Location-Specific Thickness Patterns in Intermediate Age-Related Macular Degeneration Reveals Anatomical Differences in Multiple Retinal Layers

Abstract

Purpose: To examine individual retinal layers’ location-specific patterns of thicknesses in intermediate age-related macular degeneration (iAMD) using optical coherence tomography (OCT).

Methods: OCT macular cube scans were retrospectively acquired from 84 iAMD eyes of 84 participants and 84 normal eyes of 84 participants propensity-score matched on age, sex, and spherical equivalent refraction. Thicknesses of the retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer + Henle's fiber layer (ONL_+HFL), inner- and outer-segment layers (IS/OS), and retinal pigment epithelium to Bruch's membrane (RPE-BM) were calculated across an 8 × 8 grid (total 24° × 24° area). Location-specific analysis was performed using cluster_(normal) and grid_(iAMD)-to-cluster_(normal) comparisons.

Results: In iAMD versus normal eyes, the central RPE-BM was thickened (mean difference ± SEM up to 27.45% ± 7.48%, P < 0.001; up to 7.6 SD-from-normal), whereas there was thinned outer (OPL, ONL_+HFL, and non-central RPE-BM, up to −6.76% ± 2.47%, P < 0.001; up to −1.6 SD-from-normal) and inner retina (GCL and IPL, up to −4.83% ± 1.56%, P < 0.01; up to −1.7 SD-from-normal) with eccentricity-based effects. Interlayer correlations were greater against the ONL_+HFL (mean |r| ± SEM 0.19 ± 0.03, P = 0.14 to < 0.0001) than the RPE-BM (0.09 ± 0, P = 0.72 to < 0.0001).

Conclusions: Location-specific analysis suggests altered retinal anatomy between iAMD and normal eyes. These data could direct clinical diagnosis and monitoring of AMD toward targeted locations.

Age-related macular degeneration (AMD) is typically characterized as an outer retinal degeneration, and, subsequently, early investigative efforts into the anatomy of AMD eyes were focused on the outer retinal layers.^1,2 With the advent of optical coherence tomography (OCT), in vivo quantification of all individual retinal layer thicknesses has become more accessible. A plethora of OCT studies have since reported varying macular thickness changes across the whole retina in the early stages of AMD including the retinal nerve fiber layer (RNFL),^3–6 ganglion cell layer (GCL),^3,6–10 inner plexiform layer (IPL),^4,5,11 inner nuclear layer (INL),^3,4,7,12 outer plexiform layer (OPL),^3,4,7 outer nuclear layer (ONL),^{3,4,7,13–16} inner- and outer-segment layers (IS/OS),^3,4,7 and retinal pigment epithelium to Bruch's membrane (RPE-BM).^3,4,16–23 

These studies challenge the pre-existing concept of AMD being a disease only of the outer retina. However, only very few studies to our knowledge have concurrently explored macular thicknesses across all individual retinal layers,^3,4,7 thus limiting extrapolation of results in a holistic context. For example, numerous studies demonstrate reduced GCL thickness in the early stages of AMD,^{3,6–9,24,25} but lack of context about whether thickness changes are also evident in the surrounding retinal layers hampers conclusions regarding whether this change may represent postreceptoral degeneration or simply be an isolated epiphenomenon. Knowledge of inter-related, concurrent thickness changes in other individual retinal layers would significantly bolster the anatomical understanding of AMD. 

Brandl et al.⁴ convincingly demonstrated that there were significant, concurrent thickness differences between AMD in its early stages and normal eyes in individual retinal layers of the macula, depending on location. This finding suggests that location-specific analysis of AMD may provide greater clinical insight into where and what retinal changes may occur. This study, however, only assessed a population above 70 years of age, which limits generalizability. Additionally, this study used the Early Treatment for Diabetic Retinopathy Study (ETDRS) sectors for location-specific analyses, which effectively confines retinal space to nine sectors (and combinations thereof) and assumes perfectly concentric anatomical topography. This is not ideal, considering known topographical variations in several layers,^26–30 particularly the RNFL, which originates nasally.^31,32 Comparison of ETDRS sectors between diseased and normal eyes may also introduce statistical bias when grouping data from the former according to topography of the latter.^33–36 Further study using more detailed spatial analyses, which accommodates for topographical variations of individual retinal layers of both diseased and normal eyes, could provide greater insight into iAMD anatomy, as well as directing attention toward specific retinal locations that may be useful for clinical diagnosis and monitoring of AMD. 

Recently, we developed a method of location-specific OCT analysis using cluster analysis that provides greater spatial detail and less variability than the ETDRS sectors.³⁷ Spatial clustering involves assigning location-specific data into groups of statistical likeness. Use of this method has enabled the formation of OCT-derived topographical thickness maps for each individual retinal layer.³⁷ These maps are reflective of retinal neuronal distributions described via histological studies more so than the standard ETDRS sectors, which assume neuronal distribution to be concentric and symmetric around the fovea.³⁷ Application of the GCL topographical thickness map to iAMD eyes using cluster analysis has subsequently led to observation of unique location-specific patterns of thinned GCL toward the central macula and thickened GCL toward the peripheral macula when compared to normal eyes, both statistically significant and large in magnitude of effect.²⁴ 

Application of cluster analysis to individual retinal layers beyond the GCL has yet to be performed. We hypothesize that analyzing location-specific retinal thicknesses in each individual retinal layer of iAMD versus normal eyes using cluster analysis may likewise reveal unique location-specific patterns yet to be described with more limited spatial templates such as the ETDRS sectors. These spatial patterns of thickness differences can help to ameliorate our understanding of AMD anatomy and guide clinical diagnosis and monitoring of iAMD toward targeted retinal locations. 

