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This work was supported by the Cryptic Masons Medical Research Foundation (Brownsburg, IN).

The animal protocols were approved by the Indiana University Institutional Animal Care and Use Committee (protocol number 24027). All studies followed the National Research Council&#8217;s Guide for the Care and Use of Laboratory Animals and were compliant with animal welfare Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Male C57BL/6J wild-type mice were received at 12 weeks of age for use in the experimental protocol. Animals were allowed to acclimate for 7 days prior to the start of the protocol. Induction surgeries are performed 14 days after animals are received. Commercial lab rodent chow and water were supplied ad libitum, and mice were housed in rooms with a 12 h light/dark cycle at 21 &#177; 3 &#176;C with 30&#8722;80% relative humidity and at least 10 changes/h of conditioned fresh air. All animal handling is performed while wearing personal protective equipment, including face masks and gloves.
1. Ultrasound imaging
<ol>
	<li>Prepare the mouse
	<ol>
		<li>Place the mouse into the anesthesia induction chamber.</li>
		<li>Administer inhaled isoflurane at 1.8&#8722;2% and oxygen until the mouse is anesthetized. Ensure that anesthetic gas canisters are connected to the anesthesia apparatus to reduce operator exposure to volatile anesthetics when isoflurane is used.</li>
		<li>Place the mouse in supine position on a stage heated to 37 &#176;C with the mouse&#8217;s nose placed in the anesthesia nose cone.</li>
		<li>Tape the hindlimbs to the stage with labeling tape.</li>
		<li>Apply a hair removal agent using a cotton-tipped applicator to the abdomen and wait 30 seconds.</li>
		<li>Remove the abdominal hair using a 5 cm x 5 cm cotton gauze pad.</li>
		<li>Place warmed ultrasound transmission gel on the mouse&#8217;s abdomen.</li>
	</ol>
	</li>
	<li>Place a 38 MHz ultrasound probe on the abdomen and identify the pelvic girdle.</li>
	<li>Identify the aorta as a tubular midline structure that is incompressible with moderate posteriorly directed pressure applied through the ultrasound probe. 
	NOTE: The inferior vena cava (IVC) will appear as a luminal structure to the mouse&#8217;s right of the aorta and is compressible with moderate posteriorly directed pressure applied through the ultrasound probe.</li>
	<li>Measure the anterior-posterior diameter of the abdominal aorta just cephalad to the point of bifurcation.</li>
	<li>Move the probe proximally along the aorta and identify the left renal vein crossing anterior to the aorta and entering the IVC.</li>
	<li>Measure the anterior-posterior diameter of the abdominal aorta just caudad to the point where the left renal vein crosses anteriorly to the abdominal aorta.</li>
	<li>Move the probe proximally and identify the diaphragm. 
	NOTE: The diaphragm can be identified as a hyperechoic band just cephalad to the liver.</li>
	<li>Measure the anterior-posterior diameter of the abdominal aorta just caudad to the point where it enters the abdomen.</li>
	<li>Place the mouse in its cage or in a recovery basket that is lined with towels and placed on a stage heated to 37 &#176;C.</li>
	<li>Monitor the mouse until it emerges from anesthesia.</li>
</ol>
2. Initial elastin sensitization
<ol>
	<li>Prepare the elastin degradation products (EDP) while wearing personal protective equipment, including gloves, eye protection, and facemasks. 
	NOTE: The murine pancreatic elastase, elastin, pipette tips, and microcentrifuge tubes are received as sterile products. The PBS is autoclaved to ensure sterility. The EDPs are prepared under a biosafety cabinet.
	<ol>
		<li>Obtain a formulation of murine pancreatic elastase in PBS suspension at 0.475 units/mL (4.75 units/mg protein).</li>
		<li>Take 168 &#956;L of the elastase and add it to 5 mg of murine elastin. Add another 200 &#956;L of 1X phosphate-buffered saline (PBS) to this mixture, then pipette to mix.</li>
		<li>Incubate the elastase and elastin mixture at 37 &#176;C for 20 minutes with intermittent gentle agitation.</li>
		<li>Add 632 &#956;L cold 1X PBS to the elastase and elastin mixture.</li>
		<li>Add 1 mL of complete Freund&#8217;s adjuvant.</li>
	</ol>
	</li>
	<li>Place the mouse into the anesthesia induction chamber.</li>
	<li>Administer inhaled isoflurane at 1.8&#8722;2% and oxygen until the mouse is anesthetized.</li>
	<li>Place the mouse in a prone position on a stage heated to 37 &#176;C with the mouse&#8217;s nose placed in the anesthesia nose cone.</li>
	<li>Inject 50 &#956;L of EDP subcutaneously into each flank for a total of 100 &#956;L EDP in each mouse.</li>
	<li>Place the mouse in a recovery basket that is lined with towels and placed on a stage heated to 37 &#176;C.</li>
	<li>Monitor the mouse until it emerges from anesthesia.</li>
</ol>
3. Preparation for surgery
<ol>
	<li>Prepare the surgical tools.
	<ol>
