Variation and Distribution of Lipofuscin Granules in Human RPE Cells Determined With Confocal Scanning Laser Microscopy

Abstract

Abstract: : Purpose: To image and determine the distribution of lipofuscin (LF) granules in human retinal pigment epithelial (RPE) cells using confocal scanning laser microscopy (cSLM). Methods: From 16 human donor eyes aged 20.9 – 81.0 (mean 48.8 ± 23.6) years trephined RPE/Bruch’s/choroid specimen from defined macular, nasal, temporal sites were obtained. Lipofuscin granules were imaged at exc 488 nm and em at 509–554 nm with a cSLM (Leica TCS SL, Leica, Germany) due to the intrinsic fluorescence. Nuclei were stained with propidiumiodide and emission recorded at 710 – 771 nm. Distribution of melanin granules was examined with light microscopy. Results: LF granules were readily visualized in the confocal planes through each RPE cell. Their predominant location was in the basal and midportions of the cell with a higher density towards the cell borders whereas melanin granules showed an apical polarisation and were more centrally located. With age there was an increase in LF granule density at all sites as well as an increase in intercellular variability. There was also an age–dependent significant increase in the LF density at the apical cell portion with age. A higher density of LF was noted at the posterior pole compared to specimen from the nasal or temporal periphery. Greater variability of RPE cell size and shape, i.e. pleomorphism, was found in the periphery compared to the central specimen in addition to an increasing number of RPE cells with two nuclei. Some of the LF granules showed a substructure at the cSLM level. Conclusions: CSLM is a suitable tool to image LF granules in human RPE cells ex vivo in high–resolution and at defined vertical confocal layers. Our findings show a considerable variation of LF distribution as well as RPE cell morphology in dependence of age and to fundus localization. This technique can be used to further elucidate the pathophysiological role of dynamic age– and disease–associated alterations in cytoplasmic LF granule accumulation and distribution. It will also be useful for interpreting findings from fundus autofluorescence (FAF) imaging in vivo obtained with confocal scanning laser ophthalmoscopes. German Research Council DFG Bonn, Germany, Grant Ho 1926/1–3, SPP 1088

Keywords: retinal pigment epithelium • aging • age-related macular degeneration