Comparison of Aortic Collagen Fiber Angle Distribution in Mouse Models of Atherosclerosis Using Second-Harmonic Generation (SHG) Microscopy

Abstract

Characterization of collagen fiber angle distribution throughout the blood vessel wall provides insight into the mechanical behavior of healthy and diseased arteries and their capacity to remodel. Atherosclerotic plaque contributes to the overall mechanical behavior, yet little is known experimentally about how collagen fiber orientation is influenced by atherogenesis. We hypothesized that atherosclerotic lesion development, and the factors contributing to lesion development, leads to a shift in collagen fiber angles within the aorta. Second-harmonic generation microscopy was used to visualize the three-dimensional organization of collagen throughout the aortic wall and to examine structural differences in mice maintained on high-fat Western diet versus age-matched chow diet mice in a model of atherosclerosis. Image analysis was performed on thoracic and abdominal sections of the aorta from each mouse to determine fiber orientation, with the circumferential (0°) and blood flow directions (axial ±90°) as the two reference points. All measurements were used in a multiple regression analysis to determine the factors having a significant influence on mean collagen fiber angle. We found that mean absolute angle of collagen fibers is 43° lower in Western diet mice compared with chow diet mice. Mice on a chow diet have a mean collagen fiber angle of ±63°, whereas mice on a Western diet have a more circumferential fiber orientation (~20°). This apparent shift in absolute angle coincides with the development of extensive aortic atherosclerosis, suggesting that atherosclerotic factors contribute to collagen fiber angle orientation.

Reduced arterial elasticity due to surgical skeletonization is ameliorated by abluminal PEG hydrogel

“…The skeletonization of the vessel likely results in the activation of adventitial fibroblasts, leading to a change in ECM production and organization, especially medial elastin, which have profound effects on vessel function . Interestingly, alterations in collagen play a significant role in vessel stiffening associated with the development of aneurysms, atherosclerosis, and hypertension . The changes in both elastin and collagen organization noted in our study suggest that the decrease in cyclic strain in skeletonized vessels is due to either a reduction in elastic recoil during diastole associated with elastin disruption, reduced distension during systole associated with alterations in collagen, or both.…”

“…Due to collagen's intrinsic properties such as optical anisotropy, fibril structure, and multifibril organization, collagen fibers and bundles were visualized in this study without labeling agents under SHG with femtosecond pulsed infrared excitation . The SHG technique offers many advantages for biomedical assessment of tissue structure, including visualization of collagen fibers without staining and deeper penetration within a sample when compared with conventional single‐photon excitation microscopy . SHG emission directionality is sensitive to the diameter of the fibrils that are bundled into collagen fibers, as well as their spacing within the fiber, and the disorder in their packing .…”

“…The 410 nm, second harmonic signals generated in the tissue were detected using a 400 ± 20 nm filter (Chroma Technology Corp., Bellows Falls, VT), and a 525 ± 50 nm filter (Chroma Technology Corp.) was used for two‐photon excited fluorescence (TPEF) detection. TPEF was used to visualize elastin autofluroescence and a nondescanned detection (NDD) system was used for detection of SHG in the forward direction using a photomultiplier tube (LSM NDD; Zeiss) and in the reverse direction using a GaAsP detector (LSM BiG; Zeiss) to visualize collagen …”

Collagen fibre characterisation in arterial tissue under load using SALS

“…Unlike confocal microscopy, multiphoton microscopy 3 with second harmonic generation (SHG) imaging requires no special preparation or staining of vessels. SHG imaging has previously been used to interrogate fibre orientation through a vessel thickness (Keyes et al, 2011;Watson et al, 2016) and in response to physiological and biaxial loading (Keyes et al, 2011) of mouse aortic tissue without the separation of layers. However, given the limited depth of penetration of such imaging techniques, they cannot be used for imaging the full intact wall, or even intact layers, of thicker vessel walls such as porcine carotid arteries.…”

Comparative measurement of collagen bundle orientation by Fourier analysis and semiquantitative evaluation: reliability and agreement in Masson's trichrome, Picrosirius red and confocal microscopy techniques

“…For example, collagen content and orientation measurement successfully differentiated two histologic types of fibroadenoma tissue and normal breast tissue in one study (Nie et al ., ), and in other study quantification of collagen structure in breast biopsies showed promising results for distinguishing malignant from nonmalignant breast tissue conditions (Ambekar et al ., ). Recent studies have also measured the orientation of collagen bundles in atherosclerotic plaques (Watson et al ., ) and in cardiac fibrosis after myocardial infarction (Hervas et al ., ). In skin research, lesional sclerodermic skin, nonlesional sclerodermic skin and control skin displayed different collagen bundle orientation and packing (de Vries et al ., ).…”