A Calcium Phosphate-Induced Mouse Abdominal Aortic Aneurysm Model

Summary

This protocol describes a calcium phosphate-induced abdominal aortic aneurysm (AAA) mouse model to study the pathological features and molecular mechanisms of AAAs.

Abstract

An abdominal aortic aneurysm (AAA) is a life-threatening cardiovascular disease that occurs worldwide and is characterized by irreversible dilation of the abdominal aorta. Currently, several chemically induced murine AAA models are used, each simulating a different aspect of the pathogenesis of AAA. The calcium phosphate-induced AAA model is a rapid and cost-effective model compared to the angiotensin II- and elastase-induced AAA models. The application of CaPO₄ crystals to the mouse aorta results in elastic fiber degradation, loss of smooth muscle cells, inflammation, and calcium deposition associated with aortic dilation. This article introduces a standard protocol for the CaPO₄-induced AAA model. The protocol includes material preparation, the surgical application of the CaPO₄ to the adventitia of the infrarenal abdominal aorta, the harvesting of aortas to visualize aortic aneurysms, and histological analyses in mice.

Introduction

An abdominal aortic aneurysm (AAA) is a lethal cardiovascular disease characterized by permanent dilation of the abdominal aorta, with high mortality rates once rupture occurs. AAA is associated with aging, smoking, male gender, hypertension, and hyperlipidemia¹. Several pathological processes have been shown to contribute to AAA formation, including extracellular matrix fiber proteolysis, immune cell infiltration, and loss of vascular smooth muscle cells. Currently, the pathological mechanisms of AAA remain elusive, and there are no proven drugs for the treatment of AAA¹. Research into human AAA is limited due to the existence of few human aortic samples; thus, several chemical modification-induced animal AAA models have been established and widely adopted, including subcutaneous angiotensin II (AngII) infusion, perivascular or intraluminal elastase incubation, and perivascular calcium phosphate application². A commonly used mouse model is the application of calcium phosphate (CaPO₄) to the adventitia of the infrarenal abdominal aorta, which is cost-effective and does not require genetic modification.

Direct periaortic application of CaCl₂ to the carotid artery of rabbits to induce aneurysmal change was initially reported by Gertz et al.³ and was later applied to the abdominal aortas of mice. The model was developed by Yamanouchi et al. to accelerate aortic dilation by using CaPO₄ crystals in mice⁴. Infiltration of CaPO₄ into mice aortas recapitulates many pathological features observed in human AAAs, including profound macrophage infiltration, extracellular matrix degradation, and calcium deposition. The risk factors of human AAA, such as hyperlipidemia, also augment CaPO₄-induced AAA in mice⁵. In contrast to AngII perfusion-induced AAA in ApoE^-/- or LDLR^-/- mice, CaPO₄-induced AAA occurs in the infrarenal aortic region, which mimics human AAA. Currently, this method has been widely applied to assess susceptibility to AAA development in genetically modified mice and evaluate the anti-AAA effects of drugs^6,7.

Protocol

Animal studies were performed in compliance with the guidelines of the Institutional Animal Care and Use Committee of Peking University Health Science Center and were approved by the Biomedical Ethics Committee of Peking University (LA2015142). All the mice for surgery were anesthetized with isoflurane (1.5%-2%), and anesthesia was carefully monitored to avoid pain or discomfort for the mice.

1. Preparation

1. Cut 0.3 cm wide strips of powder-free rubber gloves and gauze.
2. Purchase 8-10-week-old C57BL/6J male mice. House the animals in an air-conditioned environment with a 12 h light-dark cycle and free access to food and water.
3. Autoclave the gauze, cotton swabs, scissors, and forceps prior to surgery.
4. Obtain betadine, 70% ethanol, and antiseptic hand wash.
5. Put on a mask, gown, and sterile gloves.

