We thank Anthony Herring and Cindy Dodson for their knowledge and technical expertise.
Sources of Funding: 
Funding for this study was provided by the National Heart, Lung, and Blood Institute of the National Institute of Health under Award No. T32HL007849 and Grant Nos. R01HL081629-07 (G.R.U.) and R01HL124131-01 (G.R.U.).

Animal protocols were approved by the University of Virginia Institutional Animal Care and Use Committee (No. 3848).
NOTE: This model has been previously published by Cullen et al. and is a modified protocol described by Hynecek et al.8,15.
1. Animals
<ol>
	<li>Use non-castrated male swine weighing 20-30 kg for the experiments.</li>
	<li>In order to maximize the proportion of weight-based doses of BAPN ingested, provide pigs with divided meals of standard chow and 0.15 g/kg of BAPN mixed with whole-milk plain yogurt or wet dog food.&#160;Start BAPN administration 7 days prior to the index operation to achieve steady state in the blood, and daily during post-operative course. 
	NOTE: BAPN has numerous side effects if ingested in large quantities. Isolation precautions for staff including cap, gowns, gloves, and shoe covers should be worn whenever interacting with animals fed BAPN or handling BAPN.</li>
	<li>Make pigs nil per os (NPO) the night prior to surgery.</li>
</ol>
2. Anesthesia
<ol>
	<li>Induce general anesthesia (GA) using tiletamine-zolzepam 6 mg/kg, xylazine (2 mg/kg), and atropine sulfate (0.04 mg/kg) administered intramuscularly.</li>
	<li>Intubate the pig using a standard endotracheal tube (ETT) and Miller blade.</li>
	<li>Obtain peripheral intravenous (IV) access using a 16 or 18 gauge IV in an ear vein and secure in place with tape.</li>
	<li>Connect the ETT to anesthesia machine and maintain GA using inhaled isoflurane (0.2 mg/kg).</li>
	<li>Apply electrocardiogram (EKG) leads and pulse oximetry to monitor vital signs during surgery. Take oral temperature at the beginning of the case. Place an electrocautery pad on dependent portion of the pig.</li>
	<li>Be sure one member of your staff is continually monitoring and regularly recording vital signs to ensure the pig is appropriately sedate, ventilated, and oxygenated, as well as to identify and appropriately intervene upon any hemodynamic instability during the surgery.</li>
</ol>
3. Surgical technique
<ol>
	<li>Perform sterilization of the surgical area using sterile gauze, povidone-iodine and 70% isopropyl alcohol. Drape the pig in the usual sterile fashion. Take a blood sample prior to incision. 
	NOTE: At this point, all equipment, including instruments, balloons, wires, etc. must be sterile.</li>
	<li>Using an eleven blade or Bovie electrocautery, perform a midline laparotomy to enter the abdominal cavity.</li>
	<li>Displace the abdominal viscera cephalad to the pig&#39;s left to expose the retroperitoneum. Cover the bowel with a moist blue towel to avoid desiccation. Make a sharp incision to enter the retroperitoneum, allowing access to the inferior vena cava (IVC) and infrarenal abdominal aorta. 
	NOTE: Identification and protection of the ureters bilaterally is crucial at this portion of the case. Swine retroperitoneal anatomy (including the course of the ureters) grossly mirrors that of humans, with subtle variations detailed below.</li>
	<li>Circumferentially dissect the aorta from the renal vessels, inferiorly to the aortic trifurcation. Take care to avoid IVC and lumbar artery injury. Once the entire infrarenal aorta is exposed, use calipers to measure the aortic diameter at the mid portion of the infrarenal aorta. 
	NOTE: Unlike humans, swine have an aortic trifurcation, not bifurcation.</li>
	<li>Identify the caudal mesenteric artery on the anterior portion of the infrarenal aorta, which usually lies a few centimeters proximal to the aortic trifurcation. This artery does not exist in humans. Dissect, clamp, and transect this artery. At this point, administer 5000 units of unfractionated heparin sulfate intravenously.</li>
	<li>Cannulate the caudal mesenteric artery with a 0.018 in stainless steel wire guide from a micropuncture introducer set. Serially dilate the artery over the wire with a 5 French (Fr) and then a 7 Fr introducer.</li>
	<li>Leaving the 7 Fr introducer in place, replace the 0.018 in wire with a 0.035 in guidewire, and then remove the 7 Fr introducer, ensuring hemostasis with a finger over the cannulation site as the introducer is removed. Insert a 0.035 in guide wire until approximately 30 cm of wire remains or resistance is encountered.</li>
