Geographic Atrophy: Confocal Scanning Laser Ophthalmoscopy, Histology, and Inflammation in the Region of Expanding Lesions

Abstract

Purpose: To describe the pathology of AMD in eyes with geographic atrophy (GA) using confocal scanning laser ophthalmoscopy (SLO) blue light autofluorescence (BAF), and near-infrared (IR) AF and to correlate it with the histology and immunohistochemistry analysis at the margins of the GA lesion.

Methods: Enucleated, fixed eyes from seventeen donors with GA were imaged and analyzed by BAF-SLO, IRAF-SLO, and by fundus macroscopy (FM). Tissue from the margins of the GA lesions was cut and processed for resin embedding and histology or cryosectioning and fluorescence in the green and far-red channels, and immunohistochemistry to assess markers of inflammation. Isolated DNA from donors was genotyped for single nucleotide polymorphisms (SNPs) previously shown to be risk factors for the development and progression of AMD.

Results: Around the leading edge of the GA lesions we observed hypertrophic RPE cells with cytoplasm filled with granules fluorescent both in the far-red and green-red channels; abundant microglia and macrophage; deposition of complement factor H (CFH) in Bruch's membrane (BM) and increased membrane attack complex (MAC) on RPE cells.

Conclusions: Fluorescence imaging of cryosections of RPE cells around the leading edge of the GA lesions suggest that IRAF-SLO visualizes mostly melanin-related compounds. In addition, medium-size GA atrophy displayed the most significant changes in inflammation markers.

Figure 1.

Ex vivo imaging of representative AMD donor eyes with GA. Fundus images (A–G), BAF-SLO (H–N), and IRAF-SLO (O–U) images were acquired. Representative images of control (A), AMD donors with small (E, G), medium (C, D), and large (B, F) GA area were obtained; GA is visible in all three imaging modalities. Visualization in BAF-SLO and IRAF-SLO identified areas of fovea (arrow), optic nerve (ON), and hyper-autofluorescence in GA edges (small arrows). D, retinal detachment. Scale bar: 0.5 cm.

Figure 2.

Histologic morphology of RPE and retina in GA borders of donors with AMD. Fundus image of a control eye (A, OD) with a schematic drawing of the GA areas harvested and processed for further analysis. These include the superior (region 1), nasal (region 4), temporal (region 3), and inferior (region 2). Toluidine blue–stained plastic sections (1 µm) of retinas from all AMD donors and an age-similar control. Morphologic study of the control retina (B) displayed typical retinal lamina, slightly “nonuniform” RPE structure, and pigmentation with small patches of early basal laminar deposit (BLamD, *) over the Bruch's membrane (red arrowheads). Histologic study in the edge of the GA atrophy of AMD donors displayed degenerated photoreceptors and RPE. Observed RPE degeneration morphology included “very nonuniform” RPE (C), “dissociated” RPE (D), “shedding” RPE (E); “bilaminar” RPE (F), and “sloughed” RPE (G). Yellow arrows show ELM (external limiting membrane) descending toward the Bruch's membrane to define the GA border; red arrows show shed RPE contents. D, drusen. Scale bar: 100 µm.

Figure 3.

Accumulation of autofluorescent granules in the RPE in GA borders of donors with AMD. Representative bright field images overlaid with epifluorescence image in the green-red (A, C, E, G, I, K) and far-red (B, D, F, H, J, L) channels. Human cryosections of a matched control (A, B) and AMD donor eyes at the GA border (C–L) were observed on epifluorescence in the green-red and far-red channels; autofluorescence was merged into bright field images. The RPE from a control eye showed the prevalence of granules fluorescent in the green-red (A) and far-red (B) channels and a several dark-pigment non-fluorescent granules aligned along the apical surface (white arrowheads). The AMD donor eyes at the GA border (C–L) displayed hypertrophic RPE cells with their cytoplasm filled with granules fluorescent both in the green-red and far-red channels; a few dark-pigment nonfluorescent granules are present and distributed through the cytoplasm. Ch, choroid. Scale bar: 100 µm. * Bruch's membrane.

Figure 4.

Immunocytochemistry of retinal sections in the GA borders of donors with AMD stained with complement antibodies. Immunofluorescence of control and AMD retinal sections labeled with antibodies to CFH (A, B, C, D) and C5b-9 (E, F, G, H). The control retina displayed CFH deposition in the choroid vessels, ganglion cell layer, and retinal vessel (white arrowheads). Minor reaction was also present in the plexiform layers and photoreceptor inner segments. The AMD donors with small- and large-size GA atrophy (B, D) displayed increased CFH deposition in the choroid vessels. On the other hand, CFH was decreased in AMD donors with medium size GA atrophy (C). The AMD donors also displayed increased CFH deposition in the plexiform layers and photoreceptor inner segments. In the control retinas immunohistologic staining with C5b-9 display weak diffuse pattern in the Bruch's membrane (BM)/basal surface of the RPE (E). Increased C5b-9 staining was observed in the retina of donors with GA atrophy (F–H). Increased distribution of C5b-9 was also observed in the RGC layer, and in plexiform layers of the donors with small and medium size GA atrophy (F, G) but not in donors with large size GA atrophy (H), where the majority of C5b-9 distributed in the RPE Bruch's membrane (BM)/basal surface and cytoplasm. For all images, the atrophy was located at the left edge of the picture; image breaks in BM (G) is an artifact from processing. Scale bar: 200 µm.

Figure 5.

Immunocytochemistry of retinal sections in the GA borders of donors with AMD stained with inflammatory cell marker antibodies. Immunofluorescence of control and AMD retinal sections labeled with antibodies to Iba-1 (A, B, C, D) and CD68 (E, F, G, H). Non-AMD donor displayed very few Iba-1+ cells in their retinas, with cells mostly localized in the inner retina, but there were a few also localized to the photoreceptor outer segments (A, white arrowheads). The AMD donors with small and medium size GA atrophy (B, C) displayed infiltration of Iba-1+ cells in the outer retina; these cells displayed an amoeboid phenotype. However, fewer Iba-1+ cells were present in the retinas of AMD donors with big size GA atrophy (D). Non-AMD donor (E) displayed very few CD68+ cells in their retinas. The AMD donors with small and medium size GA atrophy displayed increased presence of CD68+ cells in the inner retina (F, G, white arrowheads). However, CD68+ cells were mostly absent from AMD donors with big size GA atrophy (D). For all images, the atrophy was located at the left edge of the picture. Scale bar: 200 µm.

Figure 6.

Schematic model of the main features identified of the RPE in and out of the GA borders. The RPE outside the GA area display apical melanin granules. Their cytoplasm is filled with lipofuscin, melanolipofuscin, or both. The RPE at the edge of the GA is hypertrophic, displays decreased melanin pigmented granules and these are disorderly distributed in the cytoplasm. There is an increase in the presence of microglia in the retina in the edge of the GA when compared with retinas of control and AMD donors outside the GA. The microglia in the edge of the GA display activated morphology. Reprinted with permission, Cleveland Clinic Center for Medical Art & Photography © 2020. All Rights Reserved.