		<li>Assemble a surgical kit with 2 toothed forceps, 1 sharp straight forceps, 1 sharp angled forceps, 1 needle driver, scissors, at least 3 self-retaining magnetic retractors, 1 skin stapler, and 1 polypropylene bottle cap.</li>
		<li>Add 4-0 polyglycolic acid braided suture to the kit in a sterile fashion immediately prior to surgery.</li>
		<li>NOTE: 3-0 or 5-0 suture sizes will also suffice.</li>
		<li>Fill the polypropylene bottle cap with sterile warmed 1X PBS. 
		NOTE: A bottle of sterile 1X PBS is kept in a warmer at 37 &#176;C for refills.</li>
	</ol>
	</li>
	<li>Prepare the elastase.
	<ol>
		<li>Dilute 27 &#956;L of 5.1 mg/mL (&#8805;4.0 units/mg protein) porcine pancreatic elastase into 73 &#956;L of 1X PBS for a final volume of 100 &#956;L for surgery on 10 mice (10 &#956;L/mouse).</li>
		<li>Prepare the surgical suite.</li>
		<li>Autoclave a beaker of cotton-tipped applicators, tapered-tip cotton-tipped applicators, and 5 cm x 5 cm gauze, and place them next to the surgical trays prior to surgery.</li>
		<li>Place a magnetic surgical stage on a heating pad set to 37 &#176;C on a microscope stage. Tape the heating pad into place on the microscope stage, but leave the magnetic component untethered to allow for repositioning during surgery.</li>
		<li>Place an absorbent pad or paper towel on the heating pad. Fold a 5 cm x 5 cm gauze in half and place it under the anesthesia nose cone.</li>
		<li>Place a 30 mL cup filled with hair removal agent, a 30 mL cup filled with 5% povidone-iodine, a tube of eye lubricant, surgical tape, non-sterile 5 cm x 5 cm gauze, non-sterile cotton tipped applicators, sealed 5 cm x 5 cm alcohol swabs, a roll of adhesive food service film, and a spray bottle of 70% ethanol near the surgical area.</li>
		<li>Draw extended release 1.3 mg/mL buprenorphine into a 1 mL syringe at a volume of 75 &#956;L per mouse, for a dose of 3.25 mg/kg in a 30 g mouse.</li>
	</ol>
	</li>
	<li>Prepare the mouse for surgery.
	<ol>
		<li>Measure and record the mouse&#8217;s weight in grams.</li>
		<li>Place the mouse into the anesthesia induction chamber.</li>
		<li>Administer inhaled isoflurane at 1.8&#8722;2% and oxygen until the mouse is anesthetized.</li>
		<li>Place the mouse in supine position on a stage heated to 37 &#176;C with the mouse&#8217;s nose placed in the anesthesia nose cone.</li>
		<li>Inject 75 &#956;L of buprenorphine subcutaneously.</li>
		<li>Tape the hindlimbs to the stage with labeling tape.</li>
		<li>Apply a hair removal agent using a cotton-tipped applicator to the abdomen and wait 30 seconds.</li>
		<li>Remove the abdominal hair using a 5 cm x 5 cm cotton gauze pad.</li>
		<li>Apply eye lubricant to the mouse&#8217;s eyes.</li>
		<li>Apply povidone-iodine to the mouse&#8217;s abdomen using a 5 cm x 5 cm gauze pad and allow it to dry.</li>
		<li>Wipe the abdomen using the 5 cm x 5 cm alcohol swabs. Reapply povidone-iodine and allow it to dry.</li>
		<li>Test the anesthesia by pinching the mouse&#8217;s toe and ensuring no response.</li>
		<li>Place a sheet of adhesive food service film over the surgical stage.</li>
		<li>Spray the surgical stage with 70% ethanol and allow it to dry.</li>
		<li>Wash your hands and don sterile surgical gloves.</li>
	</ol>
	</li>
</ol>
4. Surgery procedure (7 days after initial elastin sensitization)
<ol>
	<li>Expose the abdominal aorta.
	<ol>
		<li>Make a skin incision using scissors or a scalpel along the abdominal midline for ~3 cm, starting 0.5&#8722;1 cm cephalad to the penis and extending to 0.5 cm caudad to the xiphoid.</li>
		<li>Grasp the skin using toothed forceps and gently pull up to identify the linea alba, which will appear as an avascular plane between the rectus muscles.</li>
		<li>Using scissors, incise the linea alba while continuing to pull up on the skin. Take care to only cut the linea alba, avoiding intra-abdominal structures.</li>
		<li>Place magnetic self-retaining retractors, with one retracting the right side of the abdominal wall, one retracting the left side of the abdominal wall, and one retracting the structures in the caudal aspect of the incision, such as the bladder and seminal vesicles.</li>
		<li>Using moistened cotton-tipped applicators, eviscerate the bowel away from yourself and cover it with a moist 5 cm x 5 cm gauze. Alternatively, pack the bowel using a rolled-up moistened 5 cm x 5 cm gauze into the left side of the mouse&#8217;s abdomen. 
		NOTE: Depending on the mouse&#39;s anatomy and the placement of the retractors, the bowel may be eviscerated or packed in the opposite direction for optimal visualization.</li>
		<li>Identify the IVC and abdominal aorta in the midline posteriorly.</li>
		<li>Using cotton-tipped applicators, enter the retroperitoneum by gently opening the tissue plane covering the posterior vascular structures. 
