Retinal pigment epithelium degeneration associated with subretinal drusenoid deposits in age-related macular degeneration

Abstract

Purpose

To test whether increased light transmission (hypertransmission) through subretinal drusenoid deposits (SDD) into the choroid in age-related macular degeneration (AMD) represented retinal pigment epithelium (RPE) degeneration.

Design

Cross-sectional study.

Methods

Nineteen eyes of 12 patients with early to intermediate stage AMD and 18 eyes of 12 normal subjects were evaluated with color fundus photography, optical coherence tomography (OCT), and high-resolution adaptive optics scanning laser ophthalmoscopy (AOSLO) at baseline and 24 months later. SDD were classified using an OCT-based 3-stage grading system. Hypertransmission beneath SDD into the choroid was examined in OCT. SDD microstructure was assessed with AOSLO. To characterize the hypertransmission associated chorioretinal degeneration, choroidal thickness and photoreceptor length were measured in OCT at 1 mm and 2 mm superior, inferior, temporal, and nasal to the foveal center.

Results

OCT disclosed hypertransmission beneath stage 3 SDD in 8 eyes. These lesions showed a distinctive regressing structure in AOSLO, compared to stage 3 lesions without hypertransmission. The phenomenon persisted at follow-up, and new hypertransmission developed as SDD advanced. In eyes with hypertransmission, choroids were thinner than those of normal eyes at all sites (by 44–56%, p ≤ 0.0028) and those of eyes with SDD but without hypertransmission at superior and temporal sites (by 31–46%, p ≤ 0.039). Photoreceptors were significantly shorter than those in normal eyes (by 6–26%, p ≤ 0.0379).

Conclusions

Hypertransmission into the choroid, accompanied with SDD regression and thinning of choroid and photoreceptor layers, indicates RPE degeneration associated with advanced stages in the SDD lifecycle.

INTRODUCTION

Extracellular lesions found between the photoreceptors and retinal pigment epithelium (RPE) layer^1–4 clinically manifest as pseudodrusen.^3–5 This entity was first described by Mimoun et al as a distinctive yellowish pattern “visible en lumière bleue” (visible in blue light) in some eyes with age-related macular degeneration (AMD).⁶ Pseudodrusen as imaged by multiple imaging modalities are highly associated with progression to neovascularization and geographic atrophy.^7–9 The cross-sectional perspectives of optical coherence tomography (OCT) and histology have converged in promoting the term subretinal drusenoid deposits (SDD), which accurately reflects the multiple morphologies of pseudodrusen.^5,10,11 SDD share some proteins with drusen and differ markedly from these sub-RPE lesions in lipidic composition.^1,12 In AMD and normal eyes SDD lesions are especially prominent superior to the macula^6,13 and surrounding the optic nerve head,^14,15 whereas soft drusen are centered within the macula.¹⁶ The disparate laminar location, composition, and topography of SDD and classic drusen imply different biogenesis mechanisms, relationships to surrounding photoreceptors, RPE, and choroid, and progression sequence to late stage AMD. Since the original description of stages in OCT,⁵ SDD morphologies of dot, ribbon (or reticular), and confluent have been demonstrated.^11,17,18 These forms appear to have their own progression sequences, with dot proceeding preferentially to neovascularization and confluent proceeding preferentially to geographic atrophy.¹⁸ With spectral-domain OCT (SD-OCT), individual SDD lesions were classified into 3 stages by Zweifei et al.⁵ Querques et al suggested a fourth stage of regression.¹⁹

Histological studies revealed deflected, shortened, and missing outer and inner segments, and fewer nuclei in the outer nuclear layer (ONL) associated with SDD.^1,3 Some of these changes are observable in vivo by adaptive optics (AO) assisted retinal imaging. These include photoreceptor loss over SDD (with an AO flood illumination fundus camera²⁰) and stage-specific perturbation of photoreceptors around lesions (with AO scanning laser ophthalmoscopy (AOSLO)²¹ and AO OCT²²). The relationship between SDD and the underlying choroid is complex, with overall thinned choroid and local fibrosis directly underlying lesions.^23–27 Paradoxically, in myopic eyes with thin choroids, few SDD or drusen are found.²⁸ Eyes with regressed SDD have thinned outer retina, loss of reflective photoreceptor bands, and decreased choroidal thickness, a constellation of effects termed outer retinal atrophy (ORA) by Spaide.²⁵