	<li>Insert a 16 mm percutaneous transluminal angioplasty balloon over the wire&#160;into the infrarenal aorta and position it at the midpoint of the dissected aorta. Inflate the balloon while intermittently measuring the diameter of the dilated aorta with calipers until maximal dilation is approximately 80% greater than your baseline measurement.</li>
	<li>After 10 min of reperfusion, cross clamp the aorta just distal to the renal vessels and proximal to the aortic trifurcation. Identify and clamp the previously dissected lumbar vessels to isolate the infrarenal aorta from systemic circulation. This is important to avoid systemic perfusion of elastase, which can cause a septic response in the acute post-operative period.</li>
	<li>Reintroduce the 7 Fr introducer over the wire and remove the wire. Flush the isolated aortic segment with saline assuring no leakage of fluid. Connect the elastase (500 units) and collagenase (8000 units) solution to the introducer and perfuse 30 mL into the isolated aorta under constant manual pressure for 10 min. The entire 30 mL of solutions should be introduced to the isolated segment. 
	NOTE: A well perfused aortic segment should be taut without leakage from the aortic wall or from the cannulation site. A vessel loop may be wrapped just proximal to the cannulation site to ensure no elastase escapes. Over the course of 10 min, elastase/collagenase solution can be observed &#34;weeping&#34;&#160;through the aortic wall.</li>
	<li>After 10 min, irrigate the solution from the aortic lumen with saline. Remove the introducer and ligate the caudal mesenteric artery stump. Release all clamps (lumbar clamps first, followed by distal clamp, then proximal clamp). 
	NOTE: Restrict the clamp time to no more than 10 min in order to prevent spinal cord ischemia. Have a repair stitch (5-0 polypropylene) loaded in case of bleeding from the caudal mesenteric artery stump after the cross clamps are released.</li>
	<li>Soak a 2 cm x 5 cm piece of surgical gauze with 20 mL of undiluted elastase (27 units/mL) and wrap around the intervened aorta for 10 min. Take a measure of the aorta after all interventions with a caliper.</li>
	<li>Irrigate the abdomen with saline, replace the bowel, and close the abdomen&#160;in three layers. BAPN inhibits wound healing, so in order to minimize the risks of wound breakdown and fascial dehiscence, use suture with a long absorptions time and be sure to take judiciously small sized bites of tissue at every layer. Utilize a running synthetic absorbable monofilament 1 looped Polydioxanone (PDS) suture for the fascia, a running braided absorbable 2-0 suture for the deep dermal later, and a running subcuticular absorbable monofilament suture (4-0) for the skin.</li>
</ol>
4. Postoperative care
<ol>
	<li>Utilize 0.2 mg/kg subcutaneous buprenorphine-SR for post-operative analgesia. Evaluate each pig three times daily for the first three post-operative days for signs of pain and discomfort and administer additional analgesia if identified.&#160;</li>
	<li>Administer postoperative antibiotics (1 g cephalexin intramuscularly) on POD 1-3.</li>
	<li>Socially house animals after POD 3.</li>
</ol>
5. Aortic tissue procurement
<ol>
	<li>Perform tissue procurement on either POD 7, 14, or 28.</li>
	<li>Induce GA as described in steps 2.1-2.5 above. 
	NOTE: Terminal aortic tissue procurement does not need to be sterile.</li>
	<li>5.3. Reopen the previous midline laparotomy incision, being cognizant of adhered bowel to the anterior abdominal wall. Reflect the bowel to expose the retroperitoneum and aorta similar to step 3.3 above.</li>
	<li>Dissect the aorta until the aneurysm is exposed and measure the external diameter of the aneurysmal segment with calipers. Calculate aortic dilation (%) using the following equation: [(harvest infrarenal diameter -&#160;initial operative infrarenal diameter) x 100%]. Once the measurement is attained, administer a lethal dose of pentobarbital-phenytoin (e.g., Euthasol) via injection into the IVC.</li>
	<li>Dissect the aorta from the trifurcation to the suprarenal aorta and explant the aneurysmal segment with a control segment of untreated aorta. Place the sample in either liquid nitrogen or formalin for histologic evaluation.</li>
</ol>

