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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session A

Poster I3 - Comprehensive Histological Evaluation of Murine Elastase Aneurysm Model for Long-Term In Vivo Analysis

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Intracranial aneurysms (IAs) represent a significant health concern due to their high risk of rupture and associated mortality. An IA is characterized by a focal dilation of a cerebral artery, forming a blister-like protrusion. These aneurysms are mainly found in the major arteries at the base of the skull. Epidemiological data indicate that 10% to 30% of individuals with an aneurysm may have multiple aneurysms, though many remain small and asymptomatic. The prevalence of IAs is higher in adults aged 30 to 60, with a notable predominance in women. Approximately 6% of the U.S. population harbors unruptured aneurysms, with around 30,000 cases of rupture reported annually [1].

While significant progress has been made in understanding the pathophysiology of IAs, there remains a disconnect between findings from animal models and the clinical presentation of human aneurysms, particularly regarding long-term studies. The murine model provides the lowest phylogenic animal model for early-stage physiologic and anatomic representation of the human patient and biological response but may lack long-term relevance. To address this, we propose a comprehensive histological evaluation of the murine elastase aneurysm model at intervals of three weeks and three months.

We hypothesize that histological techniques will elucidate the long-term applicability of this model, thereby facilitating research into disease progression. Key areas of focus include inflammation, extracellular matrix remodeling, and vascular wall degeneration.

Materials and

Methods:  Immunohistochemistry (IHC) and immunofluorescence (IF) staining were employed for tissue analysis. Samples were embedded in an OCT compound and cryo-sectioned at 5 μm. Sections were mounted on glass slides and stored at -80°C.

Aneurysms were induced in 8 to 10-week-old C57BL/6 mice through elastase infusion into the left common carotid artery (CCA). The mice were anesthetized, and a midline neck incision exposed the CCA. Elastase (10 U/mL) was infused for 5 minutes, after which the incision was sutured. Aneurysms developed over 14 days.

IHC Staining: Sections were equilibrated to room temperature, underwent antigen retrieval, and were blocked with hydrogen peroxide. After PBS washes, a blocking solution was applied for 3 hours, followed by overnight incubation with primary antibodies at 4°C. Secondary antibodies were applied for 1 hour, followed by DAB staining and hematoxylin counterstaining. Slides were then dried, mounted, and sealed.

IF Staining: Sections underwent antigen retrieval and blocking. Primary antibodies αSMA, MECA-32, FSP-1, CD68, CD206, and NIMP-R14 were used targeting smooth muscle cells (SMCs), endothelial cells (ECs), fibroblasts, macrophages, M1 and M2, and neutrophils, respectively, and incubated overnight. Secondary antibodies were applied, and sections were mounted with DAPI. Slides were sealed and stored at room temperature.

Results, Conclusions, and Discussions: The aneurysm sac displayed strong brown staining for αSMA, indicating the presence of SMCs crucial for vascular integrity. A gradient of SMC density was noted, suggesting structural changes in different parts of the aneurysm wall. CD68, a macrophage marker, showed strong staining, indicating significant inflammatory activity. The presence of macrophages varied, reflecting differences in immune response within the aneurysm. CD80, indicating activated antigen-presenting cells, showed pronounced regional variation, with strong staining on one side of the sac.

The M2 macrophage marker CD206 had weak staining, suggesting a low presence of M2 macrophages, which are associated with anti-inflammatory responses. Moderate staining for fibroblasts indicated potential fibroblastic activity, possibly contributing to structural remodeling of the aneurysm wall. MECA staining was weak, indicating reduced endothelial integrity or neovascularization. No neutrophils were observed indicated by the absence of brown staining for the NIMP marker.

The observed staining patterns align with the known pathophysiology of intracranial aneurysms, characterized by complex remodeling processes, inflammation, and immune responses. The presence of macrophages, fibroblasts, and SMCs is consistent with typical aneurysm characteristics. Regional variations in marker expression highlight localized pathological processes such as inflammation and tissue remodeling, potentially contributing to vessel wall weakening.

Recent advancements in intracranial aneurysm treatment include novel biomaterials for vascular embolization and coil systems. Studies on injectable hydrogels and bioabsorbable, elastomer-coated magnesium alloy coils offer promising new therapeutic options, providing mechanical support and localized treatment. These innovations represent significant progress in biomaterial engineering, aiming to enhance the safety and efficacy of aneurysm treatments.

Our study aims to provide a detailed understanding of the murine aneurysm model through IHC and immunofluorescence (IF) analyses at 3 weeks and 3 months post-induction, evaluating both short-term and long-term cellular responses.

Acknowledgements (Optional):  The research is supported by the Swanson School of Engineering and the Office of the Provost at the University of Pittsburgh and the Brain Aneurysm Foundation Grant (AWD00008508)