		NOTE: Posteriorly directed pressure on the tissue of interest, followed by pulling the tips of the cotton applicators apart from each other, applies greater degrees of traction. For gentler traction, rotate the tips of the cotton applicators in opposite directions.</li>
		<li>Place the lateral retractors deeper to obtain better traction and visualization of the aorta.</li>
		<li>Continue using cotton-tipped applicators to gently separate the IVC and infrarenal abdominal aorta, avoiding release of the lateral attachments of the aorta at this stage.</li>
		<li>Continue developing the plane between the IVC and aorta until a triangular space becomes visible between the aorta and IVC (Figure 1). 
		NOTE: This step is typically easiest to accomplish just caudad to the left renal vein.</li>
		<li>Using blunt dissection or sharp forceps, release the lateral aortic attachments by pulling the aorta apart from the connective tissue and lumbar musculature on the mouse&#8217;s left.</li>
		<li>Place the angled sharp forceps in the space developed between the aorta and the IVC, and gently open and close the forceps in a repeated fashion to open the posterior aortic attachments.</li>
		<li>Using the same opening and closing technique, develop the posterior plane along the aorta, working from cephalad to caudal.</li>
		<li>Obtain a video micrometry image of the entire aorta and save it for analysis.</li>
	</ol>
	</li>
	<li>Apply perivascular elastase.
	<ol>
		<li>Using a pipette, drop 10 &#956;L of elastase solution along the length of the exposed aorta. 
		NOTE: By only dissecting the tissues medial and lateral to the aorta, a gutter will emerge in which the elastase pools.</li>
		<li>Place moistened 5 cm x 5 cm gauze pads over the open abdomen and allow the elastase solution to incubate for 10 minutes.</li>
		<li>Remove the gauze pad covering and irrigate the abdomen with sterile 1X PBS three times to remove residual elastase solution.</li>
	</ol>
	</li>
	<li>Close the abdomen.
	<ol>
		<li>Return abdominal organs to their original positions. Observe for and remove any twisting or kinking of bowel.</li>
		<li>Using toothed forceps, peel the rectus muscle and fascia away from the skin for a 0.25 cm skin flap.</li>
		<li>Close the abdominal fascia using a 4-0 braided polyglycolic acid (or similar) suture in a simple running fashion, taking care to incorporate the corners of the incision and traveling no more than 2 mm between bites.</li>
		<li>Staple the overlying skin together using 3&#8722;4 staples.</li>
	</ol>
	</li>
</ol>
5. Post-operative care
<ol>
	<li>Remove the mouse from the surgery stage and place it in a recovery basket that is lined with towels and placed on a stage heated to 37 &#176;C.</li>
	<li>Monitor the mouse until it emerges from anesthesia.</li>
	<li>Monitor the mouse daily postoperatively for 4 days, noting general health and the status of the abdominal incision.</li>
	<li>Postoperatively, perform a repeat ultrasound as described above to monitor the growth of the aneurysm.</li>
	<li>Remove the staples one week after surgery or at the time of the week 1 ultrasound.</li>
</ol>
6. Repeat elastin sensitization (7 days after aneurysm induction surgery)
<ol>
	<li>Perform the same steps as described above for the initial elastin sensitization by preparing the EDP solution in the same fashion, with a single substitution of incomplete Freund&#8217;s adjuvant instead of complete Freund&#8217;s adjuvant.</li>
</ol>
7. Tissue harvest procedures
<ol>
	<li>Harvest tissues on postoperative day 21 for various analyses.</li>
	<li>Prepare the mouse in the same fashion as was done for the induction surgery.</li>
	<li>Expose the aorta again in the same fashion as was done during the induction surgery, taking care not to damage any specimens to be collected for histological analyses. 
	NOTE: At this point, the mouse will have developed adhesive scarring, so more use of sharp dissection with forceps rather than blunt dissection may be necessary.</li>
	<li>Obtain video micrometry images.</li>
	<li>Perform a cardiac puncture to collect blood for analysis and euthanize the mouse.</li>
	<li>Perfuse the mouse for analysis while wearing appropriate personal protective equipment, including gloves, eye protection, and facemasks.
	<ol>
		<li>If the tissues are designated for flow cytometry (or similar) analysis, perfuse the left ventricle with 1X PBS without a vascular dilator at 100&#8722;120 mm Hg for a volume of 8 mL.</li>
		<li>If the tissues are designated for PCR analyses, perfuse the left ventricle with 1X PBS with vascular dilator (0.1 mM adenosine and 0.01 mM sodium nitroprusside) followed by RNA stabilization solution.</li>
		<li>If the tissues are designated for histological analyses, perfuse the left ventricle with 1X PBS with a vascular dilator, followed by 10% buffered formalin phosphate.</li>
	</ol>
	</li>
	<li>Harvest the desired tissues, taking care not to structurally damage any tissues intended for histological analysis. Mark orientation with sutures or marking agents.</li>
</ol>

