Microscopy

Purpose: Phenotype alterations of the retinal pigment epithelium (RPE) are a main characteristic of age-related macular degeneration (AMD). Individual RPE cell shape descriptors may help to delineate healthy from AMD-affected cells in early diseasestages. Methods: Twenty-two human RPE flat mounts (7 eyes with AMD [early, 3; geographicatrophy, 1; neovascular, 3); 15 unaffected eyes [8 aged ≤51 years; 7 aged >80 years)]were imaged at the fovea, perifovea, and near periphery (predefined sample locations)using a laser-scanning confocal fluorescence microscope. RPE cell boundaries were manually marked with computer assistance. For each cell, 11 shape descriptors were calculated and correlated with donor age, cell autofluorescence (AF) intensity, and retinal location. Statistical analysis was performed using an ensemble classifier based on logistic regression. Results: In AMD, RPE was altered at all locations (most pronounced at the fovea),with area, solidity, and form factor being the most discriminatory descriptors. In the unaffected macula, aging had no significant effect on cell shape factors; however, with increasing distance to the fovea, area, solidity, and convexity increased while form factor decreased. Reduced AF in AMD was significantly associated with decreased roundness and solidity. Conclusions: AMD results in an altered RPE with enlarged and deformed cells that couldprecede clinically visible lesions and thus serve as early biomarkers for AMD onset. Our data may also help guide the interpretation of RPE morphology in in vivo studies utilizing high-resolution single-cell imaging. Translational Relevance: Our histologic RPE cell shape data have the ability to identify robust biomarkers for the early detection of AMD-affected cells, which also could serve as a basis for automated segmentation of RPE sheets.