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Retina  |   February 2014
The Association of Retinal Structure and Macular Pigment Distribution
Author Affiliations & Notes
  • Verena Meyer zu Westrup
    Institute of Epidemiology and Social Medicine, Medical Faculty, Westfälische Wilhelms University Münster, Münster, Germany
  • Martha Dietzel
    Department of Ophthalmology, St. Franziskus Hospital, Münster, Germany
  • Daniel Pauleikhoff
    Department of Ophthalmology, St. Franziskus Hospital, Münster, Germany
  • Hans-Werner Hense
    Institute of Epidemiology and Social Medicine, Medical Faculty, Westfälische Wilhelms University Münster, Münster, Germany
  • Correspondence: Hans-Werner Hense, Institute of Epidemiology and Social Medicine, Medical Faculty, Westfälische Wilhelms University, Albert-Schweitzer-Campus 1, D 3, 48129 Münster, Germany; hense@uni-muenster.de
Investigative Ophthalmology & Visual Science February 2014, Vol.55, 1169-1175. doi:10.1167/iovs.13-12903
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      Verena Meyer zu Westrup, Martha Dietzel, Daniel Pauleikhoff, Hans-Werner Hense; The Association of Retinal Structure and Macular Pigment Distribution. Invest. Ophthalmol. Vis. Sci. 2014;55(2):1169-1175. doi: 10.1167/iovs.13-12903.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Macular pigment optical density (MPOD) and age-related macular degeneration (AMD) are thought to be associated; however, the details are not yet understood clearly. This study aimed at investigating how retinal anatomic structures relate with the spatial MPOD distribution in single eyes.

Methods.: In a subgroup of the third follow-up examination of the Münster Aging and Retina Study (MARS) cohort (mean age, 78.4 years), 124 single eyes of 79 participants with early AMD were examined. The MPOD was assessed using 2-wavelength autofluorescence (AF). Retinal thickness (RT) and fovea pit profile slopes were measured using optical coherence tomography (OCT). The results were analyzed for interocular correlation in 58 pairs of eyes, and for the association of MPOD distribution patterns with RT using uni- and multivariate statistical methods.

Results.: The interocular correlations for several measures of RT and RT layers were high (P < 0.001). The RT was inversely and significantly related to MPOD at 1.0° and at 2.0° from the foveal center, but not to central MPOD. After controlling for sex, age, smoking, and spherical equivalent, RT was significantly thinner (−39.7 μm, P < 0.001) in eyes with ring-like compared to normal MPOD distribution. In particular, a thinner layer between internal and external limiting membrane showed strong associations with ring-like structures.

Conclusions.: Higher values of MPOD at 1° and 2°, as well as a ring-like distribution of MPOD were associated significantly with thinner maculae, due to thinner inner retinal layers. The MPOD distribution was unrelated to the slope of the foveal pit or the choroidal thickness. Our results suggest that the retinal section between the internal and external limiting membrane is involved in the spatial distribution of MPOD.

