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C.-M. Li, M. E. Clark, M. Rudolf, L. Wang, C. A. Curcio; Distribution and Composition of Esterified and Unesterified Cholesterol in Extra-Macular Drusen. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3019.
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© ARVO (1962-2015); The Authors (2016-present)
To elucidate aspects of cholesterol distribution within drusen that would inform models of their biogenesis and new technologies for ocular imaging.
Eyes obtained from donors within 6 hr of death were preserved in paraformaldehyde. Drusen encased with retinal pigment epithelium (RPE) were isolated manually from 10 donors (age, 66-86 yr) with grossly normal macula. Pellets containing extra-macular drusen were cryosectioned at 10 µm. Sections were stained with filipin. Three images (differential interference contrast, autofluorescence, and filipin) were taken at the same exposure times for each image type. Filipin fluorescence intensity was measured using IP Lab Spectrum 3.5 software. A total of 193 drusen stained for both UC and EC were used to investigate distinctive filipin staining patterns (shell, core, lake, and inclusion). From other drusen/RPE and RPE samples alone lipids were extracted using chloroform and methanol (2:1). An enzymatic fluorimetric assay was performed to determine total cholesterol (TC) and unesterified cholesterol (UC) in lipid extracts.
Cores that were UC-rich and EC-poor were observed in drusen. Some core-bearing drusen had distinctive concentrically laminated cores that contained a central region, either deeply dark when stained for EC or intensely bright when stained for UC. At a mean diameter of 13.0 µm, the dark regions (reflecting EC paucity) were significantly smaller than the bright regions (17.1 µm, reflecting UC abundance in the same druse. Drusen with highly fluorescent EC-rich shells lack a UC-rich shell. Drusen may have spots representing lakes of neutral lipid visible only when stained for EC but not UC. Some drusen had small, refractive spherical inclusion visible by DIC imaging that stained neither for UC nor EC. Of 193 drusen examined, 32% had a UC-rich core, 35% had a EC-poor core, 31% had an EC-rich shell, 25% had EC-rich lakes, and 4-5% had UC-, EC-poor inclusions. Shells and cores occurred in significantly non-overlapping druse populations. TC recovered ranged from 26.1-109.5 nmoles/mg for drusen/RPE and 5.9-15.1 nmoles/mg for RPE. The percentage of EC ranged from 32-66% for drusen/RPE and 5-21% for RPE.
The core center may reflect the activity of invading cellular process. The greater size of UC-rich centers relative to the EC-poor centers may reflect a declining gradient of enzymatic activity with increased radial distance from the putative invaders. An EC-rich shell may account a hydrophobic shell of drusen postulated by others. It is possible that this shell will be visible in the living fundus.
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