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	<li>Kent KC. Clinical practice. Abdominal aortic aneurysms. N Engl J Med. 2014;371(22):2101-8.</li>
	<li>Macura KJ, Corl FM, Fishman EK, Bluemke DA. Pathogenesis in acute aortic syndromes: aortic aneurysm leak and rupture and traumatic aortic transection. AJR Am J Roentgenol. 2003;181(2):303-7.</li>
	<li>Hoornweg LL, Storm-Versloot MN, Ubbink DT, Koelemay MJ, Legemate DA, Balm R. Meta analysis on mortality of ruptured abdominal aortic aneurysms. Eur J Vasc Endovasc Surg. 2008;35(5):558-70.</li>
	<li>Beckman JA, Isselbacher EM, Preventza O, Hamilton Black Iii J, Augoustides JG, Beck AW, et al. 2022 ACC/AHA Guideline for the Diagnosis and Management of Aortic Disease: A Report of the American Heart Association/American College of Cardiology Joint Committee on Clinical Practice Guidelines. J Am Coll Cardiol. 2022;80(24):e223-e393.</li>
	<li>Claridge R, Arnold S, Morrison N, van Rij AM. Measuring abdominal aortic diameters in routine abdominal computed tomography scans and implications for abdominal aortic aneurysm screening. J Vasc Surg. 2017;65(6):1637-42.</li>
	<li>Senemaud J, Caligiuri G, Etienne H, Delbosc S, Michel JB, Coscas R. Translational Relevance and Recent Advances of Animal Models of Abdominal Aortic Aneurysm. Arterioscler Thromb Vasc Biol. 2017;37(3):401-10.</li>
	<li>Allaire E, Guettier C, Bruneval P, Plissonnier D, Michel JB. Cell-free arterial grafts: morphologic characteristics of aortic isografts, allografts, and xenografts in rats. J Vasc Surg. 1994;19(3):446-56.</li>
	<li>Wang Y, Krishna S, Golledge J. The calcium chloride-induced rodent model of abdominal aortic aneurysm. Atherosclerosis. 2013;226(1):29-39.</li>
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	<li>Nabseth DC, Martin DE, Rowe MI, Gotlieb LS, Deterling RA, Jr. Enzymatic destruction of aortic elastic tissue and possible relationship to experimental atherosclerosis. J Cardiovasc Surg (Torino). 1963;4:11-7.</li>
	<li>Norman PE, Curci JA. Understanding the effects of tobacco smoke on the pathogenesis of aortic aneurysm. Arterioscler Thromb Vasc Biol. 2013;33(7):1473-7.</li>
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</ol>

