Assessing Large-Vessel Endothelial Permeability Using Near-Infrared Fluorescence Imaging

Objective— Loss of endothelial barrier function in arterial blood vessels is characteristic of vascular pathologies, including atherosclerosis. Here, we present a near-infrared fluorescence (NIRF) imaging methodology for quantifying endothelial permeability and macromolecular uptake in large arteries in the mouse and evaluate its applicability for studying mechanisms of vascular inflammation. Approach and Results— To validate the NIRF methodology, macrovascular inflammation was induced in C57bl/6 mice by local tumor necrosis factor-α stimulation of the carotid artery or in apolipoprotein E–deficient ( APOE –/– ) mice by Western diet for 4 weeks. Evan Blue dye, serving as plasma protein marker and fluorescent in the near-infrared spectrum, was given intravenously at different doses. Carotids and aorta were excised, and Evan Blue dye fluorescence was assessed through whole vessel scan in an infrared imaging system. NIRF correlated to extraction–absorbance methodology for Evan Blue dye quantification and was superior at discriminating plasma protein accumulation in tumor necrosis factor-α–stimulated carotids. NIRF allowed for focal quantification of increased arterial wall Evan Blue dye uptake in APOE –/– mice. Importantly, NIRF left vessels intact for subsequent histological analysis or quantification of leukocyte subpopulations by flow cytometry. Conclusions— The described NIRF methodology provides a sensitive and rapid tool to locate and quantify macromolecular uptake in the wall of arterial blood vessels in vascular pathologies in mice.