3D-Reconstructed Retinal Pigment Epithelial Cells Provide Insights into the Anatomy of the Outer Retina

Abstract

The retinal pigment epithelium (RPE) is located between the neuroretina and the choroid, and plays a critical role in vision. RPE cells internalise outer segments (OS) from overlying photoreceptors in the daily photoreceptor renewal. Changes to RPE structure are linked with age and retinopathy, which has been described in the past by conventional 2D electron microscopy. We used serial block face scanning electron microscopy (SBF-SEM) to reconstruct RPE cells from the central mouse retina. Three-dimensional-reconstructed OS revealed the RPE to support large numbers of photoreceptors (90–216 per RPE cell). Larger bi-nucleate RPE maintained more photoreceptors, although their cytoplasmic volume was comparable to smaller mono-nucleate RPE supporting fewer photoreceptors. Scrutiny of RPE microvilli and interdigitating OS revealed the angle and surface area of contact between RPE and photoreceptors. Bi-nucleate RPE contained more mitochondria compared to mono-nucleate RPE. Furthermore, bi-nucleate cells contained larger sub-RPE spaces, supporting a likely association with disease. Use of perfusion-fixed tissues ensured the highest possible standard of preservation, providing novel insights into the 3D RPE architecture and changes linked with retinopathy. This study serves as a benchmark for comparing retinal tissues from donor eyes with age-related macular degeneration (AMD) and other retinopathies.

Serial block face scanning electron microscopy reveals novel organizational details of the retinal pigment epithelium

“…An important finding was that RPE cells maintained far more photoreceptors than had been previously thought. 3D-imaging revealed that each RPE cell interacted with 90-216 photoreceptors (Keeling et al, 2020b). This is in contrast to conventional EM studies carried out in the rhesus monkey retina that showed each RPE supporting between 39-45 photoreceptors (Young, 1971).…”

“…These microvilli were angled at ~143.0° and intimately associated with the outer segments of overlying photoreceptors. Footprints of the outer segments could be observed on the microvilli bed, indicating where the distal ends of photoreceptors terminated (Keeling et al, 2020b). We speculate that the unidirectional organization of microvilli could help optimize RPE-photoreceptor interactions.…”

“…We used this technique on perfusion-fixed C57BL/6 mouse eyes to image the outer retina. Our findings revealed novel organizational details of the retinal pigment epithelium (RPE) (Keeling et al, 2020b); a specialized cell monolayer that maintains the overlying photoreceptors and also forms the outer blood-retinal-barrier. RPE cells were found to look after far more photoreceptors than was widely assumed.…”

“…In our recent work, we used SBF-SEM to investigate the architecture of healthy RPE cells from the central mouse retina Perspective J. Arjuna Ratnayaka * , Eloise Keeling (Keeling et al, 2020b). To our knowledge, the study was the first to fully reconstruct a section of the RPE monolayer in 3D.…”

“…When considered from a 3D perspective, it appears that microvilli are present around as well as in-between single or clusters of outer segments to maximize this interaction. The extent of this association can be fully appreciated by viewing a supplementary movie of a SBF-SEM stack published in the original research article (Keeling et al, 2020b). Conventional EM studies have shown how the orderly arrangement of outer segments becomes disrupted with retinopathy.…”