Fundus Autofluorescence Variation in Geographic Atrophy of Age-Related Macular Degeneration: A Clinicopathologic Correlation

Abstract

Purpose: The purpose of this study was to develop ground-truth histology about contributors to variable fundus autofluorescence (FAF) signal and thus inform patient selection for treating geographic atrophy (GA) in age-related macular degeneration (AMD).

Methods: One woman with bilateral multifocal GA, foveal sparing, and thick choroids underwent 535 to 580 nm excitation FAF in 6 clinic visits (11 to 6 years before death). The left eye was preserved 5 hours after death. Eye-tracked ex vivo imaging aligned sub-micrometer epoxy resin sections (n = 140, 60 µm apart) with clinic data. Light microscopic morphology corresponding to FAF features assessed included drusen-driven atrophy, persistent hyperautofluorescence (hyperFAF) islands and peninsulas within atrophy, and hyperFAF and hypoautofluorescence (hypoFAF) inner junctional zone (IJZ) and outer junctional zone (OJZ) relative to descent of external limiting membrane (ELM). Atrophy growth rate was calculated.

Results: HypoFAF atrophic spots appeared in association with drusen, and then expanded and coalesced. Over drusen (n = 45, all calcified), RPE was continuous and thin, photoreceptors were short or absent, and initially intact ELM descended where RPE was absent. In persistent hyperFAF within atrophy and in the OJZ, the RPE was continuous and dysmorphic, photoreceptors were present and short, and BLamD was thick. In the IJZ, mottled FAF corresponded to dissociated RPE atop persistent BLamD. Overall linear growth rate (0.198 mm/ year) typified multifocal GA.

Conclusions: FAF in GA is locally multifactorial, with photoreceptor shortening potentially promoting hyperFAF by increasing incoming excitation light available to RPE fluorophores. RPE dysmorphia may lead to either longer or shorter pathlength for excitation light. At both atrophy initiation and expansion Müller glia are major participants.

Figure 1.

Representative patterns of fundus autofluorescence. Magnified fundus autofluorescence from the last clinic visit 6 years before death shows characteristic features. The green lines indicate the areas for which comparable histology was explored, as shown in subsequent figures. Drusen exhibit defined stages of FAF^, (Figs. 2, 7). Peninsulas and islands of persistent hyperFAF in the atrophic area are illustrated in Figure 7. The hyperFAF outer junctional zone (OJZ), hypoFAF inner junctional zone (IJZ) with puncta of FAF signal, and hypoFAF atrophic area are illustrated in Figure 8. For comparison, iso FAF uninvolved retina is found in the upper right corner.

Figure 2.

Stages of drusen-driven atrophy in fundus autofluorescence (FAF). Schematic is derived from previous clinicopathologic correlation (stages 1–4; inserted step 3’²) Top row: Affected retinal layers: + = present; – = decreased or not present; –/+ = mixed effects (see Methods and Table 1). Panels a and b refer to an FAF annulus and center, respectively. Middle row: FAF patterns (stage 1 = visible in color only; stage 2 = uniform hyperFAF; stage 3 = annulus of hyperFAF (a) around a center of hypoFAF (b); stage 3’ = larger hypoFAF center; and stage 4 = uniform hypoFAF). Bottom row: One RPE-capped druse and overlying external limiting membrane (ELM, dashed line) are shown. The color scale indicating progressive replacement of lipoprotein-rich soft druse material with hydroxyapatite nodules is qualitative. As photoreceptors shorten and disappear (first OS, and then IS), the ELM approaches the druse apex (stage 2). When photoreceptors are absent, the ELM is formed by reactive Müller glia alone and skims the druse ape. The RPE layer begins to disappear beneath it (stage 3). The ELM forms a circle partway down the druse slope coincident with RPE absence (stage 3’), then further down to the druse base (stage 4). Basal laminar deposit (BLamD) is present at all stages. IS, inner segment; OS, outer segment; RPE, retinal pigment epithelium.

