We thank the Unit for Laboratory Animal Medicine of the University of Michigan for their help with animal feeding and breeding. This study is supported by NIH RO1 HL138139, NIH RO1 HL153710 to&#160;J. Zhang, NIH RO1 HL109946, RO1 HL134569 to Y.E. Chen, and the American Heart Association grant 20POST35110064 to G. Zhao.

The animal protocols were approved by the University of Michigan Institutional Animal Care and Use Committee (PRO00010092). Male and female C57BL/6J wild-type (WT) mice, ~7 weeks of age, were used for the experiments.
1. Animal preparation
<ol>
	<li>Feed the mice with the standard chow diet (see Table of Materials) before and after the surgery. 
	&#8203;NOTE: Different strains and ages of mice can be used. However, age ranging from 5.5-12 week-old is recommended to achieve maximal incidence rate.</li>
	<li>For each mouse, administer 5 mg/kg of Carprofen subcutaneously 30 min before the induction of anesthesia.</li>
	<li>After 30 min, administer 100 mg/kg of Ketamine and 5mg/kg of Xylazine via intraperitoneal injection to induce anesthesia.</li>
</ol>
2. Preparation for surgery
<ol>
	<li>Prepare the surgical material.
	<ol>
		<li>Cut the nitrile gloves into strips of 4 cm x 4 mm. Cut the cotton pads into pieces of 3 cm x 2 mm. Autoclave these with other surgical instruments, including surgical scissors, tissue forceps, and Halsted-Mosquito hemostats (see Table of Materials).</li>
	</ol>
	</li>
	<li>Place the mouse in the supine position on a sterile absorbent dressing pad. Immobilize the front and hind paws with surgical tape.</li>
	<li>Use cotton-tipped applicators (see Table of Materials) to brush hair remover lotion over the middle and lower abdominal area, then swab the area with surgical gauze to remove the hair.</li>
	<li>Disinfect the surgical area&#160;at least three times in a circular motion with alternating applications of 70% alcohol and an iodine-based or chlorhexidine-based scrub. Allow to dry.</li>
</ol>
3. Surgery procedure
<ol>
	<li>Perform the following steps to access the abdominal cavity.
	<ol>
		<li>Test the mouse for the lack of toe-pinch response before skin incision.</li>
		<li>Make a 2.5 cm longitudinal incision on the skin along the midline of the middle and lower abdomen using surgical scissors.</li>
		<li>Gently pull up the underlying muscle and make a 2.5 cm longitudinal incision along linea alba to access the abdominal cavity.</li>
	</ol>
	</li>
	<li>Expose the abdominal aorta.
	<ol>
		<li>Use wetted cotton-tipped applicators to move the intestines and stomach to the right side of the mouse. 
		NOTE: Ideally, this will expose the infrarenal segment of the aorta. If the aorta is hard to locate, the right kidney and right renal artery can identify the aorta (as the right kidney has a slightly lower anatomical location than the left kidney).</li>
		<li>Use forceps to gently remove the connective tissue covering the abdominal aorta and inferior vena cava (IVC). 
		NOTE: The abdominal aorta and IVC are contained within the same vessel sheath. It is unnecessary to remove all the connective tissue as complete removal would increase the risk of damaging these two vessels.</li>
		<li>Use forceps to gently dissect the backside of the abdominal aorta and IVC from the underlying muscles. 
		NOTE: Tips of the forceps should go into the backside of the sheath transversely and create a hole in the fascia that connects the sheath to the underlying muscles. Once the hole is made, extend its size by slowly releasing the forceps.</li>
		<li>Place a piece of the 4 cm x 4 mm glove stripe (as mentioned earlier, step 2.1.1) through the backside of the abdominal aorta and IVC, then straighten the stripe. Place the stripe ~0.5 cm away from the right renal artery. 
		NOTE: Ensure that the hole is big enough so that the surrounding fascia will not twist the stripe.</li>
		<li>Above the stripe, place a piece of 3 cm x 2 mm cotton pad through the backside of the abdominal aorta and IVC, then straighten the cotton pad.</li>
	</ol>
	</li>
	<li>Incubate the elastase.
	<ol>
		<li>Use a pipette to drop 30 &#181;L of porcine pancreatic elastase (total enzymatic activity of 1.8 unit, see Table of Materials) onto the aorta segment above the cotton pad, then wrap the cotton pad and stripe around the aorta and IVC. Rinse a piece of 10 cm x 10 cm gauze with sterile 0.9% saline and place it on the abdomen. 
		&#8203;NOTE: The gauze needs only partial rinsing as overdoing would risk diluting the elastase beneath.</li>
		<li>After 30 min, remove the stripe and cotton pad with forceps.</li>
	</ol>
	</li>
	<li>Close the abdominal cavity following the steps below.
	<ol>
		<li>Irrigate the aorta and abdominal cavity with 500 &#181;L of sterile 0.9% saline. Use a 10 cm x 10 cm gauze to absorb the remaining saline.</li>
		<li>Reapproximate the muscle layers with a running 6-0 non-absorbable monofilament suture.</li>
		<li>Close skin with 3-4 interrupted 6-0 non-absorbable monofilament sutures.</li>
	</ol>
	</li>
</ol>
4. Post-operative care
<ol>
	<li>Administer 5mg/kg of Carprofen subcutaneously on the post-operative Day 1.</li>
	<li>Remove skin sutures on the post-operative Day 10.</li>
</ol>
5. Measurement of abdominal aorta aneurysm diameter
<ol>
	<li>Euthanize the mice by CO2 overdosing on post-operative Day 14. This represents the time point of maximal dilatation.</li>
	<li>Access the abdominal cavity as described in step 3.1.</li>
	<li>Perform vascular perfusion by injecting 10 mL of 0.9% saline into the circulation through the left ventricle.</li>
	<li>Expose the infrarenal segment of the abdominal aorta as described in steps 3.1-3.2. Carefully remove the surrounding connective tissue and separate the abdominal aorta from IVC.</li>
	<li>Measure the diameter of the abdominal aorta with a caliper.</li>
</ol>

