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

Summary

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.

Abstract

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.

Introduction

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 rupture^1,2,3. Currently, open surgical repair and endovascular aortic repair (EVAR) are the only available interventions for AAA patients^4,5,6. 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 studies^5,7,8,9,10.

A stable AAA animal model is vital for investigating the pathogenesis of AAA, and numerous methods have been introduced to establish such a model^11,12,13. Currently, calcium chloride (CaCl₂), elastase, angiotensin II (Ang II), xenografts, and transgenic models are used in establishing rodent AAA models^11,12,13. Among these, Ang II is the most commonly used in mice, while CaCl₂ and elastase are also widely used in mice and rats^11,12,13,14. 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 laparotomy^11,12,13. 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 rupture^11,12,13,14. 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 high^13,15.

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 mice^16,17. 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 rupture^12,18. In addition, the formation rate and diameter increase percentage of periaortic CaCl₂ application remain unstable^13,19. 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.'s report on periadventitial PPE infiltration in a murine model^20,21. Nowadays, the majority of elastase-induced AAA animal models utilize Bhamidipati'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 spontaneously^{11,12,20,21,22,23}.

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% ± 8.9% on day 5, which further increased to 86.5% ± 28.7% on day 15 and significantly escalated to 203.6% ± 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.

Protocol

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

1. Obtain the animals and feed them a normal chow diet with clean water under standard conditions (temperature 24 °C, humidity 40%-50%, 12-h light-dark cycle).
2. Cut a cotton pad into 40 mm x 4 mm strips and sterilize it along with scissors, forceps, and other surgical equipment.
3. Place the animal into the induction chamber of the anesthesia machine and use 3%-3.5% isoflurane to quickly induce anesthesia (following institutionally approved protocols). Additionally, prescribe butorphanol (1 mg/kg, intramuscular injection) as pre-emptive analgesia medication.
   1. Remove the mouse from the chamber when it no longer responds to pain stimulation, then place a mask on the mouse and maintain the depth of anesthesia with 1%-1.5% isoflurane. Monitor respiratory rate and depth every 5-10 min.
4. Place the mouse in a supine position on the heating pad, use adhesive tape to immobilize the limbs, and apply eye ointment to prevent dryness.
5. Use an electric shaver or hair removal lotion to completely remove the hair in the abdominal area, then disinfect this area with povidone-iodine and ethanol-soaked cotton swabs in a circular motion at least three times.

2. Surgical procedure

1. Place the mouse under a stereomicroscope. Use scissors to make a longitudinal incision of approximately 2-2.5 cm along the midline of the abdomen.
2. Enter the peritoneal cavity through the linea alba (transabdominal approach), place a retractor to expose the peritoneal cavity, and pack the intestines and colon into the right half of the abdomen using normal saline-soaked gauze.
3. Perform blunt separation of connective and adipose tissue adjacent to the infrarenal abdominal aorta and inferior vena cava (IVC) using forceps and cotton swabs. Gently dissect the abdominal aorta and expose the gap between the abdominal aorta and the overlying psoas major, as previously referenced²⁵.
   1. Insert the sterilized cotton pad strips into that gap, being extremely careful not to damage the lumbar artery or vein. It is not necessary to isolate the abdominal aorta from the IVC.
4. Soak the cotton ball with 0.9 M CaCl₂ and place it on the adventitia of the abdominal aorta for 15 min. Use sterilized gauze to cover the abdominal cavity during the process of infiltration.
5. Gently remove the CaCl₂-soaked cotton ball and wash the infiltrated segment with 0.9% saline solution.
6. Dilute the elastase to 2 mg/mL (8 U/mL) with ddH₂O. Use an insulin syringe to apply 50 µL of diluted elastase to the infrarenal segment of the abdominal aorta and wrap the cotton pad strips to cover the abdominal aorta and IVC. Use sterilized gauze to cover the abdominal cavity during the process of infiltration. In the Sham group, use 0.9% normal saline to replace CaCl₂ and PPE.
7. 15 min later, carefully remove the gauze and cotton pad strips. Move the intestines and colon back to their original positions, cover the treated aorta with the mesentery, and moisten the intestines to prevent adhesion.
8. Close the muscle layer and skin separately using 6-0 nonabsorbable polypropylene suture. Disinfect the surgical area with povidone-iodine and ethanol.
9. Place the mouse on a heating pad and monitor its respiratory status until it regains consciousness. Record the operation time and the number of experimental animals. Prescribe carprofen (5 mg/kg, subcutaneously) as post-operative analgesia for at least 3 days after the surgery, according to veterinary recommendation.

3. Ultrasound examination

1. 21 days after the surgery, conduct an ultrasound examination to evaluate the incidence rate of AAA.
2. Anesthetize the mouse (following institutionally approved protocols) and remove the abdominal hair as previously described.
3. Place the mouse on the heating pad of the rodent ultrasound machine. Apply ultrasonic couplant to the abdomen and measure the maximum diameter of the aneurysm and the diameter of the normal segment of the abdominal aorta.
   1. Calculate the increased percentage of the infrarenal abdominal aorta. The criterion for successful AAA formation is that the maximum diameter of the AAA has increased by 50% or more compared to the diameter of the normal segment of the infrarenal abdominal aorta.
4. Use a napkin to wipe off the remaining ultrasonic couplant after the examination, and place the mouse on the heating pad until it has fully recovered.

4. Abdominal aorta harvest and pathological experiments

1. Sacrifice the mouse by isoflurane overdose on the day following the ultrasound examination (following institutionally approved protocols).
2. Open the thoracic and abdominal cavities and perform perfusion with 1x PBS through the left ventricle until the lungs and liver turn white, following the procedure described in previous literature²⁷.
3. Use a digital caliper to measure the external diameter of the infrarenal aorta. Harvest the infrarenal abdominal aorta under a stereomicroscope and store the vessel in 4% paraformaldehyde for 24-48 h.
4. Remove the connective and adipose tissue surrounding the abdominal aorta. Embed the aorta in paraffin to prepare sections, cutting the sections into 3-5 µm slices as previously described^18,28.
5. Perform hematoxylin and eosin (H&E), Masson's trichrome, and Elastic-Van Gieson (EVG) staining on the deparaffinized slices using respective staining kits according to the manufacturer's instructions.
   1. Analyze the vessel structure and inflammation with H&E staining, observe extracellular matrix (ECM) degradation and collagen fibers with Masson's trichrome staining, and examine elastic fibers with EVG staining.

Results

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 ± standard deviations unless otherwise stated. The average operation time was 55.67 min ± 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.

Discussion

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...

Materials

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