This work was supported by the National Natural Science Foundations of China (No. 82300542, 81770471), Sichuan Science and Technology Program (No. 2022YFS0359, 2019JDRC0104) and Post-Doctor Research Project, West China Hospital, Sichuan University (No. 2023HXBH108). The funding bodies played no role in the design of the study, the collection, analysis, and interpretation of the data, and the writing of the manuscript.

The animal experiment protocol complied with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Pub No. 86/23, 1985) and was approved by the Institutional Animal Care and Use Committee of West China Hospital, Sichuan University (Approval Number 202311300012). Eight to ten-week-old C57/B6J male mice were used for this study. The details of the reagents and equipment used are listed in the Table of Materials.
1. Preoperative preparation<...

Investigating the Molecular Mechanisms Underlying Murine Abdominal Aortic Aneurysm

<ol>
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Subcommittee on reporting standards for arterial aneurysms, ad hoc committee on reporting standards, society for vascular surgery and north American chapter, international society for cardiovascular surgery.</a> J Vasc Surg. 13 (3), 452-458 (1991).</li><li>Bown, M. J., Sutton, A. J., Bell, P. R. F., Sayers, R. 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The task force for the diagnosis and treatment of aortic diseases of the European Society of Cardiology (ESC).</a> Eur Heart J. 35 (41), 2873-2926 (2014).</li><li>Wanhainen, A., 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=Editor's+choice+-+European+Society+for+Vascular+Surgery+(ESVS)+2019+clinical+practice+guidelines+on+the+management+of+abdominal+aorto-iliac+artery+aneurysms.">Editor's choice - European Society for Vascular Surgery (ESVS) 2019 clinical practice guidelines on the management of abdominal aorto-iliac artery aneurysms.</a> Eur J Vasc Endovasc Surg. 57 (1), 8-93 (2019).</li><li>Veith, F. J., Ohki, T., Lipsitz, E. C., Suggs, W. D., Cynamon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+ruptured+abdominal+aneurysms+with+stent+grafts:+A+new+gold+standard.">Treatment of ruptured abdominal aneurysms with stent grafts: A new gold standard.</a> Semin Vasc Surg. 16 (2), 171-175 (2003).</li><li>Golledge, J., Thanigaimani, S., Powell, J. T., Tsao, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+management+of+abdominal+aortic+aneurysm.">Pathogenesis and management of abdominal aortic aneurysm.</a> Eur Heart J. 44 (29), 2682-2697 (2023).</li><li>Wanhainen, A., 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=Editor's+choice+--+European+Society+for+Vascular+Surgery+(ESVS)+2024+clinical+practice+guidelines+on+the+management+of+abdominal+aorto-iliac+artery+aneurysms.">Editor's choice -- European Society for Vascular Surgery (ESVS) 2024 clinical practice guidelines on the management of abdominal aorto-iliac artery aneurysms.</a> Eur J Vasc Endovasc Surg. 67 (2), 192-331 (2024).</li><li>Golledge, 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=Lack+of+an+effective+drug+therapy+for+abdominal+aortic+aneurysm.">Lack of an effective drug therapy for abdominal aortic aneurysm.</a> J Intern Med. 288 (1), 6-22 (2020).</li><li>Li, R., Liu, Y., Jiang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+advances+in+drug+therapy+for+abdominal+aortic+aneurysms+over+the+past+five+years:+An+updated+narrative+review.">Research advances in drug therapy for abdominal aortic aneurysms over the past five years: An updated narrative review.</a> Int J Cardiol. 372, 93-100 (2023).</li><li>Lysgaard Poulsen, J., Stubbe, J., Lindholt, J. 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M., Wang, Y. <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+for+abdominal+aortic+aneurysm.">Mouse models for abdominal aortic aneurysm.</a> Br J Pharmacol. 179 (5), 792-810 (2022).</li><li>Deng, G. 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=Urokinase-type+plasminogen+activator+plays+a+critical+role+in+angiotensin+II-induced+abdominal+aortic+aneurysm.">Urokinase-type plasminogen activator plays a critical role in angiotensin II-induced abdominal aortic aneurysm.</a> Circ Res. 92 (5), 510-517 (2003).</li><li>Gertz, S. D., Kurgan, A., Eisenberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneurysm+of+the+rabbit+common+carotid+artery+induced+by+periarterial+application+of+calcium+chloride+in+vivo.">Aneurysm of the rabbit common carotid artery induced by periarterial application of calcium chloride in vivo.</a> J Clin Invest. 81 (3), 649-656 (1988).