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<li>Lysgaard Poulsen, J., Stubbe, J., Lindholt, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+used+to+explore+abdominal+aortic+aneurysms%3A+A+systematic+review%5BTitle%5D)+AND+%22European+Journal+of+Vascular+and+Endovascular+Surgery%22%5BJournal%5D)">Animal models used to explore abdominal aortic aneurysms: A systematic review.</a> European Journal of Vascular and Endovascular Surgery. 52 (4), 487-499 (2016).</li>
<li>Tsui, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+models+of+abdominal+aortic+aneurysms%5BTitle%5D)+AND+%22Open+Cardiovascular+Medicine+Journal%22%5BJournal%5D)">Experimental models of abdominal aortic aneurysms.</a> Open Cardiovascular Medicine Journal. 4, 221-230 (2010).</li>
<li>Manning, M. W., Cassi, L. A., Huang, J., Szilvassy, S. J., Daugherty, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Abdominal+aortic+aneurysms%3A+Fresh+insights+from+a+novel+animal+model+of+the+disease%5BTitle%5D)+AND+%22Vascular+Medicine%22%5BJournal%5D)">Abdominal aortic aneurysms: Fresh insights from a novel animal model of the disease.</a> Vascular Medicine. 7 (1), 45-54 (2002).</li>
<li>Chiou, A. C., Chiu, B., Pearce, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Murine+aortic+aneurysm+produced+by+periarterial+application+of+calcium+chloride%5BTitle%5D)+AND+%22Journal+of+Surgical+Research%22%5BJournal%5D)">Murine aortic aneurysm produced by periarterial application of calcium chloride.</a> Journal of Surgical Research. 99 (2), 371-376 (2001).</li>
<li>Allaire, E., Guettier, C., Bruneval, P., Plissonnier, D., Michel, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell-free+arterial+grafts%3A+Morphologic+characteristics+of+aortic+isografts%2C+allografts%2C+and+xenografts+in+rats%5BTitle%5D)+AND+%22Journal+of+Vascular+Surgery%22%5BJournal%5D)">Cell-free arterial grafts: Morphologic characteristics of aortic isografts, allografts, and xenografts in rats.</a> Journal of Vascular Surgery. 19 (3), 446-456 (1994).</li>
<li>Anidjar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elastase-induced+experimental+aneurysms+in+rats%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Elastase-induced experimental aneurysms in rats.</a> Circulation. 82 (3), 973-981 (1990).</li>
<li>Sun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mast+cells+modulate+the+pathogenesis+of+elastase-induced+abdominal+aortic+aneurysms+in+mice%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice.</a> Journal of Clinical Investigation. 117 (11), 3359-3368 (2007).</li>
<li>Bhamidipati, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+novel+murine+model+of+aortic+aneurysms+using+peri-adventitial+elastase%5BTitle%5D)+AND+%22Surgery%22%5BJournal%5D)">Development of a novel murine model of aortic aneurysms using peri-adventitial elastase.</a> Surgery. 152 (2), 238-246 (2012).</li>
<li>Pyo, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+gene+disruption+of+matrix+metalloproteinase-9+%28gelatinase+B%29+suppresses+development+of+experimental+abdominal+aortic+aneurysms%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms.</a> Journal of Clinical Investigation. 105 (11), 1641-1649 (2000).</li>
<li>Tanaka, A., Hasegawa, T., Chen, Z., Okita, Y., Okada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+rat+model+of+abdominal+aortic+aneurysm+using+a+combination+of+intraluminal+elastase+infusion+and+extraluminal+calcium+chloride+exposure%5BTitle%5D)+AND+%22Journal+of+Vascular+Surgery%22%5BJournal%5D)">A novel rat model of abdominal aortic aneurysm using a combination of intraluminal elastase infusion and extraluminal calcium chloride exposure.</a> Journal of Vascular Surgery. 50 (6), 1423-1432 (2009).</li>
<li>Busch, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Four+surgical+modifications+to+the+classic+elastase+perfusion+aneurysm+model+enable+haemodynamic+alterations+and+extended+elastase+perfusion%5BTitle%5D)+AND+%22European+Journal+of+Vascular+and+Endovascular+Surgery%22%5BJournal%5D)">Four surgical modifications to the classic elastase perfusion aneurysm model enable haemodynamic alterations and extended elastase perfusion.</a> European Journal of Vascular and Endovascular Surgery. 56 (1), 102-109 (2018).</li>
<li>Liang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((KLF11+protects+against+venous+thrombosis+via+suppressing+tissue+factor+expression%5BTitle%5D)+AND+%22Thrombosis+and+Haemostasis%22%5BJournal%5D)">KLF11 protects against venous thrombosis via suppressing tissue factor expression.</a> Thrombosis and Haemostasis. , (2021).</li>
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<li>Shannon, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Porcine+model+of+infrarenal+abdominal+aortic+aneurysm%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Porcine model of infrarenal abdominal aortic aneurysm.</a> Journal of Visualized Experiments. (153), e60169(2019).</li>
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The authors have nothing to disclose.

