surgical induction and evaluation of abdominal aortic aneurysm in mice

Investigating the Molecular Mechanisms Underlying Murine Abdominal Aortic Aneurysm

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.

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

This study presents a protocol for establishing a mouse abdominal aortic aneurysm model using calcium chloride and porcine pancreatic elastase, which can be used to investigate the pathophysiological mechanism under abdominal aortic aneurysm. Our protocol combines advantages of periadventitial PPE and the calcium chloride infiltration, which is a safe, stable, and reproducible mouse AAA model, demonstrating a high incidence rate of AAA, significant dilation of abdominal aorta, and pathophysiological changes resembling those observed in human AAA. In future, our research team is committed to dedicated engagement in clinical and basic research pertaining to aortic and peripheral arterial disease, with a particular focus on investigating the pathogenesis of these conditions and the development of novel therapeutic approaches.

Surgical Induction and Evaluation of Abdominal Aortic Aneurysm in Mice

To begin, place the anesthetized mouse in a supine position on the heating pad. After shaving and disinfection, place the mouse under the stereo microscope. Using scissors, make a longitudinal incision of approximately 2 to 2.5 centimeters along the midline of the abdomen.Enter the peritoneal cavity through the linea alba using the transabdominal approach. Place a retractor to expose the peritoneal cavity. Pack the intestines and colon into the right half of the abdomen using normal saline soaked gauze.Perform blunt separation of connective and adipose tissue adjacent to the infrarenal abdominal aorta and inferior vena cava using forceps and cotton swabs. Gently dissect the abdominal aorta and expose the gap between the abdominal aorta and the overlying psoas major. Insert the sterilized cotton pad strips into the gap without damaging the lumbar artery or vein.Place the cotton balls soaked in 0.9 molar calcium chloride on the adventitia of the abdominal aorta. After 15 minutes, remove the cotton ball. Dilute the elastase to two milligrams per milliliter with double distilled water.Using an insulin syringe, apply 50 microliters of diluted elastase to the infrarenal segment of the abdominal aorta. Wrap the cotton pad strips to cover the abdominal aorta and inferior vena cava. Cover the abdominal cavity during the process of infiltration with sterilized gauze.After 15 minutes, 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 with 0.9%saline to prevent adhesion.Using a 6-O non-absorbable polypropylene suture, close the muscle layer and skin. Place the mouse on the heating pad of the rodent ultrasound machine. Apply ultrasonic coupling to the abdomen and measure the maximum diameter of the aneurysm and the diameter of the normal segment of the abdominal aorta.In an Excel sheet, calculate the increased percentage of the infrarenal abdominal aorta. After the examination, use a napkin to wipe off the remaining ultrasonic couplet and place the mouse on the heating pad until fully recovered. In the PPE plus calcium chloride, 91.67%of mice developed abdominal aortic aneurysm at 21 days after surgery, whereas no aneurysm formation was observed in the Sham group.The average maximum internal diameter of the infrarenal abdominal aorta was significantly larger in the PPE plus calcium chloride group compared to the Sham group. The average maximum external diameter of the infrarenal abdominal aorta was significantly larger in the PPE plus calcium chloride group compared to the Sham group. Histological examination revealed thickening of the aortic wall, inflammatory cell infiltration, extracellular matrix degradation, collagen fiber deposition, and elastic fiber breakdown in the PPE plus calcium chloride group compared to the Sham group.

surgical-induction-and-evaluation-of-abdominal-aortic-aneurysm-in-mice

Research

JoVE Journal

Medicine

24 조회수. To begin, place the anesthetized mouse in a supine position on the heating pad. After shaving and disinfection, place the mouse under the stereo microscope. Using scissors, make a longitudinal incision of approximately 2 to 2.5 centimeters along the midline of the abdomen.Enter the peritoneal cavity through the linea alba using the transabdominal approach. Place a retractor to expose the peritoneal cavity. Pack the intestines and colon into the right half of the abdomen using normal sa...