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 saline, which will replace the blood in the vasculature in a water bath set to 37 &#176;C.</li>
		<li>Prepare the pressure-controlled pump by connecting the required tubing and 2 empty 10-mL syringes, 1 for the heparinized-saline buffer and 1 for the contrast agent.</li>
		<li>Fill the tubing with the warm heparinized phosphate buffered saline and remove the air bubbles from the tubing of the pressure pump.</li>
	</ol>
	</li>
	<li>Contrast agent 
	NOTE: Please refer to the Table of Materials for the contrast agent kit constituents.
	<ol>
		<li>Mix a pigmented compound with a diluent to achieve a 1:6 dye to diluent ratio.</li>
		<li>Immediately prior to use (step 2.3.12), add 200 &#181;L of a curing agent to each 5-mL aliquot of the diluted pigmented compound and mix them well (4% by volume). 
		NOTE: The manufacturer reported the working time is 40 min. As the silicone-rubber contrast agent begins to polymerize 20 min after the addition of the curing agent, it is important to prepare the solution immediately prior to its infusion.</li>
	</ol>
	</li>
	<li>BAPN drinking water
	<ol>
		<li>Dissolve &#946;-aminopropionitrile (BAPN) in drinking water to create a final concentration of 3 g/L (adapted from protocols previously described in the literature)9,16,17.</li>
		<li>Administer the BAPN-containing drinking water to a group of mice once they are 4 weeks of age until the time of perfusion for micro-CT.</li>
	</ol>
	</li>
</ol>
2. Surgical Procedure
<ol>
	<li>Animal preparation
	<ol>
		<li>Wean the mice at 3 weeks of age, maintain them on a 12 h light/12 h dark cycle, and feed them standard rodent chow. For the BAPN-treated group, administer freshly prepared BAPN drinking water for 16 - 26 weeks ad libitum. Provide the control animals with standard drinking water ad libitum.</li>
	</ol>
	</li>
	<li>Anesthetic technique 
	NOTE: 24 h prior to the CT analysis, the following procedure is performed. Surgical procedures are enacted to prepare the specimen for a postmortem intracardiac perfusion.
	<ol>
		<li>Induce an anesthesia via an induction tank with 100% O2 and 3% isoflurane delivered via a precision vaporizer. After the anesthesia induction, discontinue the isoflurane and flush the chamber with O2. Maintain the anesthesia with 2 - 2.5% isoflurane and 1 L/min of O2 via a nose cone.</li>
		<li>Attach both the induction chamber and the facemask to a charcoal scavenger for waste gas adsorption to protect personnel. Ensure an adequate anesthetic plane by demonstrating that there is no response to noxious stimuli (toe pinch).</li>
		<li>Prepare an operative field consisting of a surgical tray and the necessary surgical instruments.</li>
		<li>Transfer the animal to the surgical field and position it in dorsal recumbency.</li>
	</ol>
	</li>
	<li>Operative technique
	<ol>
		<li>Using scissors, make a midline incision through the skin and soft tissue from midway between the pubic symphysis to the sternal notch, extending through the skin and soft tissue overlying the sternum.</li>
		<li>Using scissors, create a hole in the diaphragm at the xiphoid process to enter the thoracic cavity.</li>
		<li>Use scissors to dissect the diaphragm of the ventral chest wall, bilaterally.</li>
		<li>Cut through the costal cartilages to separate the ribs from the sternum at the right sternal border.</li>
		<li>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. This will retract the thymus and sternum away from the heart, exposing the heart and great vessels for further manipulation.</li>
		<li>Sharply dissect any attachments between the heart and the chest wall.</li>
		<li>Connect the 27-gauge IV catheter needle to a syringe pre-loaded with 10 mL of heparinized phosphate buffered saline (5 U/mL) and fill all tubing with the buffer in order to remove the air bubbles from the tubes of the pressure pump.</li>