The elastase-induced AAA model was first reported by Anidjar et al. using rats in 199017. A variety of modified versions have been introduced in the past thirty years, along with significant improvement in the surgical techniques19,20,21,22. Hundreds of institutes use elastase-induced AAA models as the second most used rodent experimental model for AAA studies12. It is only natural that different groups will perform the surgery for this model slightly differently. As a result, one caveat of this model is the inevitable variations of how the surgery is performed, which makes the comparison of data from different groups using this model very difficult. The variations include but are not limited to how infrarenal segment of the aorta is exposed, how extensively the fascial layer covering the aorta is cleaned, how long the incubation period is, as well as the material used for aorta wrapping and elastase incubation and the volume of elastase added onto the wrapping material. These, however, are all essential determinants that affect the eventual incidence rate and diameter of AAA.A standardized surgical procedure is needed to solve the issue of reproducibility caused by these variations. A standardized surgical protocol is presented here for the elastase-induced AAA model through direct application of PPE to the adventitia of the infrarenal abdominal aorta in the mouse.
Aside from the consistent induction rate of AAA, this protocol owns the advantages of low expenses and short operation time. The surgical material used in this protocol can all be easily obtained at low costs. The average operation time of this protocol is 50 min, including 30 min of PPE incubation. These advantages will allow the easier conduction of many animal studies, contributing to more substantial statistical power and reproducibility.
Exposure of the abdominal aorta is the most critical step in this protocol. It is essential to remove the connective tissue in the vessel sheath that surrounds the abdominal aorta and IVC so that elastase will have a better chance of penetrating the medial layer of the aorta. However, completely removing these connective tissues is time-consuming and risks damaging the underlying aorta and IVC. This is the step from which most surgical mortality is caused. If IVC or aorta is damaged, most animals will not survive the following 48 h even if they survive the surgery, possibly due to the continuous bleeding after surgery leading to exsanguination. For this reason, a delicate balance between exposing the aorta to the greatest extent and not damaging the big vessels needs pondering over. Leaving a small amount of connective tissue on the vessel sheath will significantly reduce perioperative mortality while not affecting AAA&#39;s occurrence. Therefore, it is not recommended to completely remove the connective tissue covering the aorta and IVC for exposure to the abdominal aorta. Still, extreme caution needs to be taken during the abdominal aorta and IVC dissection from adjacent connective tissue.
Anatomically, the abdominal aorta and IVC are closely attached via a thin fascia layer and contained together in the same vessel sheath. Some experimental models such as venous thrombosis may require separating the aorta from adjacent IVC. However, it is unnecessary to do so in the elastase-induced AAA model23. Some may prefer to rotate the vessel sheath clockwise so that the aorta will be right above IVC, and elastase can then be applied directly to the aorta. This is unnecessary as the cotton pad beneath is saturated with elastase, and the aorta will be continuously exposed to elastase regardless of its anatomic relationship to IVC. It is recommended that the backside of the aorta and IVC be adequately separated from the underlying muscles via forceps. In case the dissection is not extensive enough in this step, tension force will be created by the surrounding fascia and muscles on the stripe and cotton pad, resulting in their twist and subsequent inefficient absorbance of elastase.
One limitation of this protocol is the eventual shape of the induced AAA. It is inevitable that when being dropped onto the aorta, some elastase will flow to other segments of the aorta that are not wrapped by the cotton pad. In addition, nearby bleeding caused by dissection, even of small amount, can wash elastase away from the cotton pad to other aorta segments during the 30 min incubation period. These will lead to elastase penetrating other segments of the aorta, thus causing the breakdown of the medial layer and dilation of the abdominal aorta not limited to the segment wrapped by the cotton pad. As a result, instead of exhibiting fusiform-shaped aneurysms caused by focal dilation, some animals demonstrate general dilation of a long segment of the abdominal aorta. However, the maximal aorta diameter in these animals that have continuous dilation of the aorta still shows a more than 50% increase when compared with controls. Meanwhile, no difference in maximal aorta diameter is observed between animals exhibiting continuous dilation and focal dilation of the abdominal aorta, suggesting that the aneurysm&#39;s shape is an indicator of neither successful AAA induction nor actual AAA size in this case protocol.
Another limitation of this protocol is applying this method to larger animals. The topical elastase-induced AAA model should be easily applied to other small animals such as rats. However, elastase alone may not be enough for larger animals like pigs or primates to induce a consistent AAA phenotype. For example, Marinov et al. reported that elastase perfusion alone in swine could cause some histologic changes, including elastin disruption, but could not induce consistent aneurysm24. Shannon A et al. reported a porcine model of infrarenal AAA induced by elastase. However, it requires a combination of balloon angioplasty, perfusion, topical elastase, and a diet containing BAPN (a lysyl oxidase inhibitor preventing collagen crosslinking) given the considerable durability of aorta25. Therefore, it seems the application of a standardized elastase-induced AAA protocol in larger animals is still quite challenging due to the difference in size and durability of the aorta between mice and larger animals, the technical challenges, and the potentially high cost.
In summary, despite some limitations, AAA can be induced consistently in both male and female mice in this standardized protocol. The consistency and reproducibility of AAA studies using the elastase-induced AAA model are expected to be significantly enhanced by establishing this standard surgical procedure.