Introduction
The macular pigment (MP) is made up of the three carotenoids lutein, zeaxanthin, and meso-zeaxanthin, all of which accumulate in the macula at high concentration. While lutein and zeaxanthin are derived exclusively from dietary uptake, meso-zeaxanthin is synthesized from lutein that already has passed the retinal barrier. With its antioxidant and filter functions, MP is believed to have a protective function against the development and progression of age-related macular degeneration (AMD). This protective effect of MP was suggested to be related to the different spatial distributions of the carotenoids in the inner retina, although the results of the recent Age-Related Eye Disease Study 2 (AREDS2) trials have cast some doubts on the relevance of MP in the development of AMD. 1 Putative mechanisms involve protection from the damaging effects of free radicals produced by blue light. Delori et al. 2 first, and recently, Dietzel et al. 3 described the occurrence of ring-like structures in the spatial distribution of MP optical density (MPOD), which are characterized by a secondary peak in the slope of the MPOD profile. Others observed a distribution pattern that was described as a “central dip.” 4 In several studies, it was suggested that the spatial distribution of MP correlates with a specific morphologic structure of the fovea, including features like the steepness of the foveal pit, its width, and depth. 46  
Despite the central pit being the fovea's most prominent anatomic feature, the surface contour of this region so far has been examined rarely. Dubis et al. 7 used an automated algorithm to extract morphologic characteristics of the foveal pit from standard optical coherence tomography (OCT) images and found that large variations exist in its morphologic architecture. Others developed similar automated techniques to quantify the foveal pit morphology; that is, its depth, diameter, and slope. They reported, for example, that retinas generally were thinner in women, African Americans, and in the elderly. 813 Especially in the eyes of elderly patients, which frequently are affected by various degenerative conditions, automated measurements are difficult. Recently, Ray et al. 14 evaluated OCT image artifacts and indicated common errors that occurred especially in eyes with prevailing pathologic conditions. They concluded that established automated algorithms could not be applied validly for the examination of the anatomic features of elderly eyes. 
Because the correlation of different MP spatial distributions with specific structures in different foveal cellular layers still is unproven, the present study aimed to examine the association between retinal architecture and MPOD levels, with a special focus on the ring-like distribution pattern that is seen in approximately 10% to 20% of all retinas. 3,15 The foveal pit profile, and the retinal (RT) and choroidal (CT) thicknesses were evaluated semiautomatically from OCT images. 
Methods
The examinations were carried out in a substudy of the Muenster Aging and Retina Study (MARS) conducted between 2010 and 2011 in elderly individuals with early AMD. The MARS is a prospective longitudinal study to investigate clinical, lifestyle, and genetic factors involved in the pathogenesis and clinical course of early AMD. The eligibility criteria and the schedule have been described in detail previously. 16,17 In brief, a convenience sample of 1060 individuals with and without early AMD was examined at baseline in the years 2001 to 2003 (MARS I). Eligibility criteria for the baseline examination were presence of early AMD in at least one eye, no or minimal lens opacity, and age between 60 and 80 years. In addition, volunteers, spouses, and companions of early AMD patients who had no signs of early AMD were included in the study base. After a median follow-up time of 2.6 years, 828 (85.5% of all eligible) participants took part in the subsequent MARS II visit between 2004 and 2007. The second follow-up examination in 2007 to 2009 (MARS III) was attended by 492 (69% of all eligible) participants after a median time of further 4.8 years. 
Study Population
Between October 2010 and May 2011, we reinvited MARS III participants to a substudy (MARSplus). Participants were eligible for MARSplus if, at the time of the MARS III examination, they had early AMD in at least one eye and no late AMD in the accompanying eye. Those participants had to be either homozygous for the single-nucleotide polymorphism (SNP) rs1061170 in the CFH gene and/or homozygous for SNP rs10490924 in the ARMS2 gene, or noncarriers for either of the two risk alleles. Of the 159 participants fulfilling these criteria in MARS III, 4 were deceased, 7 could not be contacted, and 39 declined participation, mostly due to their fragile general health. Thus, 109 individuals participated and 218 study eyes were examined. Three eyes were excluded due to the absence of a fundus image or other relevant data, a further 11 had to be discarded for newly contracted ophthalmo-pathological reasons not related to AMD (such as retinal vein occlusion). Another 31 eyes had progressed in the meantime to late AMD. Of the remaining study eyes, 22 showed no sign of early AMD, 19 lacked data on macular pigment, and 8 had no evaluable OCT images. Phakic eyes with spherical refractive error values ≤ −4 or ≥+3 diopters and cylindrical refractive error values of ≤ −3 or ≥+2 diopters, as well as spherical equivalent refractive error of ≤−6.00 diopters were excluded as well. This left us with 124 study eyes (of the initial 218) from 79 patients with early AMD available for analysis. 
The recruitment and research protocols were reviewed and approved by the Institutional Review Board of the University of Muenster, and written informed consent was obtained from all study participants in compliance with the Declaration of Helsinki. 
Study Examinations
Study participants were interviewed by trained staff using a standardized questionnaire. Detailed information was obtained on anthropometric measurements, and demographic characteristics, smoking history, lifestyle, medical history including the current and past use of medications, and vitamin supplements, in particular those containing lutein and/or zeaxanthin. The genotyping of risk variants in CFH (rs1061170) and ARMS2 genes (rs10490924) was performed as described previously. 18  
The spherical equivalent refractive error in each eye and the number of pseudophakic eyes also were determined. Distant and near vision was determined with best adjustment adhering to Early Treatment Diabetic Retinopathy Study (ETDRS) criteria. Clinical examinations with the slit-lamp in mydriasis involved assessment of the anterior segment of the eye and indirect funduscopy. 
We took and digitally stored 35° fundus photos (Topcon TRC.50EX; Topcon Deutschland, Willich, Germany) of all study eyes. The fundus photos were used to assess the presence of AMD features according to the International Classification and Grading System for AMD 19 and were graded by specifically trained observers according to the Rotterdam AMD stage classification system. 20  
OCT Measurements.
The OCT is a noninvasive imaging technique using low-coherence interferometry to create high-resolution cross-sectional and topographic images of optically accessible tissue, 14 visualizing the microscopic anatomy of the retina split up into functional layers. 21 We performed spectral domain (SD) OCT examinations with the Spectralis HRA+OCT (Heidelberg Engineering, Heidelberg, Germany). The examinations consisted of a horizontal and a vertical scan, both intersecting at the foveal center, automatically averaging 100 scans with width and height of 20°; each scan was taken in conventional and in enhanced-depth-imaging (EDI) mode 22 that allowed for better distinction of the choroid. The results presented in this study refer to the central horizontal scan. 
The SD-OCT images were evaluated by one investigator (VM) masked to the subjects' ophthalmologic and baseline data. Scans of poor quality were excluded from the study based on the consensus of two investigators (MD and VM). The evaluations and measurements of the SD-OCT scans were performed using Heidelberg Eye Explorer (Version 1.7.0.0; Heidelberg Engineering). Measurements were performed semiautomatically. The cursors were set manually to mark the boundaries, while the software then calculated and displayed the corresponding measurement values. Subfoveal CT was measured as the distance between the outer limit of the hyperreflective RPE layer and the inner surface of the sclera in automatic real-time enhanced depth imaging (ART-EDI) mode, while all other parameters were measured using the automatic real-time (ART) mode. 
We measured RT as the distance between the inner border of the nerve fiber layer (NFL) and the outmost edge of the RPE. The RT was measured at five locations: the foveal center, and at 75 and 300 μm nasally and temporally from the fovea. Additionally, the central RT was subdivided into two sections: the distance NFL-ELM, which ranged from the NFL to the outmost edge of the external limiting membrane (ELM), and the distance ELM-RPE between the ELM and the outmost edge of the RPE. 
To increase accuracy and repeatability of the measurements, we started by identifying a point that represented the foveal center in each OCT image and by positioning a vertical line through this point. At this position, the subfoveal central RTs and the distances NFL-ELM and ELM-RPE were measured. We then constructed an auxiliary line that was perpendicular to the central line and that intersected with the central line at the ground mark defining the RPE boundary. This auxiliary line served as base mark for the measurement of the RT distances 75 and 300 μm nasally and temporally from the foveal center. Following the approach described by Kirby et al., 6 we also calculated three foveal pit profile slopes (FPPS). The measurement procedures and the FPPS formulas are displayed in the Figure
Figure
 