		<li>Use care while preparing the fluid as bubbles in the fluid line may impede the filling of smaller vessels. Limit the number of vessels that are damaged during the animal preparation, as this will cause the contrast agent to leak out of the severed vessels, changing the volume required for a complete filling and introducing artifacts to the final imaging.</li>
		<li>Puncture the left ventricle with a 27-gauge needle that is stabilized with a right-angle clamp. Immediately incise the right ventricle or the inferior vena cava to drain the heparin solution and blood. 
		NOTE: Heparin is used as an anticoagulant to prevent the blood from clotting in the vessels after the animal&#39;s death.</li>
		<li>Perfuse the animal at a constant rate of 2 mL/minute using a single syringe pump. Note the visible blanching of the organs. Continue the perfusion until the perfusate draining from the venous circulation is free of blood (about 5 - 6 mL). Stop the pump.</li>
		<li>Disconnect the IV catheter tubing from the 10-mL syringe, taking care not to disrupt the needle&#39;s position in the left ventricle.</li>
		<li>Immediately after the complete exsanguination, separate the contrast agent solution into 5-mL aliquots and add the curing agent at this time (see step 1.2). Mix them well. Draw up 5 mL of the contrast agent mix into a 10-mL syringe and perfuse the animal with it.</li>
		<li>For a complete filling of the vessels (arteries and veins), continue the infusion past the point when it can be seen exiting the venous solution. Look for signs of a successful perfusion including the visualization of a casting agent in the coronary arteries, pulmonary arteries, intestine, and liver vasculature.</li>
		<li>The contrast agent will cure after approximately 20 min at room temperature. Upon curing, harvest the individual organs, as needed, and fix them in 10% neutral buffered formalin. Fix whole carcasses if the samples are not used for micro-CT scanning the following day. If the carcasses will be used the subsequent day, position them on a metal tray and place them in a refrigerator at 4 &#176;C to cure overnight.</li>
	</ol>
	</li>
</ol>
3. Micro-CT Scanning and Parameters
NOTE: The specific image acquisition parameters will be dependent upon the machine in use.
<ol>
	<li>Acquire X-ray computed tomography images of each mouse the day following the perfusion using a micro-CT scanner using an X-ray tube voltage of 55 kVp, a current of 150 &#956;A, a system magnification factor of 2.19, and a CCD camera pixel binning factor of 2. This yields an effective pixel size of 29 &#181;m.
	<ol>
		<li>Lay the mouse carcass supine on the micro-CT scanner table and obtain a scout X-ray scan.</li>
		<li>Focus the detector field of view of 57.4 mm (axial) x 37.1 mm (transaxial) on the torso to image the full length of the aorta.</li>
		<li>Acquire 180 image projections with a rotation increment of 2 degrees and a time per projection of 2800 ms.</li>
	</ol>
	</li>
	<li>Reconstruct the images using a modified Feldkamp algorithm; the reconstructed voxel size is 29 x 29 x 29 &#181;m3 (slice thickness = 29 &#181;m) using the Multimodal 3D Visualization plug-in for the software used here.</li>
</ol>
4. Post-processing and Rendering
<ol>
	<li>Convert the CT data to a DICOM format using the appropriate software.</li>
	<li>Analyze the images to identify whether an aneurysm was present. Measure the minor axis diameter at the widest point of the aortic arch, descending thoracic aorta, and abdominal aorta as previously described18 (Figure 1). 
	NOTE: In our study, images were analyzed by two independent observers (one blinded) utilizing a DICOM viewer to identify whether an aneurysm was present. The minor axis diameter was measured at the widest point of the aortic arch, descending thoracic aorta, and abdominal aorta as previously described19 (Figure 1). Mean non-aneurysmal arterial segments of BAPN-untreated mice established normal vessel diameters serving as the age-matched control values.</li>
	<li>Aneurysms are defined as a localized or diffuse dilation of the aortic segments to diameters greater than 50% of the reference diameter. Locate these based on the above measurements.</li>
</ol>