Abdominal aortic aneurysm (AAA) is defined as a segmental dilatation of the abdominal aorta with at least a 50% increase of vessel diameter1. AAA is potentially fatal, as the rupture can result in an extremely high mortality rate, even with intervention2,3,4. It has been reported that AAA is responsible for approximately 13,000 deaths annually in the USA, which makes it the 10th leading cause of death1,5.
The pathogenesis of AAA is not yet wholly understood6,7,8. To investigate the molecular mechanism of AAA and test potential therapeutic targets, several experimental AAA models have been established9,10. Rodent models of AAA include elastase, calcium chloride, angiotensin II and xenograft models, among which the elastase-induced AAA model is the second most used model10,11,12,13,14,15,16,17. This model involves direct infusion or application of porcine pancreatic elastase (PPE) to the infrarenal segment of the aorta. The penetration of elastase into the medial layer of the aorta will cause degradation of elastic lamellae and infiltration of inflammatory cells, leading to the loss of aortic wall integrity and subsequent dilation of abdominal aorta7,18. The elastase-induced AAA model was first reported by Anidjar et al. in 1990 using rats, in which an isolated segment of the aorta was perfused with elastase17. Later in 2012, a modified model using a periadventitial application of PPE was reported by Bhamidipati et al.19. Nowadays, most surgeries for the elastase-induced AAA model are inspired by Bhamidipati&#39;s group and are performed with the external application rather than intraluminal perfusion of PPE. Although the external application has less requirement on fine surgical skills, the incidence rate of AAA is relatively lower and size somewhat smaller than that of intraluminal perfusion11,19.
Although widely used in AAA studies, the elastase-induced AAA model possesses certain limitations. One caveat of this model is the inevitable variations of how the surgery is performed, which can lead to the issue of reproducibility. For example, the difference may exist in the surgical procedure regarding how the infrarenal segment of the aorta is isolated and which part of the segment is selected for PPE application among different laboratories. The enzymatic activity of PPE and the time duration of PPE incubation can also vary. These, however, are all essential determinants that affect the eventual AAA formation rate and aneurysm diameter.The variation of these critical determinants makes data comparison of AAA studies from different groups using this model very difficult. Therefore, a standardized surgical procedure is needed as a tool to get comparable results from various institutions.
This article describes a standardized surgical protocol for the elastase-induced AAA model through direct application of PPE to the adventitia of infrarenal abdominal aorta in mice. Details about surgical material and procedures essential for successful and robust generation of AAA in mice using this model will also be discussed.

