The Project MACULA Retinal Pigment Epithelium Grading System for Histology and Optical Coherence Tomography in Age-Related Macular Degeneration

Abstract

Purpose: To seek pathways of retinal pigment epithelium (RPE) fate in age-related macular degeneration via a morphology grading system; provide nomenclature, visualization targets, and metrics for clinical imaging and model systems.

Methods: Donor eyes with geographic atrophy (GA) or choroidal neovascularization (CNV) and one GA eye with previous clinical spectral-domain optical coherence tomography (SDOCT) imaging were processed for histology, photodocumented, and annotated at predefined locations. Retinal pigment epithelial cells contained spindle-shaped melanosomes, apposed a basal lamina or basal laminar deposit (BLamD), and exhibited recognizable morphologies. Thicknesses and unbiased estimates of frequencies were obtained.

Results: In 13 GA eyes (449 locations), 'Shedding,' 'Sloughed,' and 'Dissociated' morphologies were abundant; 22.2% of atrophic locations had 'Dissociated' RPE. In 39 CNV eyes (1363 locations), 37.3% of locations with fibrovascular/fibrocellular scar had 'Entombed' RPE; 'Sloughed,' 'Dissociated,' and 'Bilaminar' morphologies were abundant. Of abnormal RPE, CNV and GA both had ~35% 'Sloughed'/'Intraretinal,' with more Intraretinal in CNV (9.5% vs. 1.8%). 'Shedding' cells associated with granule aggregations in BLamD. The RPE layer did not thin, and BLamD remained thick, with progression. Granule-containing material consistent with three morphologies correlated to SDOCT hyperreflective foci in the previously examined GA patient.

Conclusions: Retinal pigment epithelium morphology indicates multiple pathways in GA and CNV. Atrophic/scarred areas have numerous cells capable of transcribing genes and generating imaging signals. Shed granule aggregates, possibly apoptotic, are visible in SDOCT, as are 'Dissociated' and 'Sloughed' cells. The significance of RPE phenotypes is addressable in longitudinal, high-resolution imaging in clinic populations. Data can motivate future molecular phenotyping studies.

Figure 1

Graphical hypothesis of RPE pathways in AMD. The schema omits normal aging changes and begins with ‘Very Nonuniform.’ RPE cells proceed to more advanced epithelial and nonepithelial grades. Three RPE-derived morphologies (grayed out) are reported separately.^, ‘Dissociated’ and ‘Entombed’ are the final steps before dissolution of the RPE layer (atrophy). ‘Dissociated’ cells likely give rise to cellular fragments and loose granules seen in the neurosensory retina. Direct and primary contributors to ‘Dissociated’ appear to be the epithelial components of ‘Sloughed’/‘Intraretinal’ and ‘Shedding.’ ‘Sloughed’ RPE can become ‘Intraretinal.’ Some enter the lumen of outer retinal tubulation from the subretinal space.^, The epithelial component of ‘Shedding’ contains intracellular granule aggregations, less readily appreciated in histology, which are released into BLamD and may represent apoptotic bodies. The relationship between ‘Bilaminar’ and ‘Entombed RPE’ is hypothetical, and the fate of rare ‘Vacuolated’ RPE cells is unknown. The term atrophy does not specify the fate of individual cells. Death is one possible outcome. Alternatives are transition to a form that does not meet our criteria for RPE (e.g., loss of granules) and emigration. Not all morphologies depigment; the principal ones are ‘Entombed’ and ‘Subducted.’ nvAMD, neovascular AMD.

Figure 2

Grades of RPE morphology in late AMD. Submicrometer epoxy resin sections were stained with toluidine blue. Epithelial RPE and RPE morphologies with epithelial components (A, B, D, E, G, I, J); nonepithelial (noncontinuous) morphologies (C, F); atrophic RPE (H, K). (A) ‘Nonuniform’ RPE: slightly ‘Nonuniform’ morphology and pigmentation with small patches of early BLamD. (B) ‘Very Nonuniform’ RPE: more nonuniformity in shape and pigmentation; melanosomes within apical processes (pink arrowhead). Subretinal drusenoid deposits (SDD) localize to RPE apical aspect. (C) ‘Dissociated’ RPE: individual RPE cells with or without nuclei in atrophic area, adherent to early BLamD. Some RPE granules are translocated among HFL fibers. (D) ‘Shedding’ RPE: basal translocation of shed RPE fragments into a thick continuous layer of BLamD (late and early forms shown by large and small yellow arrowheads, respectively); BLinD (black arrowheads). (E) ‘Intraretinal’ RPE: anterior migration through ELM. Epithelial component remains atop BLamD (bottom), which in turn overlies an artifactually empty soft druse. Photoreceptors have degenerated. Retina is artifactually detached. (F) Cells ‘Entombed’ by a subretinal scar (s) together with nonpigmented cells. Persistent BLamD divides subretinal fibrocellular scar in the subretinal space from fibrovascular scar (fv.s) in sub-RPE space. (G) ‘Sloughed’ RPE: release of spherical cells into the subretinal space; the epithelial component overlies BLamD (blue) and BLinD (gray). (H) ‘Atrophy with BLamD’: absent RPE and persistent BLamD. Photoreceptors have atrophied. ELM delimits end-stage outer retinal tubulation. (I) ‘Bilaminar’: double layers of epithelial RPE (delimited by dotted line) adherent to BLamD. (J) ‘Vacuolated’ RPE: cells with a single large vacuole delimited apically by extremely effaced cytoplasm. (K) ‘Atrophy without BLamD’: absent RPE, absent BLamD. Photoreceptors have atrophied. Yellow arrowheads: BLamD; red arrowheads: calcification in BrM; green arrowheads: ELM. BLamD, basal laminar deposits; BLinD, basal linear deposits; ELM, external limiting membrane; HFL, Henle fiber layer; INL, inner nuclear layer; RPE, retinal pigment epithelium.

