Three-Dimensional Imaging Provides Detailed Atherosclerotic Plaque Morphology and Reveals Angiogenesis After Carotid Artery Ligation

Abstract

Rationale: Remodeling of the vessel wall and the formation of vascular networks are dynamic processes that occur during mammalian embryonic development and in adulthood. Plaque development and excessive neointima formation are hallmarks of atherosclerosis and vascular injury. As our understanding of these complex processes evolves, there is a need to develop new imaging techniques to study underlying mechanisms. Objective: We used tissue clearing and light-sheet microscopy for 3-dimensional (3D) profiling of the vascular response to carotid artery ligation and induction of atherosclerosis in mouse models. Methods and Results: Adipo-Clear and immunolabeling in combination with light-sheet microscopy were applied to image carotid arteries and brachiocephalic arteries, allowing for 3D reconstruction of vessel architecture. Entire 3D neointima formations with different geometries were observed within the carotid artery and scored by volumetric analysis. Additionally, we identified a CD31-positive adventitial plexus after ligation of the carotid artery that evolved and matured over time. We also used this method to characterize plaque extent and composition in the brachiocephalic arteries of ApoE-deficient mice on high-fat diet. The plaques exhibited inter-animal differences in terms of plaque volume, geometry, and ratio of acellular core to plaque volume. A 3D reconstruction of the endothelium overlying the plaque was also generated. Conclusions: We present a novel approach to characterize vascular remodeling in adult mice using Adipo-Clear in combination with light-sheet microscopy. Our method reconstructs 3D neointima formation after arterial injury and allows for volumetric analysis of remodeling, in addition to revealing angiogenesis and maturation of a plexus surrounding the carotid artery. This method generates complete 3D reconstructions of atherosclerotic plaques and uncovers their volume, geometry, acellular component, surface, and spatial position within the brachiocephalic arteries. Our approach may be used in a number of mouse models of cardiovascular disease to assess vessel geometry and volume. Visual Overview: An online visual overview is available for this article.

Three-Dimensional Imaging of Intraplaque Neovascularization in a Mouse Model of Advanced Atherosclerosis

“…2) and has been successfully used to image three-dimensional structures, including intact mouse organs such as the brain, kidney, intestine, eye, and even whole embryos [18,[22][23][24][25]. Recently, Becher et al [26] applied the iDIS-CO technology for 3D profiling of atherosclerotic plaques and arterial remodeling after carotid artery ligation. We optimized iDISCO for mouse atherosclerotic tissue using endothelial cell-specific CD31 antibodies, and we show here for the first time a 3D reconstruction of IP neovascularization in carotid plaques of ApoE −/− Fbn1 C1039G+/− mice.…”

Dare to Compare. Development of Atherosclerotic Lesions in Human, Mouse, and Zebrafish

“…Nevertheless, interpretation of the acquired data requires even more experience. To date, the best imaging techniques with the highest resolution, such as microCT and light-sheet microscopy, are still lethal or only applicable to one model ( 137 – 139 ). While in vivo imaging of mice can be achieved by adapting human imaging technologies and vice versa , imaging of zebrafish larvae can be achieved with simple microscopes.…”

Deep learning reveals 3D atherosclerotic plaque distribution and composition

“…Using light-sheet microscopy to study plaque formation in different blood vessels has very recently been suggested by Becher et al 19 . Optical cross-sections obtained by LSFM were shown to correlate well with traditional histological stained tissue sections.…”

“…LSFM is a non-destructive imaging technique that makes paraffin-embedding and sectioning unnecessary, requiring little hands-on sample preparation time and offering great scaling capability (Supplemental Tables 1 , 2 ). The technology has been applied to image parts of the cardiovascular system before 13 , 16 – 18 Becher et al have recently shown that this imaging technology can successfully be applied to detect plaque in brachiocephalic, carotid and radial arteries 19 . However, 3D light sheet imaging generates large data volumes that pose unique and so far unanswered challenges for rapid image analysis in particular in preclinical drug discovery studies.…”