Fundus Autofluorescence in Neovascular Age-Related Macular Degeneration: A Clinicopathologic Correlation Relevant to Macular Atrophy

Abstract

Purpose: Macular atrophy (MA) of retinal pigment epithelium (RPE) and photoreceptors leads to vision loss in neovascular age-related macular degeneration (nAMD) despite successful treatment with antiangiogenic agents. To enhance understanding of MA, fortify the cellular basis of fundus autofluorescence (FAF) imaging, and inform management of nAMD, we performed histologic analysis of an eye with multimodal clinical imaging and apparent prior exudation due to nAMD.

Design: Case study and clinicopathologic correlation.

Participant: A White woman in whom age-related macular degeneration (AMD) findings of inactive subretinal fibrosis (right eye) were followed for 9 years using FAF and OCT, with no detectable subretinal fluid or other recurrent exudation and no intravitreal injections before her death at age 90 years.

Methods: The right eye was preserved 6.25 hours after death, postfixed in osmium tannic acid paraphenylenediamine, and prepared for submicrometer epoxy resin sections (n = 115), with 19 matched to clinical OCT B-scans.

Main outcome measures: Light microscopic morphology of a hyperautofluorescent (hyperFAF) area due to prior exudation ("floodplain" hyperFAF), hypoautofluorescent (hypoFAF) spots of MA, and areas of unremarkable FAF.

Results: Floodplain hyperFAF was visible throughout the 9 years of follow-up, with several hypoFAF atrophic spots expanding within it over time. The hyperFAF pattern corresponded to outer retinal atrophy (ORA) on OCT and photoreceptor loss over dysmorphic yet continuous RPE in histology. The hypoFAF spots inside the floodplain corresponded to complete RPE and outer retinal atrophy (cRORA) on OCT and loss of both photoreceptors and RPE in histology. In contrast, areas of unremarkable FAF showed continuous RPE accompanied by full-length photoreceptors and a thick outer nuclear layer.

Conclusions: This direct clinicopathologic correlation for FAF imaging is the first for nAMD. Fundus autofluorescence is a projection image that involves optical signal modulation by photoreceptors as well as emission signal sources in RPE. Hyperautofluorescence due to an exudative floodplain signifies loss of photoreceptors over continuous RPE. Hypoautofluorescence in MA signifies loss of both cell layers. For maximal value, fundus autofluorescence imaging should be interpreted with the multilayer perspective provided by OCT. Prevention of exudation in nAMD may preserve photoreceptors.

Keywords: Age-related macular degeneration; Autofluorescence; Histopathology; Lipofuscin; Neovascularization; Photoreceptors; Retinal pigment epithelium.

Figure 1.. Baseline multimodal imaging of an eye with advanced neovascular age-related macular degeneration.

All images were acquired at presentation, 9 years before patient death. A. Color fundus photograph (CFP) shows subretinal fibrosis (green asterisk) with spotted black pigment (blue arrowhead). There is a grayish region (white asterisk) inferior temporal to the fovea, and several drusen (yellow arrowhead) mostly superior temporal to the fovea. B. Fundus autofluorescence (FAF) shows a central round area of hypoFAF (green asterisk) and an inferior temporal region (white asterisk) of mild hyperFAF believed to represent a floodplain subsequent to exudation. C,D. Fluorescein angiography (FA) in mid and late phases shows central hyperfluorescence of subretinal fibrosis (green asterisk) with poorly defined staining (occult choroidal neovascularization) at its inferior temporal border (white asterisk).

Figure 2.. Macular regions with defined autofluorescence patterns, over time.

A,B. Color fundus photograph (CFP) and fundus autofluorescence (FAF) were acquired 4 years before patient death (Topcon camera). C,D. Optos multicolor and FAF were acquired at the last clinic visit, 16 months before patient death. All regions were defined based on FAF in panel D and numbered in all panels. Region 1, central hypoFAF region at the fibrotic scar (colored green for illustrative clarity); region 2, hyperFAF attributed to an exudative floodplain; region 3, hypoFAF at the atrophic spots; region 4, isoFAF in the non-floodplain region; region 5, hyperFAF attributed to peripapillary outer retinal atrophy; region 6, hyperFAF rings at the sites of drusen. Areas of all defined regions changed little during 3 years of follow-up, except for the enlargement of hypoFAF atrophic spots in region 3.

Figure 3.. Optical coherence tomography (OCT) B-scans of retinal regions with known fundus autofluorescence (FAF) characteristics.

OCT B-scans acquired 16 months before death. Near-infrared reflectance (NIR, 1^st column) have green arrows indicating location and direction of the corresponding OCT B-scans (2^nd column). A. NIR shows a central hyperreflective area (green asterisk) surrounded temporally by several drusen (yellow arrowheads). B-scan shows calcified drusen (yellow) temporally and a shallow irregular RPE elevation (SIRE, pink arrowhead) nasally; histology of the SIRE is shown in Figure 4. B. Green dashed contour indicates the fibrotic scar (region 1 of Figure 2) with a thin fibrotic scar by OCT (green arrowheads). White dashed contour indicates the region with floodplain FAF (region 2 of Figure 2) with complete outer retinal atrophy (cORA) by OCT (white frame and inset). White arrowheads indicate outer retinal tubulation. Teal arrowheads indicate choroidal hypertransmission due to complete RPE and outer retinal atrophy (cRORA). C. White dashed contour indicates atrophic spots (region 3 of Figure 2) with cRORA by OCT (white frame and inset). Yellow dashed contour shows area of non-floodplain FAF (region 4 of Figure 2) and a visible EZ (yellow frame and inset). Green arrowheads indicate the central fibrotic scar. Teal arrowheads indicate choroidal hypertransmission due to cRORA.

