Mikroskopie

Purpose : Phenotypic alterations of retinal pigment epithelium (RPE) cells are central in age-related macular degeneration (AMD). Shape descriptors for individual RPE cells based on RPEs cytoskeleton may help to delineate healthy from AMD affected cells, even in early stages of disease. Here, we quantified differences in RPE morphology between donors with unaffected macula and with AMD. Methods : Twenty-two human RPE flatmounts (7 AMD, mean age 85 ± 3 yrs (early: 3, geographic atrophy: 1; neovascular: 3;); 15 unaffected (≤51 yrs, n = 8; >80 yrs, n = 7)) were imaged at different locations (fovea, perifovea, near periphery) using a laser scanning confocal fluorescence microscope (exc. 488 nm; emission: 490-695 nm; z-stack: 390 nm steps). Hypoautofluorescent gaps between adjacent RPE cells were formed by the absence of lipofuscin/melanolipofuscin granules between the F-actin cytoskeletons. These gaps were manually marked with computer-assistance and considered borders between cells. Shape descriptors including form factor (measure for circularity), area, solidity, convexity, and roundness for each cell were calculated using customized software. Total autofluorescence (AF) per cell was defined as the integrated density of intensity from all pixels within the cell. Statistical analysis was performed using an ensemble classifier based on logistic regression. Results : Comparing young vs healthy aged eyes, there was a trend to increased area and reduced form factor. In AMD eyes, RPE shape was significantly altered at all locations with area, solidity and form factor being the most discriminatory descriptors. In samples from healthy donors, with increasing distance to the fovea, area, solidity, and convexity increased, while form factor decreased. Finally, reduced RPE cell AF in AMD was significantly associated with decreased roundness and solidity of the RPE. Conclusions : Morphological RPE cell alterations in presence of AMD can be accurately quantified using cell shape descriptors. Enlarged and deformed cells in AMD are assumed to be indicative of structurally and functionally impaired RPE cells. The results of this study may help guide the interpretation of RPE morphology in in vivo studies utilizing high-resolution single cell imaging, e.g., adaptive optics scanning laser ophthalmoscopes or transscleral optic phase imaging.