2. Surgical procedure

1. Feed chewable carprofen tablets (5 mg/kg dose) to an 8-10-week-old C57BL/6J mouse 2-4 h before surgery. Then, place the mouse in an induction chamber (206 mm x 210 mm x 140 mm) with isoflurane at a flow rate of 1.5%-2%.
   1. Monitor the mouse for about 5 min until respiration is visibly slowed. Ensure that the mouse has no response to pain stimulation before surgery.
2. Apply an ophthalmic ointment to the eyes and provide thermal support with a heating pad or blanket. Confirm the depth of anesthesia with a toe pinch every 15 minutes during the surgical procedure.
3. Shave the abdominal hair of the mouse using an electric clipper or hair removal cream. Swab and wipe the shaved area with betadine, followed by 70% ethanol, several times in a circular motion. Change gloves to maintain sterility.
4. Use scissors to make a ~1.5 cm incision at the lower abdomen along the midline of the abdomen.
5. Use a sterile cotton swab moistened with normal saline to carefully remove the intestine until the infrarenal aorta is visible.
6. Dissect the connective tissue and fat from the infrarenal aorta for an approximately 0.5 cm section. Note the small vessels to the dorsal side and avoid tearing them. There is no need to separate the abdominal aorta from the abdominal main vein.
7. Pack a piece of the saline-soaked rubber glove strip under the abdominal aorta and abdominal main vein. Use a cotton swab to wipe off excess liquid.
8. Pack a piece of gauze soaked with 0.5 M CaCl₂ on the adventitia of the infrarenal abdominal vasculature for 10 min. For the sham mouse group, replace the 0.5 M CaCl₂ with normal saline.
9. Remove the gauze and pack another piece of gauze soaked with PBS solution for 5 min to generate CaPO₄ crystals in situ in the adventitia of the aorta.
10. Carefully remove the rubber glove strip and gauze. Reset the intestinal tract of the mouse.
11. Suture the abdominal incision and skin with a 5-0 suture.
12. Place the mouse on a heating pad until the mouse regains consciousness. Provide postsurgical pain recovery housing and analgesia, according to the local animal ethics committee.
13. House the mouse for another 14 days. Monitor the mouse closely after the surgery and observe at least 1x every day subsequently. Perform a necropsy immediately if any mice die during this period.

3. Harvesting for imaging of aortas

1. 14 days after the surgery, sacrifice the mice using CO₂.
2. Cut open the mouse thoracic and abdominal cavities ventrally and cut open the right atrium.
3. Perfuse the mice with PBS buffer through the left ventricle of the heart to remove blood in the aorta, and then perfuse with 4% paraformaldehyde as previously described⁸.
4. Harvest the aorta under the stereoscope.
5. Place harvested aortas in tubes containing 5 mL of 4% paraformaldehyde for 48 h.
6. Remove adventitial tissue and fat carefully under the stereoscope and pin the aorta on a black wax plate with insect needles.
7. Acquire aortic images.

4. Evaluation of the degradation of the elastic fibers

1. Cut the aortic aneurysm tissues into serial cryosections (7 µm thick).
2. Analyze the elastic fibers using a commercial elastic van Gieson (EVG) staining kit according to the manufacturer's protocol.
3. Grade the elastin degradation. Grade 1: <25% degradation; grade 2: 25% to 50% degradation; grade 3: 50% to 75% degradation; or grade 4: >75% degradation.

Results

14 days after CaPO₄ application, the C57BL/6J male mice were euthanized, and their aortas were harvested and cleaned. The morphology of the aortas was imaged to visualize AAA formation. As shown in Figure 1A-B, the application of CaPO₄ led to dilation of the infrarenal abdominal aorta. Histologically, CaPO₄ resulted in a dramatic degradation of elastic fibers, as illustrated by elastin breaks (Figure 1C

Discussion

Periaortic application of CaPO₄ is a robust approach to induce AAA in mice. Several studies have used the CaPO₄ model and consistently reported that this is a rapid and reproducible method to study AAA in mice^7,9. This model is considered to recapitulate part of the features of human aortic aneurysm and provide mechanistic insights into AAA pathogenesis, including inflammation and extracellular matrix degradation.

Materials

Name	Company	Catalog Number	Comments
CaCl2	MECKLIN	C805225
NaCl	Biomed	SH5001-01
PBS	HARVEYBIO	MB5051
Small animal ventilator	RWD	H1550501-012