</li><li>Chiou, A. C., Chiu, B., Pearce, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+aortic+aneurysm+produced+by+periarterial+application+of+calcium+chloride.">Murine aortic aneurysm produced by periarterial application of calcium chloride.</a> J Surg Res. 99 (2), 371-376 (2001).</li><li>Zhang, S., Cai, Z., Zhang, X., Ma, T., Kong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+calcium+phosphate-induced+mouse+abdominal+aortic+aneurysm+model.">A calcium phosphate-induced mouse abdominal aortic aneurysm model.</a> J Vis Exp. (189), e64173 (2022).</li><li>Isenburg, J. C., Simionescu, D. T., Starcher, B. C., Vyavahare, N. 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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=Development+of+a+novel+murine+model+of+aortic+aneurysms+using+peri-adventitial+elastase.">Development of a novel murine model of aortic aneurysms using peri-adventitial elastase.</a> Surgery. 152 (2), 238-246 (2012).</li><li>Xue, C., Zhao, G., Zhao, Y., Chen, Y. E., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+abdominal+aortic+aneurysm+model+induced+by+perivascular+application+of+elastase.">Mouse abdominal aortic aneurysm model induced by perivascular application of elastase.</a> J Vis Exp. 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X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+new+abdominal+aortic+aneurysm+model+in+rats+by+a+retroperitoneal+approach.">Establishment of a new abdominal aortic aneurysm model in rats by a retroperitoneal approach.</a> Front Cardiovasc Med. 9, 808732 (2022).</li><li>Bi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+novel+rabbit+model+of+abdominal+aortic+aneurysm+via+a+combination+of+periaortic+calcium+chloride+and+elastase+incubation.">Development of a novel rabbit model of abdominal aortic aneurysm via a combination of periaortic calcium chloride and elastase incubation.</a> PLoS One. 8 (7), e68476 (2013).</li><li>Gage, G. J., Kipke, D. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+experimental+aortic+aneurysms+in+dogs.">Persistent experimental aortic aneurysms in dogs.</a> Surgery. 47, 21-28 (1960).</li><li>Freestone, T., Turner, R. J., Higman, D. J., Lever, M. J., Powell, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+hypercholesterolemia+and+adventitial+inflammation+on+the+development+of+aortic+aneurysm+in+rabbits.">Influence of hypercholesterolemia and adventitial inflammation on the development of aortic aneurysm in rabbits.</a> Arterioscler Thromb Vasc Biol. 17 (1), 10-17 (1997).</li><li>Carsten, C. 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=Elastase+is+not+sufficient+to+induce+experimental+abdominal+aortic+aneurysms.">Elastase is not sufficient to induce experimental abdominal aortic aneurysms.</a> J Vasc Surg. 33 (6), 1255-1262 (2001).</li><li>Yue, J., Yin, L., Shen, J., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+murine+abdominal+aortic+aneurysm+rupture+model+using+elastase+perfusion+and+angiotensin+II+infusion.">A modified murine abdominal aortic aneurysm rupture model using elastase perfusion and angiotensin II infusion.</a> Ann Vasc Surg. 67, 474-481 (2020).</li><li>Ailawadi, 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=Gender+differences+in+experimental+aortic+aneurysm+formation.">Gender differences in experimental aortic aneurysm formation.</a> Arterioscler Thromb Vasc Biol. 24 (11), 2116-2122 (2004).</li><li>Furubayashi, K., 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+significance+of+chymase+in+the+progression+of+abdominal+aortic+aneurysms+in+dogs.">The significance of chymase in the progression of abdominal aortic aneurysms in dogs.</a> Hypertens Res. 30 (4), 349-357 (2007).</li><li>Bi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+a+modified+rabbit+model+of+abdominal+aortic+aneurysm+induced+by+topical+application+of+porcine+elastase:+5-month+follow-up+study.">Performance of a modified rabbit model of abdominal aortic aneurysm induced by topical application of porcine elastase: 5-month follow-up study.</a> Eur J Vasc Endovasc Surg. 45 (2), 145-152 (2013).</li><li>Wu, X. 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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Research into the molecular mechanisms of AAA requires a stable animal model. Consequently, numerous AAA animal models have been established since its initial development by Economou et al. in the 1960s29. Among these models, CaCl2 is frequently employed in rodents due to its cost-effectiveness, technical simplicity, and reliable reproducibility. However, perivascular CaCl2 infiltration has been shown to be unstable in establishing AAA. Bi et al. and Freestone et al. reported...

