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K. Seckerdieck, C. A. Curcio, S. Grisanti, M. Rudolf; Esterified-Cholesterol Containing Extra-Macular Drusen in Human Bruch’s Membrane Wholemounts: Substructures and Local Interactions. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4929.
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To evaluate the distribution of esterified cholesterol, an important druse constituent (Li et al. 2007), in extra-macular drusen in wholemount (WM) preparations of Bruch’s membrane (BrM).
BrM-choroid WMs of 3 donor eyes (76-86 years) fixed with 4% paraformaldehyde were prepared including cautious RPE-removal. WMs were stained with filipin for esterified cholesterol (Curcio et al. 2006) and extra-macular drusen were evaluated by wide-field epifluorescence microscopy with parameters previously used for drusen in histological sections by Li et al. (2007).
A total of 368 drusen were judged: 150 drusen (40.8%) demonstrated an intense and evenly distributed fluorescence throughout the entire druse (full body staining); 68 (18.5%) showed a hypofluorescent core, 10 large drusen (2.7%) had 3 cores (probably after advanced confluence); 38 drusen (10.3%) had shells; 233 drusen (63.3%) had lakes (comma-shaped hyperfluorescent areas), abundantly in 155 drusen (42.1%) and scattered in 78 drusen (21.2%). We found clusters containing up to 7 merging drusen. It is noteworthy that all merging drusen of a group exhibited the same staining characteristics.
The WM perspective reveals more information about cholesterol deposition in drusen, because sample size is large and an entire drusen can be seen as well their neighbors. Our data on druse substructure prevalence differs somewhat from our previous data obtained from sections of extra-macular drusen, as we found esterified cholesterol-rich lakes more frequently and shells less frequently than previously. Judgment of drusen confluence is more reliable in WMs than in sections. All merging drusen of a cluster exhibited the same staining pattern which might indicate that local mechanisms might initiate certain sequences of drusen biogenesis.
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