic aortic dissection in C57BL/6 mice.</a> Scientific Reports. 6, 28149 (2016).</li><li>Kanematsu, Y., 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=Pharmacologically-induced+thoracic+and+abdominal+aortic+aneurysms+in+mice.">Pharmacologically-induced thoracic and abdominal aortic aneurysms in mice.</a> Hypertension. 55 (5), 1267-1274 (2010).</li><li>Weyers, J. J., Carlson, D. D., Murry, C. E., Schwartz, S. M., Mahoney, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+Perfusion+and+Filling+of+Mouse+Coronary+Vasculature+as+Preparation+for+Micro+Computed+Tomography+Imaging.">Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging.</a> J Vis Exp. (60), e3740 (2012).</li><li>Cortell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silicone+rubber+for+renal+tubular+injection.">Silicone rubber for renal tubular injection.</a> Journal of Applied Physics. 26 (1), 158-159 (1969).</li><li>Bentley, M. D., Ortiz, M. C., Ritman, E. L., Romero, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+microcomputed+tomography+to+study+microvasculature+in+small+rodents.">The use of microcomputed tomography to study microvasculature in small rodents.</a> American Journal of Physiology - Regulatory, Integrative and Comparative Physiology. 282 (5), R1267-R1279 (2002).</li><li>Marxen, M., 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=MicroCT+scanner+performance+and+considerations+for+vascular+specimen+imaging.">MicroCT scanner performance and considerations for vascular specimen imaging.</a> Medical Physics. 31 (2), 305-313 (2004).</li><li>Yang, J., Yu, L. X., Rennie, M. Y., Sled, J. G., Henkelman, R. 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X., 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=Mechanical+stretch-induced+endoplasmic+reticulum+stress,+apoptosis,+and+inflammation+contribute+to+thoracic+aortic+aneurysm+and+dissection.">Mechanical stretch-induced endoplasmic reticulum stress, apoptosis, and inflammation contribute to thoracic aortic aneurysm and dissection.</a> The Journal of Pathology. 236 (3), 373-383 (2015).</li><li>Kurihara, T., 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=Neutrophil-derived+matrix+metalloproteinase+9+triggers+acute+aortic+dissection.">Neutrophil-derived matrix metalloproteinase 9 triggers acute aortic dissection.</a> Circulation. 126 (25), 3070-3080 (2012).</li><li>Dillavou, E. D., Buck, D. G., Muluk, S. C., Makaroun, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+versus+three-dimensional+CT+scan+for+aortic+measurement.">Two-dimensional versus three-dimensional CT scan for aortic measurement.</a> Journal of Endovascular Therapy. 10 (3), 531-538 (2003).</li><li>Muratoglu, S. C., 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=LRP1+protects+the+vasculature+by+regulating+levels+of+connective+tissue+growth+factor+and+HtrA1.">LRP1 protects the vasculature by regulating levels of connective tissue growth factor and HtrA1.</a> Arterioclerosis, Thrombosis, and Vascular Biology. 33, 2137-2146 (2013).</li><li>Badea, C. T., Dragova, M., Holdsworth, D. W., Johnson, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+small+animal+imaging+using+micro-CT+and+digital+subtraction+angiography.">In vivo small animal imaging using micro-CT and digital subtraction angiography.</a> Phys Med Biol. 53 (19), R319-R350 (2008).</li><li>Zhou, Y. Q., 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=Ultrasound-guided+left-ventricular+catheterization:+A+novel+method+of+whole+mouse+perfusion+for+microimaging.">Ultrasound-guided left-ventricular catheterization: A novel method of whole mouse perfusion for microimaging.</a> Laboratory Investigation. 84, 385-389 (2004).</li></ol>

Micro-CT imaging can be used to provide highly detailed and three-dimensional reconstructions of vascular pathology in animal models. Through the use of intravascular contrast-enhanced media, non-enhanced soft tissues, such as the lumen of a blood vessel, can be differentiated from those that are enhancing. While laser Doppler, microangiography, magnetic resonance angiography, histology with confocal, or two-photon microscopy may be used to assess vascular beds, they typically focus on a limited area of study and/or are ...