Abdominal aortic aneurysm (AAA) is defined as a diameter increase of more than 50% or a maximum aortic diameter exceeding 3 cm in the abdominal aorta. This condition poses a significant threat to life, with around a 90% mortality rate upon aneurysm rupture1,2,3. Currently, open surgical repair and endovascular aortic repair (EVAR) are the only available interventions for AAA patients4,5,6. However, there is insufficient evidence to support the effectiveness of medical treatments in inhibiting aneurysm formation or slowing the growth rate of the abdominal aorta in patients who do not have surgical indications7. Nevertheless, non-specific treatments, including persistent surveillance of maximum aneurysm diameter, blood pressure control, antiplatelet therapy, and statins, are used to reduce the risk of sudden aneurysm rupture and potential cardiovascular and neurological events as much as possible. Despite this, the role of antiplatelet medications and statins in preventing aneurysm rupture remains controversial and requires further studies5,7,8,9,10.
A stable AAA animal model is vital for investigating the pathogenesis of AAA, and numerous methods have been introduced to establish such a model11,12,13. Currently, calcium chloride (CaCl2), elastase, angiotensin II (Ang II), xenografts, and transgenic models are used in establishing rodent AAA models11,12,13. Among these, Ang II is the most commonly used in mice, while CaCl2 and elastase are also widely used in mice and rats11,12,13,14. The Ang II-induced AAA model is the sole animal model capable of inducing atherosclerosis that closely resembles human AAA pathology, which is simple and reproducible and obviates the need for laparotomy11,12,13. However, in contrast to the models induced by CaCl2 or elastase, the site of aneurysms induced by Ang II is uncertain and frequently observed in the descending aorta or suprarenal abdominal aorta, with a heightened risk of rupture11,12,13,14. The aneurysm incidence rate of the Ang II-induced AAA model in gene-deficient mice can be as high as 100%, while only 39% of C57BL/6 mice developed aneurysms, and the time and economic cost of obtaining gene-deficient mice are high13,15.
Initially introduced by Gertz et al., CaCl2 was utilized to induce aneurysm formation in the carotid artery and later modified by Chiou et al. to establish an AAA model in mice16,17. However, despite its ability to induce elastic fiber breakdown, vessel inflammation, and extracellular matrix degradation, CaCl2 lacks several human AAA features, including atherothrombosis, intraluminal thrombus (ILT), and aneurysm rupture12,18. In addition, the formation rate and diameter increase percentage of periaortic CaCl2 application remain unstable13,19. The initial intraluminal porcine pancreatic elastase (PPE) perfusion model for inducing AAA was developed by Anidjar et al. in 1990, followed by Bhamidipati et al.&#39;s report on periadventitial PPE infiltration in a murine model20,21. Nowadays, the majority of elastase-induced AAA animal models utilize Bhamidipati&#39;s protocol due to its feasibility and minimally invasive nature. However, it is important to note that the incidence rate and maximum diameter of AAA can vary and are generally lower than intra-aortic PPE perfusion. Moreover, aneurysms induced by periaortic application of elastase present a tendency to heal spontaneously11,12,20,21,22,23.
To address these limitations, Tanaka et al. proposed a combination approach involving intraluminal PPE perfusion and CaCl2 infiltration to establish an AAA model in rats, which yielded satisfactory results24. In addition, Zhu et al. demonstrated that the PPE + CaCl2 model offers advantages such as higher survival rates and increased aneurysm formation rates compared to both the single PPE group and the PPE + BAPN group25. This combined approach also exhibits good stability and reproducibility. Moreover, Bi et al. successfully established a rabbit AAA model via the combination of periaortic CaCl2 and elastase incubation. The average dilation ratio was 65.3% &#177; 8.9% on day 5, which further increased to 86.5% &#177; 28.7% on day 15 and significantly escalated to 203.6% &#177; 39.1% on day 3026. However, there is currently no existing literature reporting the induction of AAA in rodents by combining periaortic CaCl2 and elastase application.