<ol>
	<li>. Leading Causes of Death Reports, National and Regional, 1999 - 2016 Available from: <a target="_blank" href="https://webappa.cdc.gov/sasweb/ncipc/leadcause.html">https://webappa.cdc.gov/sasweb/ncipc/leadcause.html</a> (2018)</li><li>Erbel, R., 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=Diagnosis+and+management+of+aortic+dissection.">Diagnosis and management of aortic dissection.</a> European Heart Journal. 22 (18), 1642-1681 (2001).</li><li>Cameron, J., Cameron, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Surgical Therapy 11th edition. 11, 777-783 (2014).</li><li>. Overview of abdominal aortic aneurysm Available from: <a target="_blank" href="https://www.uptodate.com/contents/overview-of-abdominal-aortic-aneurysm">https://www.uptodate.com/contents/overview-of-abdominal-aortic-aneurysm</a> (2017)</li><li>Pearce, W. H., Zarins, C. K., Bacharach, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerotic+Peripheral+Vascular+Disease+Symposium+II:+controversies+in+abdominal+aortic+aneurysm+repair.">Atherosclerotic Peripheral Vascular Disease Symposium II: controversies in abdominal aortic aneurysm repair.</a> Circulation. 118 (25), 2860-2863 (2008).</li><li>Daugherty, A., Cassis, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+abdominal+aortic+aneurysms.">Mouse models of abdominal aortic aneurysms.</a> Arterioscler Thromb Vasc Biol. 24 (3), 429-434 (2004).</li><li>Anidjar, 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=Elastase-induced+experimental+aneurysms+in+rats.">Elastase-induced experimental aneurysms in rats.</a> Circulation. 82 (3), 973-981 (1990).</li><li>Hynecek, R. L., 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=The+creation+of+an+infrarenal+aneurysm+within+the+native+abdominal+aorta+of+swine.">The creation of an infrarenal aneurysm within the native abdominal aorta of swine.</a> Surgery. 142 (2), 143-149 (2007).</li><li>Lu, G., 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=A+novel+chronic+advanced+stage+abdominal+aortic+aneurysm+murine+model.">A novel chronic advanced stage abdominal aortic aneurysm murine model.</a> Journal of Vascular Surgery. 66 (1), 232-242 (2017).</li><li>Barrow, M. V., Simpson, C. F., Miller, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lathyrism:+a+review.">Lathyrism: a review.</a> The Quarterly Review of Biology. 49 (2), 101-128 (1974).</li><li>Coulson, W. F., Linker, A., Bottcher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lathyrism+in+swine.">Lathyrism in swine.</a> Archives of Pathology &amp; Laboratory Medicine. 87 (4), 411-417 (1969).</li><li>McCallum, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Lathyrism+in+Mice.">Experimental Lathyrism in Mice.</a> The Journal of Pathology and Bacteriology. 89, 625-636 (1965).</li><li>Behmoaras, J., 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=Differential+expression+of+lysyl+oxidases+LOXL1+and+LOX+during+growth+and+aging+suggests+specific+roles+in+elastin+and+collagen+fiber+remodeling+in+rat+aorta.">Differential expression of lysyl oxidases LOXL1 and LOX during growth and aging suggests specific roles in elastin and collagen fiber remodeling in rat aorta.</a> Rejuvenation Research. 11 (5), 883-889 (2008).</li><li>Davies, I., Schofield, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connective+tissue+ageing:+the+influence+of+a+lathyrogen+(beta-aminopropionitrile)+on+the+life+span+of+female+C57BL/Icrfat+mice.">Connective tissue ageing: the influence of a lathyrogen (beta-aminopropionitrile) on the life span of female C57BL/Icrfat mice.</a> Experimental Gerontology. 15 (5), 487-494 (1980).</li><li>Cullen, J. 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=A+novel+swine+model+of+abdominal+aortic+aneurysm.">A novel swine model of abdominal aortic aneurysm.</a> Journal of Vascular Surgery. , (2018).</li><li>Marinov, G. R., 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=Can+the+infusion+of+elastase+in+the+abdominal+aorta+of+the+Yucatan+miniature+swine+consistently+produce+experimental+aneurysms.">Can the infusion of elastase in the abdominal aorta of the Yucatan miniature swine consistently produce experimental aneurysms.</a> Journal of Investigative Surgery. 10 (3), 129-150 (1997).</li><li>Pope, N. H., 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=Interleukin-6+Receptor+Inhibition+Prevents+Descending+Thoracic+Aortic+Aneurysm+Formation.">Interleukin-6 Receptor Inhibition Prevents Descending Thoracic Aortic Aneurysm Formation.</a> Annals of Thoracic Surgery. 100 (5), 1620-1626 (2015).</li><li>Johnston, W. F., 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=Genetic+and+pharmacologic+disruption+of+interleukin-1beta+signaling+inhibits+experimental+aortic+aneurysm+formation.">Genetic and pharmacologic disruption of interleukin-1beta signaling inhibits experimental aortic aneurysm formation.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 33 (2), 294-304 (2013).</li><li>Johnston, W. F., 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+interleukin-1beta+decreases+aneurysm+formation+and+progression+in+a+novel+model+of+thoracic+aortic+aneurysms.">Inhibition of interleukin-1beta decreases aneurysm formation and progression in a novel model of thoracic aortic aneurysms.</a> Circulation. 130 (11), 51-59 (2014).</li><li>Ruddy, J. M., Jones, J. A., Spinale, F. G., Ikonomidis, J. 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A novel model of infrarenal AAA in swine was created using a combination of balloon angioplasty, perfusion and topical elastase, and dietary as BAPN. Using this model, aortic dilation of &#62;100% was achieved with gross and histologic characteristics of chronic human aneurysmal disease. This model provides a gateway to further understand the complex pathophysiology of AAA and translate potential therapies to human disease.
Prior models of AAA in swine have been achieved with modest success. M...