Methods

Study Population

Participant data were obtained through retrospective review of records from July 12, 2010, to January 13, 2020, of patients attending the Centre for Eye Health (CFEH) in Sydney, Australia. CFEH is a referral-only clinic providing advanced ocular diagnostic testing and disease management by specially trained optometrists and ophthalmologists.³⁸ All participants in this study provided prior written informed consent to use of their de-identified data for research in accordance with the Declaration of Helsinki and approved by the Biomedical Human Research Ethics Advisory Panel of the University of New South Wales. 

Inclusion criteria for normal eyes were defined as visual acuity better than 0.1 logMAR (20/25 Snellen) for participants younger than 60 years old or 0.2 logMAR (20/32 Snellen) for participants older than 60 years old; intraocular pressure <22 mm Hg in both eyes; and no evidence of macular-involving disease including but not limited to AMD, diabetic retinopathy, and glaucoma, nor any signs of significant macular preretinal abnormalities such as vitreo-macular traction, nor any significant macular intraretinal or subretinal deposits, fluid, pigment, or vascular changes. 

Inclusion criteria for iAMD eyes were defined as age of 50 years or older, diagnosis of iAMD,³⁹ and no evidence of macular-involving disease or significant structural abnormalities as described above unrelated to iAMD. Classification of iAMD was based on fundus photography between at least two nonblinded investigators using a modified Beckman Initiative classification,³⁹ that is, participants 50 to 54 years of age are still considered to have iAMD if they followed all phenotypic criteria in the classification system (as done so in other notable studies).^40–43 Specifically, eyes were classified as iAMD based on the presence of large drusen (>125 µm) or pigmentary abnormalities with at least medium drusen (63–125 µm), without evidence of late AMD signs such as neovascularization or geographic atrophy. No eyes had reticular pseudodrusen. Scanning laser ophthalmoscopy photography and OCT were used to confirm non-late AMD classification and exclude eyes with other posterior ocular disease. 

Propensity-Score Matching

Selection of the included normal and iAMD eyes were performed via propensity-score matching using multivariable logistic regression based on age, sex, and spherical equivalent refraction. Rather than exact matching, which can lead to larger bias due to individuals being unmatched in a limited sample pool,^44,45 potential predictor covariables were balanced between groups via propensity-score matching.^46–49 IAMD eyes were propensity-score matched with normal eyes using fuzzy matching without replacement.^50,51 Iterative random draws were performed with increasing match tolerance until all iAMD eyes were matched with normal eyes. This resulted in relatively balanced propensity-scores (logistic regression predicted probabilities) of 0.46 ± 0.02 (mean ± SEM) and 0.42 ± 0.02, respectively, and propensity-score match tolerance of 0.2.⁵² 

Image Acquisition and Retinal Layer Segmentation

OCT macular cube scans, covering an area of 8600 µm × 7167 µm or 30° × 25° across 61 B-scans, were acquired with Spectralis SD-OCT (Heidelberg Engineering, Heidelberg, Germany) as previously described.^37,53,54 In the presence of multiple scans per participant, the earliest scan meeting inclusion criteria without significant artefacts and with signal strength above 15dB were selected. Scans were corrected for ocular tilt and automatic segmentation applied to each individual retinal layer using the HRA/Spectralis Viewing Module 6.9.5.0 (Heidelberg Engineering, Heidelberg, Germany). 

Segmentation for the RNFL, GCL, IPL, INL, OPL, ONL + Henle's fiber layer (ONL_+HFL), IS/OS, and RPE-BM for iAMD eyes were reviewed across all 61 B-scans and corrected where necessary by authors M.T. and V.K. according to previous studies.^37,53,54 One of two randomized blocks of all participants had their scans reviewed independently by M.T. and V.K. Upon completion, scans from the alternate block of participants were then independently reviewed by the other author. Any segmentation adjustments required from the second block review were achieved through discussion and consensus between M.T. and V.K.. All segmentations were agreed upon by M.T. and V.K. after one session of discussion. Manual correction of segmentation for iAMD eyes were performed in approximately 75% of all B-scans with approximately 10% of the contours in each B-scan undergoing minor corrections. Notably, segmentation boundaries were corrected to continue through large vasculature to mitigate their effect on inner retinal layer thicknesses (Figs. 1A, 1B, magenta, asterisk), Henle's fiber layer were corrected to be part of the ONL as commonly done so in OCT studies due to its inconsistent reflectivity (despite anatomically being part of the OPL; Figs. 1A, 1B, cyan, arrowhead), and mis-segmentation of the inner- and outer-RPE-BM boundaries surrounding drusen were corrected (Figs. 1A, 1B, cyan, dagger). Manual correction of segmentation by authors M.T. and V.K. were regarded as the “ground-truth” in concordance with other studies that have compared manual OCT segmentation versus automatic OCT segmentation protocols.^55–57 Manual correction of segmentation for iAMD eyes in this study were performed to the same standard as manual correction of segmentation for normal eyes, which were completed in previous studies.^37,53,54 

Figure 1.

 

Retinal layers segmented within the HRA/Spectralis Viewing Module (A) automatically and with (B) manual correction. Note the manual correction of segmentation applied to the: distortion surrounding large vasculature (magenta, asterisk); ambiguity at Henle's fiber layer (cyan, arrowhead); and mis-segmentation surrounding drusen (cyan, dagger). The boundaries of each individual retinal layer are displayed in the yellow box.