Measurements of RT, NFL-ELM, and ELM-RPE layers, and definition of foveal pit slopes. The calculations were performed according to the following formulas: Slope 1 (75–300 μm) m 1 = (y2-y1)/(x2-x1), Slope 2 (0–75 μm) m 2 = (y1-y0)/(x1-x0), and Slope 3 (0–300 μm) m 3 = (y2-y0)/(x2-x0). y0, central subfoveal RT; y1, subfoveal RT 75 μm nasal/temporal from center; y2, subfoveal RT 300 μm nasal/temporal from center; x0, central measuring point; x1, measuring point 75 μm nasal/temporal from center; x2, measuring point 300 μm nasal/temporal from center.
Figure
 
Measurements of RT, NFL-ELM, and ELM-RPE layers, and definition of foveal pit slopes. The calculations were performed according to the following formulas: Slope 1 (75–300 μm) m 1 = (y2-y1)/(x2-x1), Slope 2 (0–75 μm) m 2 = (y1-y0)/(x1-x0), and Slope 3 (0–300 μm) m 3 = (y2-y0)/(x2-x0). y0, central subfoveal RT; y1, subfoveal RT 75 μm nasal/temporal from center; y2, subfoveal RT 300 μm nasal/temporal from center; x0, central measuring point; x1, measuring point 75 μm nasal/temporal from center; x2, measuring point 300 μm nasal/temporal from center.
Repeatability of the method was assessed in a masked remeasurement of 10 randomly selected eyes. The intermeasurement mean differences were less than 2% for any of the measurements taken, and the variation was less than ±5% except for subfoveal RT (±7.5%). 
Macular Pigment Measurement.
We performed measurements of the macular pigment optical density with a Heidelberg Retina Angiograph (HRA 1; Heidelberg Engineering) using the two wavelengths method, as described previously in detail. 3,15,23 Alignment and focusing of the central retina were performed under 488 nm wavelength light of the HRA 1 for at least 30 seconds. After retinal bleaching, sequences of 20° images were captured at 488 and 514 nm, and MP density maps were generated by digital subtraction of the log AF images, upon which mean MPOD values were calculated for circles centered on the fovea. In the present analyses, these circles were located at eccentricities of 0.25°, 0.5°, 1.0°, and 2.0°. The reference location was selected at an eccentricity of 6° as described previously. 24  
MP Density Profile and Ring-Like Structures.
To investigate the spatial distribution of MP, we analyzed the MP density maps and the radial density profiles as displayed in detail in our previous publication. 3 The latter were generated and displayed graphically by plotting the mean MPOD values that were calculated for each radius around the fovea, against the distance to the fovea. As described previously, 3 the ring-like structure was defined as a density profile showing a bimodal pattern, consisting of a central peak of MPOD followed by a decline and a secondary peak of increased density on the slope of the profile. In cases where the distribution of MPOD showed no strictly monotonic decline from the center of the fovea to the periphery, and no explicit ring-like pattern of MP (but, for example, an implied plateau on the slope of the profile), the eyes were marked as having “intermediate MP distributions.” The absence of a ring-like structure was defined as a strictly monotonous decline of the density profile graph from the center of the fovea to the periphery without any plateauing or bimodal pattern. 3  
The MP density profiles were analyzed for the eccentricity at which the maximum and minimum of the ring occurred, and for the respective MPOD measured at these eccentricities; the “maximum” of the ring was defined thereby as the secondary maximum of MP forming the ring, and the “minimum” as the minimum density at the nadir between the central peak of MPOD and the second peak. Furthermore, all MPOD profiles were analyzed for the “half width,” which indicates the eccentricity from the fovea where half of the peak of MPOD is present. 3  
Statistical Analysis
The statistical data analysis comprised measurements of MPOD, RT, CT, spherical equivalent refractive error, age, sex, smoking history, status of the lenses (phakic, pseudophakic), risk allele status in CFH and ARMS2 genes, and classification of AMD. We averaged the nasal and temporal RT measurements in each eye. Spearman's rank correlation coefficients were computed to assess the interophthalmic association of the OCT measurements in 58 pairs of eyes. The χ2 tests were used to compare categorical and t-tests for continuous variables for analyses of the 124 single study eyes. The impact of influential factors and confounders was evaluated by multivariable linear regression models, and adjusted mean differences with their 95% confidence intervals (95% CI) are reported. All statistical analyses were performed using the software package SAS for Windows, version 9.2 (SAS Institute, Inc., Cary, NC). 
Results
The baseline characteristics of the 124 study eyes are shown in Table 1. They belonged to patients with a mean age of approximately 78 years and were slightly more often from female (n = 65) than male (n = 59) participants. Approximately one in five eyes showed a ring-like structure in the spatial distribution of MPOD. Table 1 also shows that, due to the sampling of study participants, the risk alleles for AMD in the CFH and ARMS2 genes were more frequent than in random samples from the general population. 
Table 1
 