The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article.

Since it was first described in rats in 1990, the elastase-based AAA model has been modified several times and used in various animals10,19,20,21,22,23. Some of the modifications include aortic cannulation, which is likely to increase mortality and introduce a traumatic component to the aortic pathology17</s...

Abdominal aortic aneurysm (AAA) affects &#62;35 million people worldwide, and 4&#8211;8% of men aged 65-801,2. Rupture, the most devastating complication of AAA, typically occurs when the mechanical wall stress of the aneurysm exceeds the strength of the aortic tissue, leading to massive hemorrhage and an overall mortality rate of 80&#8722;90%3,4. Current management is aimed at prevention, with ultrasound screening of high-risk individuals and surgical repair when the risk of rupture exceeds the risk of repair, typically at 5.5 cm in diameter5. However, most AAAs are asymptomatic, and many are diagnosed incidentally6. As such, efforts should be made to identify high-risk patients and develop non-surgical treatments to reduce AAA growth and, therefore, the risk of rupture.
To develop medical treatments for AAA, the pathogenesis must be better understood. Several experimental models are currently used, including small-animal models utilizing angiotensin II infusion7, xenografting8, calcium chloride application9, and infusion10 and/or topical application of elastase11. Each of these models uses a different understanding of the pathologic basis of AAA, and most have been modified, improved, or combined with other methods since inception.
Importantly, AAA histology demonstrates loss of the normal lamellar elastin matrix, which is also seen in lung tissue in chronic obstructive pulmonary disease12,13. Elastin degradation via matrix metalloproteinase activity provides an important linkage between AAA pathogenesis and smoking, the most important modifiable risk factor for AAA formation12,14. Notably, reduced aortic elastin, elastin fiber disruption, and increased circulating elastin peptides are observed in human AAA and are associated with increased AAA wall distensibility15,16. Therefore, stimulating elastin degradation is crucial in creating a translatable AAA model. Existing AAA models that rely on elastin degradation, while effective, can be complicated by high animal mortality rates, especially in models that use aortic cannulation17, and by inconsistent aneurysm formation, particularly in models that rely solely on topical elastase application18. Furthermore, all models relying on induction surgeries are subject to operator learning curves and technical variation.
To address these issues, we leverage the host immune response to sensitize animals to elastin by injecting elastin degradation products (EDP). One week later, aneurysm induction surgery occurs in a standardized stepwise fashion, using periaortic application of elastase to create EDP moieties for the sensitized host to mount an immune response against. Subsequent &#8220;booster&#8221; EDP sensitization, a week after induction surgery, ensures the host immune response continues. Repetitive immune sensitization enables reliable aortic dilation. We utilize a murine AAA model due to lower cost, ease of maintenance, rapid disease development, and technical consistency.
This model does not reliably result in aortic rupture and is therefore not intended for the study of factors associated with AAA rupture. However, this model does reliably produce progressive aortic dilation to 21 days post-induction surgery. As such, the model is best suited to studying therapeutic modalities that may reduce or reverse aortic dilation as the aneurysm is forming.