<table><tbody><tr><td>6-0 non-absorbable monofilament suture</td><td>Pro Advantage</td><td>P420697</td><td></td></tr><tr><td>Carprofen</td><td>Zoetis Inc.</td><td>NDC: 54771-8507</td><td></td></tr><tr><td>Chow Diet</td><td>LabDiet</td><td>3005659-220</td><td>PicoLab 5L0D</td></tr><tr><td>Cotton Applicator</td><td>Dynarex</td><td>4303</td><td></td></tr><tr><td>Cotton Pad</td><td>Rael</td><td>UPC: 810027130969</td><td></td></tr><tr><td>GraphPad Prism 8</td><td>GraphPad Software Inc.</td><td></td><td>Version 8.4.3</td></tr><tr><td>Grarfe Forceps</td><td>Fine Science Tools</td><td>11051-10</td><td></td></tr><tr><td>Halsted Mosquito Hemostats</td><td>Fine Science Tools</td><td>13009-12</td><td></td></tr><tr><td>Ketamine</td><td>Par Pharmaceutical</td><td>NDC: 42023-0115-10</td><td></td></tr><tr><td>Nitrile gloves</td><td>Fisherbrand</td><td>19-130-1597</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher</td><td>15140122</td><td></td></tr><tr><td>Porcine pancreatic elastase</td><td>Sigma-Aldrich</td><td>E1250-100MG</td><td></td></tr><tr><td>Scissors</td><td>Fine Science Tools</td><td>14068-12</td><td></td></tr><tr><td>Sterile 0.9% saline solution</td><td>Baxter</td><td>2B1324X</td><td></td></tr><tr><td>Xylazine</td><td>Akorn</td><td>NDC: 59399-110-20</td><td></td></tr></tbody></table>

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.

A total of twenty-three 7-week-old wild-type (WT) mice, including 12 females and 11 males, were operated following the presented protocol. The survival rate was 100% (surgical mortality excluded). Maximal abdominal aorta diameter was measured by a caliper.
AAA was defined as dilating the abdominal aorta with a 50% vessel diameter increase. Therefore, a 50% increase in the maximal abdominal aorta diameter was selected as the cut-off point for successful AAA induction. Based upon this criterion, the incidence rate of AAA on post-operative Day 14 was 91.7% for female animals, with 11 out of 12 that underwent surgery developed AAA; while the incidence rate for male animals was 72.7%, with 8 out of 11 that underwent surgery developed AAA (Figure 1A). Among the 19 animals in which AAA occurs, there is a 1.7-fold and 1.6-fold increase of maximal abdominal aorta diameter in females and males compared to non-surgery controls, respectively (Figure 1B). Representative images of the aortas from non-surgery and surgery groups on post-operative Day 14 are shown in Figure 1C.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63608/63608fig01.jpg" /> 
Figure 1: Characterization of AAA in mice undergone surgery. All measurement is done on post-operative Day 14. (A) The incidence rate of AAA in mice that underwent surgery. (B) Maximal diameter of the infrarenal aorta in mice from both non-surgery (n = 4 for both female and male) and surgery groups (n = 12 for female; n = 11 for male). Data are mean &#177; SEM. *p &#60; 0.05. (C) Representative images of aortas in mice from both non-surgery and surgery groups. Scale bar = 2 mm. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63608/63608fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

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. ...

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Universidad San Sebastian

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JoVE Journal

Biology

5.2K Views.  University of Michigan, Ann Arbor. Dit is een gestandaardiseerd protocol voor elastase geïnduceerd abdominaal aorta-aneurysma, wat de reproduceerbaarheid en de consistentie van studies met behulp van dit model aanzienlijk zou verbeteren. Deze techniek heeft een consistente inductiesnelheid van abdominaal aorta-aneurysma. Het bezit ook het voordeel van lage kosten en korte bedrijfstijd, wat zal resulteren in een sterkere statistische kracht en de reproduceerbaarheid.U moet ervoor zorgen dat de bindweefsels die de aorta ...

Video: Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

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 ...

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 ...

Medicine

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

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In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

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The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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. ...

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In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

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Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

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Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

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In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

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

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

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Mouse Abdominal Aortic Aneurysm Model Induced by Perivascular Application of Elastase

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