<ol><li>Meszaros, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidemiology+and+clinicopathology+of+aortic+dissection%5BTitle%5D)+AND+%22CHEST%22%5BJournal%5D)">Epidemiology and clinicopathology of aortic dissection.</a> CHEST. 117 (5), 1271-1278 (2000).</li>
<li>Kochanek, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deaths%3A+final+data+for+2009%5BTitle%5D)+AND+%22National+Vital+Statistics+Reports%22%5BJournal%5D)">Deaths: final data for 2009.</a> National Vital Statistics Reports. 60 (3), 1-116 (2011).</li>
<li>Li, J. S., Li, H. Y., Wang, L., Zhang, L., Jing, Z. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+beta-aminopropionitrile-induced+aortic+dissection+model+in+rats+by+different+administration+and+dosage%5BTitle%5D)+AND+%22Vascular%22%5BJournal%5D)">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/pubmed?term=((Functional+importance+of+connective+tissue+repair+during+the+development+of+experimental+abdominal+aortic+aneurysms%5BTitle%5D)+AND+%22Surgery%22%5BJournal%5D)">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/pubmed?term=((Molecular+mechanisms+of+thoracic+aortic+dissection%5BTitle%5D)+AND+%22Journal+of+Surgical+Research%22%5BJournal%5D)">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/pubmed?term=((Inhibition+of+cross-links+in+collagen+is+associated+with+reduced+stiffness+of+the+aorta+in+young+rats%5BTitle%5D)+AND+%22Atherosclerosis%22%5BJournal%5D)">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/pubmed?term=((Lysyl+oxidase+induces+vascular+oxidative+stress+and+contributes+to+arterial+stiffness+and+abnormal+elastin+structure+in+hypertension%3A+Role+of+p38MAPK%5BTitle%5D)+AND+%22Antioxidants+%26+Redox+Signaling%22%5BJournal%5D)">Lysyl oxidase induces vascular oxidative stress and contributes to arterial stiffness and abnormal elastin structure in hypertension: Role of p38MAPK.</a> Antioxidants & 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/pubmed?term=((Allylamine+and+beta-aminopropionitrile+induced+aortic+medial+necrosis%3A+Mechanisms+of+synergism%5BTitle%5D)+AND+%22Toxicology%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4916438/">β-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/pubmed?term=((Pharmacologically-induced+thoracic+and+abdominal+aortic+aneurysms+in+mice%5BTitle%5D)+AND+%22Hypertension%22%5BJournal%5D)">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. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retrograde+Perfusion+and+Filling+of+Mouse+Coronary+Vasculature+as+Preparation+for+Micro+Computed+Tomography+Imaging%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Silicone+rubber+for+renal+tubular+injection%5BTitle%5D)+AND+%22Journal+of+Applied+Physics%22%5BJournal%5D)">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/pubmed?term=((The+use+of+microcomputed+tomography+to+study+microvasculature+in+small+rodents%5BTitle%5D)+AND+%22American+Journal+of+Physiology+-+Regulatory%2C+Integrative+and+Comparative+Physiology%22%5BJournal%5D)">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/pubmed?term=((MicroCT+scanner+performance+and+considerations+for+vascular+specimen+imaging%5BTitle%5D)+AND+%22Medical+Physics%22%5BJournal%5D)">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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+structural+and+hemodynamic+analysis+of+vascular+trees%5BTitle%5D)+AND+%22American+Journal+of+Physiology+-+Heart+and+Circulatory+Physiology%22%5BJournal%5D)">Comparative structural and hemodynamic analysis of vascular trees.</a> American Journal of Physiology - Heart and Circulatory Physiology. 298 (4), H1249-H1259 (2010).</li>
<li>Jia, L. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+stretch-induced+endoplasmic+reticulum+stress%2C+apoptosis%2C+and+inflammation+contribute+to+thoracic+aortic+aneurysm+and+dissection%5BTitle%5D)+AND+%22The+Journal+of+Pathology%22%5BJournal%5D)">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/pubmed?term=((Neutrophil-derived+matrix+metalloproteinase+9+triggers+acute+aortic+dissection%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">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/pubmed?term=((Two-dimensional+versus+three-dimensional+CT+scan+for+aortic+measurement%5BTitle%5D)+AND+%22Journal+of+Endovascular+Therapy%22%5BJournal%5D)">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/pubmed?term=((LRP1+protects+the+vasculature+by+regulating+levels+of+connective+tissue+growth+factor+and+HtrA1%5BTitle%5D)+AND+%22Arterioclerosis%2C+Thrombosis%2C+and+Vascular+Biology%22%5BJournal%5D)">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/pubmed?term=((In+vivo+small+animal+imaging+using+micro-CT+and+digital+subtraction+angiography%5BTitle%5D)+AND+%22Phys+Med+Biol%22%5BJournal%5D)">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/pubmed?term=((Ultrasound-guided+left-ventricular+catheterization%3A+A+novel+method+of+whole+mouse+perfusion+for+microimaging%5BTitle%5D)+AND+%22Laboratory+Investigation%22%5BJournal%5D)">Ultrasound-guided left-ventricular catheterization: A novel method of whole mouse perfusion for microimaging.</a> Laboratory Investigation. 84, 385-389 (2004).</li>
</ol>