While the impact of SDD on overlying photoreceptors and underlying choroid has been disclosed by advanced retinal imaging technology, the impact of SDD on subjacent RPE is less well studied. Alten and co-workers found that SDD were common in eyes with pigment epithelium detachment in AMD.²⁹ Recently, we observed increased light transmission into the choroid underlying some dot SDD using SD-OCT, a phenomenon we herein call hypertransmission. Increased light transmission below the RPE is considered as a reliable indicator of absence of the RPE layer in geographic atrophy.^30,31 We hypothesize that the hypertransmission associated with SDD indicate RPE degeneration and tested this hypothesis by determining whether features known to be associated with AMD progression were also associated with the presence of hypertransmission. Our purpose was 2-fold: to investigate the relationship between the RPE and overlying SDD and to correlate RPE degeneration with the structure of adjoining photoreceptors and choroid. Our data advances the understanding of AMD pathophysiology while exploring the limits of cellular-level in vivo imaging of outer retina.

METHODS

The study followed the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board at the University of Alabama at Birmingham. Written informed consent was obtained from participants after the nature and possible consequences of the study were explained. The study complied with the Health Insurance Portability and Accountability Act of 1996.

Patients and controls

Subjects were a subgroup from our previous study.²¹ Patients previously diagnosed with AMD were recruited from the clinical research registry of the Department of Ophthalmology of the University of Alabama at Birmingham and through the Retina Service between October 2010 and April 2013. Disease severity was determined by a masked, experienced grader using the Age-Related Eye Disease Study (AREDS) severity scale for AMD using color digital 30° fundus photog raphs.³² As described,²¹ subjects with diabetes, history of retinal vascular occlusions, any signs or history of hereditary retinal dystrophy, malignant hypertension, high myopia (< −6 D), retinal detachment, central serous chorioretinopathy, glaucoma, and prior vitreoretinal surgery, laser treatment, or photodynamic therapy were excluded. Subjects were also excluded for reasons that might prevent successful imaging. Normal controls were age-similar subjects at AREDS grade 1 in both eyes, with no clinically significant cataract, and best-corrected visual acuity (BCVA) of 20/50 or better. A total of 24 subjects were enrolled, including 12 patients with early to intermediate stage AMD and 12 normal control subjects (grades 3–8 and grade 1 on the AREDS 9-step scale, respectively). All subjects were white and non-Hispanic.

Longitudinal follow-up

To examine if the hypertransmission beneath SDD persisted over time, 11 eyes of 7 patients were examined with the same protocol at baseline and 24 months later. Due to development of other conditions (e.g., neovascularization), old age (average 79.8 years old for patients who had SDD and hypertransmission), and moving away, only some patients studied at baseline returned for follow-up study. These included 4 eyes of 2 patients with hypertransmission and 7 eyes of 5 patients without hypertransmission at baseline as defined below.

Image acquisition and analysis

Color digital 30° fundus photographs were taken with a FF450 Plus fundus camera (Carl Zeiss Meditec, Dublin, CA) after pupil dilation. En face infrared (IR) (λ = 830 nm) and autofluorescence (AF) (excitation, 488 nm; emission, ≥ 600 nm) images were acquired with the confocal scanning laser ophthalmoscope (SLO) of the Spectralis (Heidelberg Engineering, Carlsbad, CA). Retinal cross-sections were imaged with the Spectralis SD-OCT (λ = 870 nm; acquisition speed, 40,000 A-scans per seconds; scan depth, 1.9 mm; digital depth resolution, 3.5 µm per pixel in tissue; lateral resolution in tissue 14 µm). In each study eye, volume scans were acquired across a 20° × 20° area of the central macula with 11 µm or 30 µm spacing between B-scans. Each B-scan was an average of 9 frames. SD-OCT bands were named by the terminology of Staurenghi et al.³³ High-resolution AOSLO imaging of the macula was performed using the UAB AOSLO developed in our laboratory.^21,22,34 AOSLO videos were recorded continuously across an area about 20° × 20°. Registered images were averaged to enhance the signal-to-noise ratio using custom software.³⁵ Images of different retinal locations were manually aligned on a cell-to-cell basis to create a montage (Photoshop, Adobe Systems Inc., Mountain View, CA). Color fundus photographs, IR, and AF images were registered manually using retinal vessels and capillaries as landmarks. These fundoscopic images were grouped, magnified, and registered with AOSLO montages.