Figure 3

‘Dissociated’ RPE in eyes with late AMD. Yellow arrowheads: BLamD; red arrowheads: calcification in BrM; green arrowheads: ELM. (A–C) Photoreceptors have atrophied, and ELM is absent. (A) RPE cells adhere to thick late BLamD. Groups of RPE granules have been shed into BLamD. Isolated granules also spread into HFL. Focal extracellular deposits of gray-stained soft druse material (basal mounds) are visible at outer BLamD (black arrowheads). (B) ‘Dissociated’ RPE cells, adherent to BLamD; pigmented granules spread in HFL and in BLamD. (C) Two RPE cells detached from BLamD. (D) Individual RPE cells on BrM at a GA border with curved ELM and reduced photoreceptor nuclei. GCL, ganglion cell layer; other abbreviations as in Figure 2.

Figure 4

‘Entombed’ RPE in eyes with CNV. Yellow arrowheads: BLamD; red arrowheads: calcification in BrM. (A) ‘Entombed’ RPE and nonpigmented cells inside a subretinal fibrovascular scar (fv.s). Thick BLamD separates subretinal fibrovascular scar from sub-RPE fibrous scar (s). A slightly pigmented monolayer on the scar internal aspect contacts photoreceptor inner segments. (B) A cluster of cells, with nuclei on two levels, adjacent to thin and discontinuous BLamD is between fibrovascular subretinal and sub-RPE scars (fv.s). (C) ‘Entombed’ RPE intermingled with fibrin (pink asterisk). BLinD, black arrowheads. Photoreceptor outer and inner segments are lost. (D) ‘Entombed’ RPE cells in a row on BrM inside a fibrovascular subretinal scar (fv.s). The nucleus of RPE cell on the right is displaced eccentrically by intracellular fluid. Abbreviations as in Figure 2.

Figure 5

Distribution of RPE grades in eyes with GA and choroidal neovascularization. Superior and Central sections of GA eyes (n = 13) and CNV eyes (n = 39). The Superior section is located 2 mm superior to the Central section. RPE morphologies are indicated by colored bars: Epithelial (blue), Nonepithelial (green), and Atrophic (orange). The numbers atop each column represent the number of affected locations. RPE morphologies in 150 Superior and 299 Central locations of eyes with GA show many locations with ‘Dissociated’ and ‘Atrophy with BLamD.’ RPE morphologies in 452 Superior and 911 Central locations in eyes with CNV show many locations with ‘Entombed RPE’ and ‘Atrophy.’

Figure 6

RPE and BLamD thickness in eyes with advanced AMD. Epithelial, Nonepithelial, and Atrophic RPE are blue, green, and orange bars, respectively. At the top of each column is mean RPE or BLamD thickness in combined Superior and Central sections. Same sample size as in Figure 5. (A) RPE thickness does not decrease with perturbed morphology. Trends for different RPE morphologies are similar between GA and CNV eyes. ‘Entombed’ RPE's thickness is >1.5 times that of Nonuniform RPE. (B) BLamD thickness is maintained with advancing pathology.

Figure 7

Clinicopathologic correlation of RPE morphologies. The right eye of a 98-year-old white woman with advanced AMD (both GA and CNV) was clinically examined with SDOCT imaging 8 months before death. (A, B) In vivo SDOCT scans match histology (C, D), respectively. GA borders are defined by the end of curved ELM (green dots) in (A, C). Bar in (A) applies to both. (A) Increased choroidal reflectivity in GA and hyperreflective scar over BrM. A dotted hyperreflective line crosses GA (pink arrowheads). It is separated from the scar by a hyporeflective layer (yellow asterisk) containing additional hyperreflective dots (yellow arrowhead). (B) Hyperreflective dots (blue arrowhead) over a continuous RPE-BrM complex band (green arrowhead) outside GA and individual hyperreflective dots inside GA (pink arrowhead). The ELM band is visible. (C, D) Low magnification of different histologic sections from this eye that match SDOCT scans in (A, B). Bar in (C) applies to both. (C) Central GA and sub-RPE fibrovascular scar. Multiple Dissociated RPE cells overlie very thick BLamD containing granule aggregates (E, F). Inner retinal layers are continuous outside the gap at the foveal depression. (D) GA border; ‘Nonuniform’ and ‘Sloughed’ RPE outside GA, two ‘Dissociated’ RPE cells inside GA. (E–G) High magnification of histology of (A, B). BLamD, yellow asterisk; fv.s, fibrovascular scar. Bar in (E) applies to all. It was possible to match classes of hyperreflective spots to classes of cells, if not individual cells and spots. However, the configurations of spots in (A) and cells in (E–G) are not identical. (E) ‘Dissociated’ nucleated RPE cells (pink arrowhead) on thick early and late BLamD with granule aggregates and two granule-containing cells (a rare finding, yellow arrowhead). (F) ‘Dissociated’ RPE cells (pink arrowhead) lying on BLamD and ‘Dissociated’ RPE cells migrating toward the INL. ‘Shedding’ RPE inside BLamD (yellow arrowhead). With ELM absent, these cells are not called ‘Intraretinal.’ Pigmented cells located within the scar are ‘Subducted’; their reflectivity may be indistinguishable from that of the surrounding scar. (G) Epithelial (green arrowhead) and nonepithelial (blue arrowhead) cells of the ‘Sloughed’ morphology, in the subretinal space outside GA. Two ‘Dissociated’ RPE cells are seen inside GA (pink arrowhead).