The incidence of aortic dissection is 3 cases per 100,000 per year1. Aortic dissection and aneurysmal diseases account for over 10,000 deaths in the United States each year, accounting for 1 - 2% of all deaths in Western countries2. Aortic dissection is initiated by a tear in the intimal layer of the vessel with the propagation of blood through the layers of the aortic wall under physiologic pressures. Elevated patient pulse pressures are associated with an increased incidence of dissection and complications. Increased wall shear stress is associated with the aortic wall expansion leading to an aneurysm formation3,4. Consequences of aortic dissection include the occlusion of blood flow to distant organs including the brain, kidneys, bowels, and limbs, the formation of chronic aneurysms, rupture, or death5,6,7.
At present, the biochemical and cellular processes involved in the initiation and progression of aortic aneurysms and dissections are still poorly understood. Reproducible animal models of aortic aneurysm and dissection are key to understanding their pathophysiology. &#946;-aminopropionitrile (BAPN) is a lysyl oxidase inhibitor, which prevents the cross-linking of elastin and collagen and has been shown to significantly alter the structure of the vessel wall extracellular matrix and its biomechanical integrity6,8. Rodents treated with BAPN have been utilized as a common animal model of aortic aneurysm and dissection9,10.
Vascular imaging modalities are instrumental in identifying vascular pathology, confirming vessel patency, and evaluating organ perfusion. Recently, micro-computed tomography (micro-CT) has been utilized to study the vasculature of mice and similarly sized animals. Unlike bone, the axial imaging of blood vessels by computed tomography is limited, as intraluminal blood is inherently relatively radiolucent. When combined with intravascular contrast agents, however, micro-CT enables detailed three-dimensional reconstructions of animal vasculatures for the study of macro-anatomic vascular pathology11.
The selected contrast agent (see the Table of Materials) is a radiopaque silicone rubber that contains lead chromate and lead sulfate. Upon perfusion in the presence of a catalyst, it quickly hardens to form a cast of the vasculature with minimal alterations in the macro-anatomic architecture of the vessels, making the vasculature highly radiopaque in contrast to the background tissues when examined radiographically. This contrast agent is advantageous because it is easy to handle and avoids the tissue degradation and vessel loss due to breakage often associated with vascular cast corrosion. As it cures with minimal shrinkage12, vessels cleared of blood remain patent and allow for an accurate assessment of the animal macro-vasculature in non-survival experiments. Previous work has successfully used radiopaque silicone rubber-contrast in a variety of animal studies. Specifically, applicability in visualizing the coronary, glomerular, placental, and cerebral circulations11,12,13,14,15 has been shown. In this paper, we detail the methodology of open left-ventricular puncture for the intravascular perfusion of lead-based radiopaque silicone rubber to quantitatively characterize BAPN-induced aortic pathology in a mouse model by micro-CT.

<table>
	<tbody>
		<tr>
			<td>Microfil</td>
			<td>Flow Tech, Inc</td>
			<td>MV-122</td>
			<td>We use yellow, a different color can be ordered as desired. Kit includes MV-Compound, MV-Diluent, and MV-Curing Agent.</td>
		</tr>
		<tr>
			<td>Heparin (1000 U/mL)</td>
			<td>Sagent Pharmaceuticals</td>
			<td>25021-400-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Vet One, MWI</td>
			<td>502017</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Aminopropionitrile fumarate salt</td>
			<td>Sigma-Aldrich</td>
			<td>A3134</td>
			<td></td>
		</tr>
		<tr>
			<td>Single syringe pump</td>
			<td>Fisher Scientific</td>
			<td>14-831-200</td>
			<td></td>
		</tr>
		<tr>
			<td>27-gauge scalp vein set needle</td>
			<td>Exel Int</td>
			<td>26709</td>
			<td>27G x 3/4&#34;, 12&#34; tube</td>
		</tr>
		<tr>
			<td>Inveon Micro-CT scanner</td>
			<td>Siemens Medical Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osirix MD</td>
			<td>Pimxmeo SARL</td>
			<td></td>
			<td>Version 8.0.2</td>
		</tr>
		<tr>
			<td>Inveon Research Workplace</td>
			<td>Siemens Medical Solutions</td>
			<td></td>
			<td>Version 4.2</td>
		</tr>
		<tr>
			<td>Rodent Chow</td>
			<td>Harlan Teklad</td>
			<td>2018sx</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In order to evaluate this protocol, 20 male adult mice, of mixed background as previously described19 and of 20 - 30 weeks of age, with or without BAPN treatment, were perfused with a lead-based radiopaque silicone rubber (see the Table of Materials) using the protocol detailed above. They underwent micro-CT scanning on the following day (Figure 1 and Figure 2). There were...

Watch this Scientific Journal Video about Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of Î²-aminopropionitrile-induced Aortic Aneurysm and Dissection at JoVE.com

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

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 ...