Figure 3.

Multimodal imaging at the last clinic visit, 6 years before death. (A, B, C) Green lines indicate level of the optical coherence tomography (OCT) B-scan (D) and tissue section (E). (A) Color photography shows multifocal areas of hypopigmented geographic atrophy with foveal sparing. The entire central area is replete with refractile drusen with soft drusen superior temporally. (B) By near-infrared reflectance, many drusen have punctate reflectivity. Atrophy is most visible superior to the fovea. A thick choroid overall reduces signal from the sclera. The green arrowhead, subretinal drusenoid deposit; these are also apparent at the top of the panel. (C) Fundus autofluorescence at 435 to 580 nm excitation shows multifocal atrophy that is confluent superior, nasal, and temporal to the fovea, which is spared. (D, E) By OCT (D) and histology (E), choroid (yellow arrowheads) is thick nasally and thin temporally. The pink arrowhead indicates a calcified druse with punctate and linear reflectivity (D) and retina detached from a large druse (E) that is not visible in this section.

Figure 4.

Progression of atrophy in 535 to 580 nm excitation fundus autofluorescence. (A–F), Images from six clinic visits are shown, with time before death in years. Images are aligned for continuous viewing in Supplementary Video S2. Selected images are magnified in Figure 4. Many individual spots of atrophy grow and coalesce over this period, beginning with superior-nasal and inferior to the fovea. As these spots expand, islands and peninsulas of hyperFAF may persist in the atrophic area for several years. A hyperFAF border is apparent first nasally, then throughout, by the end of this period. Original circular images were cropped to ovals showing identical regions.

Figure 5.

Fundus autofluorescence (FAF) showing stages over drusen. A progression through stages over drusen are shown in Figure 2. Time points in years before the patient’s death are indicated. Variations in illumination cause changes in the FAF signal and the appearance of atrophic areas. Scale bar = 200 µm (A–E). A melt through these images is shown in Supplementary Video S2. (A) Multifocal geographic atrophy with foveal sparing and predominantly stages 4 and 2 FAF over drusen in the central area and near-periphery, as defined. (B) Geographic atrophy progresses toward the fovea, with coalescence in the nasal, superior, and inferior sectors. FAF over drusen is stage 3 to 3’. (C) As atrophy expands, islands and a peninsula emerge, with FAF over drusen progressing (black arrowhead). FAF remains stable in some regions (yellow/orange arrowheads). (D) Teal arrowheads show the transition between stages 2 and 3 FAF over drusen (teal dotted box magnified in F). Superior and inferior regions reach stages 3 (brown arrowheads) and 4 (black arrowheads). (E) Most FAF over drusen progresses to stage 4 (black arrowheads), except for a stable patch (orange arrowheads). (F) Magnified inset of D, highlights 2 drusen transitioning from stage 2 to 3 (upper teal arrowhead) and stage 3 to 3’ (lower teal arrowhead, scale bar = 100 µm).

Figure 6.

Cross-sectional histology of a large druse complex and overlying retinal layers. Five histological sections (60 µm apart from superior to inferior) through one druse complex. This complex, located inferior temporal to the fovea, has two atrophic areas bounded by external limiting membrane descents (ELMd) (A, B, D, E) and an area with photoreceptors and intact ELM (C). (A–E) The Henle fiber layer/ outer nuclear layer (HFL/ ONL) exhibits dyslamination (ONL depopulation, inward migration of photoreceptor cell bodies across the HFL, and gliosis of interleaved Müller cell processes which are pale-stained. A thick basal laminar deposit (BLamD) encapsulates the complex. Druse content of numerous calcific nodules is dislodged in processing. (A) ELMd are at the druse base, and RPE is absent (stage 4). The orange arrowhead shows most visible lumen of a diminished choriocapillaris. (B) At the druse apex, RPE absence is encircled by an ELMd (stage 3’). The druse base shape verticalizes the RPE layer, for longer pathlength of incoming excitation light (arrow). (C) The druse is encapsulated by dysmorphic RPE (stages 2 and 3). The ELM does not descend but skims over the druse top, because photoreceptors are present. Sloughed RPE cells (arrow) increase the vertical pathlength for excitation light. (D) A short region of RPE absence underlies a shorter region of ELM absence (stages 2 and 3). The druse base shape verticalizes the RPE layer (arrow). (E) Regions of absent RPE and ELM are larger than in D. BrM, Bruch membrane; Ch, choroid; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segments; OPL, outer plexiform layer; OS, outer segments; RPE, retinal pigment epithelium; SDD, subretinal drusenoid deposits.