Identification and grading of SDD

SDD identification has been described in a previous study from our group,²¹ in which each SDD was identified on multimodal imaging including infrared scanning laser ophthalmoscopy, autofluorescence, blue reflectance, AOSLO, and SD-OCT. SDD were identified based on their presence in at least 2 standard en face imaging modalities and in SD-OCT.^36,37 In our prior study SDD was considered present if the number of unambiguously identified subretinal lesions in the macula was greater than 5. SDD was staged as described.⁵ All analyzed lesions in the current study were of the ‘dot’ type.¹¹

Analysis of hypertransmission beneath SDD

The SDD associated hypertransmission was defined as localized stripes of increased brightness in the choroid/sclera complex that were directly external to SDD, as imaged in SD-OCT B-scans. In general, the width of a hypertransmission stripe corresponded to the base width of the associated SDD, measured along the RPE layer. These hypertransmission stripes (Figure 1), were surveyed in SD-OCT B-scans across the macula in relation to the presence of SDD. Three authors (XX, XW, and Zhang) identified these hypertransmission stripes after extensive discussion and analysis (a trial and error process). The en face structure of specific lesions with or without associated hypertransmission was examined by AOSLO. After the hypertransmission stripes were identified, quantitative measurements of the chorioretinal structure were conducted by 2 authors (XX, XW) independently.

Figure 1.

Subretinal drusenoid deposits (SDD) with and without hypertransmission revealed by multimodal imaging. Rows 1 and 2, an 84-year-old woman with intermediate stage age-related macular degeneration (AMD) (Age-Related Eye Disease Study (AREDS) grade 7, best corrected visual acuity (BCVA) 20/20) (subject 1, AMD-022). Rows 3 and 4, a 73-year-old man with intermediate stage AMD (AREDS grade 6, BCVA 20/25 (subject 2, AMD-041). In en face imaging, SDD appear as interlacing of yellow-white dots (color fundus photography, rows 1 and 3), a pattern of hyporeflective or hyper-reflective spots (infrared (IR) reflectance, middle panels of rows 1 and 3), a pattern of small hypo-autofluorescent areas against a background of mild hyper-autofluorescence (right panels of rows 1 and 3). In spectral domain optical coherence tomography (SD-OCT), SDD are hyper-reflective mounds internal to the RPE (color arrows, rows 2 and 4). SD-OCT scans were taken along the green lines in the IR images. (Row 2, left) SD-OCT revealed distinctive hypertransmission stripes (indicated by yellow arrowheads) in the choroid/sclera directly beneath 5 stage-3 SDD (yellow arrows). Meanwhile, (right, row 2) hypertransmission stripes (red arrowheads) also appeared underneath regressed SDD (red arrows) as examined in en face imaging. These hypertransmission stripes were not seen beneath the SDD (arrows in white, green and magenta in the IR image in row 3 and the SD-OCT in row 4) seen in subject 2.

Chorioretinal structure measurement

To examine if subjects had the characteristic features of chorioretinal degeneration associated with SDD progression and regression, and to investigate if retinas with SDD associated hypertransmission had severer degeneration than those with SDD but lacking hypertransmission, retinal thickness, choroidal thickness, and photoreceptor length were measured in OCT at 1 mm and 2 mm superior, inferior, temporal and nasal from the foveal center. Following the method of Spaide,²⁵ choroidal thickness was measured from the outer limit of the RPE-Bruch’s membrane band to the inner surface of the sclera. Eccentricities were referenced to the external fovea,^33,38 an inward bowing of the external limiting membrane (ELM) and ellipsoid zone (EZ) bands. Photoreceptor length was defined as the distance from the inner boundary of the outer plexiform layer (OPL) to the midpoint of the RPE-Bruch’s membrane band.²⁵ When we measured photoreceptor length at the selected sites, we carefully examined the corresponding OCT B-scan to ensure that measurements were done where SDD was absent and the RPE band-Bruch’s membrane band could be clearly delineated. In each eye with SDD associated hypertransmission, ten (n=10) individual SDD lesions with hypertransmission and ten (n=10) lesions nearby without hypertransmission were chosen for measurement of lesion diameter in the baseline IR images using the integrated software package (Heidelberg Eye Explorer software, version 1.7.0.0; Heidelberg Engineering). Two authors (XX and XW) conducted these measurements independently.

