3D confocal microscope imaging of macromolecule uptake in the intact brachiocephalic artery

Abstract

Background and aims Elevated uptake of plasma macromolecules by the arterial wall is an early event in atherogenesis. Existing optical techniques for detecting macromolecular tracers in the wall have poor depth penetration and hence require en face imaging of flattened arterial segments. Imaging uptake in undistorted curved and branched vessels would be useful in understanding disease development. Methods Depth penetration was increased by applying optical clearing techniques. The rat aorto-brachiocephalic junction was imaged intact by confocal microscopy after it had been exposed to circulating rhodamine-labelled albumin in vivo, fixed in situ, excised and then cleared with benzyl alcohol/benzyl benzoate. Tracer uptake was mapped onto a 3D surface mesh of the arterial geometry. Results Tracer fluorescence was detectable throughout the wall closest to the objective lens and, despite a vessel diameter of c. 1 mm, in the wall on the other side of the artery, across the lumen. By tile scanning, tracer concentrations were mapped in the aorta, the brachiocephalic artery and their junction without opening or flattening either vessel. Optical clearing was also shown to be compatible with immunofluorescent staining and imaging of experimental atherosclerosis. Conclusions The technique obviates the need for labour-intensive sample preparation associated with standard en face imaging. More importantly, it preserves arterial geometry, facilitating co-localisation of uptake maps with maps of biomechanical factors, which typically exist on 3D surface meshes. It will permit the correlation of haemodynamic wall shear stress with macromolecule permeability more accurately in regions of high curvature or branching, such as in the coronary arteries.