Visualizing lipid behind the retina in aging and age-related macular degeneration, via indocyanine green angiography (ASHS-LIA)

Abstract

Age-related macular degeneration (AMD) causes legal blindness in older adults worldwide. Soft drusen are the most extensively documented intraocular risk factor for progression to advanced AMD. A long-standing paradox in AMD pathophysiology has been the vulnerability of Asian populations to polypoidal choroidal vasculopathy (PCV) in the presence of relatively few drusen. Age-related scattered hypofluorescent spots on late phase indocyanine green angiography (ASHS-LIA) was recently proposed as precursors of PCV. Herein, we offer a resolution to the paradox by reviewing evidence that ASHS-LIA indicates the diffuse form of lipoprotein-related lipids accumulating in Bruch's membrane (BrM) throughout adulthood. Deposition of these lipids leads to soft drusen and basal linear deposit (BLinD), a thin layer of soft drusen material in AMD; Pre-BLinD is the precursor. This evidence includes: 1. Both ASHS-LIA and pre-BLinD/BLinD accumulate in older adults and start under the macula; 2. ASHS-LIA shares hypofluorescence with soft drusen, known to be physically continuous with pre-BLinD/BLinD. 3. Model system studies illuminated a mechanism for indocyanine green uptake by retinal pigment epithelium. 4. Neither ASHS-LIA nor pre-BLinD/ BLinD are visible by multimodal imaging anchored on current optical coherence tomography, as confirmed with direct clinicopathologic correlation. To contextualize ASHS-LIA, we also summarize angiographic characteristics of different drusen subtypes in AMD. As possible precursors for PCV, lipid accumulation in forms beyond soft drusen may contribute to the pathogenesis of this prevalent disease in Asia. ASHS-LIA also might help identify patients at risk for progression, of value to clinical trials for therapies targeting early or intermediate AMD.

Fig. 1. Retinal layers, regions, and the role of lipid in AMD progression.

Lower left: The schematic shows the posterior pole of the ocular fundus. Black oval is the optic nerve, with arteries and veins of the retinal circulation shown. The Early Treatment of Diabetic Retinopathy Study grading grid and its dimensions are shown. The grid consists of the central subfield, inner ring, and outer ring (1, 3, and 6 mm in diameter, respectively). The central subfield is mostly all cones with a few rods around its outer rim. The black stippling represents a central bouquet of very thin cone photoreceptors and supporting Müller glia. The 3 mm-diameter inner ring has prominent xanthophyll carotenoid pigments, shown as yellow, in the neurosensory retina, representing the macula lutea. The 6 mm diameter area is called central area or macular region. Upper: the trilaminar BrM consists of inner collagenous (ICL), elastic (EL), and outer collagenous (OCL) layers. With aging, lipoprotein particles (yellow circles) accumulate in the BrM, forming a transport barrier between the ChC and the outer retina. Pre-BLinD, a thin lipid layer between RPE-BL and the ICL of the BrM forms a cleavage plane. BLinD, continuous with pre-BLinD, contains ultrastructural evidence for lipid fusion and pooling and peroxidized lipids capable of promoting cytotoxic and proinflammatory response in vivo. Into this milieu, Type 1 neovascularization invades (mauve up-arrow). Soft drusen, a lump of the same extracellular material as in BLinD, could contribute to ischemia of the overlying RPE, which may migrate intraretinally (seen as hyperreflective foci). BLamD is stereotypically thickened basement membrane proteins (green, in middle and right) between the RPE plasma membrane and native RPE-BL, either replacing or incorporating infoldings of basal RPE. Basal mounds are soft drusen material trapped within BLamD. Between the RPE and photoreceptors are SDDs (first called reticular pseudodrusen), extracellular material that is directly disruptive to photoreceptors. Eyes with SDDs are at risk for type 3 MNV (mauve down-arrow). The black and gray columns at the right and left represent reflective bands of current optical coherence tomography. AMD age-related macular degeneration, BLinD basal linear deposit, BLamD basal laminar deposit, ChC choriocapillaris, ChC-BL ChC basal lamina, OS outer segments of photoreceptors, M melanosome, ML melanolipofuscin, Mt mitochondria.

Fig. 2. Molecular circuitry for production of Bruch’s membrane lipoproteins.