Baseline Description of the Study Eyes
Table 1
 
Baseline Description of the Study Eyes
n Mean (SD) %
Age, y 124 78.4 (4.8)
MPOD, density units
 Peak 0.73 (0.22)
 0.25° 0.68 (0.22)
 0.5° 0.60 (0.22)
 1.0° 0.47 (0.19)
 2.0° 0.18 (0.09)
Halfwidth, degrees 1.19 (0.38)
Genetic pattern
 Homozygous rs1061170, CFH 60 50.0
 Homozygous rs10490924, ARMS2 27 22.3
Ring structure in the MPOD distribution
 Absent 80 55.7
 Intermediate 34 21.0
 Present 32 23.4
In 58 pairs of eyes we observed a very strong intraindividual, between-eye correlation of RT throughout all of our measurements (Table 2). The crude average of RT and CT, as well as the foveal pit slopes is displayed in Table 3
Table 2
 
Interocular Symmetry of the Foveal Morphology by SD-OCT (n = 58 Pairs of Eyes)
Table 2
 
Interocular Symmetry of the Foveal Morphology by SD-OCT (n = 58 Pairs of Eyes)
Rank Correlation P Value
Slopes
 Slope1 0.56254 <0.0001
 Slope3 0.52475 <0.0001
RT
 Central 0.62375 <0.0001
 75 μm 0.65465 <0.0001
 300 μm 0.67545 <0.0001
 NFL-ELM 0.60590 <0.0001
 ELM-RPE 0.33601 0.0099
CT 0.76771 <0.0001
Table 3
 
Results of the OCT Measurements for RT, CT, and Slopes
Table 3
 
Results of the OCT Measurements for RT, CT, and Slopes
n Crude Mean (SD)
RT central, subfoveolar, μm 124 245.0 (30.6)
RT 75 μm eccentric, μm 123 251.4 (31.8)
RT 300 μm eccentric, μm 123 291.2 (34.7)
Distance NFL-ELM, μm 123 136.8 (27.1)
Distance ELM-RPE, μm 123 107.5 (21.9)
CT, μm 124 200.1 (77.8)
Slope 1 124 0.177 (0.066)
Slope 2 124 0.085 (0.054)
Slope 3 124 0.154 (0.055)
Correlation of RT With MPOD
The RT in the central fovea was not correlated with central MPOD at 0.0°, but an inverse correlation was apparent with MPOD at 1.0° and 2.0° (Table 4). Likewise, RT measured at 75 and 300 μm from the fovea also was related only to MPOD measured at the more peripheral locations, but not to central MP. Interestingly, NFL-ELM was correlated significantly with lower MPOD at 2.0°, while no such association was found for ELM-RPE or any of the foveal pit profile slopes. Furthermore, MPOD at any foveal location was unrelated to CT. 
Table 4
 
Correlations Between RT and CT, and MPOD at Various Eccentricities From the Fovea
Table 4
 
