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

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Abstract

Multiple lines of evidence suggest that intraplaque (IP) neovascularization promotes atherosclerotic plaque growth, destabilization, and rupture. However, pharmacological inhibition of IP neovascularization remains largely unexplored due to the limited number of animal models that develop IP neovessels and the lack of reliable methods for visualizing IP angiogenesis. Here, we applied 3D confocal microscopy with an optimized tissue-clearing process, immunolabeling-enabled three-dimensional imaging of solvent-cleared organs, to visualize IP neovessels in apolipoprotein E-deficient (ApoE<sup>−/−</sup>) mice carrying a heterozygous mutation (C1039+/−) in the fibrillin-1 gene. Unlike regular ApoE<sup>−/−</sup> mice, this mouse model is characterized by the presence of advanced plaques with evident IP neovascularization. Plaques were stained with antibodies against endothelial marker CD31 for 3 days, followed by incubation with fluorescently labeled secondary antibodies. Subsequent tissue clearing with dichloromethane (DCM)/methanol, DCM, and dibenzyl ether allowed easy visualization and 3D reconstruction of the IP vascular network while plaque morphology remained intact.

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Douglas S Richardson, Jeff W. Lichtman (2015)

Biological specimens are intrinsically three dimensional; however, because of the obscuring effects of light scatter, imaging deep into a tissue volume is problematic. Although efforts to eliminate the scatter by "clearing" the tissue have been ongoing for over a century, there have been a large number of recent innovations. This Review introduces the physical basis for light scatter in tissue, describes the mechanisms underlying various clearing techniques, and discusses several of the major advances in light microscopy for imaging cleared tissue.

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Author and article information

Journal

Journal ID (publisher-id): JVR

Journal ID (nlm-ta): J Vasc Res

Journal ID (doi): 10.1159/issn.1018-1172

Title: Journal of Vascular Research

Publisher: S. Karger AG

ISSN (Print): 1018-1172

ISSN (Electronic): 1423-0135

Publication date Subscription-year: 2020

Publication date (Print): November 2020

Publication date (Electronic): 01 July 2020

Volume: 57

Issue: 6

Pages: 348-354

Affiliations

_aLaboratory of Physiopharmacology, University of Antwerp, Antwerp, Belgium

_bLaboratory of Cell Biology and Histology, University of Antwerp, Antwerp, Belgium

_cDepartment of Surgery, Leiden University Medical Center, Leiden, The Netherlands

_dEinthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands

Author notes

*Wim Martinet, Laboratory of Physiopharmacology, University of Antwerp, Universiteitsplein 1, BE–2610 Antwerp (Belgium), wim.martinet@uantwerpen.be

Article

Publisher ID: 508449 Other ID: J Vasc Res 2020;57:348–354

DOI: 10.1159/000508449

PubMed ID: 32610324

License:

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