<table><tbody><tr><td>1 mL Tuberculin Syringes</td><td>Becton Dickinson</td><td>309628</td><td></td></tr><tr><td>10% Buffered Formalin Phosphate</td><td>Fisher Chemical</td><td>SF100-4</td><td></td></tr><tr><td>70% Isopropyl Alcohol Swabs</td><td>Cardinal Health</td><td>S-16183</td><td></td></tr><tr><td>Angled sharp forceps</td><td>Dumont</td><td>I I25 I-35</td><td></td></tr><tr><td>AutoClip 9mm (Skin stapler)</td><td>MikRon</td><td>427630</td><td></td></tr><tr><td>Betadine</td><td>Atlantis Consumer Healthcare</td><td>67618-155-16</td><td></td></tr><tr><td>Cotton gauze pad</td><td>Dynarex Corporation</td><td>3223</td><td></td></tr><tr><td>Cotton tipped applicator</td><td>Puritan Medical Products</td><td>25-806 2WC</td><td></td></tr><tr><td>Elastase from porcine pancreas (&amp;ge;4.0 units/mg protein)</td><td>Sigma-Aldrich</td><td>E1250</td><td>Stored in aqueous solution at 4 &amp;deg;C</td></tr><tr><td>Elastase from mouse pancreas (4.75 units/mg protein)</td><td>Elastin Products Co., Inc.</td><td>MS838</td><td>Stored in aqueous solution (0.1 mg in 1 mL PBS)at 4 &amp;deg;C</td></tr><tr><td>Elastin</td><td>MP Biomedicals</td><td>101636</td><td>Stored in aqueous solution at 4 &amp;deg;C</td></tr><tr><td>Enersight Desktop Software</td><td>Leica</td><td>https://www.leica-microsystems.com/products/microscope-software/p/enersight/</td><td></td></tr><tr><td>Ethiqa XR (extended release buprenorphine)</td><td>Fidelis</td><td>NDC 86084-100-30</td><td></td></tr><tr><td>Eye lubricant</td><td>OptixCare</td><td>Opx-4242</td><td></td></tr><tr><td>Flexacam i5 (Stereo)</td><td>Leica</td><td>https://www.leica-microsystems.com/products/microscope-cameras/p/12730536-leica/</td><td></td></tr><tr><td>Freund&amp;rsquo;s complete adjuvant</td><td>MP Biomedicals</td><td>642851</td><td>Stored in aqueous solution at 4 &amp;deg;C</td></tr><tr><td>Freund&amp;rsquo;s incomplete adjuvant</td><td>MP Biomedicals</td><td>642861</td><td>Stored in aqueous solution at 4 &amp;deg;C</td></tr><tr><td>Gloves (Sterile)</td><td>Encore</td><td>7824PF</td><td></td></tr><tr><td>Hypodermic Needles (23 G)</td><td>Becton Dickinson</td><td>305145</td><td></td></tr><tr><td>Hypodermic Needles (27 G)</td><td>Becton Dickinson</td><td>305109</td><td></td></tr><tr><td>Isospire (Isoflurane)</td><td>Dechra Veterinary</td><td>07-894-8943</td><td></td></tr><tr><td>Labeling Tape</td><td>Fisher Scientific</td><td>15959</td><td></td></tr><tr><td>Masks</td><td>Dukal Corp.</td><td>1540</td><td></td></tr><tr><td>Needle driver</td><td>Scanlan</td><td>6006-36</td><td></td></tr><tr><td>Omnicon F/air Anesthetic Gas Canister</td><td>A.M. Bickford Inc.</td><td>80120</td><td></td></tr><tr><td>Press&#039;N Seal</td><td>Glad</td><td>12587704417</td><td></td></tr><tr><td>RC2 Rodent Circuit Controller</td><td>VetEquip Incorporated</td><td>922100</td><td></td></tr><tr><td>RNAlater Stabilization solution</td><td>ThermoFisher Scientific</td><td>AM7021</td><td></td></tr><tr><td>Scissors</td><td>Fine Science Tools</td><td>14005-12</td><td></td></tr><tr><td>Skin staples (clips)</td><td>MikRon</td><td>205016</td><td></td></tr><tr><td>Small Rodent Warmer Heater</td><td>Stoelting</td><td>53851</td><td></td></tr><tr><td>Small Rodent Warmer Stage</td><td>Stoelting</td><td>53812</td><td></td></tr><tr><td>Stereo Microscope M60</td><td>Leica</td><td>https://www.leica-microsystems.com/products/light-microscopes/stereo-microscopes/p/leica-m80/</td><td></td></tr><tr><td>Straight sharp forceps</td><td>Dumont</td><td>I I 25 I-30</td><td></td></tr><tr><td>SurgiSuite self-retaining magnetic retractor set and stage</td><td>Kent Scientific</td><td>13-005-180</td><td></td></tr><tr><td>Tahoe Portable Oxygen Concentrator</td><td>Vetland Medical</td><td>595-2300A</td><td></td></tr><tr><td>Toothed forceps</td><td>Fine Science Tools</td><td>11043-08, 11042-08</td><td></td></tr><tr><td>Vevo 2100 Ultrasound</td><td>FUJIFILM Visualsonics</td><td>EDU00496</td><td></td></tr><tr><td>Visorb polyglycolic acid suture</td><td>CP Medical</td><td>397A</td><td></td></tr><tr><td>VWR Absorbents</td><td>VWR</td><td>95057-860</td><td></td></tr></tbody></table>

reliable creation of a murine abdominal aortic aneurysm model using elastin sensitization and perivascular elastase

Reliable Creation of a Murine Abdominal Aortic Aneurysm Model Using Elastin Sensitization and Perivascular Elastase