Data Extraction

An 8 × 8 grid (total 6880µm × 6880µm or 24° × 24° area) centered on the fovea was placed over macular cube scans (8600 µm × 7167µm or 30° × 25° area). Each grid was 0.74 mm² in size (approximately 860 µm × 860 µm or 3° × 3° area). Individual retinal layer thicknesses for iAMD eyes were then extracted as 64 average measurements across the 8 × 8 grid as described from previous studies.^37,53,54 The 8 × 8 grid pattern were selected as it incorporated the highest preset number of B-scans (61 B-scans spaced 120µm apart) using the Spectralis SD-OCT without significant compromise in image quality.⁵⁸ 

Based on known retinal anatomy, inner retinal cellular displacement and a sharp decline in cellular densities toward the fovea produces highly variable GCL, IPL, INL, and OPL thicknesses. As such, the central four grids of these layers were excluded. The total number of grids analyzed for each layer was 64 in the RNFL, ONL_+HFL, IS/OS, and RPE-BM, and 60 in the GCL, IPL, INL, and OPL. 

Global Analysis

Global analyses were performed between the total 6880 µm × 6880 µm or 24° × 24° area mean thickness of iAMD versus normal eyes in each individual retinal layer, expressed as mean difference ± SEM in micrometers and as a percentage to account for varying layer thicknesses. To confirm results, multi-variable linear regression including age, sex, spherical equivalent refraction, and AMD status (i.e., iAMD or normal eye) against mean thickness was also performed in each individual retinal layer. Multi-variable linear regression was then repeated using the simplified AREDS severity score⁵⁹ instead of AMD status to explore potential dose-response relationships. That is, whether increased AMD severity was associated with greater magnitude of global thickness difference. Scores ranged from zero to two per eye, with one point assigned for the presence of either large drusen or pigmentary abnormalities, and two points assigned for the presence of both. 

Location-Specific Analysis

Clusters were predefined as groups of data with statistical likeness, derived from normal OCT grid-wise thicknesses. Normal cluster patterns for each retinal layer formed meaningful, in vivo topographical thickness maps^37,53,54 (Fig. 2A), which corresponded to histological neuronal distributions more so than the standard ETDRS sectors, which assume concentricity and symmetry around the fovea.³⁷ To address the modifiable areal unit problem (MAUP) for iAMD data, which states that statistical bias may be introduced based on how data are spatially grouped,^34,35 we used two main methods for location-specific analyses: cluster_(normal) comparison and grid_(iAMD)-to-cluster_(normal) comparison. 

Figure 2.

 

Schematic example of location-specific analyses using cluster_(normal) and grid_(iAMD)-to-cluster_(normal) comparisons. (A) OCT normative topographical thickness maps for each individual retinal layer, whereby each cluster is represented by a distinct color and defines groups of location-specific data with statistical likeness. (B) Cluster_(normal) comparison was performed by comparing mean thickness within each cluster area of each individual retinal layer between iAMD versus normal eyes, expressed as mean difference ± SEM. (C) Grid_(iAMD)-to-cluster_(normal) comparison was performed for further spatially detailed analysis, comparing individual grid-wise thicknesses of iAMD eyes to mean cluster thicknesses of normal eyes from the same 10-yearly age group and forming qualitative and quantitative difference plots from normal. Size scale and color scale at the bottom right. All images are in right eye format as demonstrated by location of the optic nerve.

First, cluster_(normal) comparison used a more traditional approach to address the MAUP by using spatial grouping that were meaningful and demonstrated inter-group separability and intra-group similarity,^33,34,36 that is, normal clusters.^37,53,54 The mean thickness of iAMD versus normal eyes in each cluster area of each individual retinal layer were expressed as mean difference ± SEM in µm and as a percentage (Fig. 2B). To determine a potential effect of eccentricity on mean differences between iAMD versus normal eyes, a linear regression slope was calculated between each individual retinal layer's mean difference values versus mean eccentricity (°) of each cluster, with exception of the RNFL. 

Second, grid_(iAMD)-to-cluster_(normal) comparison further addressed the MAUP by using unmodifiable basic grouping of iAMD data,^34,36 that is, grids, and provided even greater spatial detail and measure of effect sizes (i.e., the strength of a relationship in practical terms).^60–62 Individual grid-wise thicknesses of iAMD eyes were compared to corresponding mean cluster thicknesses of normal eyes from the same 10-yearly age group (Fig. 2C). For example, a 63-year-old participant with iAMD would have each grid-wise thickness compared with the 60- to 69-year-old normal group's corresponding mean cluster thicknesses. Qualitative and quantitative difference plots from normal were formed for each iAMD eye, represented in SD units to account for varying thicknesses across the macula in each retinal layer. Values of 1.96 SD were selected as scale endpoints to represent measurements outside the 95% distribution limits (top and bottom 2.5% of thickness values from normal). 

To confirm results derived from comparison of propensity-score matched groups, multivariable linear regression including age, sex, spherical equivalent refraction, and AMD status against mean thickness was performed in each cluster in each individual retinal layer. Multivariable linear regression was repeated using the simplified AREDS severity score⁵⁹ instead of AMD status to explore potential location-specific dose-response relationships. 

Interlayer Correlation Analysis

To examine whether a rudimentary spatial model could be formed linking the retinal layers of iAMD eyes, we performed grid-to-grid correlational analysis between layers that showed spatial patterns of SD-from-normal thickness differences. To account for Henle's fibers’ displacement, the mean eccentricity (mm) of each grid (Supplementary Fig. S1A) alongside additional displacements to these eccentricities according to Drasdo et al.⁶³ (Supplementary Fig. S1B) were calculated. Comparisons between the outer and inner retinal grids were then adjusted (Supplementary Figs. S1C, S1D), that is, the central four grids of the outer retinal layers would be correlated against the paracentral four grids of the GCL and IPL along the same angular plane from the fovea. 

