The spatial relation of diabetic retinal neurodegeneration with diabetic retinopathy.

PURPOSE: Diabetic retinal neurodegeneration (DRN) has been demonstrated in eyes of patients with diabetes mellitus (DM), even in the absence of diabetic retinopathy (DR). However, no studies have looked at the rate of change in retinal layers and presence/development of DR over time per quadrant of the macula. In this longitudinal study, we aimed to clarify whether the rate of DRN is associated with the development/presence of DR within 4 different quadrants of the retina.
METHODS: 80 eyes of 40 patients with type 1 DM and no/minimal DR were included. At 4 visits over 6 years, SD-OCT and fundus images were acquired. Thickness of the Retinal Nerve Fiber Layer (RNFL), Ganglion Cell Layer (GCL) and Inner Plexiform Layer (IPL) was measured in a 1-6mm circle around the fovea overall and for each quadrant (superior, nasal, inferior, temporal). Fundus images were scored for the presence/absence of DR in these areas. Multilevel analyses were performed to determine the rate of change for each layer overall and per quadrant for eyes/quadrants without and with DR during the follow-up period.
RESULTS: RNFL and GCL showed significant thinning over time, IPL significant thickening. These changes were more pronounced for GCL and IPL in eyes/quadrants with DR during the follow-up period.
CONCLUSIONS: RNFL and GCL both showed thinning over time, which was more pronounced in eyes with DR for GCL. This holds true even in regional parts of the retina, as quadrant analyses showed similar results, showing that structural DRN is associated with DR per quadrant independently.

Introduction

Diabetic retinopathy (DR) is one of the leading causes of low vision and blindness in the Western World [1, 2]. Approximately one-third of the 285 million people with Diabetes Mellitus (DM) worldwide suffer from DR, and the prevalence of Diabetes Mellitus (DM) is expected to increase to about 552 million in 2030 [3]. This makes DR a serious health problem in ophthalmology, which is only expected to increase in the oncoming years [4]. Early detection and frequent monitoring of the retina is necessary to ensure adequate and timely treatment [4].

Since its development in 1991, optical coherence tomography (OCT) has become an indispensable diagnostic tool within the ophthalmological field [5, 6]. Based on the interference of reflected light, a detailed cross-sectional image of the retina can be obtained [5]. This enables visualization of pathological processes such as fluid or epiretinal membranes, but also permits measuring of the thickness of the retina and its layers [7].

Several studies have shown that retinal thickness is reduced in individuals suffering from DM, even in the absence of vascular DR, a process we have called Diabetic Retinal Neurodegeneration (DRN) [2, 8–12]. It is theorized this may be because chronic hyperglycaemia (or alternatively fluctuating levels of serum glucose), oxidative stress and accumulation of advanced glycation end products leads to an increase in glutamate and a loss of neuroprotective factors, which in turn causes neurodegeneration [2, 8, 9, 11, 12]. Especially the inner (neuronal) layers of the retina seem to be affected in DRN, in particular the retinal nerve fiber layer (RNFL), ganglion cell layer (GCL) and inner plexiform layer (IPL) [8, 9]. A classification scheme that combines all three retinal complications of diabetes—retinopathy proper, DRN, and diabetic macular edema—is under development, and a pilot study showed that DRN can be slowed down with intravitreal steroids [13, 14]. These studies showed that DRN in fact precedes any form of clinically detectable vasculopathy related to DR, and has the potential to serve as a very early detection method for retinal damage in DM, provided there is a causal or epiphenomenal relationship between DRN and vasculopathy. However, many of these studies are of a cross-sectional type, making relation statements about cause and effect difficult. It also has to be noted that the EUROCONDOR study found that, while there was indeed a clear association between DRN and vasculopathy, there is also a significant amount of diabetic patients not expressing this association [15].

Up till now, no studies have looked into the temporal and spatial relation between the onset of structural DRN and the onset of DR. However, such a relation has been shown for functional DRN, reporting a local change in mfERG with subsequent local DR development by Bearse et al [16]. Does the development of increased DRN imply DR is to be expected as a next or simultaneous step, or will this also occur in individuals who never develop DR? Are there regional differences in the relationship between DRN and DR? To gain more insight into these topics, we performed a longitudinal study in patients with type 1 DM and no or very early DR, to explore if the development of DRN is associated with the development or presence of DR, in 4 different quadrants (superior, nasal, inferior, and temporal) of the central retina.