According to the Center for Disease Control (CDC), aortic aneurysms (AA) are a leading cause of death in the United States and represent a significant disease burden1. An aortic aneurysm is defined as a dilation of a discrete portion of the vessel lumen by over 50%2. A subset of AA in the abdomen, referred to as abdominal aortic aneurysms (AAA) are a growing concern. AAA remain clinically silent until impending rupture or dissection, with acute onset, severe abdominal pain generally being the only presenting symptom3,4. Rupture of AAA is almost always fatal with a mortality rate of 90%5. Open or endovascular surgery is the only therapeutic option for patients, and can be a highly morbid procedure. Importantly, AAA are one of the few cardiovascular diseases with no medical therapy for cure.
To date, much of the research on AAA pathogenesis has focused on rodent models, using elastase, which is an enzyme that degrades elastin found within the aortic media, to induce aneurysms.6,7 However, the clinical translatability of small animal models to human aneurysmal disease is restricted, as evaluation of structural changes in the aorta, and altered hemodynamics are limited due to size. Because of anatomical and size similarity, the porcine circulatory system correlates better with human biology than rodents8. Large animal models allow further understanding of cellular mechanisms of the disease process, can be used develop novel treatments at therapeutic doses for large mammals, and test mechanical repair devices, which would not be feasible in small animal models. Additionally, the acute nature of rodent models does not replicate the chronicity and pathologic characteristics of human aneurysmal disease.
The combination of elastase and a compound called &#946;-aminopropionitrile (BAPN) has revolutionized murine AAA models, by creating aneurysms that are larger and contain sequela of chronic aneurysmal disease, including mural thrombus, dissection, and rupture9. BAPN is an inhibitor of lysyl oxidase, which is essential for collagen crosslinking, a crucial component of the aortic wall10,11,12. Lysyl oxidase activity decreases with aging and given the association of age and the chronic nature of complicated AA, BAPN has great potential to experimentally mimic the effects of aging9,13,14. The use of BAPN and its ability to replicate chronic disease in a subacute setting offers a novel advantage over alternative large animal models of AAA. Compared to other established porcine AAA models, this model creates the largest aneurysms with hallmarks of end-stage disease, and the results have been previously published8,11,15.
While conferring certain advantages, significant resources and investment are required to successfully complete this model that may deter some investigators. Among these resources include access to operating rooms, qualified surgeons and anesthesia providers, animal housing, and veterinary staff to assist with post-operative care. Additionally, the cost of BAPN may be prohibitively expensive for some labs.
Few large animal models exist to study the complex pathophysiology of AAA formation and translate to human disease. Large animal models of AAA are critical to help assess the viability of novel technologies and treatments for human disease. Therefore, the purpose of this study was to create a reproducible model of advanced stage infrarenal AAA in swine. The rationale for the use of BAPN and elastase swine model is to better understand the pathophysiology of AAA by mimicking the chronic nature and sequela of human aneurysmal disease in an acute or subacute setting, as well as to test novel therapies and devices for AAA treatment.

