Reconstruction of apical processes (AP) originating from human retinal pigment epithelium (RPE) cell bodies by 3D connectomics technology

Abstract

Purpose : To determine for the first time the number, dimensions, branching points, and signal generating organelle contents of apical processes (AP) of human RPE cells using 3D connectomics technology; these membrane specializations function in photoreceptor phagocytosis (PMID 16301) and retinoid processing (PMID 15336505).

Methods : A whole globe of a 21-year-old male Caucasian organ donor with an unremarkable macula was processed via rapid organ recovery. Perifoveal epoxy-embedded RPE was imaged by serial block-face scanning electron microscopy. In our ongoing study the image stack is annotated manually with computer assistance by expert readers using TrakEM (ImageJ).

Results : The 3D dataset displayed complete ultrastructural preservation with fully attached neurosensory retina and allowed the reconstruction of RPE cells. Our initial results from this tissue sample indicate 313 APs originating from the apical part of one RPE cell body and extending into the interphotoreceptor matrix. These interact with outer segments of 38 rod photoreceptors (cones were not accessible). 57% of APs show branching with 21% having one branch, 13% two branches, 8% three branches and 14% four branches or more resulting in a total of 814 endtips of APs. The mean length of a single AP branch was 1694 ± 1558 (standard deviation) nm. APs without branching showed a mean length of 1423 ± 1237 nm from start to endtips. The diameter of an apical process ranged from 135 to 615 nm. APs of one single RPE cell contained 67 melanosomes and 3 lipofuscin granules, which is in range of previously published data by our group.

Conclusions : Our 3D connectomics approach revealed that hundreds of APs derive from the apical part of a single RPE cell body, ensheath photoreceptors and contain the majority of RPE cell melanosomes; the latter are known contributors to OCT reflectivity via Mie scattering.
Results will impact interpretation of clinical OCT images. They will also help elucidate an ultrastructural basis of how rod-mediated dark adaptation is modulated. This functional biomarker for incident age-related macular degeneration localizes to the photoreceptor-RPE-choriocapillary interface (PMID 33344065).

This is a 2021 ARVO Annual Meeting abstract.