Methods

Participants

This prospective cohort study consists of 40 patients with type 1 DM, also described previously [9, 10, 17–19]. Patient recruitment and follow-up took place from 2004 to 2014 at the outpatient clinic of Internal Medicine at the Amsterdam UMC, location AMC. In 2007 the original OCT device (Zeiss Stratus, time domain OCT) was replaced by a spectral domain OCT (SD-OCT) device (Topcon). The present study is based on the SD-OCT measurements made during the last 7 years of follow-up only, and used 4 visits of each participant, covering a 5 year follow-up period for each individual participant. Some participants only underwent 3 visits over this period, causing the number of participants per visit moment to fluctuate slightly (all participants underwent the baseline visit). The study was approved by the Medical Ethics committee of the Amsterdam UMC, location AMC. The study followed the Tenets of the Declaration of Helsinki and written informed consent was obtained from all participants.

Inclusion criteria were: patients suffering from type 1 DM with no or mild non-proliferative DR (microaneurysms only, classified as no or mild DR according to the ICDR) as determined by a retinal specialist through indirect fundoscopy and fundus images [20].

Exclusion criteria were: more than mild DR (moderate, severe or very severe non-proliferative DR, proliferative DR, diabetic macular edema), refractive error >+5D or <-8D, best corrected visual acuity (BCVA) ≥0.1 LogMAR, severe media opacities interfering with scan quality, a history of ocular surgery other than cataract extraction, glaucoma, (a history of) uveitis or other retinal diseases interfering with retinal thickness (e.g. vitreoretinal interface pathology).

Study visits

All participants underwent the following ophthalmological examinations at the study visits: best corrected visual acuity, intra-ocular pressure, refraction data, slit lamp examination, indirect fundoscopy, fundus photography and SD-OCT (Topcon 1000). Tropicamide 0.5% was used for pupil dilation to enable these examinations. Date of diagnosis of diabetes was obtained from charts, and time since diagnosis was calculated from this. HbA1C was calculated per visit from all HbA1C measurements in the preceding year. For final analyses, these HbA1C measurements were averaged to obtain 1 value for the whole follow-up period for each participant.

Optical coherence tomography

SD-OCT (Topcon 3D OCT-1000, Topcon Medical Systems, Inc., Oakland, CA, USA) was used to image the participants. A 3-D volume scan protocol (6 × 6 × 2.3 mm), consisting of 128 B-scans, with 512 A-scans, each consisting of 650 measuring points, was used. All scans were segmented using the 3D segmentation algorithm developed by the Iowa Institute for Biomedical Imaging [21, 22]. Iowa Reference Algorithms is a free software program available in the public domain at https://www.iibi.uiowa.edu/content/shared-software-download. Individual layer thickness of RNFL, GCL and IPL in all 4 quadrants (superior, nasal, inferior, and temporal) in both the inner and the outer ring according to the Early Treatment Diabetic Retinopathy Study (ETDRS) macular grid were obtained for analyses. Values in quadrants of the inner and outer ring were averaged to obtain a single value for 4 regions from 1-6mmm in the macular area: superior, nasal, inferior an temporal (Fig 1). Because the surface area differs between the inner and outer ring (with a factor of around 3), this average calculation emphasized the changes in the inner ring, where, according to literature, DNR is most present [23].

Fig 1

Macular ETDRS grid used for defining the 4 quadrants for a right eye.

For a left eye, the T and N quadrants are vertically mirrored. S = Superior quadrant, N = Nasal quadrant, I = Inferior quadrant, T = Temporal quadrant.

Fundus photography

Fundus images were obtained from both eyes with a 50° field of view using a TRC-50IX fundus camera; (Topcon Corporation, Tokyo, Japan). All images were graded for the presence or absence of DR by 2 trained graders (JAvdK and JMPLCPY). DR presence or absence was also graded in each quadrant (superior, nasal, inferior and temporal) around the fovea, as defined in the OCT images (Fig 1). From the DR scores over 4 time points, a dichotomous variable was established consisting of either no DR (i.e. no DR on baseline or any of the subsequent visits) or development/presence of DR (i.e. no DR on baseline and DR on any of the subsequent visits, or DR on baseline and none or only some of subsequent visits). This was done for both the entire retinal region captured in the image and in each of the four quadrants individually within that image.