Degradation of the elastic lamellar matrix in the abdominal aortic wall is a histopathologic feature of abdominal aortic aneurysms (AAA). Growing evidence implicates elastin degradation and related host immune responses in the pathogenesis of AAA. Effective modeling of these processes is important for improving the understanding and treatment of this common disease. While previous AAA models have used elastin-disrupting techniques for aneurysm induction, the single use of elastase in many of these models does not account for the ongoing immune response against elastin and its degradation products (EDP). To model this ongoing immune response, we developed a model that sensitizes C57BL/6 mice to EDP by subcutaneously injecting EDP 7 days prior to aneurysm induction surgery. Aneurysm induction surgery proceeds with exposure of the abdominal aorta and application of topical elastase in the periaortic compartment, with the intention to expose elastin and EDP on the mouse aorta. With these moieties exposed, the animal should mount an immune response against these antigens to which it was previously sensitized. Repeat &#8220;booster&#8221; EDP injection 7 days later serves to propagate this immune response. Herein, we describe the protocol for the reliable creation of AAA in C57BL/6 mice via multiple sensitizations with EDP, followed by AAA induction surgery with perivascular elastase application. This model can be created quickly and at moderate cost, while inducing &#62;30% aortic dilation in 82% of mice by video micrometry and 73% by ultrasound measurement, and &#62;50% dilation in 64% by video micrometry and 27% by ultrasound, as measured at day 21 post-induction surgery. This technique is also associated with a 95% intraoperative survival rate.

A total of 28 male C57BL/6J mice have undergone this surgical protocol, with one perioperative mortality from an IVC injury (3.6%). There were three additional delayed mortalities, one of which was due to an incisional hernia and the other two of which were for unknown reasons, for a total 21-day survival of 85.7%. The average time from induction of anesthesia to the end of anesthetic administration was 46.4 &#177; 8.0 minutes. Sham-surgery mice underwent a nearly identical protocol to the aneurysm group, including initi...

This protocol provides a repeatable and reliable method for inducing abdominal aortic aneurysms (AAAs) in C57BL/6 mice using elastin sensitization and perivascular elastase application, as an AAA model that incorporates an immune-sensitization component.

Degradation of the elastic lamellar matrix in the abdominal aortic wall is a histopathologic feature of abdominal aortic aneurysms (AAA). Growing evidence ...

