Abstract
Abstract: :
Purpose: To identify ultrastructural forms of esterified and unesterified cholesterol in drusen and basal deposits of aged and ARM eyes with emphasis on MemD. Classically described MemD consists of "coiled membranes" in BlinD, drusen, and in linear tracks or aggregates within basal laminar deposits. Methods: Eyes from 44 human donors (age 71–96 yr) were preserved <6 hr post mortem in 2.5% glutaraldehyde and 1% paraformaldehyde. Maculas were divided through the fovea with 1–2 blocks post–fixed in 2% osmium or 1% osmium /1.5% potassium ferri– or ferrocyanide and 1–2 blocks post–fixed in osmium– tannic acid– paraphenylenediamine (OTAP) for neutral lipid. One µm thick toluidine–blue–O–stained sections were evaluated for histopathology and to guide electron microscopic (EM) examination. Solid particles identified in OTAP preparations were considered closest to the in vivo state of extracellular lipids. Micrographs were examined for intermediate forms, with greatest weight given to comparable images from different preparations of the same eye. Results: Twenty eyes (8 ARM, 4 late exudative ARM, 8 with small basal deposits or drusen) had adequately preserved extracellular lipid. The exterior surface of MemD was thicker and more electron–dense than basal infoldings of retinal pigment epithelium (RPE) cells. In OTAP preparations, individual MemD profiles were solid, ranged in diameter from 80–200 nm, and formed tracks across or aggregations within basal laminar deposits. Solid particles and/or pools of neutral lipid were visible in BlinD and drusen. Conclusions: When lipid preserving methods are used, MemD is distinguishable from membranes of surrounding cells and vesicles possessing aqueous interiors. Solid particles which would have been extracted during conventional EM processing are visible within MemD. Pooled neutral lipid may contribute to structural instability of BlinD rendering it permissive to dissecting choroidal neovascularization. We previously showed that aged human Bruch’s membrane (BrM) features esterified cholesterol–containing 80–100 nm diameter particles, drusen and basal deposits contain apolipoprotein B (apoB), and the RPE expresses genes and proteins required for lipoprotein assembly. The current data support the hypothesis that an RPE–secreted apoB–lipoprotein with a neutral lipid core is a plausible mechanism for producing abundant esterified and unesterified cholesterol in aging BrM, drusen, and basal deposits.
Keywords: age–related macular degeneration • lipids • drusen