Correlations Between RT and CT, and MPOD at Various Eccentricities From the Fovea
OCT Measurement MPOD Measurement Correlation Coefficient P Value
Retinal thickness
 central Halfwidth −0.19 0.03
Peak 0.04 0.65
MPOD at 0.25° 0.08 0.36
MPOD at 0.5° 0.08 0.34
MPOD at 1.0° −0.18 0.04
MPOD at 2.0° −0.23 0.009
 75 μm eccentric Halfwidth −0.19 0.37
Peak 0.05 0.58
MPOD at 0.25° 0.06 0.36
MPOD at 0.5° 0.09 0.33
MPOD at 1.0° −0.18 0.05
MPOD at 2.0° −0.23 0.009
 300 μm eccentric Halfwidth −0.13 0.16
Peak 0.08 0.33
MPOD at 0.25° 0.13 0.16
MPOD at 0.5° 0.13 0.14
MPOD at 1.0° −0.12 0.17
MPOD at 2.0° −0.20 0.026
NFL-ELM central Halfwidth −0.15 0.09
Peak 0.11 0.21
MPOD at 0.25° 0.13 0.15
MPOD at 0.5° 0.13 0.15
MPOD at 1.0° −0.12 0.19
MPOD at 2.0° −0.18 0.05
ELM-RPE central Halfwidth −0.10 0.23
Peak −0.06 0.45
MPOD at 0.25° −0.06 0.52
MPOD at 0.5° −0.05 0.56
MPOD at 1.0° −0.14 0.11
MPOD at 2.0° −0.12 0.19
Choroidal thickness central Halfwidth −0.03 0.74
Peak −0.02 0.89
MPOD at 0.25° −0.00 0.98
MPOD at 0.5° −0.01 0.87
MPOD at 1.0° −0.2 0.85
MPOD at 2.0° −0.9 0.29
Comparison of the RT measurements in the three groups of eyes that displayed different MPOD distributions revealed that the crude RT was consistently lower in eyes with a ring-like MPOD distribution compared to eyes with no ring (difference = 39.7 μm, P < 0.001, Table 5). Eyes with an intermediate MP distribution showed RTs that ranged between those from eyes with and those without a ring. In particular, the distance NFL-ELM showed strong and statistically significant associations with ring-like structures, while ELM-RPE, the three foveal pit profile slopes, and CT were unrelated to MPOD distributions. Of note, adjustment for sex, age, body height, and smoking did not materially alter these associations. Further inclusion of the spherical equivalent had no impact on the observed highly significant differences (Table 5). Inclusion of the CFH and ARMS2 genotype information had also no effect. 
Table 5
 
Crude and Adjusted Mean Values of RT and CT Measurements in Three Groups of MPOD Distribution
Table 5
 