Statistical Analysis

Statistical analyses were performed using GraphPad Prism Version 8, SPSS Version 25, and Microsoft Excel Version 2012. Default significance was considered as P < 0.05. Single comparisons between continuous variables were performed using the Student's t-test or Mann-Whitney U-test, depending on the relevant data assumptions. Multiple cluster_(normal) comparisons were not adjusted because each comparison was considered important⁶⁴ and instead contextualized alongside grid_(iAMD)-to-cluster_(normal) and multivariable linear regression analyses. Single comparisons between categorical variables were performed using Fisher's exact test where available or χ² test. SD-from-normal were interpreted according to Cohen's effect sizes, that is, ≥0.2 = small, ≥0.5 = medium, and ≥0.8 = large.⁶² Multivariable linear regression analyses were performed with backward stepwise elimination, removing nonsignificant covariables from the regression model in a stepwise manner until all remaining variables were P < 0.1 to allow transparency of β values with borderline significance (0.05 ≤ P < 0.1).⁶⁵ Dichotomous values, that is, females and males, AMD status, and normal eye status, were encoded as 1 and 0, respectively. Correlational analyses were performed using Pearson's r.⁶² Multiple comparisons between correlations were performed using Brown-Forsythe and Welch ANOVA for unequal SDs or one-way ANOVA and Tukey's multiple comparisons test for equal SDs. 

Results

Participant Demographics

Eighty-four eyes with iAMD from 84 participants and 84 normal eyes from 84 participants were propensity-score matched and included in this study. There were no significant differences with means or distributions of age, sex, spherical equivalent refraction, or visual acuity between the two groups when compared in total or as 10-yearly age groups (Table 1). To explore potential dose-response relationships, further subdivision of the 84 iAMD eyes into simplified AREDS severity scores revealed 60 eyes with a score of one (presence of large drusen or pigmentary abnormalities) and 24 eyes with a score of two (presence of large drusen and pigmentary abnormalities). 

Table 1.

 

Normal and iAMD Participant Demographics

 

Global Analysis in All Retinal Layers

To first determine whether there were differences in total macular thickness between iAMD and normal eyes, each individual retinal layer's total mean thickness (across the total 24° × 24° area) was compared between groups. The mean difference between iAMD and normal eyes showed a significantly thinned mean GCL (−5.03 ± 1.41 %, P < 0.0001), IPL (−5.93 ± 1.4 %, P < 0.0001), OPL (−8.36% ± 1.16%; P < 0.0001), and ONL_+HFL (−4.08% ± 1.53%; P < 0.01), whereas the RPE-BM were significantly thickened (11.55% ± 2.11%; P < 0.0001). There were no significant differences in RNFL, INL, and IS/OS thicknesses between iAMD and normal eyes (Fig. 3). 

Figure 3.

 

Global analysis in each individual retinal layer represented as total mean thickness difference (%) ± SEM of iAMD versus normal eyes. P values are noted above data points. Significant P values are denoted by: *P < 0.05, **P < 0.01, ***P < 0.001.

Multivariable linear regression adjusted for all covariables in propensity score matching (age, sex, and spherical equivalent refraction) showed that AMD status was significantly associated with mean thickness in the GCL (β = 1.27 [0.47, 2.07]; P < 0.01), IPL (β = 1.29 [0.63, 1.96]; P < 0.0001), OPL (β = 2.2 [1.6, 2.79]; P < 0.0001), ONL_+HFL (β = 2 [0.32, 3.69]; P < 0.05), and RPE (β = −1.56 [−2.12, −1]; P < 0.0001; Supplementary Table S1). Repeated multivariable linear regression using a simplified AREDS severity score instead of AMD status confirmed the significant associations between AMD with GCL, IPL, OPL, ONL_+HFL, and RPE-BM global thicknesses (Supplementary Table S3). There was greater slope with increased AMD severity in some layers (notably the RPE-BM) suggesting potential dose-response relationships, although statistical comparisons between slopes of severity scores were precluded because of limited sample size. 

Location-Specific Analysis in the RNFL and GCL

Location-specific analysis (for each 3° × 3° grid in the 24° × 24° area) was then performed in each individual retinal layer to assess spatial distribution of retinal thicknesses. In the RNFL, cluster_(normal) comparison was performed, whereby the mean thickness within each cluster area derived from the topographical thickness map (Fig. 4A) between iAMD and normal eyes were compared. This demonstrated thinned RNFL in most clusters for iAMD versus normal eyes (−2.61% ± 2.24% to −0.03% ± 2.37%; Fig. 4B). No differences in any cluster, however, reached statistical significance. 

Figure 4.

 

Location-specific analysis in the RNFL and GCL. Previously developed topographical thickness maps in the (A) RNFL and (D) GCL are represented by spatial clusters of distinct colors, whereby clusters are in order of greatest to lowest mean thicknesses (color scale below). Central black shading in the GCL represents the excluded area containing the fovea. Cluster_(normal) comparison of iAMD versus normal eyes in the (B) RNFL and (E) GCL are displayed via mean difference (%) graphs ± SEM. Mean difference values in µm are included below the x-axis. P values are noted above data points. Significant P values are denoted by *P < 0.05, **P < 0.01. Grid_(iAMD)-to-cluster_(normal) comparison of iAMD versus normal eyes in the (C) RNFL and (F) GCL are displayed via difference plots in SD units. Size scale and color scale at the bottom right. All images are in right eye format as demonstrated by location of the optic nerve in the underlying fundus photo.