Statistical Analysis

Subjects were classified into 3 groups. Group 1 included eyes in normal chorioretinal health (AREDS grade 1), group 2 consisted of eyes with SDD but without associated hypertransmission, and group 3 consisted of eyes with SDD with hypertransmission. BCVA was converted to logarithm of the minimum angle of resolution (LogMAR). Age and BCVA were assessed with nonparametric (distribution-free) methods. Intra-observer and inter-observer repeatability were assessed by intra-class correlation coefficients (ICC) with 95% confidence interval (CI).

Choroidal thickness and photoreceptor length in all groups were compared using generalized estimating equations (GEE) to account for the within-person correlation, as some subjects had both eyes involved in the study. Spearman’s correlation coefficient was calculated to evaluate correlation between choroidal thickness and photoreceptor length at each corresponding site. Statistical analyses used SAS (SAS Institute, Cary, NC). For all statistical tests, p-values of less than 0.05 were considered significant.

RESULTS

Of 19 eyes of 12 AMD patients, 14 eyes had both SDD and conventional drusen. Eighteen eyes of 12 control subjects did not meet a 5-lesion criterion⁵ for SDD presence. Some of these eyes may have had SDD presence by different criteria.¹⁵

Group 1 had 12 normal subjects (4 females and 8 males). Six subjects had 1 eye measured, and 6 subjects had both eyes measured. Group 2 included 7 patients with diagnosed AMD (5 females and 2 males). Three patients had 1 eye measured, and 4 subjects had both eyes examined. Group 3 involved 5 patients with AMD (2 females and 3 males). Two subjects had 1 eye measured, and 3 subjects had both eyes measured. Subject age and BCVA are listed in Table 1.

TABLE 1.

Subject characteristics

Subjects	Normal Eyes (Group 1)	Eyes with stage 3 subretinal drusenoid deposits
		No hypertransmission (Group 2)	Hypertransmission (Group 3)	F	p
Age (years old)a	73.6 ± 5.9	73.6 ± 7.8	79.8 ± 4.4	4.32	0.115
BCVA (LogMAR)b	0.13 ± 0.17	0.21 ± 0.17	0.13 ± 0.10	1.01	0.381
AREDS scalec	1	3–8	5–7

^aAge difference was examined using Kruskal-Wallis Test

^bBest corrected visual acuity (BCVA) (Logarithm of the minimum angle of resolution (LogMAR)) was tested with mixed procedure.

^cAge-Related Eye Disease Study severity scale

Hypertransmission stripes underneath stage 3 SDD in SD-OCT scans (2nd row, Figure 1) were observed in 8 eyes of 5 Group 3 patients. All stripes were found beneath stage 3 SDD. It was apparent that the widths of the stripes corresponded to the widths of the individual stage 3 SDD at their bases. These stripes were not seen in 11 eyes of 7 Group 2 patients with stage 3 SDD (bottom row, Figure 1).

Light transmission underneath SDD in the choroid was disclosed with 3 distinct transmission patterns by SD-OCT (Figure 2). Pattern 1 featured a relative homogeneous light transmission across the extent of the choroid without discernable hypertransmission beneath a stage 3 SDD. The ELM band was visible and continuous, the EZ band was interrupted, the interdigitation zone (IZ) was diffuse but still identifiable, and the RPE-Bruch’s membrane band appeared continuous. Pattern 2 was characterized by discrete hypertransmission stripes in the choroid beneath stage 3 SDD. The ELM, EZ, and IZ were similar to those in pattern 1. Pattern 3 had hypertransmission stripes like those in pattern 2, but lesions overlying the RPE were faded and missing their typical architecture. The ELM was present, the EZ was disrupted, the IZ could not be distinguished from the RPE-Bruch’s membrane band, and the RPE-Bruch’s membrane band was continuous yet fuzzy, relative to patterns 1 and 2. A total of 319 hypertransmission stripes (40 ± 16 per eye, mean ± standard deviation) were examined by SD-OCT.