Left Circles 1–3 are detailed in the right panel. Circle 1 shows accumulation of Bruch’s membrane lipoproteins, resulting in pro-inflammatory peroxidized lipids. Circle 2 shows generation of a primordial lipoprotein at the endoplasmic reticulum, with MTTP-mediated transfer of lipid as in liver and intestines. Circle 3 shows that in rough endoplasmic reticulum at apical RPE, lipid droplets destined for lipoproteins are formed from components originating in diet » outer segments » endogenous synthesis. Lutein, zeaxanthin, vitamin A, and some unesterified cholesterol (UC) enter RPE via receptor-mediated uptake of plasma lipoproteins. These are transferred to photoreceptors by mechanisms that may involve interphotoreceptor retinol-binding protein (IRBP, center), a hypothetical apolipoprotein (apo) E-based small HDL particle (right), or diffusion (not shown). Because lipids in Bruch membrane lipoproteins are rich in the fatty acid linoleate (and not docosahexaenoate), soft drusen are proposed as consequent to constitutive dietary delivery of lipophilic essentials to macular cells, and the RPE- mediated recycling of these unneeded lipids and those from outer segments to plasms via large lipoprotein particles. Lipoproteins accumulate between RPE-basal lamina and inner collagenous layer of Bruch’s membrane. ABCA1 ATP-binding-cassette A1, apoE apoB, apoA-I, apolipoproteins E, B, A-I; BrM Bruch’s membrane, C cones, CD36 cluster of differentiation 36, CETP cholesteryl ester transfer protein, DHA docosahexaenoate, EC esterified cholesterol, ELM external limiting membrane, FA fatty acid, HMGCR HMG co-A reductase, target of statins, IS inner segment, LDLR LDL-receptor, M Müller cells, MTTP microsomal triglyceride transfer protein, ONL outer nuclear layer, OS outer segment, R rods, SR-BI scavenger receptor BI, SR-BII scavenger receptor B2, TG triglyceride, UC unesterified cholesterol.

Fig. 3. Homogeneous background fluorescence in late phase ICGA and the formation of ASHS-LIA.

Scale bar in (A) applied to all panels. A–C Normal fundus fellow eye from a 38-year-old woman whose left eye was diagnosed with idiopathic choroidal neovascularization (not shown). D–F Normal fundi fellow eye from a 61-year-old man whose left eye was diagnosed with polypoidal choroidal vasculopathy (not shown). Time after ICG dye injection was indicated in the top right-hand corner. A ICG dye was located within retinal and choroidal vessels in the early-phase after injection; B ICG dye was extravasated into the choroidal stroma and accumulated within the RPE over time; C Homogeneous background fluorescence (no ASHS-LIA) was observed in late phase ICGA. D ICG dye was located within retinal and choroidal vessels in the early-phase after injection; E Scattered hypofluorescent spots (green arrowheads) were appreciable in the mid-phase after injection. F Hypofluorescent spots were obvious in late phase ICGA (ASHS-LIA) and distributed in the macular region (green arrowheads).

Fig. 4. The grades of ASHS-LIA based on the involved region.

Different grades of ASHS-LIA were shown in late phase ICGA (30 min after dye injection). Scale bar in (A) applied to all panels. A Grade 1: ASHS-LIA distributed in the macular region (green arrowheads); B Grade 2: ASHS-LIA distributed in the macular region (green arrowhead) and around the optic disc (yellow arrowheads); C ASHS-LIA distributed throughout the whole posterior pole (Teal arrowheads); D ASHS-LIA distributed in the macular region with partial confluence (green asterisk); E ASHS-LIA distributed in the macular region and around the optic disc, with partial confluence (green asterisk); F ASHS-LIA distributed throughout the whole posterior pole, with partial confluence (green asterisks).

Fig. 5. ASHS-LIA are invisible on other imaging modalities.

Normal fundi fellow eye from a 64-year-old man whose right eye was diagnosed with neovascular age-related macular degeneration (not shown). Scale bar in (A) applied to (A–D). A ASHS-LIA (green arrowheads) were distributed in the macular region in late phase ICGA. No corresponding changes were present in the color fundus photography (B), blue autofluorescence (C), and fluorescein fundus angiography (D). E Spectral-domain optical coherence tomography (SD-OCT) at green arrow in (A) showed that the retinal structure was generally normal, and the RPE band was intact and smooth, with no intraretinal, subretinal or sub-RPE visible deposition corresponding to ASHS-LIA. F Magnified SD-OCT image showed the white frame region in (E).

Fig. 6. The frequency of ASHS-LIA increases with age after age 30 years.

Patients were divided into seven subgroups by age in decades. The percentage of eyes with ASHS-LIA increased markedly with aging. No ASHS-LIA was observed in the age group of ≤30. The percentage of eyes with ASHS-LIA were 1.4%, 4.8%, 23.1%, 49.3%, 45.1% and 64.7% respectively in the age groups of 31–40, 41–50, 51–60, 61–70, 71–80 and 81–90. Data are replotted from Chen et al. Clin Exp Ophthalmol 2018 [14].

Fig. 7. The distribution relationship of ASHS-LIA and soft & hard drusen.

