Murine Abdominal Aortic Aneurysm Intraluminal Thrombus Composition and Structure

Introduction: : An abdominal aortic aneurysm (AAA) is a dilation or ballooning of the aortic wall in the abdominal cavity. While some AAAs are asymptomatic and do not increase in size, others will continue to enlarge until rupture, frequently causing uncontrolled hemorrhage into the peritoneal space. In men over the age of 65, the occurrence of ruptured AAAs is estimated to be 3-5% [1]. These ruptured aneurysms have high mortality rates, between a mortality rate of 75-90%, and account for more than 10,000 deaths per year in the United States [1, 2]. AAAs are typically found through annual screening in high-risk populations, and treatment for AAAs is determined based on aneurysm size and growth rate. While larger aneurysms are more likely to rupture, there are still instances of both smaller aneurysm rupture and larger aneurysm stability. Additionally, 75% of patients with AAAs also develop intraluminal thrombus (ILT) within the expansion, but the role of ILT in aneurysm progression and rupture is not well understood [3, 4]. Developing a better understanding of what factors contribute to aneurysm development and expansion could help improve patient-specific surgical treatment timing and aid in the creation of pharmaceutical treatments of AAA.
[1] Lindholt et al. British Medical Journal, 2005.
[2] Kent. New England Journal of Medicine. 2014.
[3] Ma et al. Biomolecules. 2022.
[4] Harter et al. Journal of Ultrasound in Medicine. 1982.

Materials and

Methods: : We induced AAAs in male C57Bl6/J mice (n=25) through a topical elastase surgical application and β-aminopropionitrile (BAPN) water-administration model. We caused initial damage to the abdominal aorta by surgically exposing the aorta, removing the surrounding connective tissue, and topically applying 5 µL of 5 or 10 mg/mL porcine pancreatic elastase (Sigma Aldritch, Saint Louis, MO) to the region ranging from the renal arteries to the iliac split. To counter ECM cross-linking repair, 0.2% BAPN (Sigma Aldritch), a lysyl oxidase inhibitor, was administered in the mouse drinking water starting two days prior to elastase application. Ultrasound images of these AAAs were collected weekly/biweekly over a period of 56 days to evaluate AAA growth over time. At the end of the experimental timeline, mice were euthanized, aortae were harvested, and histology and scanning electron microscopy images were collected. In Movat’s pentachrome stained histological samples, we semi-quantitatively assessed the degree of elastin degradation in samples with and without ILT. In H&E-stained samples, we used similar semi-quantitative approaches to evaluate the presence of mononuclear infiltrate (indicating chronic inflammation), polymorphonuclear infiltrate (indicating acute inflammation), and circumferential involvement (indicating how much of the arterial wall experienced inflammation).

Results, Conclusions, and Discussions:: We found that mice subjected to the topical elastase surgery + oral BAPN additive show significant and continuous growth over time. Mice with the presence of ILT had significantly greater expansion earlier in the study (significance beginning at Day 14, p< 0.05) as well as more significant expansion over the length of the study compared to the samples without ILT (significance beginning at Day 21, p< 0.05). From histology, samples with ILT showed more elastin disorganization than samples without intraluminal thrombus. Samples with ILT showed the presence of greater inflammation occurring across all three categories. From scanning electron microscopy, we were able to confirm the presence of layered sheets of fibrin and abnormally shaped red blood cells (polyhedrocytes) within the ILT deposits.

In this study, murine aortas with the presence of ILT also had increased average maximum diameter, greater damage to elastin fibers, and more severe inflammatory processes. Elastase causes injury to the aorta by degrading the elastin fibrils present in the aortic wall, reducing the ability of the aorta to contract and maintain its shape during high-pressure blood flow. Further damage to the extracellular matrix within the wall is often driven by subsequent acute and chronic inflammation. Here we observed the tissue samples displaying greater acute-on-chronic inflammation are correlated with more damage to elastin, allowing for greater expansion of the aorta. Further, larger aortic expansions were correlated with slower blood flow, likely due to the increased cross-sectional area. Increased aortic expansion and damage to the aortic wall may be more likely to allow for the hemodynamic conditions that are conducive to the initiation of ILT deposition: damage to the aortic endothelium and reduced blood flow. Understanding the relationship between ILT formation, aortic wall degradation, and inflammation could help refine clinical strategies for monitoring and treating AAAs. Based on this understanding, potential treatments to slow the progression of AAA development could include platelet-targeting therapy. Future work focused on modulating inflammation and ILT formation could offer promising pathways for non-surgical intervention in AAA treatment.

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