Grid_(iAMD)-to-cluster_(normal) comparison was then performed, whereby grid-wise thicknesses of iAMD eyes were compared to corresponding mean cluster thicknesses of normal eyes from the same 10-yearly age group. The resultant difference plot confirmed a scattered distribution of thickness differences in the RNFL (Fig. 4C). Although some locations demonstrated a large magnitude of thinned and thickened RNFL (−2.5 SD- to 3 SD-from-normal), there was no distinguishable spatial pattern of differences. 

In the GCL, cluster_(normal) comparison using the topographical thickness map (Fig. 4D) displayed thinned GCL in clusters 1 to 4 for iAMD versus normal eyes (−4.37% ± 1.63% to −3.74% ± 1.61%; P < 0.01 to P < 0.05; Fig. 4E). Differences in the peripheral macula clusters 5 and 6 did not reach statistical significance. Linear regression slope of GCL mean differences versus mean eccentricity (°) of each cluster were calculated. The linear regression was significant (β = 0.38 ± 0.09, P < 0.05; Supplementary Table S2), implying decreased magnitude of thinned GCL with increasing retinal eccentricity. 

Grid_(iAMD)-to-cluster_(normal) comparison of the GCL of iAMD versus normal eyes confirmed up to a large magnitude of thinned GCL across most macula locations (up to −1.7 SD-from-normal). Some locations toward the peripheral macula, particularly inferiorly, demonstrated large magnitude of thickened GCL (up to 1.1 SD-from-normal; Fig. 4F). 

Multivariable linear regression adjusted for age, sex, and spherical equivalent refraction showed that AMD status were significantly associated with thicknesses in GCL clusters 1 to 5 (β = −3.51 [−5.01, −2.01] to −0.77 [−1.45, −0.09]; P < 0.0001 to P < 0.05), and no clusters in the RNFL (Supplementary Table S3). Repeated multivariable linear regression using a simplified AREDS severity score confirmed the associations between AMD GCL clusters 1 to 5 thicknesses (score 1 β = −3.09 [−4.73, −1.45] to −0.71 [−1.46, 0.04], P < 0.001 to P < 0.1; score 2 β = −4.54 [−6.78, −2.31] to −0.91 [−1.94, 0.11], P < 0.0001 to P < 0.1; Supplementary Table S3), and almost no RNFL clusters. There was consistently greater slope with increased AMD severity in GCL clusters 1 to 5, which may have indicated a dose-response relationship. 

Location-Specific Analysis in the IPL, INL, and OPL

In the IPL, cluster_(normal) comparison indicated thinned IPL in clusters 1 to 5 in iAMD versus normal eyes (−4.83% ± 1.56% to −3.42% ± 1.56%; P < 0.01 to P < 0.05; Figs. 5A, 5B). Differences in the peripheral macular clusters 6 and 7 did not reach statistical significance. The linear regression slope of all IPL clusters was non-significant (β = 0.2 ± 0.08, P = 0.07; Supplementary Table S2), implying no significant effect of eccentricity on IPL thicknesses in iAMD eyes versus normal eyes. 

Figure 5.

 

Location-specific analysis in the IPL, INL, and OPL. Presentation as in Figure 4. Previously developed topographical thickness maps are displayed for the (A) IPL, (D) INL, and (G) OPL. Cluster comparison of iAMD versus normal eyes are displayed for the (B) IPL, (E) INL, and (H) OPL. Significant P values are denoted by ***P < 0.001, ****P < 0.0001. Grid_(iAMD)-to-cluster_(normal) comparison of iAMD versus normal eyes are displayed for the (C) IPL, (F) INL, and (I) OPL.

Grid_(iAMD)-to-cluster_(normal) comparison confirmed thinned IPL across most locations with up to −1.3SD-from-normal (Fig. 5C). Consistent with the linear regression analysis, some locations toward the peripheral macula showed up to large magnitude of thickened IPL (up to 0.8 SD-from-normal). 

Meanwhile, the INL cluster_(normal) comparison showed nonsignificant mean thickness differences of iAMD versus normal eyes in all clusters (−0.99% ± 1.33% to −0.2% ± 1.22%; Figs. 5D, 5E). The linear regression slope of all INL clusters was also nonsignificant (β = 0.05 ± 0.05; P = 0.37; Supplementary Table S2). 

Grid_(iAMD)-to-cluster_(normal) comparison of iAMD versus normal eyes indicated a scattered distribution of up to medium magnitude of thinned INL (up to −1 SD-from-normal) and up to large magnitude of thickened INL (up to 1.3 SD-from-normal) forming no distinguishable spatial pattern (Fig. 5F). 

In the OPL, cluster_(normal) comparison revealed thinned OPL in all clusters (−6.76% ± 2.47% to −3.16% ± 1.02%; P < 0.001 to P < 0.01). The linear regression slope of all OPL clusters was significant (β = 0.38 ± 0.03; P < 0.0001; Supplementary Table S2) and implied less magnitude of thinned OPL with increasing retinal eccentricity. 

Grid_(iAMD)-to-cluster_(normal) comparison confirmed up to large magnitude of diffusely thinned OPL (up to −1.5 SD-from-normal; Fig. 5I), although the aforementioned slope describing less thinned OPL with increasing eccentricity was not appreciable likely because of intercluster differences in SD. 