A, B The right eye from a 78-year-old man with PCV in his left eye (not shown). A Late phase ICGA showed that soft drusen presented as dense hypofluorescence in the macular region (yellow arrowhead), and ASHS-LIA were present in its surround (green arrowhead). B SD-OCT at green arrow in (A) showed drusenoid pigment epithelium detachment corresponding to the soft drusen. B1 Magnified SD-OCT image showed the yellow frame region in (B). sd, soft drusen. C, D The right eye from a 66-year-old woman with PCV in her left eye (not shown). C Late phase ICGA showed ASHS-LIA (green arrowhead) throughout the posterior pole, with remarkable confluence (asterisk). Hard drusen presented as hyperfluorescent spots (teal arrowhead) and were located outside the ASHS-LIA. D SD-OCT at green arrow in (C) showed focal high reflective deposits underneath the RPE, corresponding to the hyperfluorescent spots in (C). D1 Magnified SD-OCT image (the teal frame region in D) showed the high reflective deposits elevated the RPE and the myoid zone (teal arrowheads).

Fig. 8. BLinD and soft drusen are diffuse and focal deposits of the same lipoprotein-derived debris located in the same plane [59].

A–C Soft drusen are visible in OCT-based multimodal imaging (yellow arrowheads), basal linear deposit is not. A Color fundus photograph shows yellowish drusen in the macular region especially inferior temporal to the fovea. B Red-free image shows drusen clearly. C SD-OCT B-scan at green arrow in (A) shows several RPE elevations with a medium and homogeneous internal hyperreflectivity representing soft drusen. D Corresponding histologic image shows soft drusen (yellow arrowheads) that correspond well with the B-scan. Green frame shows a region magnified in (E). Retina is artifactually detached at the inner segment myoids (bacillary layer detachment). E Three soft drusen with finely granular, lightly osmophilic contents (d) are continuous with BLinD (red arrowheads) containing the same material, both underneath the RPE. Sixty-nine-year-old white old man with early age-related macular degeneration.

Fig. 9. Proposal of correlation between ASHS-LIA and BLinD.

A Normal structure of the RPE, BrM, and choroidal capillaries (CC). Five layers of connective tissue are visible: basal lamina of the RPE (RPE-BL); inner collagenous layer (ICL); elastic layer (EL); outer collagenous layer (OCL); and basal lamina of the choriocapillaris endothelium (CC-BL). Under normal conditions, ICG dye could extravasate into the choroidal stroma, pass through BrM and eventually be taken up by the RPE. Therefore, homogeneous background fluorescence is observed in late phase ICGA (a). B With aging, the BLinD (dark yellow) formed between the ICL of BrM and the RPE basal lamina, containing mainly neutral lipids, could reduce ICG dye through BrM into the RPE. Hence, hypofluorescence is observed (b). Blue material represents the BLamD. C BLinD could be confluent and further reduced ICG dye through BrM into the RPE. Therefore, confluent hypofluorescence is observed (c). D Both soft drusen and the BLinD is located in precisely the same sub-RPE space and contain mainly neutral lipids. Soft drusen could impede ICG dye through BrM into the RPE to a higher degree than BLinD because of thicker deposits. Hence, soft drusen present as dense hypofluorescence in late phase ICGA.

Fig. 10. Distribution of sub-RPE-BL lipid in early AMD eye [63].

A A panoramic view of a section showing categories of the sub-RPE-BL lipid. Black dashed frames are magnified in (B–E). B No sub-RPE-BL lipid. C Pre- BLinD is a flat layer of finely granular material in gray (green arrowheads). D Basal linear deposit is an undulating layer of the same extracellular material as in soft drusen (yellow arrowheads), usually continuous with pre-BLinD. E Soft drusen are lump version of the same extracellular material, usually continuous with BLinD. F Sub-RPE-BL lipid distribution in fellow eyes. AMD age-related macular degeneration, BL basal lamina, BLinD basal linear deposit, BLamD basal laminar deposit, Ch choroid, ChC choriocapillaris, d drusen, OS outer segment. All lipid, drusen + BLinD + pre-BLinD. Scale bar in (B) applies to (B–E).

Fig. 11. Continuity of basal linear deposit and soft drusen with type 1 neovascularization and exudation [59].

A Panoramic view of a section that passes through the edge of the foveal floor, judging from the rod-free zone and single row of ganglion cell bodies. Teal frame shows a region magnified in (B). B One continuous compartment contains BLinD and soft druse (frame C), fluid (frame D), and type 1 neovascularization (frame E), magnified in (C–E), respectively. C Druse continuous with BLinD (yellow arrowheads) and fringe of BLinD persists at a site of artifactual separation of BLamD from the inner collagenous layer of BrM. Yellow asterisk, basal mound. D Fluid in the same sub-RPE-basal lamina compartment (fuchsia arrowheads). E Choroidal neovessels with patent lumens (fuchsia asterisks) pass through a break in BrM (orange arrowheads), accompanied by pericytes (teal arrowheads) and fibrous material. An 81-year-old female donor. INL inner nuclear layer. Green arrowheads, ELM external limiting membrane. Scale bar in (D) applies to (C and D).