Figure 2.

Three light transmission patterns associated with subretinal drusenoid deposits (SDD) revealed by spectral domain optical coherence tomography (SD-OCT) and adaptive optics scanning laser ophthalmoscopy (AOSLO). The subject was an 85-year-old woman (AMD-022) with intermediate stage AMD (Age-Related Eye Disease Study grade 7, best corrected visual acuity 20/20). (Top row) High-resolution AOSLO retinal image montage is overlaid on color fundus photograph. The fundus appearance of SDD lesions with 3 light penetration patterns revealed by SD-OCT (bottom row) is shown in the magnified yellow box. SDD with patterns 1 (green arrowhead) and 2 (yellow arrowhead) appear as dot-like yellow-white lesions in color fundus photography, whereas the area with regressed SDD in pattern 3 (aqua arrowhead) presents a pigmentary change. (Middle row) In AOSLO, pattern 1 lesion (4 green arrowheads) shows a hyporeflective annulus surrounded the reflective core of the SDD. Pattern 2 lesion (4 yellow arrowheads) shows a dark split inside the hyperreflective core of the lesion. Pattern 3 lesion (4 aqua arrowheads) is a regressed SDD, showing dispersed lesion material and reflective spots without legible cone mosaic. Green arrow lines in the AOSLO images indicate the levels of SD-OCT B-scans shown in the top row. (Bottom row) SD-OCT revealed 3 light penetration patterns. Pattern 1 (left), there is no stripe of hypertransmission beneath the SDD (pointed by the green arrowhead). Pattern 2 (middle), the imaging light transmits through the SDD and forms a distinct hypertransmission stripe underneath the SDD (pointed by the yellow arrowhead) through the RPE into the choroid (Ch). The ellipsoid zone (EZ) of the photoreceptors has been broken by the lesion, but the retinal pigment epithelium (RPE) band is still visible. Pattern 3 (right), a hypertransmission stripe is underneath a regressed SDD (pointed by the aqua arrowhead), with a disrupted EZ and a fuzzy RPE band.

The typical en face microstructure of SDD without beneath hypertransmission, as revealed by AOSLO, is a hyporeflective annulus containing indistinct photoreceptors surrounding a central reflective area of the lesion itself (green arrowheads, middle row of Figure 2). Outside the annulus are preserved cones, albeit at variable reflectivity levels relative to normal photoreceptors. Over stage 3 SDD with hypertransmission, AOSLO revealed a dark split in the central area (yellow arrowheads, middle row of Figure 2). In eyes with regressed SDD (aqua arrowheads, middle row of Figure 2), AOSLO disclosed dispersed hyperreflective spots, presumably remains of the SDD lesions, without detectable photoreceptor mosaic. Pigmentary changes were visible at the corresponding location in the color fundus photograph (magnified panel, top row, Figure 2). SDD were not visible in IR and AF images of eyes with faded SDD on SD-OCT.

The hypertransmission associated with SDD persisted at follow-up, with slight variation of size and brightness. New hypertransmission stripes developed with lesion progression, as determined by examining every single SD-OCT scan through the affected areas (Figure 3). In the 4 eyes with both baseline and 24-month follow-up data, we observed 237 hypertransmission stripes at baseline. At follow-up, 211 hypertransmission stripes persisted, 9 disappeared in association with regressed SDD, 17 could not be identified due to poor image quality, and 9 hypertransmission stripes newly appeared.

Figure 3.

