Method Article

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

DOI:

10.3791/66674

⸱

August 2nd, 2024

 ,  ,  ,  ,  ,  , 
1Division of Vascular Surgery, Department of General Surgery, West China Hospital, Sichuan University, 2Key Laboratory of Birth Defects and Related Diseases of Women and Children of MOE, State Key Laboratory of Biotherapy, West China Second University Hospital, Sichuan University

* These authors contributed equally

In This Article

Summary

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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.

Abstract

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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

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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.'s report on periadventitial PPE infiltration in a murine model20,21. 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 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% ± 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

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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 described18,28.
  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

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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.

In the PPE + CaCl2 group, 11 out of 12 mice (91.67%) developed AAA at 21 days after surgery, while no aneurysm formation was observed in the Sham group. The average maximum internal diameter of the infrarenal abdominal aorta was 1.29 mm ± 0.18 mm in the PPE + CaCl2 group, compared to 0.50 mm ± 0.04 mm in the Sham group. The average percentage increase in internal diameter of the infrarenal abdominal aorta in the PPE + CaCl2 group was 99.50% ± 25.42%. The average maximum external infrarenal aortic diameter was 1.49 mm ± 0.19 mm in the PPE + CaCl2 group, compared to 0.61 mm ± 0.04 mm in the Sham group.

Representative images of the abdominal aorta in the Sham and PPE + CaCl2 groups under a stereomicroscope and ultrasound were shown in Figure 1A,B. Statistical analysis results of the average infrarenal abdominal aorta maximum internal and external diameter in the PPE + CaCl2 and Sham groups were presented in Figure 1C,D. Further histological examination with H&E staining revealed thickening of the aortic wall and infiltration of inflammatory cells in the PPE + CaCl2 group, while Masson's trichrome staining showed extracellular matrix degradation and collagen fiber deposition in the aorta wall. EVG staining demonstrated elastic fiber breakdown (Figure 1E).

Abdominal aorta analysis: macrophotographs, ultrasound, histology (H&E, Masson, EVG), PPE+CaCl₂ effect.
Figure 1: Outcomes of mice AAA model established by periadventitial CaCl2 and elastase infiltration. (A) Representative images of the normal abdominal aorta and AAA under stereo microscope. (B) Transverse and longitudinal ultrasound images of the abdominal aorta in Sham and PPE + CaCl2 group. (C) Statistical analysis of maximum internal diameter of the infrarenal abdominal aorta in Sham and PPE + CaCl2 group, ****p < 0.0001. (D) Comparison of the external infrarenal aortic diameter of Sham and PPE + CaCl2 group, ****p < 0.0001. (E) H&E, Masson, and EVG staining images of abdominal aorta. Scale bars: 50 µm. Please click here to view a larger version of this figure.

Discussion

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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 that only around 60% of rats developed AAA even when the criterion for AAA formation was defined as a 20% increase compared to the normal segment of the abdominal aorta. Both studies noted that no aneurysm formation was observed after a single periaortic incubation of 0.5 mol/L CaCl2 for 20 min13,19,26,30.

Moreover, there is currently no consensus in existing literature regarding the optimal concentration of CaCl2 infiltration, exposure time, and follow-up duration11. As such, the elastase-induced AAA model stands as the second most commonly utilized rodent model for studying AAA. However, intra-aortic PPE infusion is associated with high peri-operative and post-operative mortality rates, up to 40%. Furthermore, notable gender discrepancies have been observed in the PPE-induced AAA model, with female mice exhibiting a much lower percentage increase in aortic diameter compared to their male counterparts11,20,25,31,32,33.

To address these limitations, a modified version of the elastase-induced AAA model involving perivascular elastase infiltration was introduced as a feasible and less invasive alternative. However, it should be noted that the aneurysm formation rate and diameter using this approach were less stable than with intraluminal PPE perfusion11,12,21,22. Therefore, researchers proposed a combination of periadventitial CaCl2 and elastase incubation to establish AAA in rabbits, which has been shown to be simple, effective, and reproducible26.

Meanwhile, another study established an AAA model in rats by combining PPE perfusion and CaCl2 incubation, demonstrating that the PPE + CaCl2 model has high survival rates, high aneurysm formation rates, and good reproducibility25. However, there is currently no literature regarding how to use peri-aortic PPE + CaCl2 infiltration to establish AAA in mice and its outcomes.

This article indicates that the AAA incidence rate and the increase in aorta diameter percentage using this protocol remained stable without peri-operative deaths, as confirmed by ultrasound and histology examinations. Moreover, this protocol effectively induces morphological and pathological changes similar to those seen in human AAA. Furthermore, the average operation time for this protocol was limited to less than 1 h, including 15 min of CaCl2 infiltration and 15 min of PPE incubation. This surgical protocol is robust and easily implemented, using readily available materials. These advantages suggest its potential application in future basic research endeavors.

Discrepancies exist in the rate of aneurysm development in PPE- or CaCl2-induced AAA animal models, and current chemically induced models cannot guarantee a 100% success rate in establishing AAA11,12,13. Although pathological changes associated with AAA, such as chronic inflammation, intima/media thickness, elastic breakdown, calcification, and intraluminal thrombus, can be observed after PPE or CaCl2 infiltration, the dilation of the aorta or formation of an aneurysm may tend to heal spontaneously because the chemical application period is limited and does not provide ongoing activity once the stimulus stops12,23. Furthermore, the surgical procedure for establishing AAA cannot be completely standardized, leading to potential variations in the extent of exposure of the abdominal aorta, which may impact aneurysm formation. However, even in mice where modeling was unsuccessful, the diameter of the abdominal aorta still exhibited a significant 44% expansion, indicating that this method can induce considerable enlargement in the abdominal aorta, although it may not always meet the diagnostic criteria for abdominal aortic aneurysm.