This method could help answer key questions in the vascular biology field, such as determining how vessel size is impacted in different animal models of vascular disease. The main advantage of this technique is that micro-CT enables detailed images of both large and small vessels, both in vivo and ex vivo, which are able to be precisely quantified. 24 hours prior to the CT analysis, perform the surgical procedures to prepare the specimen for a postmortem intracardiac profusion.For anesthesia, use 3%isoflurane, and be sure to have charcoal scavenger in use. Then, maintain the anesthesia with two to 2.5 percent isoflurane from a face mask, also attached to a scavenger with one liter of oxygen per minute. Prior to beginning the surgery, perform a toe pinch to check for the absence of a pedal reflex.Then, set up an operative field with instruments and so forth. Next, position the animal in dorsal recumbency and secure the limbs. Now, using scissors, make a midline incision through the skin and soft tissue over the sternum between the pubic symphysis to the sternal notch.Next, use the scissors to create a hole in the diaphragm at the xiphoid process to enter the thoracic cavity. Then, continue using the scissors to dissect the diaphragm of the ventral chest wall bilaterally. Cut through the costal cartilages to separate the ribs from the sternum at the right sternal border.Now, apply a fine hemostatic clamp to the tip of the sternum near the xiphoid process and move the hemostat cranially so that it is positioned over the head of the mouse. Then, sharply dissect any attachments between the heart and the chest wall. Limit the number of vessels damaged during the animal preparation, as this will cause the contrast agent to leak out of the severed vessels, which will change the volume required for a complete filling, and will introduce artifacts to the final imaging.Now, connect a 27 gauge IV catheter needle to a syringe preloaded with 10 milliliters of heparinized PBS and fill all the tubing with the buffer, removing all of the air bubbles. Removal of all bubbles from the lines is essential. Then, immediately incise the right ventricle, or the inferior vena cava, to drain the heparin solution and blood.To proceed, use the cannula to puncture the left ventricle, stabilized by the clamp. Conduct the profusion at a constant two milliliters per minute using a syringe pump. There should be visible blanching of the organs.In a few minutes, the vena's circulation should be free of blood. Then, stop the pump and disconnect the catheter tubing from the syringe, but do not move the catheter needle. Now, separate the contrast agent solution into five milliliter aliquots, add the curing agent and mix well.Then, load five milliliters of the mixture into a 10 milliliter syringe and profuse the animal with the contrast agent at a steady two milliliter per minute. Keep the infusion going past the point of seeing the casting agent exiting the animal. The contrast agent will cure in 20 minutes.Once cured, harvest the organs of interest and fix them in 10%neutral buffered formalin. Next, it the samples are not used for micro-CT scanning, fix the whole carcass. Otherwise, position the carcass on a metal tray and place it in a refrigerator at four degrees Celsius to cure overnight.On the following day, acquire x-ray computed tomography images of each mouse using a micro-CT scanner. Set the scanner parameters as follows;An x-ray tube peak voltage of 55 kilovolts, a current of 150 microamps, a system magnification factor of 2.19, and a CCD camera pixel binning factor of two. This binning factor will yield an effective pixel size of 29 microns.Next, analyze the images to identify aneurysms using a standardized approach. Measure the minor axis diameter at the widest point of three locations;of the aortic arch, the descending thoracic aorta, and the abdominal aorta. Ideally, two observers, one of which is blinded, should analyze each image.Using the measurements, identify aneurysms as localized or diffused dilations of the aortic segments to diameters greater than 50%of the reference diameter. 20 male adult mice of mixed background and 20 to 30 weeks old were analyzed with or without BAPN treatment. Each was profused with a lead-based radiopaque silicone rubber.The mean diameter of the ascending aorta in the BAPN-treated mice was significantly larger than that of the untreated age-matched controls. Data points from mice determined to have aneurysms are in red. By contrast, the inhibition of lysyl oxidase with BAPN did not have a significant effect on the mean descending thoracic, nor did this treatment affect abdominal aortic diameters.An aneurysm was defined as being 1.5 times the mean diameter of the untreated group. The treatment lead to a significant increase in aneurysms by this definition. Aneurysms in the BAPN-treated mice were identified exclusively in the aorta, with the majority identified in the thoracic aorta.After watching this video, you should have a good understanding of how to profuse the vasculature for radiographic imaging. Once mastered, the profusion and image analysis can be done in two hours if it is performed properly. While attempting this procedure, it's important to remember that CT imaging protocols can be adjusted with the assistance of radiologic staff to optimize image acquisition for the specific purposes of individual experiments.

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