<table><tbody><tr><td>Arrow Ergo Pack System</td><td>Arrow</td><td>CDC-21242-X1A</td><td>Just need 7 Fr dilator</td></tr><tr><td>Atlas PTA Balloon dilation catheter</td><td>Bard</td><td>AT-120184</td><td>16 mm x 4 cm x 120 cm</td></tr><tr><td>Bovie electrocautery</td><td>Bovie Medical</td><td>A2350</td><td></td></tr><tr><td>Collagenase Type 1 (5 gm)</td><td>Worthington</td><td>LS004196</td><td></td></tr><tr><td>Crile Needle drviers</td><td>MFI medical</td><td>61-2201</td><td></td></tr><tr><td>DeBakey Atraumatic Forceps</td><td>MFI medical</td><td>52-4977</td><td></td></tr><tr><td>DeBakey Peripheral Vascular Clamp</td><td>Medline</td><td>MDS1318119</td><td></td></tr><tr><td>Glidewire</td><td>Terumo Interventional Systems</td><td>GS3506</td><td>outer Wire diameter 0.035 mm, Length 150 cm</td></tr><tr><td>GraphPad Prism 6</td><td>GraphPad Software Inc. La Jolla, Calif)</td><td></td><td>statistical software</td></tr><tr><td>Metzenbaum Scissors</td><td>MFI medical</td><td>61-0004</td><td></td></tr><tr><td>Mayo-Hegar Needle Holder</td><td>tiger medical</td><td>N407322</td><td></td></tr><tr><td>Micropuncture Introducer Set</td><td>Cook</td><td>G47946</td><td></td></tr><tr><td>Mixter Forceps, Standard Grade, Right angle</td><td>Cole-Parmer</td><td>UX-10818-16</td><td></td></tr><tr><td>Monocryl suture</td><td>Ethicon</td><td>Y496G-BX</td><td>4-0 monocryl</td></tr><tr><td>PDS II suture</td><td>Ethicon</td><td>D8926</td><td>Number 1 looped</td></tr><tr><td>Porcine Pancreatic Elastase</td><td>Sigma-Aldrich</td><td>E1250</td><td></td></tr><tr><td>Satinsky Vascular Clamps</td><td>Medline</td><td>MDs5632515</td><td></td></tr><tr><td>Suction canister</td><td>Cardinal Health</td><td>65651212</td><td></td></tr><tr><td>Schuco Aspirator</td><td>MFI medical</td><td>S430A</td><td></td></tr><tr><td>Vicryl suture</td><td>Ethicon</td><td>J789D-SD</td><td>2-0 vicryl</td></tr><tr><td>Yankauer Suction tube</td><td>Sklarcorp</td><td>07-1801</td><td></td></tr></tbody></table>

porcine model of infrarenal abdominal aortic aneurysm

Large animal models to study abdominal aortic aneurysms are sparse. The purpose of this model is to create reproducible, clinically significant infrarenal abdominal aortic aneurysms (AAA) in swine. To achieve this, we use a combination of balloon angioplasty, elastase and collagenase, and a lysyl oxidase inhibitor, called &#946;-aminopropionitrile (BAPN), to create clinically significant infrarenal aortic aneurysms, analogous to human disease.
Noncastrated male swine are fed BAPN for 7 days prior to surgery to achieve a steady state in the blood. A midline laparotomy is performed and the infrarenal aorta is circumferentially dissected. An initial measurement is recorded prior to aneurysm induction with a combination of balloon angioplasty, elastase (500 units)/collagenase (8000 units) perfusion, and topical elastase application. Swine are fed BAPN daily until terminal procedure on either postoperative day 7, 14, or 28, at which time the aneurysm is measured, and tissue procured. BAPN + surgery pigs are compared to pigs that underwent surgery alone.
Swine treated with BAPN and surgery had a mean aortic dilation of 89.9% &#177; 47.4% at day 7, 105.4% &#177; 58.1% at day 14, and 113.5% &#177; 30.2% at day 28. Pigs treated with surgery alone had significantly smaller aneurysms compared to BAPN + surgery animals at day 28 (p &#60; 0.0003). The BAPN + surgery group had macroscopic and immunohistochemical evidence of end stage aneurysmal disease.
Clinically significant infrarenal AAA can be induced using balloon angioplasty, elastase/collagenase perfusion and topical application, supplemented with oral BAPN. This model creates large, clinically significant AAA with hallmarks of human disease. This has important implications for the elucidation of AAA pathogenesis and testing of novel therapies and devices for the treatment of AAA. Limitations of the model include variation in BAPN ingested by swine, quality of elastase perfusion, and cost of BAPN.&#160;