Progression of subretinal drusenoid deposit (SDD) with hypertransmission, seen with adaptive optics scanning laser ophthalmoscopy (AOSLO) and multiple spectral domain optical coherence tomography (SD-OCT) scans, at baseline and 24 months follow-up. The subject was a 73-year-old man (AMD-041) with intermediate AMD (Age-Related Eye Disease Study grade 6 at baseline, best corrected visual acuity 20/25). (Rows 1 and 2) AOSLO images of the same retinal areas taken at baseline and 24-month follow-up. Color arrowheads indicate 4 exemplary stage 3 SDD at baseline and their progression. By AOSLO stage 3 SDD has a hyporeflective annulus signifying non-visualized photoreceptors and a central reflective core signifying the lesion material. (Rows 3–6) Paired B-scans from the SD-OCT volumes taken at baseline and follow-up, as indicated by the green lines with arrowheads on the AOSLO images. The spacing between adjacent B-scans is 30 µm. Over 24 months, one regressing bi-lobed SDD (aqua arrowheads) has completely disappeared at the follow-up (Rows 1 and 3), associated with clearance of lesion material from the subretinal space and persistence of associated hypertransmission (Panel pairs B1–F1 to B5–F5, showing pattern 2 described in Figure 2). Another small regressing lesion (magenta, Rows 1 and 2) diminished in size and exhibited hypertransmission over time (panel pairs B5–F5 to B8–F8), exemplifying a Pattern 2 lesion progressing to Pattern 3. A large SDD (yellow arrowheads) without discernable hypertransmission at baseline shrank (Rows 1 and 2) and developed hypertransmission (panel pairs B5–F5 to B8–F8) at follow-up, demonstrating a Pattern 1 lesion progressing to Pattern 2. An adjacent small SDD (green arrowheads) associated with hypertransmission at baseline expanded at follow-up (Row 1), with hypertransmission persisting over the follow-up period. A version of these images without labelling is presented in Supplemental Figure 1.

There were no significant differences between stage 3 SDD base diameters in eyes with (88.90 ± 32.30 μm, range 40–179 μm) and without hypertransmission (92.33 ± 33.04 μm, range 43–176 μm) (p = 0.372).

Choroids of eyes with SDD associated hypertransmission were significantly thinner than those of normal eyes at all measurement sites (by 44–56%, p ≤ 0.0028) and thinner than those of eyes with SDD but without hypertransmission at the superior and temporal sites (by 31%–46%, p ≤ 0.039). Choroidal thickness in eyes with SDD but without hypertransmission was not significantly different from that in normal control eyes (p ≥ 0.1112), as shown in Table 2.

TABLE 2.

Choroidal thickness measurements (mean ± standard deviation; µm) and comparison

Location (mm)	Normal Eyes (Group 1, n=18)	Eyes with stage 3 subretinal drusenoid deposits		pa
		No hypertransmission (Group 2, n=11)	Hypertransmission (Group 3, n=8)	Group 1 vs. Group 2	Group 1 vs. Group 3	Group 2 vs. Group 3
Superior 1	258 ± 88	211 ± 86	114 ± 68	0.2635	0.0002	0.0257
Temporal 1	259 ± 95	205 ± 71	115 ± 59	0.1570	0.0002	0.0166
Inferior 1	248 ± 98	198 ± 89	112 ± 73	0.2793	0.0013	0.0677
Nasal 1	234 ± 95	158 ± 99	102 ± 66	0.1112	0.0014	0.2468
Superior 2	244 ± 69	201 ± 69	119 ± 44	0.2097	<0.0001	0.0102
Temporal 2	228 ± 64	197 ± 71	127 ± 50	0.2939	0.0002	0.0390
Inferior 2	220 ± 95	179 ± 97	124 ± 54	0.4169	0.0172	0.2221
Nasal 2	171 ± 78	132 ± 103	79 ± 48	0.4006	0.0028	0.2475

^aGeneralized estimating equations (GEE)

Photoreceptors were significantly shorter at most sites in eyes with SDD and hypertransmission than those in normal eyes (by 6–26%, p ≤ 0.0379). They were not significantly shorter than those in eyes with SDD but without hypertransmission (p ≥ 0.0671). Photoreceptor length in eyes with SDD and lacking hypertransmission was not significantly different from that in normal control eyes (p ≥ 0.0967), as shown in Table 3. Except for the temporal sites, choroidal thickness and photoreceptor length at corresponding sites were significantly and positively correlated (r ≥ 0.39), shown in Table 4.

TABLE 3.