Multivariable linear regression adjusted for age, sex, and spherical equivalent refraction showed that AMD status were significantly associated with thicknesses in all IPL clusters (β = −2.58 [−3.56, −1.61] to −0.74 [−1.36, −0.12]; P < 0.0001 to 0.05), all OPL clusters (β = −3.99 [−5.58, −2.4] to −1.26 [−1.69, −0.83], P < 0.0001), and no INL clusters (Supplementary Table S3). Repeated multivariable linear regression using a simplified AREDS severity score confirmed the associations between AMD with all IPL and OPL cluster thicknesses (scores 1 and 2 β = −4.4 [−6.79, −2.01] to −0.78 [−1.46, −0.09], P < 0.0001 to P < 0.05; Supplementary Table S3), and no INL clusters. However, there appeared to be no consistent patterns of dose-response relationships in these layers. 

Location-Specific Analysis in the ONL_+HFL, IS/OS, and RPE-BM

The ONL_+HFL cluster_(normal) comparison displayed nonsignificant mean differences of iAMD versus normal eyes in the foveal cluster and clusters 1 and 2 (−1.6% ± 1.68% to 0.1% ± 1.68%). The ONL_+HFL was borderline thinned in peripheral macula cluster 3 (−3.03% ± 1.56%; P = 0.05) and significantly thinned in peripheral macula cluster 4 (−5.37% ± 1.71%; P < 0.01; Figs. 6A, 6B). Linear regression slope of all ONL_+HFL clusters was significant (β = −0.47 ± 0.14; P < 0.05; Supplementary Table S2), implying greater magnitude of thinned ONL_+HFL with increasing retinal eccentricity. 

Figure 6.

 

Location-specific analysis in the ONL_+HFL, IS/OS, and RPE-BM. Presentation as in Figure 4. Previously developed topographical thickness maps are displayed for the (A) ONL_+HFL, (D) IS/OS, and (G) RPE-BM. Cluster_(normal) comparison of iAMD versus normal eyes are displayed for the (B) ONL_+HFL, (E) IS/OS, and (H) RPE-BM. Significant P values are denoted by: ***P < 0.001, ****P < 0.0001. Grid_(iAMD)-to-cluster_(normal) comparison of iAMD versus normal eyes are displayed for the (C) ONL_+HFL, (F) IS/OS, and (I) RPE-BM. F, foveal.

Grid_(iAMD)-to-cluster_(normal) comparison toward the central macula of iAMD eyes highlighted small magnitude of thinned ONL_+HFL (up to −0.3SD-from-normal) and up to medium magnitude of thickened ONL_+HFL (up to 0.5 SD-from-normal). At the peripheral macula, there were up to large magnitude of thinned ONL_+HFL (up to −1.6 SD-from-normal; Fig. 6C). 

In the IS/OS, cluster_(normal) comparison exhibited no significant differences in any cluster for iAMD versus normal eyes (Figs. 6D, 6E). Linear regression of IS/OS mean differences versus mean eccentricity (°) of each cluster were also nonsignificant (β = 0.09 ± 0.01; P = 0.08; Supplementary Table S2). 

Grid_(iAMD)-to-cluster_(normal) comparison demonstrated small magnitude of thinned IS/OS toward the central macula (up to −0.4SD-from-normal) and the up to large magnitude of thickened IS/OS toward the peripheral macula (up to 0.8 SD-from-normal; Fig. 6F) of iAMD versus normal eyes. 

Finally in the RPE-BM, cluster_(normal) comparison showed significantly thickened RPE-BM centrally in the foveal cluster and clusters 1 to 3 (27.45% ± 7.48% to 6.11% ± 2.33%; P < 0.001 to P < 0.01; Figs. 6G, 6H). No differences in clusters 4 to 6 reached statistical significance. Linear regression of RPE-BM mean differences versus mean eccentricity (°) of each cluster was significant (β = −2.53 ± 0.39; P < 0.01; Supplementary Table S2), implying reduced thickening of the RPE-BM with increasing retinal eccentricity. 

Grid_(iAMD)-to-cluster_(normal) comparison was consistent with the linear regression analysis, demonstrating a large magnitude of thickened RPE-BM toward the central macula (up to 7.6 SD-from-normal) and up to large magnitude of thinned RPE-BM toward the peripheral macula (up to −0.8 SD-from-normal; Fig. 6I) for iAMD eyes. 

Multivariable linear regression adjusted for age, sex, and spherical equivalent refraction showed that AMD status was associated with cluster thicknesses in ONL_+HFL clusters 3 and 4 (β = −2.35 [-4, −0.71] and −4.29 [−5.83, −2.75]; P < 0.01 and < 0.0001, respectively; Supplementary Table S3), and RPE-BM foveal cluster and clusters 1 to 4 (β = 9.43 [−6.29, 12.57] to 0.73 [−0.22, 1.24]; P < 0.0001 to P < 0.01). AMD status was not significantly associated with thicknesses in any IS/OS clusters. Repeated multivariable linear regression using a simplified AREDS severity score confirmed the associations between AMD with ONL_+HFL clusters 3 and 4 (scores 1 and 2 β = −4.57 [−6.26, −2.89] to −2.09 [−4.55, 0.38]; P < 0.0001 to P < 0.1), and RPE-BM foveal cluster and clusters 1-4 (score 1 β = 7.36 [3.99, 10.72] to 1.45 [0.61, 2.28]; P < 0.0001 to P < 0.001; score 2 β = 14.62 [10.01, 19.22] to 1.63 [0.89, 2.37]; P < 0.0001; Supplementary Table S3), and no IS/OS clusters. There was a consistently greater slope with increased AMD severity in RPE-BM foveal cluster and clusters 1 to 4, which may have indicated a dose-response relationship. 