All statistical analyses were performed using Fisher exact test or chi squared test as appropriate. Data values are reported as mean aortic dilation (%) &#177; standard deviation (%). Statistical significance was set P &#60; 0.05. The combination of BAPN and surgery providing elastase treatment (surgery/elastase) creates more robust and reproducible AAA in swine at day 28 compared to those treated with surgery and elastase alone (mean aortic dilation (%) &#177; standard deviation (%): 113.5% &#177; 30.2% (n = 8) versus 5...

Watch this Scientific Journal Video about Porcine Model of Infrarenal Abdominal Aortic Aneurysm at JoVE.com

Porcine Model of Infrarenal Abdominal Aortic Aneurysm

This novel model creates robust infrarenal abdominal aortic aneurysms in swine using a combination of balloon angioplasty, elastase/collagenase perfusion, topical elastase application, and oral compound &#946;-aminopropionitrile administration, which interferes with collagen cross-linking.

Large animal models to study abdominal aortic aneurysms are sparse. The purpose of this model is to create reproducible, clinically significant infrarenal ...

porcine-model-of-infrarenal-abdominal-aortic-aneurysm

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JoVE Journal

Medicine

9.3K Views.  University of Virginia School of Medicine. Er bestaan weinig grote diermodellen om abdominale aorta-aneurysma's te bestuderen, maar deze modellen zijn van cruciaal belang voor het beoordelen van de levensvatbaarheid van nieuwe technologieën en behandelingen voor menselijke ziekten. De combinatie van elastase en een verbinding genaamd BAPN creëert aneurysma's die groot zijn en bevatten sequelae van chronische aneurysmale ziekte, zoals trombi, dissecties, en breuken. Het aantonen van de procedure met mij zal artsen Jolian Dahl en Erik...

Video: Porcine Model of Infrarenal Abdominal Aortic Aneurysm

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase 

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of abdominal aortic aneurysm (AAA) ever since it was first described in rats by Anidjar and colleagues.1 The rationale for its development was based on the disrupted nature of elastin observed in AAAs. This rat model has been modified to produce AAAs in the infrarenal aortic region of mice.2 The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice. Moreover, it is a viable platform to test potential therapeutic agents for AAA. In this video, we demonstrate the elastase infusion AAA procedure used in our laboratory. 
Mice are anesthetized using 2.5% isoflurane, and a laparotomy is performed under sterile conditions. The abdominal aortais isolated with the assistance of an operating stereomicroscope (Leica). After placing temporary ligatures around the proximal and distal aorta, an aortotomy is created at the bifurcation with the tip of a 30-gauge needle. A heat-tapered segment of PE-10 polyethylene tubing is introduced through the aortotomy and secured. The aortic lumen is subsequently perfused for 5-15 minutes at 100 mm Hg with saline containing type I porcine pancreatic elastase (4.5 U/mL; Sigma Chemical Co.). After removing the perfusion catheter, the aortotomy is repaired without constriction of the lumen.

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

This video shows how to induce abdominal aortic aneurysms (AAA) in mice via transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta. The model has the ability to add broad insight into the pathobiology of AAA due to the emergence of numerous transgenic and gene knockout mice.

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of ...

Biology

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

The Helsinki Rat Microsurgical Sidewall Aneurysm Model

Experimental saccular aneurysm models are necessary for testing novel surgical and endovascular treatment options and devices before they are introduced into clinical practice. Furthermore, experimental models are needed to elucidate the complex aneurysm biology leading to rupture of saccular aneurysms.
Several different kinds of experimental models for saccular aneurysms have been established in different species. Many of them, however, require special skills, expensive equipment, or special environments, which limits their widespread use. A simple, robust, and inexpensive experimental model is needed as a standardized tool that can be used in a standardized manner in various institutions.
The microsurgical rat abdominal aortic sidewall aneurysm model combines the possibility to study both novel endovascular treatment strategies and the molecular basis of aneurysm biology in a standardized and inexpensive manner. Standardized grafts by means of shape, size, and geometry are harvested from a donor rat&#39;s descending thoracic aorta and then transplanted to a syngenic recipient rat. The aneurysms are sutured end-to-side with continuous or interrupted 9-0 nylon sutures to the infrarenal abdominal aorta.
We present step-by-step procedural instructions, information on necessary equipment, and discuss important anatomical and surgical details for successful microsurgical creation of an abdominal aortic sidewall aneurysm in the rat.