Crude and Adjusted Mean Values of RT and CT Measurements in Three Groups of MPOD Distribution
Ring-Like MPOD Distribution n Crude Mean Value P Value Adjusted* Mean Value P Value
Retinal thickness
 Central Absent 69 258.0 <0.001 258.3 <0.0001
Intermediate 29 238.5 237.9
Present 26 218.1 219.1
 75 μm Absent 69 264.5 <0.001 265.0 <0.0001
Intermediate 29 245.2 244.9
Present 25 222.6 223.1
 300 μm Absent 69 305.9 <0.001 305.9 <0.0001
Intermediate 29 284.4 284.2
Present 25 258.5 259.8
NFL-ELM Absent 69 146.5 <0.001 147.0 <0.0001
Intermediate 29 133.4 131.9
Present 25 114.3 114.9
ELM-RPE Absent 69 111.2 0.08 111.2 0.08
Intermediate 29 105.0 105.9
Present 25 100.3 100.5
Choroidal thickness Absent 69 193.1 0.451 193.8 0.61
Intermediate 29 205.8 203.8
Present 26 212.2 210.8
Slope 1 Absent 69 0.184 0.33 0.181 0.52
Intermediate 29 0.174 0.174
Present 25 0.161 0.163
Slope 2 Absent 69 0.088 0.31 0.088 0.37
Intermediate 29 0.089 0.091
Present 25 0.071 0.072
Slope 3 Absent 69 0.160 0.24 0.158 0.39
Intermediate 29 0.153 0.158
Present 25 0.138 0.141
Discussion
This study was conducted with the aim of investigating how the choroidoretinal architecture impacts on the amount and spatial distribution of macular pigment. Our results provided some new insights related to these questions. First, the very high symmetry of foveal pit morphology measured by SD-OCT in pairs of eyes indicated that the foveal configuration and the retinal structure appear to be constitutional features of an individual person rather than an individual eye. This would imply that, if RT were related causally to MPOD distribution, the latter also would be expected to be similar in pairs of eyes. In fact, previous work in our group has confirmed that MPOD measured at different eccentricities from the fovea 23 as well as presence and shape of ring-like MPOD distribution are highly concordant in pairs of eyes. 3  
Second, the present results suggested that reduced RT is not associated with higher MPOD at the fovea center, but more peripherally at 1° or 2° away from the center. This finding concurs with the association of RT and MPOD distribution: Eyes with intermediate or ring-like distribution tend to have a broader distribution of MPOD with higher levels of MPOD in the periphery compared to eyes with a monotonously declining MPOD, which accumulate their macular pigments mainly close to the fovea center. 3 Thus, we supposed that the inverse correlations reported in Table 4 essentially reflect the same association as the results of Table 5, which show that ring-like MPOD distributions are associated with a lower RT. 
In this study, RT at the foveal center, and at 75 and 300 μm from the center, related inversely with the peripheral MPOD at 1° and 2°. Interestingly, this inverse association was present exclusively for the layer from NFL to ELM, but not for the stratum ranging from ELM to Bruch's membrane. Furthermore, the retina of eyes displaying a ring-like distribution of MPOD was significantly thinner than in eyes that showed a monotonous decline of MPOD toward the periphery. Of note, the layer NFL-ELM, rather than ELM-RPE, accounted for most of this reduced thickness These findings were independent of covariates, including the spherical equivalent. 
Third, we could not confirm the hypothesis recently put forward that the foveal shape is related to distribution patterns of MPOD. 4,6 The foveal width was suggested as another potentially influential factor; however, precise methods of foveal width measurements are missing. In our study sample of elderly patients, it often was difficult to identify the rims, pit depth, and foveal width due to AMD-related changes, such as drusen. These age-related problems in the evaluation of OCT scans also were the reason why we decided to perform all OCT measurements semiautomatically. This decision was backed by a recent discussion 14,25,26 highlighting common calculation errors and the limitations of automated techniques of OCT image measurements that affect interpretation in elderly eyes. 
In this context, we also emphasized that our study is different from many previous reports 2,4,7,8,1013,2730 in that our study eyes originated only from subjects who were 65 years and older. Using partly similar methods, Liew et al. 27 also had measured RT with MPOD and collected results different from ours, in particular regarding correlations of RT and MPOD at varying degrees from the center. An important difference between the studies lies in the fact that Liew et al. 27 recruited female eye-healthy subjects between 17 and 50 years of age, while the participants of our study were men and women averaging 78 years of age diagnosed with early AMD. Thus, the observed differences may be at least partly attributable to different age, technique, and sample composition of the study eyes. While at first sight these almost opposite findings look contradictory, it must be considered that, in contrast to the study of Liew et al., 27 our investigation was able to differentiate and separate layers within the central RT that reflect the structural architecture of the retina. Especially, the finding that the inverse association with RT in eyes with rings is attributable largely to a thinner NFL-ELM layer, may enable a better understanding of these complex alterations. Furthermore, Liew et al. 27 seem to label as “low central MPOD” those eyes that show an intermediate or a ring distribution of MPOD, but they do not report their proportion in the study sample. Of note, this proportion amounted to 45% in our study. Nevertheless, Liew et al. 27 demonstrate very clearly in their Figure 3, and they report it explicitly in their results section, that central RT was low in eyes with low central MPOD: Figure 3 in their publication reveals that these eyes had a ring-like MPOD distribution (according to our definition). Hence, despite being quantitatively different, the results by Liew et al. 27 do not refute our finding of RT being significantly thinner in eyes with ring-like MPOD distribution. 
Nolan et al. 4 suggested that the anatomic structure of a subject's fovea has an important role in the way MP is distributed. Our findings suggested that a thinner retina, due to a thinner layer between inner and external limiting membrane, is associated with a ring-like distribution of MPOD. According to earlier studies with microspectroscopy, MP is known to accumulate primarily in the inner plexiform layer and the long cone receptor axons (Henle's fibers). 5 More recent studies have shown that Müller cells probably are involved in the trafficking and storage of MP within the retina as well. 31 Gass 32 hypothesized that a layer of Müller cells resides between the internal limiting membrane and Henle's fiber layer, and that a lump of Müller cells (named Müller cell cone) accumulates immediately under the inner limiting membrane at the bottom of the foveal pit depression. Interestingly, reduction of Müller cells related directly to central MP depletion was observed recently in eyes with macular telangiectasia type 2. 33 Thus, we hypothesized that, as the packing density of the cone axons is low at the foveal center, much of the central peak of MPOD actually is attributable to this cone of Müller cells, while the more peripheral amount of MPOD probably is contributed by the long axons, which are packed more densely in Henle's fiber layer, which, in turn, is covered by only a thin layer of Müller cells. 32 The monotonous decline of MPOD from the center to the periphery may be thought of as being created by the superposition of two MP-containing structures, Müller cells in the center and long cone axons in the periphery. 
Of note, central RT was unrelated in our study to central MPOD, that is, according to our hypothesis, to the Müller cell cone. By contrast, thinning of the NFL-ELM layer was related to higher MPOD at 1° and 2°. Of note, this layer is composed largely by Henle's fibers, in particular in the more eccentric parts of the fovea. We hypothesize that ring-like MPOD distributions, which notably do not commonly show decreased central peaks, 3 are the result of spatial arrangements of axon fibers, which reflect a thinner NFL-ELM layer. When such a fiber arrangement is present, it results in the clearer decomposition of the total MPOD volume into one component that is attributable to the Müller cell cone (central peak) and another one reflecting Henle's fibers (shoulder peak), and it then presents itself as a ring-like distribution. 
Nolan et al. 4 and Kirby et al. 6 hypothesize that wider foveas may lead to longer cone axons and, therefore, accumulate more MP. They found no association of RT with MPOD; however, their MPOD measurements were confined to 0.25° and 0.5° eccentricity, thereby excluding the foveal parts, where our study found an inverse association. Likewise, they reported that eyes with steep foveal depression slopes were associated with a steeper MPOD decline in 15 subjects, 6 a finding that we could not confirm with our data of 124 eyes, where foveal pit slopes showed no relation to MPOD. 
The question was raised whether ring-like MPOD distributions remain stable with increasing age. 3,23 Interestingly, central dips in the MP spatial profile were found to be associated with age and smoking in a group of 484 healthy individuals, and were suspected to represent an undesirable feature of macular pigmentation. 34 By contrast, our study was confined to a narrow age range of elderly individuals and the study outcome was ring-like MPOD rather than central dips. In fact, as also outlined by Dietzel et al., 23 central MPOD dips are not a feature of the ring-like distributions, which consistently show central peaks that are higher than the shoulder peaks. Moreover, based on the assumptions of our hypothesis, central dips might rather be related to alterations of the Müller cell cone, 32 whereas the ring-like structures might represent spatial fiber arrangements. The very high bilateral eye symmetry for ring-like MPOD distributions may even be seen as an indication that the underlying anatomic arrangement features are constitutional rather than acquired during lifetime. This would suggest further that central dips may, indeed, represent damage to Müller cells and dysfunction, eventually even promoting AMD, while ring-like MPOD distribution reflect an individual's congenital eye build. This differentiation between central dip and ring-like MPOD also may help to resolve the apparent contradiction found in the reports from Kirby et al. 34 and our group, where we found that ring-like MPOD was more common in women and those without age-related maculopathy 3 : a combination of constitutional (RT) and acquired (central dip) factors could result in individual MP spatial profiles that emerge from the superposition of MPOD patterns attributed by Müller cells and nerve fibers. A closer look at the anatomic microstructure might provide a better insight to the spatial setup of the fovea and improve our understanding of the processes involved. Interpreting the aforementioned AREDS2 trials, it seems that at least the progression of AMD appears to be only weakly affected by supplementing macular pigments once the conventional AREDS medication (zinc, copper, vitamins C and E, and beta carotenoids) are provided. This leads to the assumption that uptake, transport, storage, and stability of MP follow a far more complicated pattern than previously thought. 
Conclusions
Our data suggested that the thickness of the internal retinal layers; that is, between nerve fiber layer and external limiting membrane, relates directly to ring-like distributions of MPOD. This distribution pattern seems different from central MPOD dips found in other studies. It will be necessary to investigate further which structures in detail are responsible for different MPOD distributions. We propose that Müller cells have a major role for central dips of MPOD, while the spatial arrangement of cone axons may be involved in the more peripheral MPOD distribution patterns. 
Acknowledgments
The authors thank Birte Claes for her excellent and steady support in data management, and all study participants for their time and effort. 
Supported in part by Deutsche Forschungsgesellschaft Grants HE 2293/5-1, 5-2, 5-3, and PA 357/7-1; the Intramural International Monetary Fund of the University of Muenster; the Pro Retina Foundation; and the Jackstaedt Foundation. 
Disclosure: V. Meyer zu Westrup, None; M. Dietzel, None; D. Pauleikhoff, None; H.-W. Hense, None 
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Figure
 