Correlating Interlayer Thickness Differences in iAMD

Finally, we examined whether a rudimentary spatial model could be formed linking the retinal layers of iAMD eyes that showed spatial patterns of SD-from-normal thickness differences. Grid-to-grid correlations between the RPE, ONL_+HFL, OPL, IPL, and GCL were performed after adjustment for Henle's fibers’ displacement regarding the GCL and IPL (Supplementary Fig. S1). Absolute correlations significantly varied between comparisons (Brown-Forsythe and Welch ANOVA; P < 0.0001; Table 2). As expected, the IPL and GCL had the greatest absolute correlation (|r| ± SEM; 0.58 ± 0.02, 61/64 grids significant), followed by the ONL_+HFL and OPL (0.24 ± 0.02, 29/60 grids significant). The ONL_+HFL provided the next greatest absolute correlations, albeit weak (mean |r| ± SEM; 0.19 ± 0.03; Tukey's multiple comparisons test P = 0.14 to P < 0.0001), whereas comparisons against the RPE-BM resulted in the poorest absolute correlations (0.09 ± 0; P = 0.72 to P < 0.0001). 

Table 2.

 

Correlations Between Inter-Layer SD-From-Normal Thickness Differences

 

To further investigate the poor correlations using the RPE-BM, we compared the coefficients of variation (CV) within each cluster of the RPE-BM between normal and iAMD eyes. CV were significantly greater in iAMD eyes (mean ± SD, 26.32% ± 16.13%; P < 0.001) compared to normal eyes (8.71 ± 0.61 %; Supplementary Table 4), suggesting highly variable levels of drusen load in the iAMD group. 

Discussion

In this study, location-specific analyses revealed various patterns of thickness differences between iAMD and normal eyes not evident in global analyses. These difference patterns were unique to each individual retinal layer and exhibited statistical significance or large effect sizes in all individual retinal layers. Results were also mostly in concordance with other OCT studies that have described thickness differences between iAMD versus normal eyes individually in the GCL,^3,6–10 IPL,^4,5,11 OPL,^3,4,7 ONL_+HFL,^{3,4,7,13–16} IS/OS,^3,4,7 or RPE-BM.^3,4,16–23 Specifically, the RPE-BM was thickened centrally, although there was thinned outer (OPL, ONL_+HFL, and RPE-BM) and inner retina (GCL and IPL) with some eccentricity-based effects. There were significant but weak correlations between the outer and inner retinal layers. These results improve anatomical understanding of iAMD and could guide clinical diagnosis and monitoring of AMD by indicating specific retinal locations more subject to change. 

Location-Specific Thickness Analysis Highlights iAMD Anatomy

Our results demonstrated concurrent thickness differences in the outer and inner retinal layers of iAMD eyes. Based on well-established anatomy of OCT reflectance profiles,⁶⁶ plausible anatomical explanations for the observed location-specific thickness patterns are provided. Specifically, locations of thinned GCL and IPL may represent reduced ganglion cell density in the GCL⁶⁷ and ganglion cell synapses in the IPL, whereas thinned ONL and OPL may represent reduced photoreceptor density in the ONL and synaptic terminals and proteins in the OPL.^2,68,69 Thinned RPE-BM toward the peripheral macula may be characteristic of RPE atrophy.^2,70–78 Alternatively, thickened GCL and IPL may reflect Müller cell hypertrophy⁷⁹ or neuronal proliferative and migratory changes as seen in outer retinal degeneration,^69,80,81 thickened IS/OS could reflect photoreceptor segment disorganization or translocation,^71,82,83 and centrally thickened RPE-BM would likely represent drusen.⁸⁴ Multiple cellular populations per OCT reflective layer can, however, confound interpretations of the underlying cellular structures affected in iAMD. Further work combining our results with other measures of retinal integrity could help strengthen interpretation of alterations in AMD retinal anatomy. 

In the GCL, OPL, and ONL_+HFL, we also observed eccentricity-based effects that may have been consequential to the greater magnitude of thickened RPE-BM centrally. Greater magnitude of thickened retina toward the peripheral macular GCL and OPL could also reflect hypertrophic glial cells or neuronal migration as commonly seen in other outer retinal degenerative diseases such as retinitis pigmentosa.⁷⁹ In particular, Müller cell volumes (including their processes that extend approximately from the inner- to the external-limiting membrane) have been proposed to depend on available space.⁸⁵ Thus it is possible that the suggested thickening toward the peripheral macula in several individual retinal layers may reflect Müller cell hypertrophy at areas of greater Müller cell density.⁸⁶ Differences between central and peripheral Müller cell morphology and transcriptome^87–89 may also explain thickening toward the peripheral retina, although linkage of these findings in vitro to AMD eyes in vivo is yet to be shown. Alternatively, the ONL_+HFL showed greater magnitude of thinned retina toward the peripheral macula. Although this may reflect rod susceptibility in AMD,^27,28,90 as has been explored via structural^2,91–96 and functional^97–102 measures, clinical OCT analyses cannot distinguish rod from cone photoreceptors without further augmentation such as adaptive-optics; further research is warranted. 

Proposed Mechanisms for “Sparing” of the RNFL and INL

Location-specific analyses suggested sparing of the RNFL and INL in iAMD eyes compared to normal eyes in concordance with other OCT studies.^3–7 The macular RNFL being genuinely spared in the early stages of AMD may reflect similar finding of radial peripapillary capillary plexus sparing in iAMD eyes.¹⁰³ Zucchiatti et al.⁹ also established that the RNFL is spared in non-neovascular (early and late atrophic stage) AMD but significantly decreased in neovascular AMD. However, these findings are counterintuitive because implied loss of ganglion cells would also allude to concomitant loss of ganglion cell axons. Alternatively, RNFL sparing may be in part an artefact of high variability in macular RNFL thicknesses. Our grid_(iAMD)-to-cluster_(normal) comparison found locations with variable thickness profiles that were masked by an indistinguishable spatial pattern. Variability in macular RNFL thicknesses may have occurred because of the relatively sparse distribution of ganglion cell axons at the macula.^{31,32,104–107} Other reasons for the irregular pattern of RNFL thickness profiles may include subclinical vitreo-macular traction, astrocyte hypertrophy, or Müller cell process proliferation in iAMD.^80,81 Further study is required to determine what cellular structures may be implicated in the RNFL of iAMD eyes, if any. 

