Three-dimensional reconstruction of human retinal pigment epithelium (RPE) permits a novel view of the RPE – photoreceptor outer segment (OS) interface

Abstract

Purpose : RPE cells and their interaction to photoreceptor OS are the first microconnectome of the visual system yet our understanding of the morphology relies on limited data from electron microscopy (EM). Our goal was to use volume EM to provide a detailed and unprecedented description of human RPE cells and their contact with OS.

Methods : A parafoveal retinal volume from a 28-year-old male Caucasian with an unremarkable macula was prepared by serial block-face scanning EM. The RPE cell membrane was reconstructed manually using TrakEM2 software (ImageJ). The cell body organelle content was reconstructed using advanced automated image segmentation methods (Dragonfly, Object Research Systems).

Results : Surface area and volume of a single complete RPE cell was 16898 μm² and 6042 μm³, respectively. AP (n=808) originate from the apical part of the CB and comprised 85% and 21% of the total RPE surface area and volume. AP are elongated (mean length±SD: 6926±4353 nm) and form specialized interactions with overlying OS of cones and rods. Diameter of an AP ranged from 135 to 615 nm. The cell contained 546 pigment granules (lipofuscin/ melanolipofuscin/ melanosome), with a total surface area and volume of 5028 μm² and 734 μm³. Details of volume coverage percentage of organelles are listed in the Table. We identified 438 mitochondria in the cytoplasm with a surface area and volume of 3607 μm² and 271 μm³. The nucleus showed a total surface area and volume of 274 μm² and 375 μm³. Membranous tubules and sacs in the cytoplasm resembling endoplasmic reticulum, yet to be confirmed by histochemical analysis, showed a total surface area and volume of 5321 μm² and 988 μm³.

Conclusions : The apical surface of RPE and its relationship to photoreceptor OS is more extensive and complex than previously appreciated from light microscopy and single section EM studies. The surface area for molecular exchange vital for vision is vast. Cytoplasmic organelles are important clinical imaging signal sources. Our data will assist the interpretation of clinical high-resolution optical coherence tomography in early detection of neurodegenerative retinal diseases including age-related macular degeneration, for explicating the viral vector route in subretinal gene therapy, and benchmarking cells used for cell-based therapy and disease modeling.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

 

Table: Volume coverage % of organelles