Measurements of RT, NFL-ELM, and ELM-RPE layers, and definition of foveal pit slopes. The calculations were performed according to the following formulas: Slope 1 (75–300 μm) m 1 = (y2-y1)/(x2-x1), Slope 2 (0–75 μm) m 2 = (y1-y0)/(x1-x0), and Slope 3 (0–300 μm) m 3 = (y2-y0)/(x2-x0). y0, central subfoveal RT; y1, subfoveal RT 75 μm nasal/temporal from center; y2, subfoveal RT 300 μm nasal/temporal from center; x0, central measuring point; x1, measuring point 75 μm nasal/temporal from center; x2, measuring point 300 μm nasal/temporal from center.
Figure
 
Measurements of RT, NFL-ELM, and ELM-RPE layers, and definition of foveal pit slopes. The calculations were performed according to the following formulas: Slope 1 (75–300 μm) m 1 = (y2-y1)/(x2-x1), Slope 2 (0–75 μm) m 2 = (y1-y0)/(x1-x0), and Slope 3 (0–300 μm) m 3 = (y2-y0)/(x2-x0). y0, central subfoveal RT; y1, subfoveal RT 75 μm nasal/temporal from center; y2, subfoveal RT 300 μm nasal/temporal from center; x0, central measuring point; x1, measuring point 75 μm nasal/temporal from center; x2, measuring point 300 μm nasal/temporal from center.
Table 1
 
Baseline Description of the Study Eyes
Table 1
 
Baseline Description of the Study Eyes
n Mean (SD) %
Age, y 124 78.4 (4.8)
MPOD, density units
 Peak 0.73 (0.22)
 0.25° 0.68 (0.22)
 0.5° 0.60 (0.22)
 1.0° 0.47 (0.19)
 2.0° 0.18 (0.09)
Halfwidth, degrees 1.19 (0.38)
Genetic pattern
 Homozygous rs1061170, CFH 60 50.0
 Homozygous rs10490924, ARMS2 27 22.3
Ring structure in the MPOD distribution
 Absent 80 55.7
 Intermediate 34 21.0
 Present 32 23.4
Table 2
 