Of particular importance, several crucial steps are involved in this protocol. First and foremost, adequately exposing the infrarenal abdominal aorta during the establishment of the mouse AAA model is imperative. Maximizing the separation of connective and adipose tissue surrounding the abdominal aorta facilitates better infiltration of CaCl2 and elastase into the middle layer of the infrarenal abdominal aorta, thereby increasing the rate of aneurysm formation22. Moreover, it may be easier to separate connective and adipose tissue after CaCl2 infiltration. Therefore, if the initial dissection of surrounding tissue is not satisfactory, it is advisable to continue separating the connective tissue adjacent to the abdominal aorta after CaCl2 infiltration. However, exposing the abdominal aorta carries inherent risks of damaging the lumbar artery and vein, potentially leading to severe bleeding and fatal outcomes during or after surgery. Therefore, utmost caution must be exercised when removing connective and adipose tissue around the abdominal aorta. Another important step in this protocol involves the separation of the abdominal aorta from the inferior vena cava (IVC). Previous studies and experimental findings consistently indicate that whether or not to dissect the IVC from the abdominal aorta has no impact on aneurysm formation, rendering it unnecessary for this procedure18,22.

This study has several limitations. Firstly, it lacks baseline data on the incidence rate of aneurysms and pathological changes in the abdominal aorta at various time points. Additionally, it did not evaluate the levels of important biomarkers involved in AAA development, such as MMP2, MMP9, TIMP1, and SMA. Secondly, this method has not been previously utilized in mice and lacks cross-sectional comparisons with other commonly used models, especially those involving PPE or CaCl2 alone, to assess aneurysm formation and pathological characteristics. Thirdly, AAA induced by this method lacks several features of human AAA, including atherosclerosis and intramural thrombosis. Furthermore, the concentrations of CaCl2 and elastase used in this method, as well as the infiltration time, differ slightly from those employed in previous studies. The authors aim to address these questions through experimental investigations in future studies.

Another limitation of this protocol is its applicability to larger animal models, such as porcine or canine. Currently, individual chemical agents like elastase, collagenase, or CaCl2 have not demonstrated the capability to induce a 50% dilation of the abdominal aorta in non-rodents, except for rabbits. Only combinations of chemicals and angioplasty or other surgical procedures have shown potential for inducing aneurysm formation in dogs and swine11,34. Previous research has also indicated that aneurysms exhibited signs of regeneration during a 5-month follow-up in a rabbit AAA model induced by elastase35. Although Bi et al. demonstrated the induction of AAA in rabbits using this protocol, further studies are needed to determine its validity and long-term outcomes in swine or dogs26.

The last limitation concerns the inclusion of only male mice, as inducing AAA in female mice using elastase has been associated with lower rates of aneurysm formation and aortic dilation percentage11,13,33,36. However, studies by Xue et al. have demonstrated that perivascular application of elastase alone resulted in a 90% incidence rate of AAA in female mice. This suggests that this protocol may also be effective in female mice22, although further studies are warranted to confirm its efficacy. It is also important to note that damage to the vessel structure and elastic fibers occurs only on the side infiltrated by CaCl2 and elastase, and no pathological changes appear on the side adjacent to the inferior vena cava (IVC) due to their close proximity. However, as previously mentioned, these factors do not impact the incidence rate of AAA or the percentage increase in abdominal aorta size during AAA development.

In conclusion, this manuscript presents a novel, safe, stable, and reproducible mouse AAA model demonstrating a high incidence rate of AAA, significant dilation of the abdominal aorta, and pathophysiological changes resembling those observed in human AAA. Despite its limitations, this mouse AAA model holds great potential for investigating the molecular mechanisms underlying AAA and for developing effective therapeutic approaches for patients with this condition.

Disclosures

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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.

Acknowledgements

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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.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Anesthesia MachineRWDR550
ButorphanolJiangsu Hengrui Pharmaceutical Co., Ltd220608BP1ml: 1mg
C57BL/6JGpt Male MiceGemPharmatechN000013
Calcium ChlorideSigma AldrichC4901100 g
CarprofenMCEHY-B1227100 mg
Chow DietDossy Experimental Animals Co.Ltd
Digital CaliperGreenerIP54
EthanolJinhe Pharmaceutical Co.Ltd53968275%/500 mL
EVG Staining KitSolarbioG1597
GraphPad PrismGraphpadVer 9.0.0
H&E Staining KitServicebioG1076
Insulin SyringeBD2143420
IsofluraneRWDR510-22-4100 mL
Masson Staining KitServicebioG1006
Normal SalineServicebioG4702500 mL
ParaformaldehydeBiosharpBL539A4%/500 mL
PBS, 1xServicebioG4202500 mL
Porcine Pancreatic ElastaseSigma AldrichE1250100 mg
Povidine IodineYongan Pharmaceutical Co.Ltd5%/100 mg
Prolene Polypropylene SutureEthicon LLC8709H
Rodent Ultrasound SystemFujifilmVevo 3100LT
Stereo MicroscopeOlympusSZ61
Ultrasonic CouplantKepplerKL-250250 g

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Mouse AAA ModelCalcium Chloride InfiltrationPeriadventitial ApplicationInfrarenal Abdominal AortaAortic Wall ThickeningExtracellular Matrix DegradationUltrasound MeasurementHistological Examination

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