This manuscript presents a standard protocol for establishing a murine AAA model through the combined use of periadventitial CaCl2 and elastase infiltration. The subsequent sections provide detailed surgical procedures and present representative results of the murine AAA model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthesia Machine</td>
			<td>RWD</td>
			<td>R550</td>
			<td></td>
		</tr>
		<tr>
			<td>Butorphanol</td>
			<td>Jiangsu Hengrui Pharmaceutical Co., Ltd</td>
			<td>220608BP</td>
			<td>1ml: 1mg</td>
		</tr>
		<tr>
			<td>C57BL/6JGpt Male Mice</td>
			<td>GemPharmatech</td>
			<td>N000013</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>C4901</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Carprofen</td>
			<td>MCE</td>
			<td>HY-B1227</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Chow Diet</td>
			<td>Dossy Experimental Animals Co.Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Caliper</td>
			<td>Greener</td>
			<td>IP54</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Jinhe Pharmaceutical Co.Ltd</td>
			<td>539682</td>
			<td>75%/500 mL</td>
		</tr>
		<tr>
			<td>EVG Staining Kit</td>
			<td>Solarbio</td>
			<td>G1597</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>Graphpad</td>
			<td></td>
			<td>Ver 9.0.0</td>
		</tr>
		<tr>
			<td>H&#38;E Staining Kit</td>
			<td>Servicebio</td>
			<td>G1076</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringe</td>
			<td>BD</td>
			<td>2143420</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD</td>
			<td>R510-22-4</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Masson Staining Kit</td>
			<td>Servicebio</td>
			<td>G1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>Servicebio</td>
			<td>G4702</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Biosharp</td>
			<td>BL539A</td>
			<td>4%/500 mL</td>
		</tr>
		<tr>
			<td>PBS, 1x</td>
			<td>Servicebio</td>
			<td>G4202</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Porcine Pancreatic Elastase</td>
			<td>Sigma Aldrich</td>
			<td>E1250</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Povidine Iodine</td>
			<td>Yongan Pharmaceutical Co.Ltd</td>
			<td></td>
			<td>5%/100 mg</td>
		</tr>
		<tr>
			<td>Prolene Polypropylene Suture</td>
			<td>Ethicon LLC</td>
			<td>8709H</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent Ultrasound System</td>
			<td>Fujifilm</td>
			<td>Vevo 3100LT</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Couplant</td>
			<td>Keppler</td>
			<td>KL-250</td>
			<td>250 g</td>
		</tr>
	</tbody>
</table>

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

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

In this study, a total of 24 mice were included and randomly assigned: 12 in the Sham group and 12 in the PPE + CaCl2 group, respectively. All data are presented as means &#177; standard deviations unless otherwise stated. The average operation time was 55.67 min &#177; 4.08 min. There were no intra-operative deaths or aneurysm ruptures, and the survival rate within 21 days after surgery was 100%. No severe intestinal adhesions or complications related to abdominal aorta dissection were observed.
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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 ...

a-mouse-abdominal-aortic-aneurysm-model-periadventitial-calcium

Research

JoVE Journal

Medicine

321 Görüntüleme.  Sichuan University. 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.