Figure 4.. Clinicopathologic correlation of type 1 MNV adjacent to a fibrotic scar.

A. Optical coherence tomography (OCT) B-scan 16 months before patient death shows large shallow irregular RPE elevations (SIRE, red arrowheads) flanking an area of choroidal hypertransmission (teal) at the border of a fibrotic scar. B. Color fundus photograph (CFP, Optos) acquired at the same time as A. The green arrow line shows the location and direction of the OCT B-scan in A. The dashed line indicates that the B-scan ends short of the histology section, which extends further nasal into the peripapillary area. The white frame (E) indicates location of histology shown in panel E. C. Panoramic view of histology shows areas of type 1 MNV (red arrowhead) and atrophy (teal arrowhead) corresponding with structures seen on OCT (A). Black dashed frame is magnified in (D). D. Magnified image shows an atrophic area (blue asterisk) bounded by ELM descent (green arrowhead), type 1 MNV on both sides, and a large BrM defect (orange arrowhead). Black dashed frame is magnified in (E). E. SIRE corresponds to Type 1 MNV, i.e., continuous RPE with BLamD atop fibrovascular tissue with patent vascular lumens in the subRPE-basal lamina space. MNV, macular neovascularization; NFL, nerve fiber layer; INL, inner nuclear layer; ONL, outer nuclear layer; PR, photoreceptors; RPE, retinal pigment epithelium; BLamD, basal laminar deposit; BrM, Bruch’s membrane; Ch, choroid; ELM, external limiting membrane.

Figure 5.. Clinicopathologic correlation of floodplain hyperautofluorescence in neovascular AMD.

A. Optical coherence tomography (OCT) B-scan 16 months before patient death shows a large fibrotic scar (red arrowheads), with hyperreflective lesions (blue arrowheads) and outer retinal tubulation (ORT, yellow arrowheads). White frame (D) indicates an area within the floodplain region, showing complete outer retinal atrophy (cORA) comprised of invisible EZ, continuous RPE layer, and almost intact ELM. Of note, no choroidal hypertransmission was observed in this area. B. Fundus autofluorescence (FAF, Optos) acquired at the same time as A. The green arrow shows the location and direction of the OCT B-scan. The dashed line indicates that the histology section extends nasal to the B-scan. Two small white frames (D and E) indicate floodplain and non-floodplain areas (regions 2 and 4 of Figure 2, respectively). C. Panoramic view of histology shows the floodplain area (black frame, D), fibrotic scar (red arrowhead), melanotic cells (blue arrowhead), and ORT (yellow arrowhead) corresponding with structures identified on in vivo OCT (A). D. Histology of the floodplain (indicated in A-C) shows dysmorphic but almost continuous RPE with a thin layer of BLamD. The RPE thickness is highly variable with numerous shed granule aggregates (orange arrowhead). Photoreceptors in this area have virtually disappeared with only one recognizable cone (pink arrowhead) and several stunted inner segments. Intact ELM is straight, condensed, and very close to the RPE. ONL and HFL are dyslaminate, and OPL and INL are disorganized. E. Histology of non-floodplain region (indicated by small white frame in B) shows continuous RPE, full-length photoreceptors, and thick ONL.

Figure 6.. Clinicopathologic correlation of hypoautofluorescent regions of macular atrophy in neovascular AMD.

A. Fundus autofluorescence (FAF, Optos), 16 months before death. Long green line indicates the location of histologic section in panel B. Short green line indicates the location of histologic section in panel C. D and E indicate the atrophic spots and fibrotic scar areas of regions 3 and 1 of Figure 2, respectively. B. Panoramic view of histology shows macular atrophy in the left and fibrovascular scar in the middle (black dashed frames D and E, magnified in panels D and E). C. Atrophy within the floodplain area is bordered by an ELM descent on the temporal (left) side (green arrowhead). Intraretinal RPE-derived material including cells can be observed inside and outside the atrophy (red arrowhead). ChC ghosts (yellow arrowhead) are apparent. D. Atrophy within the floodplain area is bordered by an ELM descent on both sides (green arrowhead) with an island of residual ELM, RPE (red arrowhead) and IS (pink asterisk) in the middle. Persistent BLamD (outer retinal corrugation) is prominent as is intense dyslamination, cellular invasion, and removal of RPE granules by Müller glia. E. Fibrovascular scar and atrophy is bordered by an ELM descent on both sides (green arrowhead). Within the atrophic area, double layers of BLamD (teal arrowhead), Müller glia invasion (trajectory indicated by blue arrow), outer retinal tubulation (red asterisk) and a cyst (black asterisk) are seen. ONL and HFL are dyslaminate, and OPL and INL are disorganized. In the sub-RPE-BL space is a fibrotic scar with vessel lumens (type 1 MNV). Orange arrowheads, BrM defects.