Photoreceptor length measurements (mean ± standard deviation; µm)

Location (mm)	Normal Eyes (Group 1, n=18)	Eyes with stage 3 subretinal drusenoid deposits		pa
		No hypertransmission (Group 2, n=11)	Hypertransmission (Group 3, n=8)	Group 1 vs. Group 2	Group 1 vs. Group 3	Group 2 vs. Group 3
Superior 1	120.2 ± 15.7	109.4 ± 19.9	91.6 ± 29.3	0.1560	0.0379	0.2311
Temporal 1	135.9 ± 8.5	125.5 ± 24.2	105.5 ± 29.9	0.1725	0.0274	0.1982
Inferior 1	122.3 ± 12.8	102.8 ± 30.2	90.6 ± 32.2	0.1178	0.0349	0.5205
Nasal 1	131.8 ± 13.0	114.6 ± 25.7	105.3 ± 33.5	0.0967	0.0897	0.6100
Superior 2	108.6 ± 9.4	111.5 ± 12.4	90.4 ± 18.2	0.5639	0.0361	0.0245
Temporal 2	114.4 ± 10.6	112.2 ± 14.9	107.5 ± 10.4	0.6787	0.2286	0.4642
Inferior 2	102.6 ± 7.7	101.2 ± 11.9	89.8 ± 12.3	0.7262	0.0251	0.0671
Nasal 2	105.7 ± 14.1	100.0 ± 23.8	87.1 ± 29.3	0.5422	0.1745	0.5400

^aGeneralized estimating equations (GEE)

TABLE 4.

Correlation between the choroidal thickness and the photoreceptor length at measured retinal locations

Location (mm)	ra	p
Superior 1	0.39	0.0197
Temporal 1	0.29	0.0883
Inferior 1	0.58	0.0002
Nasal 1	0.38	0.0199
Superior 2	0.47	0.0066
Temporal 2	0.09	0.6163
Inferior 2	0.49	0.0040
Nasal 2	0.53	0.0010

^aSpearman’s correlation coefficient

The inter-observer ICCs in choroidal and retinal measurements were 0.956 (0.889–0.983, 95% CI) and 0.884 (0.725–0.954), respectively. The intra-observer ICCs in choroidal thickness measurements and retinal thickness measurements were 0.970 (0.924–0.988) and 0.987 (0.966–0.995), respectively. Because of the good intra- and inter-rater reproducibility, the data reported are from one observer only (XX).

DISCUSSION

In this study, we examined increased light transmission (i.e., hypertransmission) into the choroid associated with advanced stage SDD and systematically assessed surrounding chorioretinal structure in eyes with intermediate AMD. Specifically, the preferential association of hypertransmission with advanced stage SDD and the progressive degradation of ELM, EZ, and IZ bands in patterns 1–3 are consistent with known stages of photoreceptor degeneration.^21,39 Further, the thinner choroid and photoreceptor layers in eyes with SDD and hypertransmission are also consistent. Hypertransmission into the choroid has been considered as a reliable indicator of loss of RPE in geographic atrophy.^{30,31,40–43} Thus, hypertransmission observed in this study appear to represent degeneration of RPE lying external to individual SDD lesions.

In a typical OCT image of intact RPE, imaging light passes through the RPE and illuminates the choroid uniformly at a moderate intensity level (Figure 1). Normal RPE cell bodies are hexagonal prisms with 3 cushions of reflective organelles from apical to basal (melanosomes, lipofuscin/melanolipofuscin, and mitochondria), with additional melanosomes in the delicate apical processes. Fewer melanosomes in the light path would allow greater passage of light transmitting into the choroid.¹⁰ Thus, hypertransmission in the choroid (Figure 2) suggests numerically fewer organelles, a re-distribution of organelles due to dysmorphia (aberrant shape) of the enclosing cells, or a combination. Recent systematized high-resolution histology of AMD eyes has newly emphasized the impact of RPE dysmorphia on OCT reflectivity.^44–46 Hypertransmission in the choroid in geographic atrophy is attributable to disintegration of the RPE layer as cells die or migrate away.^30,31,41,43 Hypertransmission in the choroid on a smaller scale than geographic atrophy was suggested by increased light penetration through cuticular drusen, attributed to effacement of RPE over the pointed tips of these lesions¹⁰. In eyes with SDD, the predominant histologic RPE phenotype underlying lesions is very non-uniform, i.e., highly variable thickness.³ Although an SDD lesion can occasionally replace an underlying RPE cell,^3,47 hypertransmission underneath SDD in our study sample is likely accounted for by dysmorphic RPE with fewer reflective organelles in the light path rather than RPE absence. To directly address this question, new histopathology on clinically imaged eyes, using techniques that reveal all RPE cells and organelles^44,48 is desirable. Nevertheless, our current observation taken with published evidence on cuticular drusen¹⁰ emphasize the subcellular RPE detail currently available through SD-OCT imaging.

