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M. Rudolf, C.-M. Li, M. F. Chimento, C. A. Curcio; Topography of Esterified Cholesterol Deposition in Human Bruch Membrane (BrM) Wholemounts. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2184. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a wholemount method for demonstrating the topography and local variations of neutral lipid deposition in human BrM.
Four human eyes (age 64-86) without grossly visible drusen or pigmentary changes in the macula were preserved in paraformaldehyde. BrM-choriocapillaris wholemounts were created by completely removing the retinal pigment epithelium, drusen, basal laminar deposits, and large choroidal vessels. Completeness of dissection was monitored on representative samples by electron microscopy. For maximum tissue flatness the wholemounts were mounted on organosilane-coated coverslips. The tissue was stained with oil red O, Nile red, BODIPY 493/503, and filipin for esterified cholesterol (FEC) following ethanol extraction and cholesterol esterase treatment. Wholemounts were evaluated by widefield epifluorescence and light microscopy. Fluorescence intensity was measured and analyzed from images of different BrM regions obtained with the same exposure parameters.
Only FEC positive material was highly localized in BrM and intercapillary pillars and revealed distinctive distribution patterns. In general the intercapillary pillars were more intensely stained than the adjacent BrM. FEC positive particle-like structures were evenly distributed in BrM, fine-grained in the periphery and more intense, more densely packed, and coarse-grained in the macula. In comparison all other stains were rather indistinct and largely affected by background artifacts. In part choriocapillaris structures were also stained and BrM specific effects could not be clearly distinguished. FEC fluorescence measurements of BrM omitting intercapillary pillars showed a gradual decline of intensity from the foveal region to the periphery. Statistical analysis confirms that the fluorescence intensity in the foveal region (144.9+/-26.1 arbitrary units) is significantly higher than in the outer macula (130.7+/-26.3; p=0.02), midperiphery (123.5+/-19.0; p<0.01), and outer periphery (77.1+/-23.7; p<0.01). The intensity of outer macula and midperiphery did not differ (p=0.24).
Esterified cholesterol is almost exclusively found in BrM and shows a distinct pattern of distribution in BrM wholemount preparations. The concentration is significantly higher in the central macula and gradually declines to the periphery. This topography contributes to the susceptibility of the macula to age-related changes and degeneration.
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