Interocular Symmetry of the Foveal Morphology by SD-OCT (n = 58 Pairs of Eyes)
Table 2
 
Interocular Symmetry of the Foveal Morphology by SD-OCT (n = 58 Pairs of Eyes)
Rank Correlation P Value
Slopes
 Slope1 0.56254 <0.0001
 Slope3 0.52475 <0.0001
RT
 Central 0.62375 <0.0001
 75 μm 0.65465 <0.0001
 300 μm 0.67545 <0.0001
 NFL-ELM 0.60590 <0.0001
 ELM-RPE 0.33601 0.0099
CT 0.76771 <0.0001
Table 3
 
Results of the OCT Measurements for RT, CT, and Slopes
Table 3
 
Results of the OCT Measurements for RT, CT, and Slopes
n Crude Mean (SD)
RT central, subfoveolar, μm 124 245.0 (30.6)
RT 75 μm eccentric, μm 123 251.4 (31.8)
RT 300 μm eccentric, μm 123 291.2 (34.7)
Distance NFL-ELM, μm 123 136.8 (27.1)
Distance ELM-RPE, μm 123 107.5 (21.9)
CT, μm 124 200.1 (77.8)
Slope 1 124 0.177 (0.066)
Slope 2 124 0.085 (0.054)
Slope 3 124 0.154 (0.055)
Table 4
 
Correlations Between RT and CT, and MPOD at Various Eccentricities From the Fovea
Table 4
 
Correlations Between RT and CT, and MPOD at Various Eccentricities From the Fovea
OCT Measurement MPOD Measurement Correlation Coefficient P Value
Retinal thickness
 central Halfwidth −0.19 0.03
Peak 0.04 0.65
MPOD at 0.25° 0.08 0.36
MPOD at 0.5° 0.08 0.34
MPOD at 1.0° −0.18 0.04
MPOD at 2.0° −0.23 0.009
 75 μm eccentric Halfwidth −0.19 0.37
Peak 0.05 0.58
MPOD at 0.25° 0.06 0.36
MPOD at 0.5° 0.09 0.33
MPOD at 1.0° −0.18 0.05
MPOD at 2.0° −0.23 0.009
 300 μm eccentric Halfwidth −0.13 0.16
Peak 0.08 0.33
MPOD at 0.25° 0.13 0.16
MPOD at 0.5° 0.13 0.14
MPOD at 1.0° −0.12 0.17
MPOD at 2.0° −0.20 0.026
NFL-ELM central Halfwidth −0.15 0.09
Peak 0.11 0.21
MPOD at 0.25° 0.13 0.15
MPOD at 0.5° 0.13 0.15
MPOD at 1.0° −0.12 0.19
MPOD at 2.0° −0.18 0.05
ELM-RPE central Halfwidth −0.10 0.23
Peak −0.06 0.45
MPOD at 0.25° −0.06 0.52
MPOD at 0.5° −0.05 0.56
MPOD at 1.0° −0.14 0.11
MPOD at 2.0° −0.12 0.19
Choroidal thickness central Halfwidth −0.03 0.74
Peak −0.02 0.89
MPOD at 0.25° −0.00 0.98
MPOD at 0.5° −0.01 0.87
MPOD at 1.0° −0.2 0.85
MPOD at 2.0° −0.9 0.29
Table 5
 
Crude and Adjusted Mean Values of RT and CT Measurements in Three Groups of MPOD Distribution
Table 5
 
Crude and Adjusted Mean Values of RT and CT Measurements in Three Groups of MPOD Distribution
Ring-Like MPOD Distribution n Crude Mean Value P Value Adjusted* Mean Value P Value
Retinal thickness
 Central Absent 69 258.0 <0.001 258.3 <0.0001
Intermediate 29 238.5 237.9
Present 26 218.1 219.1
 75 μm Absent 69 264.5 <0.001 265.0 <0.0001
Intermediate 29 245.2 244.9
Present 25 222.6 223.1
 300 μm Absent 69 305.9 <0.001 305.9 <0.0001
Intermediate 29 284.4 284.2
Present 25 258.5 259.8
NFL-ELM Absent 69 146.5 <0.001 147.0 <0.0001
Intermediate 29 133.4 131.9
Present 25 114.3 114.9
ELM-RPE Absent 69 111.2 0.08 111.2 0.08
Intermediate 29 105.0 105.9
Present 25 100.3 100.5
Choroidal thickness Absent 69 193.1 0.451 193.8 0.61
Intermediate 29 205.8 203.8
Present 26 212.2 210.8
Slope 1 Absent 69 0.184 0.33 0.181 0.52
Intermediate 29 0.174 0.174
Present 25 0.161 0.163
Slope 2 Absent 69 0.088 0.31 0.088 0.37
Intermediate 29 0.089 0.091
Present 25 0.071 0.072
Slope 3 Absent 69 0.160 0.24 0.158 0.39
Intermediate 29 0.153 0.158
Present 25 0.138 0.141
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