Sparing of the INL alternatively may be attributed to several factors. Histology studies have revealed a complex array of inner retinal remodeling events secondary to photoreceptor degeneration, including outgrowth of rod bipolar dendrites¹⁰⁸ and horizontal and amacrine cell neurites in hereditary retinal degeneration models¹⁰⁹; inner INL apoptosis (possibly representing amacrine cells) in non-neovascular AMD eyes⁷¹; and proliferation and upregulation of Müller cells in non-neovascular AMD eyes.^69,80,81 These epiphenomena have been suggested to occur to maintain synaptic functionality with retracted photoreceptor axons in AMD and stimulate adjacent axonal regeneration.^{69,71,80,81,108,109} Studies using rodent models of retinal degeneration have also shown reduced number and impaired functionality of bipolar, horizontal, and amacrine cells^110–113; increased synaptic activity of bipolar and amacrine cells¹¹⁴; bipolar and amacrine cell migration^115,116; and reorganization of Müller cell processes.¹¹⁵ Although translatability of results from animal studies to human eyes is equivocal, these models underscore the complex machinations possible within the INL that may explain its variable thickness profiles. 

Postreceptoral Degeneration in AMD

An emerging pathophysiological theory in AMD suggests that AMD insults may arise from the outer retina or choroid and then propagate via anterograde degeneration within the inner retina. This process is known as anterograde trans-synaptic or postreceptoral degeneration in AMD.^117,118 Although this theory is well discussed in other OCT studies,^3,5–7,9,10 we provided more holistic insight by examining the spatial relationship between retinal layers rather than focusing only on a few specific retinal layers.^{5,6,8–10,12–15,15,17–23,119–135} Correlations were poorest when compared against the RPE-BM, possibly because heterogeneous levels of drusen load in the iAMD group may have spatially unpredictable retinal effects. This unpredictability may be exacerbated by the nonlinear association between drusen load and AMD severity as seen with drusen regression.^136,137 Alternatively, correlations were greater, albeit weak compared against the ONL_+HFL. It is possible that the spatial areal unit of sampled thicknesses may have been too large and subsequently hampered correlational values.³³ This was particularly notable when considering that Henle's fibers’ displacements are much smaller in size than the 3° × 3° grids.⁶³ Nonetheless, there is still limited evidence to support the theory of postreceptoral degeneration in AMD and future works using location-specific and longitudinal designs could help clarify this theory. 

Limitations

The primary limitation of this study was related to the interpretation of OCT data. Although OCT provides an accessible, in vivo measure of retinal reflectance profiles, the cross-sectional location-specific retinal thickness patterns we describe do not specify which cellular structures are affected and only imply a possible cause-effect relationship with iAMD. In our repeated multivariable regression models, statistical comparisons between slopes of AREDS severity scores were precluded because of limited sample size. Future works using a larger AMD group with broader severities, and possibly more comprehensive grading such as the (non-simplified) AREDS severity scale,¹³⁸ could explore potential dose-response relationships such as the greater magnitude of thinned GCL and thickened RPE-BM with increased AMD severity that we described. Further extension of this work, including longitudinal analysis and direct comparison to other measures of retinal cellular integrity such as adaptive-optics OCT or visual function, could also significantly strengthen the putative cause-effect relationship.^139,140 

Additionally, there were slight discrepancies between our results and those of Brandl et al.⁴ and Lamin et al.³; they found no significant differences in OPL thickness or volume in contrast to our observations of thinned OPL in iAMD eyes. These studies did not, however, clearly account for Henle's fiber layer during segmentation, which may explain the disparity. Our comparative areal units, that is, normal clusters, were also different to the above studies, which may have contributed to minor discrepancies in results. However, we ensured that our comparative areal units were meaningful and had proven intergroup separability and intragroup similarity³⁷ to mitigate any potential bias.^33–36 

Conclusion

Location-specific analyses of each individual retinal layer revealed various patterns of thickness differences between iAMD and normal eyes not evident in global analyses. The central RPE-BM was thickened, whereas there was thinned outer (OPL, ONL_+HFL, and non-central RPE-BM) and inner retina (GCL and IPL) with some eccentricity-based effects. There were significant but weak correlations between the thinned outer and thinned inner retinal layers. These results improve anatomical understanding of iAMD and could guide clinical diagnosis and monitoring of AMD by indicating specific retinal locations more subject to change. 

Acknowledgments

The authors thank Gordon Doig (Centre for Eye Health, Sydney, Australia) for methodological advice. 

Supported, in part, by research grants from the Rebecca Cooper Foundation and the National Health and Medical Research Council of Australia (NHMRC grant no. 1174385) awarded to L.N.S. M.T. and V.K. are supported by the Australian Research Training Program scholarship. Guide Dogs NSW/ACT provides support for the Centre for Eye Health (the clinic of recruitment), a top-up scholarship for V.K., and salary support for M.K. and L.N.S. 

Disclosure: M. Trinh, None; V. Khou, None; M. Kalloniatis, None; L. Nivison-Smith, None