Microsurgical sidewall aneurysms in rats are created by end-to-side anastomosis of an aortic graft to the abdominal aorta. We present step-by-step instructions and discuss anatomical and surgical details for successful experimental saccular aneurysm creation.

Experimental saccular aneurysm models are necessary for testing novel surgical and endovascular treatment options and devices before they are introduced ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

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

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

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

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

Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. An aneurysm forms as a result of progressive weakening and eventual dilation of the aorta, which can rupture or tear once it reaches a critical diameter. Aneurysms of the descending aorta in the chest, called descending thoracic aortic aneurysms (dTAA), make up a large proportion of aneurysm cases in the United States. Uncontained dTAA rupture is almost universally lethal, and elective repair has a high rate of morbidity and mortality. The purpose of our model is to study dTAA specifically, to elucidate the pathophysiology of dTAA and to search for molecular targets to halt the growth or reduce the size of dTAA. By having a murine model to study thoracic pathology precisely, targeted therapies can be developed to specifically test dTAA. The method is based on the placement of porcine pancreatic elastase (PPE) directly on the outer murine aortic wall after surgical exposure. This creates a destructive and inflammatory reaction, which weakens the aortic wall and allows for aneurysm formation over weeks to months. Though murine models possess limitations, our dTAA model produces robust aneurysms of predictable size. Furthermore, this model can be used to test genetic and pharmaceutical targets which may arrest dTAA growth or prevent rupture. In human patients, interventions such as these could help avoid aneurysm rupture, and difficult surgical intervention.

We describe a surgical protocol to consistently induce robust descending thoracic aortic aneurysms in mice. The procedure involves left thoracotomy, thoracic aorta exposure, and placement of a sponge soaked in porcine pancreatic elastase on the aortic wall.

According to the Center for Disease Control, aortic aneurysms (AAs) were considered a leading cause of death in all races and both sexes from 1999-2016. ...

Swine Models of Aneurysmal Diseases for Training and Research

Large animal models, specifically swine, are widely used to research cardiovascular diseases and therapies, as well as for training purposes. This paper describes two different aneurysmal swine models that may help researchers to study new therapies for aneurysmal diseases. These aneurysmal models are created by surgically adding a pouch of tissue to carotid arteries in swine. When the model is used for research, the pouch must be autologous; for training purposes, a synthetic pouch suffices.
First, the right external jugular vein (EJV) and right common carotid artery (CCA) must be surgically exposed. The EJV is ligated and a vein pouch fashioned from a short segment. This pouch is then sutured to an elliptical arteriotomy performed in the CCA. Animals must be kept heparinized during model creation, and local vasodilators may be used to decrease vasospasms. Once the suture is completed, correct blood flow should be inspected, checking for bleeding from the suture line and vessel patency. Finally, the surgical incision is closed by layers and an angiography performed to image the aneurysmal model.
A simplification of this aneurysmal carotid model that decreases invasiveness and surgical time is the use of a synthetic, rather than venous, pouch. For this purpose, a pouch is tailored in advance with a segment of a polytetrafluoroethylene (PTFE) prosthesis, one end of which is sutured close using polypropylene vascular suture and sterilized prior to surgery. This "sac" is then attached to an arteriotomy performed in the CCA as described. 
Although these models do not reproduce many of the physiopathological events related to aneurysm formation, they are hemodynamically similar to the situation found in the clinical setting. Therefore, they can be used for research or training purposes, allowing physicians to learn and practice different endovascular techniques in animal models that are close to the human system.

Description of two different aneurysmal swine models for neuroradiology training courses and research studies. This study provides evidence of the feasibility of these aneurysm porcine model creations and the reproducible methods that are close to the clinical setting.

Large animal models, specifically swine, are widely used to research cardiovascular diseases and therapies, as well as for training purposes. This paper ...

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. ...

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

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

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

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

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

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

Porcine Model of Infrarenal Abdominal Aortic Aneurysm

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