Distribution and composition of esterified and unesterified cholesterol in extra-macular drusen

More details about the distribution of esterified and unesterified cholesterol (EC, UC), abundant druse components, would inform models of druse biogenesis and new technologies for ocular imaging. From donors with grossly normal maculas (n=10, 66-86years), whose eyes were preserved in paraformaldehyde within 6h of death, extra-macular drusen encased with retinal pigment epithelium (RPE) were isolated manually. Cryosections of pelleted drusen, stained with filipin for UC and EC, were used to investigate filipin staining patterns within single drusen (n=193) and to quantify fluorescence (n=146). From lipid extracts of other drusen/RPE and RPE samples, total cholesterol (TC) and UC were determined by enzymatic fluorimetry. Drusen contained cores, basally located regions that were intensely bright when stained for UC or deeply dark when stained for EC; many were surrounded by concentric lamellae. Within the same cores, the EC-poor regions were significantly smaller (13.0mum) than UC-rich regions (17.1mum). Drusen with highly fluorescent EC-rich shells lacked UC-rich shells. Small spots representing lakes were visible only in drusen stained for EC. Some drusen had small, refractive spherical inclusions lacking both UC and EC. Of drusen examined, 32% had a UC-rich core, 35% had an 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. The percentage of TC that was esterified ranged from 32-66% for drusen/RPE and 5-21% for RPE. The disposition of cholesterol in cores may reflect the activity of invading cellular process. The greater size of UC-rich cores relative to EC-poor cores may reflect a declining gradient of enzymatic activity with increased radial distance from the putative invaders. The relative sizes of sub-domains defined by cholesterol composition are compared to sub-domains detected in drusen by in vivo imaging methods.