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Journal of Histochemistry and Cytochemistry, Vol. 45, 743-754, Copyright © 1997 by The Histochemical Society, Inc.

TECHNICAL NOTE

A Novel Technique For Mapping the Lipid Composition of Atherosclerotic Fatty Streaks by En Face Fluorescence Microscopy

Anne M. Klinknera, Peter J. Bugelskia,b, C. Robbie Waitesa, Calvert Loudena, Timothy K. Harta, and William D. Kernsa
a Department of Toxicology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania
b Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Correspondence to: Anne M. Klinkner, Cellular Pathology UE0462, SmithKline Beecham Pharmaceuticals, PO Box 1539, King of Prussia, PA 19406.

We introduce here a new fluorescence microscopy technique for en face analysis of the atherosclerotic fatty streaks (FS). This technique is semiquantitative and has the sensitivity and resolution to map lipids to individual cells in FS less than 100 µm in diameter. New Zealand White rabbits were fed an atherogenic diet for up to 26 weeks. Aortas were fixed in formalin and stained en bloc with the fluorescent dyes Nile red and filipin. Fluorescent staining was validated by correlating microfluorimetric and biochemical measurements of the lipid content in FS. To determine the cell types associated with the different staining patterns, FS were also evaluated by transmission electron microscopy (TEM) and immunohistochemistry (IH). Correlation of microfluorimetry, TEM, IH, and biochemical data indicated that regions rich in non-esterified cholesterol stained with filipin and fluoresced blue owing to accumulations of lipid vessicles and/or cholesterol crystals. Regions rich in neutral and polar lipids stained with Nile red and fluoresced yellow or orange, respectively, owing to accumulations of lipids in both macrophages and smooth muscle cells (SMC). Digital overlays of the filipin and Nile red images revealed that larger lesions (>0.5 mm diameter) had a "nested" distribution of lipids, with a blue (filipin) fringe surrounding an orange (Nile red) fringe surrounding a yellow (Nile red) center. (J Histochem Cytochem 45:743-753, 1997)

Key Words: filipin, Nile red, computer-assisted image, processing


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