Our data showing that some SDD-associated hypertransmission newly appeared and disappeared at 24 months follow-up supports the concept that RPE degeneration is a dynamic and observable in vivo at its earliest stages. RPE can enlarge to fill in gaps, as some cells degenerate,^44,49 and thus thin and present fewer reflective organelles. Eyes with geographic atrophy exhibit features suggestive of motile non-epithelioid RPE, as revealed by AO-assisted IR imaging.⁵⁰ Recent imaging-histology correlations have demonstrated cells with a full complement of reflective RPE organelles within the subretinal space and retina,^46,51,52 consistent with anterior migration. Thus the range of RPE cellular activities observable in vivo has expanded in step with new visualization technologies. Our follow-up data from a limited number of patients should spur future studies with more patients and objective methods for quantifying the number and intensity of hypertransmission associated with SDD over time.

The RPE degeneration associated with advanced dot SDD, as indicated by hypertransmission, was also corroborated by characteristic chorioretinal degeneration associated with SDD progression and regression, such as thinning of choroid and shortening of photoreceptors.²⁵ By examining AMD eyes with and without SDD-associated hypertransmission, as well as eyes considered normal, we found that the choroid was thinnest in eyes with SDD associated hypertransmission. Interestingly, for this study sample, choroidal thickness in eyes having SDD but without hypertransmission and the normal eyes were not significantly different at most sites. The difference may imply a progression of choroidal degeneration or involvement in the disease progression. Not surprisingly, our examination confirmed that shorter photoreceptors correlate with thinner choroid, as reported by Spaide.²⁵ Thus, the RPE degeneration observed in this study may be part of the overall chorioretinal degenerating process that is associated with SDD progression and regression. Future studies should investigate the correlation between changes in choroidal thickness and photoreceptor length in larger a population, followed over longer time.

High resolution AOSLO provided new insights into the lesion status associated with hypertransmission, beyond those shown by SD-OCT. We found the hypertransmission beneath advanced (stage 3) lesions only, without relation to SDD base diameter, suggesting that hypertransmission occurs during lesion regression. Some hypertransmission stripes are indeed associated with apparently regressing lesions, which Querques and co-workers described as faded SDD material and disrupted or absent EZ band.¹⁹ By AOSLO, SDD without hypertransmission is a typical stage 3 lesion, i.e., a hyporeflective annulus surrounding a reflective core (Figure 2 middle left panel).²¹ Also by AOSLO, most lesions with hypertransmission have dark veins or splits across the reflective core, suggesting lesion dilapidation by processes that remain to be determined. AOSLO, due to its high resolution and high magnification, advances the descriptions of Querques et al¹⁹ by revealing additional SDD microstructure and lifecycle details.

A strength of this study is the use of high-resolution AOSLO and SD-OCT to examine each of many individual SDD. Limitations include the small sample size, the inability to observe every participant at follow-up, and the absence of imaging technologies that could provide special insight into RPE health and integrity, such as near infrared autofluorescence to visualize melanosomes.⁵³ Nevertheless, we can conclude that hypertransmission beneath SDD indicates RPE degeneration in patients with AMD. RPE integrity is critical for the health and normal function of the photoreceptors. Given the high risk of advanced AMD associated with SDD, the awareness of the clinical significance of this phenotype is important. If extended with longitudinal data in more patients, these results would deepen our understanding of the pathophysiology of SDD and its role in AMD progression.

Multimodal retinal imaging featuring AOSLO reveals increased light transmission (hypertransmission) through subretinal drusenoid deposits (SDD) into the choroid in age-related macular degeneration, accompanied with SDD regression and thinning of choroid and photoreceptor layers, indicating retinal pigment epithelium degeneration associated with distinct phases in the SDD lifecycle.