We developed a model of aortic aneurysm that includes an immune sensitization component to study novel preventative therapies. These models are complicated due to high mortality rates or inconsistent aortic dilation. We use elastin sensitization to limit these complications.To begin, put on personal protective equipment, including gloves, eyewear, and a face mask. Obtain murine pancreatic elastase at 4.75 units per milligram of protein. Add 168 microliters of elastase to five milligrams of murine elastin.Then, add 200 microliters of 1X PBS to the mixture. Incubate the elastase and elastin mixture at 37 degrees Celsius for 20 minutes with intermittent gentle agitation. Next, add 632 microliters of cold PBS to the elastase and elastin mixture, followed by one milliliter of complete Freund's adjuvant.Place the anesthetized 13-week-old male C57BL/6J mouse, prone on a stage preheated to 37 degrees Celsius with the anesthesia nose cone positioned over its snout. Inject 50 microliters of the prepared EDP mixture subcutaneously into each of the mouse flanks. Place the mouse in a recovery basket lined with towels on a stage heated to 37 degrees Celsius.Monitor the mouse until it emerges from anesthesia. Seven days after the initial elastin sensitization, set up the surgical station for the aneurysm induction surgery. Prepare a working elastase solution by mixing 27 microliters of 5.1 milligrams per milliliter porcine pancreatic elastase with 73 microliters of PBS.Position the anesthetized mouse supine on a heated stage at 37 degrees Celsius. Position the mouse's nose in the anesthesia nose cone. Inject extended release buprenorphine subcutaneously at 3.25 milligrams per kilogram body weight.Secure the hind limbs to the stage with a labeling tape. Apply a lubricant to the eyes and confirm the depth of anesthesia by pinching the animal's toe. Then, remove the abdominal hair and prepare the surgical site.Make an approximately three-centimeter midline abdominal incision using a surgical blade. Start 0.5 to one centimeter cephalad to the penis and extend to 0.5 centimeter caudad to the xiphoid. Using toothed forceps, grasp the skin and gently pull up.Identify and incise the linea alba with scissors while continuing to pull the skin up. Avoid injury to the intraabdominal structures. Place magnetic self-retaining retractors to retract the abdominal wall and the caudal structures, such as the bladder and seminal vesicles.Using moistened cotton-tipped applicators, eviscerate the bowel away. Cover the bowel with a moist 5x5 centimeter gauze. Identify the inferior vena cava and abdominal aorta in the posterior midline.Using cotton-tipped applicators, gently open the tissue plane over the posterior vascular structures to enter the retroperitoneum. Then, place the lateral retractors deeper to improve traction and aortic visualization. Use cotton-tipped applicators to separate the inferior vena cava from the infrarenal abdominal aorta.Avoid releasing the lateral aortic attachments at this stage. Develop the plane between the inferior vena cava and the aorta until the triangular space becomes visible. Using blunt dissection or sharp forceps, separate the lateral aortic attachments from the connective tissue and lumbar musculature on the mouse's left.Next, place angled sharp forceps into the triangular space. Gently open and close the forceps repeatedly to release the posterior aortic attachments. Use the same opening and closing motion to develop the posterior plane along the aorta, from cephalad to caudal, then obtain a video micrometry image of the entire aorta and save it for analysis.Using a pipette, drop 10 microliters of the solution containing 13.8 micrograms of elastase along the length of the exposed aorta. Place a moistened 5x5 centimeter gauze pad over the open abdomen and wait for 10 minutes. Then, remove the gauze and irrigate the abdomen with sterile PBS three times to remove the residual elastase solution.Return the abdominal organs to their original positions and correct any bowel twisting or kinking. Using toothed forceps, peel the rectus muscle and fascia away from the skin to create a 0.25-centimeter skin flap. Close the abdominal fascia with a 4-0 braided polyglycolic acid suture in a simple running pattern.Incorporate the corners of the incision and space the bites less than two millimeters apart. Staple the overlying skin together using three to four staples. Place the mouse in a recovery basket lined with towels on a stage heated to 37 degrees Celsius.Monitor the mouse until it emerges from anesthesia. Seven days after the surgery, perform an ultrasound to monitor the aneurysm growth. Then, prepare a fresh EDP mixture using incomplete Freund's adjuvant and repeat the elastin sensitization procedure as demonstrated earlier.On postoperative day 21, collect blood and tissue samples for morphological and molecular analyses. At day 21, the mean aortic diameter ratio relative to baseline was significantly higher in the aneurysm group than in sham surgery controls by both ultrasound and video micrometry. Representative histological images of aortic sections after tissue harvest on day 21 showed cellular infiltration with a thickened intima.Substantial collagen deposition was seen surrounding an aneurysmal aorta. The aneurysm model showed thinned, fragmented, or absent lamellar elastin, whereas the non-aneurysmal section showed thick, well-defined elastin layers. Compared to the normal aorta, the aneurysmal aorta was grossly larger in the entire infrarenal segment with an area of greater dilation near the aortic bifurcation.This protocol allows the study of aortic dilation, inflammatory changes, immune responses, and the effects of preventative therapies in aortic aneurysm. Balancing adequate exposure of the abdominal aorta with survival of the animal is a key judgment step in the protocol. Future studies can evaluate various elastin degradation product preparations, cell therapies, and biologic therapies to limit the progression of aortic aneurysms.

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Research

JoVE Journal

Biology

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.

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase 

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.

A New Murine Model of Endovascular Aortic Aneurysm Repair

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.

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

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.

Murine Surgical Model of Topical Elastase Induced Descending Thoracic Aortic Aneurysm

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.

Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

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.

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

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 ß-aminopropionitrile through drinking water.

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

The present protocol describes a standardized surgical method for the induction of carotid aneurysms in a murine model through a single surgical operation.

A Murine Model of Carotid Aneurysm Formation

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

Murine Model of Hindlimb Ischemia 

Description of the surgical obliteration of a cerebral aneurysm utilizing an ultrasonic flow probe to assess arterial flow prior to and after aneurysm clip placement.

Microsurgical Clip Obliteration of Middle Cerebral Aneurysm Using Intraoperative Flow Assessment

Indiana University School of Medicine.  This protocol provides a repeatable and reliable method for inducing abdominal aortic aneurysms (AAAs) in C57BL/6 mice using elastin sensitization and perivascular elastase application, as an AAA model that incorporates an immune-sensitization component.

Reliable Creation of a Murine Abdominal Aortic Aneurysm Model Using Elastin Sensitization and Perivascular Elastase

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