The authors have nothing to disclose.

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 limited to two-dimensional assessments. Micro-CT provides a cost-effective means of obtaining detailed imaging of vascular structures, which can aid in gaining an understanding of the underlying angiogenesis and vascular biology. Additional methods of imaging small animals include in vivo micro-CT and digital subtraction angiography. Similar to the technique described here, in vivo micro-CT relies upon an exogenous contrast agent to increase the resolution. Fenestra VC and Isovue-370 are 2 such blood-pool contrast agents that are available for micro-CT. Fenestra VC has been used and is reported to have a maximum mean enhancement of ~620 Hounsfield Units in the aorta 20 min following injection20. Other nanoparticle-based contrast agents have also shown potential in both CT and MRI. Left ventricular catheterization without opening the chest is another method of whole mouse perfusion for microimaging that has been described21. Ultrasound imaging for the longitudinal measurements of a vessel diameter is a final method that can be used in survival studies.
During this procedure, adequate perfusion requires a high degree of attention to detail. It is important for the vasculature to be completely exsanguinated prior to the perfusion with a contrast agent. If the liver does not blanch and remains its dark red color, additional flushing with a heparinized saline solution may be needed prior to the perfusion with a contrast. Incomplete exsanguination of the vasculature may affect the final image quality, appearing as filling defects in the vasculature or as artifactual distal vascular occlusions. Further, the insertion of the needle into the left ventricle is a delicate procedure that can tear the ventricular wall or cut through into the right ventricle. If the needle is inserted into the right ventricle, the lungs will be perfused quickly and will turn yellow early during the perfusion, leading to an incomplete vascular perfusion. A vessel rupture is a final complication that leads to an incomplete perfusion. The chance of a vessel rupture can be minimized by ensuring adequate drainage from the venous circulation prior to initiating the perfusion via the arterial tree. Retrograde injection is an alternative method to perfusing a vascular bed, which has previously been described to image the coronary vasculature11. We chose an antegrade method of perfusion as it more accurately represents physiologic blood flow in the thoracic and abdominal aorta. The quality of the image obtained with micro-CT does limit the ability to assess the aorta for intramural thrombus or hematoma. However, the soft tissue can be visualized quite well depending on how the CT protocol is formulated. For our studies, we noticed segments of the aorta with filling defects presumably due to stenoses from intraluminal thrombus. The technique as described is best for assessing the intravascular space.
In summary, this protocol provides a safe technique for the high-resolution examination of the vasculature of a murine model of aortic aneurysm and dissection. Quantitative analyses can be easily achieved using micro-CT images, which are able to provide a precise cast of the vasculature. For the purposes of the experiments illustrated in this publication, we chose to measure the largest aortic diameter to mimic the measurement of aortic aneurysms in clinical practice. A combination of anatomic landmarks and slice location distances can be utilized to measure specific segments of the aorta if appropriate. It is also shown that the perfused animal vasculature can be imaged and digitally separated from the skeletal background to the desired amount to create a three-dimensional representation of the major blood vessels and its smaller distal branches. This protocol preserves the vascular tissues of interest, which can be embedded in paraffin and used for histological sectioning for further examination.
Micro-CT is a promising imaging modality for the study of vascular biology. With its high spatial resolution, it offers an opportunity to evaluate the structure, organization, and pathology of blood vessels. The procedure described in this manuscript requires multiple skills and techniques including animal handling and preparation, image acquisition, and quantification of the results. This article has successfully demonstrated one application of vascular perfusion to identify aortopathies in mice. Further studies are needed to assess its applicability in other animal models and disease processes.

