Multi-nucleate retinal pigment epithelium cells of the human macula exhibit a characteristic and highly specific distribution

Abstract

Background: The human retinal pigment epithelium (RPE) is reportedly 3% bi-nucleated. The importance to human vision of multi-nucleated (MN)-RPE cells could be clarified with more data about their distribution in central retina.

Methods: Nineteen human RPE-flatmounts (9 ≤ 51 years, 10 > 80 years) were imaged at 12 locations: 3 eccentricities (fovea, perifovea, near periphery) in 4 quadrants (superior, inferior, temporal, nasal). Image stacks of lipofuscin-attributable autofluorescence and phalloidin labeled F-actin cytoskeleton were obtained using a confocal fluorescence microscope. Nuclei were devoid of autofluorescence and were marked using morphometric software. Cell areas were approximated by Voronoi regions. Mean number of nuclei per cell among eccentricity/quadrant groups and by age were compared using Poisson and binominal regression models.

Results: A total of 11,403 RPE cells at 200 locations were analyzed: 94.66% mono-, 5.31% bi-, 0.02% tri-nucleate, and 0.01% with 5 nuclei. Age had no effect on number of nuclei. There were significant regional differences: highest frequencies of MN-cells were found at the perifovea (9.9%) and near periphery (6.8%). The fovea lacked MN-cells almost entirely. The nasal quadrant had significantly more MN-cells compared to other quadrants, at all eccentricities.

Conclusion: This study demonstrates MN-RPE cells in human macula. MN-cells may arise due to endoreplication, cell fusion, or incomplete cell division. The topography of MN-RPE cells follows the topography of photoreceptors; with near-absence at the fovea (cones only) and high frequency at perifovea (highest rod density). This distribution might reflect specific requirements of retinal metabolism or other mechanisms addressable in further studies.

Keywords: Bi-nucleate; Cell nucleus; Multi-nucleate; Retinal pigment epithelium.

Figure 1. Computer-assisted manual marking of cell nuclei

A Single image from z-stack before marking. B Single image from z-stack with Voronoi regions (surrogate for cells, thin green borders around cells) displayed and cell nuclei identified (white dots). All data within yellow edging were used for analysis. As previously described, (Ach et al., 2014) Voronoi regions (collection of regions that tessellate a plane) were generated from corresponding RPE cytoskeleton images (not shown). Scale bar: 20 μm.

Figure 2. RPE cells with one (A) or multiple (B–D) cell nuclei in normal human macula

There are mono- (A) and fewer bi-nucleate (B) RPE cells visible throughout the macula. Only a few cells with ≥3 nuclei were detectable: tri-nucleate (C) and penta-nucleate (D). Cells in regions with a high percentage of bi-nucleate RPE cells are pleomorphic in size and shape. Of note in (D), cell nuclei differ in size (blue arrow: nucleus size comparable to nuclei in surrounding cells; magenta arrow: a noticeably smaller nucleus). Foveal cells in panel A have abundant, apically located melanosomes which can be mistaken for large nuclei. A Temporal perifovea, female, 41 years. B Nasal near periphery, female, 37 years. C Superior near periphery, male, 88 years. D Superior perifovea, female, 50 years. Scale bar 20 μm

Figure 3. Percentages of multi-nucleate RPE cells as a function of eccentricity and quadrant

The percentage of multi-nucleate RPE cells at each location, i.e., quadrant at each eccentricity, is presented in this multi pie illustration: as number and area (red) of each pie. Cells were pooled across all tissues. Quadrant (T = temporal, S = superior, N = nasal, I = inferior)