Three-dimensional vessel wall normal microarchitecture and remodeling with abdominal aortic aneurysms quantified using immunofluorescent array tomography [electronic resource]

Abdominal aortic aneurysms (AAA) are described by a pathological dilation of at least 150% in the abdominal aorta. Aneurysm pathogenesis is characterized by extracellular matrix (ECM) remodeling, smooth muscle cell (SMC) apoptosis, and inflammatory cell infiltration. Overall, the structural organization and integrity of the vessel wall is lost. In order to better understand the mechanism of AAA development, a novel microscopy technology, in combination with an AAA animal model, were used to visualize and quantify the microstructural and cellular changes in the vessel wall. Therefore, the goals of this work were to introduce Immunofluorescent Array Tomography (IAT), a novel three-dimensional high resolution microscopy technology, to the field of cardiovascular research, and to apply IAT to describe the vessel wall microarchitecture of a healthy aorta, as well as to investigate the spatial and temporal changes in tissue and cellular content, structure, and organization during AAA development. The purpose of the initial studies was two-fold: to develop the methods needed to enable the application of IAT to murine blood vessels and to investigate the microarchitecture of the healthy murine aorta. The anterior and posterior regions of the infrarenal aorta of 8 to 10 week old C57BL6 mice were evaluated. Staining and custom image analysis methods were developed. Antibody selection, primary antibody concentration, co-staining with multiple primary antibodies, and the multi-cycle staining design were optimized to produce positive and specific staining of elastin, smooth muscle cell actin (SMCA), and collagen type I. Algorithms were developed and applied to the healthy murine aorta to quantify volume fractions (VF) of medial elastin (27.5 ± 0.99%), SMCA (18.4 ± 0.67%), and nuclei (6.1 ± 0.14%), as well as adventitial collagen type I (22.3 ± 1.7%). Elastin thickness (1.6 ± 0.35 [Mu]m), spacing between elastin lamellae (3.5 ± 0.13 [Mu]m), elastin fragmentation (4.2 x 10-3 ± 1.6 x 10-4 # of objects/elastin area ([Mu]m)), media wall thickness (20.4 ± 3.1 [Mu]m), nuclei aspect ratio (3.2 ± 0.21 [Mu]m), and nuclei amount (17.3 ± 0.69 nuclei) were also quantified. The 3D microstructure and cellular morphology of the anterior and posterior infrarenal murine aorta were qualitatively and quantitatively described using IAT. IAT was then used to investigate the spatial and temporal remodeling of vessel wall microarchitecture and cellular morphology during AAA development in the murine elastase perfusion model. Infrarenal aortas of C57BL6 mice (N=20) were evaluated at 0, 7 and 28 days after elastase or heat-inactivated elastase perfusion. Custom algorithms quantified VFs of elastin, SMCA, and adventitial collagen type I, as well as elastin thickness, elastin fragmentation, media thickness, and nuclei amount. 3D renderings depicted elastin and collagen type I degradation, and dynamic changes in SMC phenotype, morphology, and amount. Elastin degradation was described by a 37.5% (p < 0.01) loss in VF, 48.9% decrease in thickness, and a 449.7% (p < 0.001) increase in fragmentation over 28 days. SMCA VF decreased 78.3% (p < 0.001) from Day 0 to Day 7, and increased 139.7% (p < 0.05) from Day 7 to Day 28. Media thickness increased 61.1% and medial nuclei amount increased 159.1% (p < 0.01) over 28 days. Adventitial collagen type I VF decreased 64.1% (p < 0.001) over 28 days. IAT and custom image analysis algorithms have enabled robust quantification of vessel wall content, microstructure, and organization to help elucidate the unique dynamics of major tissue constituent remodeling during AAA development. In conclusion, the methodologies needed to enable the application of IAT to cardiovascular research have been developed, the healthy murine vessel wall microarchitecture has been qualitatively and quantitatively described, and the unique dynamics of vascular remodeling during aneurysm development have been investigated.

Keywords: Bioengineering.
Advisor: Taylor Charles A. (Charles Anthony)
Yock Paul G. primary advisor. Thesis advisor
Tsao Philip advisor. Thesis advisor