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&quot;, 12&quot; 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 no significant differences in the ages of the mice among any of the compared groups.
Minor axis diameters were then quantified in these mice. The mean diameter of the ascending aorta in the BAPN-treated mice was significantly larger than that of the untreated age-matched controls (1.43 &#177; 0.56 mm vs. 0.93 &#177; 0.11 mm; p = 0.023, unpaired Students t-test). The inhibition of lysyl oxidase with BAPN did not have a significant effect on the mean descending thoracic or abdominal aortic diameters compared to the age-matched controls (p &#62;0.082,unpaired Students t-test, Figure 3).
An aneurysm was defined as being 1.5x the mean diameter of the untreated group. There was a significant increase in the number of mice with aneurysms in the BAPN-treated compared with the BAPN-untreated controls (50% of the BAPN-treated mice vs. 0% of the untreated mice; P = 0.042) by Fisher&#39;s exact test. Aneurysms in the BAPN-treated mice were identified exclusively in the aorta, with the majority identified in the thoracic aorta (8 out of 10 aneurysms identified). 4 mice developed more than 1 aneurysm with the BAPN-treatment (Table 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57589/57589fig1.jpg" /> 
Figure 1: Representative cross-sectional micro-CT images. A. This is a cross-sectional imaging of the BAPN-untreated mice. B. This is a cross-sectional imaging of the BAPN-treated mice. The minor axis diameter measurements are made at the level of the aortic arch.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57589/57589fig2.jpg" /> 
Figure 2: Three-dimensional reconstruction of mouse aortic aneurysm and dissection identified by micro-CT. A. This is a representative 3-D reconstruction of a BAPN-treated mouse with a saccular abdominal aortic aneurysm and an aortic arch aneurysm. B. This panel shows a representative descending thoracic aortic dissection in a BAPN-treated mouse. The true and false lumen are separated by the intimal flap. The true lumen (TL) is the normal passageway of blood and the false lumen (FL) is the newly created passageway.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57589/57589fig3.jpg" /> 
Figure 3: Effect of BAPN treatment on aortic diameters. These panels show the mean minor axis vessel diameters measured by micro-CT in BAPN-untreated and BAPN-treated male mice. Those mice determined to have an aneurysm are highlighted in red. The mean of the untreated group is represented as a dotted line in each panel. a. This is the ascending aorta/arch (AAo/Arch); 0.93 &#177; 0.11 vs. 1.43 &#177; 0.56 mm; p = 0.023. b. This is the descending thoracic aorta (DTA); 0.82 &#177; 0.04 vs. 1.11 &#177; 0.43 mm; p = 0.0817. c. This is the abdominal aorta; 0.66 &#177; 0.11 vs. 0.89 &#177; 0.58 mm; p = 0.296. All data are presented as mean &#177; standard deviation. <a href="/files/ftp_upload/57589/57589fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>No BAPN 
			(n = 8)</td>
			<td>BAPN 
			(n = 12)</td>
		</tr>
		<tr>
			<td>Total Animals with Aneurysm</td>
			<td>0</td>
			<td>6&#160; *</td>
		</tr>
		<tr>
			<td>Total Number of Aneurysms</td>
			<td>0</td>
			<td>10</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; AAO/ARCH&#160;&#160;</td>
			<td>0</td>
			<td>5</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; DTA</td>
			<td>0</td>
			<td>3</td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ABD AORTA&#160;</td>
			<td>0</td>
			<td>2</td>
		</tr>
	</tbody>
</table>
Table 1. Anatomic distribution of identified aneurysms in male mice with and without BAPN treatment. This table shows the incidence of aneurysms as identified by micro-CT (n = 20). AAO = ascending aorta; DTA = descending thoracic aorta; ABD AORTA = abdominal aorta. * p &#60;0.05.

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 ...

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Nanyang Technological University

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9.6K Views.  University of Maryland School of Medicine. 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.

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

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

Immunology and Infection

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.