Figure 7.

Islands and peninsulas of persistent hyperautofluorescence in atrophy. In an area of persistent hyperFAF, RPE (orange) is continuous, overall thick, with some ectopic pigmented cells, and a thin continuous basal laminar deposit. The presence of RPE distinguishes this formation from the outer retinal tubulation. Overlying photoreceptors have outer segments. Whether debris between outer segments and RPE contains subretinal drusenoid deposits or shed inner segments 111 awaits ultrastructural investigation. The ELM is intact (green arrowheads) and separates this region from atrophy at the right and left. Atrophic area at right has dissociated RPE atop persistent basal laminar deposit and a druse from which calcified nodules were dislodged. Ch, choroid; HFL/ONL, Henle fiber layer and outer nuclear layer combined due to dyslamination; INL, inner nuclear layer; IPL, inner plexiform layer; OPL, outer plexiform layer; RPE-BL-BrM, RPE-BLamD-Bruch membrane complex; Sc, sclera, v, vein. The arrow indicates most visible lumen of a diminished choriocapillaris.

Figure 8.

Histology of fundus autofluorescence patterns at the border of atrophy. (A) Optical coherence tomography (OCT) B-scan through drusen-driven atrophy contextualizes histology of junctional zones. This image was captured at the last clinic visit (ART 12, Quality 37) and expanded axially to reveal detail. It is located approximately 1620 µm superior to where histology is taken 6 years later but is nevertheless representative because the atrophy expanded inferiorly. Six green arrowheads indicate descents of the external limiting membrane (ELM). A central area of atrophy is delimited by green arrowheads 3 and 4. Within that area, the OPL subsidence occurs at the pink arrowheads 1 and 2. To the right of pink arrowhead 2, where the OPL is still visible, are junctional zones comparable to the histology in panels B and C. Reflective “crownlike elevations” in the atrophic area are calcified drusen with persistent overlying basal laminar deposit (BLamD). Paired green arrowheads 1 and 2 and 5 and 6 indicate small atrophic spots outside the central area which show ELM descents over individual deposits. The ELM is barely visible over one retinal pigment epithelium (RPE)-capped druse at the far right (no arrowheads). (B) The ELM descent divides a hyper fundus autofluorescence (FAF) outer junctional zone on the right, from the inner junctional zone of atrophy, where hypoFAF is mottled due to autofluorescent puncta (see Figure 1). The remainder of the atrophic zone, at the left, is hypoFAF. The yellow frame indicates the area magnified in panel C. d, druse; green arrowheads, ELM; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; HFL/ONL, Henle fiber layer and outer nuclear layer combined due to dyslamination; RPE-BL-BrM, RPE-BLamD-Bruch membrane complex; Ch, choroid. C. HyperFAF, photoreceptors lacking outer segments, continuous dysmorphic RPE, thick BLamD; Mottled FAF, absent photoreceptors, dissociated RPE (orange arrowhead), thin BLamD; HypoFAF, absent photoreceptors, absent RPE, thin BLamD. Yellow arrowhead, horizontally oriented processes of Müller glia in a subretinal membrane. White arrowhead, BLamD protuberances, attached to (left) and detached from (right) continuous BLamD; White arrow indicates most visible lumen of a diminished choriocapillaris.