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 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 ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

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

A Calcium Phosphate-Induced Mouse Abdominal Aortic Aneurysm Model

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the abdominal aorta. Currently, several chemically induced murine AAA models are used, each simulating a different aspect of the pathogenesis of AAA. The calcium phosphate-induced AAA model is a rapid and cost-effective model compared to the angiotensin II- and elastase-induced AAA models. The application of CaPO4 crystals to the mouse aorta results in elastic fiber degradation, loss of smooth muscle cells, inflammation, and calcium deposition associated with aortic dilation. This article introduces a standard protocol for the CaPO4-induced AAA model. The protocol includes material preparation, the surgical application of the CaPO4&#160;to the adventitia of the infrarenal abdominal aorta, the harvesting of aortas to visualize aortic aneurysms, and histological analyses in mice.

This protocol describes a calcium phosphate-induced abdominal aortic aneurysm (AAA) mouse model to study the pathological features and molecular mechanisms of AAAs.

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Isolation and Analysis of Aortic Arch and Root Lesions in an Atherosclerotic Mouse Model

Atherosclerosis, a leading cause of cardiovascular diseases, necessitates a detailed examination of lesion development and progression. This study introduces a comprehensive protocol for the isolation and histological analysis of aortic arch and root lesions in a widely used atherosclerotic mouse model, low-density lipoprotein receptor knock-out (Ldlr-/-) mice. The aortic arch and root are key sites for atherosclerotic lesions, and their examination is critical for assessing the onset, progression, or regression of atherosclerosis, predicting cardiovascular event risks, and identifying potential therapeutic targets. This protocol outlines methods for quantifying atherosclerotic burden in the aortic arch and root, including tissue isolation, fixation, Oil Red O staining, aortic root sectioning, Hematoxylin and Eosin (HE) staining, Verhoeff-Van Gieson (VVG) staining, and image analysis. Oil Red O staining measures plaque area in the aortic arch, evaluating the severity of atherosclerosis, while HE staining of the aortic root reveals plaque components such as the lipid core and fibrous cap, facilitating the assessment of plaque stability and rupture risk. VVG staining can stain collagen fibers within tissues, providing further insights into plaque composition and related information. This thorough analysis offers valuable insights into the mechanisms of lesion development and may guide the creation of novel therapeutic strategies for preventing and treating atherosclerosis.

This protocol provides a comprehensive methodology for the quantitative analysis of atherosclerotic plaques to facilitate more in-depth research into the mechanisms and progression of atherosclerosis.

Atherosclerosis, a leading cause of cardiovascular diseases, necessitates a detailed examination of lesion development and progression. This study ...

Murine Model of Thoracic Aortic Dissection Induced by Oral &#946;-Aminopropionitrile and Subcutaneous Angiotensin II Infusion

Thoracic aortic dissection (TAD) is a highly fatal cardiovascular disease that lacks efficient medical treatment. Replication of animal models of TAD pathophysiology is essential for studying the intrinsic mechanisms of TAD. The widely used TAD model induced by &#946;-aminopropionitrile (BAPN, an irreversible and orally active lysyl oxidase inhibitor) in mice has the limitation of an inconsistent success rate. This protocol describes in detail a reported modified murine model of TAD induced by oral BAPN combined with subcutaneous angiotensin II (Ang II) infusion. After four weeks of BAPN administration followed by 24 h of Ang II infusion, a murine model with characteristics similar to human TAD was reliably induced, and the success rate of TAD model construction was significantly improved. Oral BAPN inhibits the cross-linking of elastin and collagen, resulting in the destruction of the aortic wall structure and inducing aortic dilation and dissection formation to a certain extent. The subsequent induction of Ang II further exacerbates the degeneration of the aortic wall, thereby promoting the occurrence of TAD. Consequently, the combination of BAPN and Ang II represents a refined approach to constructing a murine TAD model, offering a valuable tool to explore the pathogenesis and potential therapeutic approaches for TAD.

This protocol provides a detailed, step-by-step procedure for the induction of thoracic aortic dissection in mice. Specifically, it includes the precise calculation of the required doses of &#946;-aminopropionitrile and angiotensin II, the procedure for osmotic pump filling, and the implantation technique of the osmotic pump.

Thoracic aortic dissection (TAD) is a highly fatal cardiovascular disease that lacks efficient medical treatment. Replication of animal models of TAD ...

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

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