Statistical analysis

To look at the differences in rate of change of retinal layer thickness over time in areas without DR versus those with development or presence of DR, we performed multilevel analyses consisting of 3 or 4 levels: quadrants were clustered within eyes (for the overall retinal layer thickness, this level was not included for the individual quadrant analyses), eyes were clustered within visits and visits were clustered within patients. Multilevel analyses enables correction for dependencies such as the expected dependencies between quadrants within eyes, eyes within the same visit and both eyes of an individual patient. We performed these analyses using both a crude and an adjusted model, corrected for age, sex, mean HbA1C and duration of DM (i.e. time since diagnosis). All analyses were performed using MLwiN (version 2.28, Center for Multilevel Modelling, University of Bristol, United Kingdom). Bar charts were drawn using Graphpad Prism (version 7.04, GraphPad Software Inc., California, USA).

Results

Table 1 shows the descriptive information for the study population at each visit.

Table 1

Demographics of the study population at each visit, data are means unless otherwise specified.

	Visit 1	Visit 2	Visit 3	Visit 4
Number of participants (N)	40	37	37	35
Number of eyes (N)	80	74	74	70
Age (years)	33.1 (±10.1)	35.4 (±10.1)	37.0 (±10.2)	38.3 (±9.9)
Sex, N female (%)	28 (70.0%)	26 (70.3%)	26 (70.3%)	24 (68.6%)
Time since baseline visit (months)	-	22.0 (±7.6)	44.1 (±14.0)	74.0 (±12.2)
DR N eyes (%)	35 (43.8%)	30 (39.0%)	28 (36.4%)	32 (42.7%)
BCVA (LogMAR)	-0.06 (±0.09)	-0.07 (±0.09)	-0.06 (±0.10)	-0.05 (±0.08)
HbA1C (mmol/mol, normal = <53)	75.7 (±11.8)	73.6 (±11.7)	65.5. (±14.6)	66.2 (±10.1)
Time since diagnosis of DM (years)	18.7 (±8.5)	20.7 (±8.5)	22.4 (±8.4)	23.6 (±7.6)

Note that the individuals returning for a subsequent visit are not necessarily the same individuals returning the next visit (i.e. some skipped visit 2 only whereas others skipped visit 3 only etc.). DR = presence of Diabetic Retinopathy, BCVA = Best Corrected Visual Acuity (both eyes averaged), DM = Diabetes Mellitus.

Tables 2 (crude) and 3 (adjusted) show the results of the multilevel analyses. In both models, RNFL and GCL showed a significant thinning over time, whereas IPL showed a slight thickening. For the GCL and IPL, more pronounced changes were associated with the presence or development of DR in those eyes/quadrants during the follow-up period, with overall and temporal GCL reaching a statistically significant difference. Fig 2 shows bar charts for the results obtained with the adjusted model.

Table 2

Multilevel analysis for change in retinal layer thickness overall and per quadrant in years for both absence or presence of Diabetic Retinopathy.

	No diabetic retinopathy			Diabetic retinopathy
	Change in μm/year	p-value	SE	Change in μm/year	p-value	SE
RNFL overall	-0.206	<0.001	0.023	-0.179	<0.001	0.035
• Superior	-0.077	0.348	0.082	-0.199	0.070	0.110
• Nasal	-0.292	<0.001	0.077	-0.386	0.002	0.124
• Inferior	-0.174	0.020	0.075	-0.092	0.483	0.131
• Temporal	-0.301a	<0.001	0.085	-0.031a	0.768	0.105
GCL overall	-0.102a	0.013	0.041	-0.292a	<0.001	0.062
• Superior	-0.127	0.140	0.086	-0.286	0.013	0.115
• Nasal	-0.151	0.059	0.080	-0.301	0.021	0.130
• Inferior	-0.121	0.121	0.078	-0.220	0.108	0.137
• Temporal	0.011a	0.901	0.088	-0.331a	0.003	0.110
IPL overall	0.084	0.016	0.035	0.166	0.001	0.052
• Superior	0.104	0.115	0.066	0.126	0.157	0.089
• Nasal	0.087	0.161	0.062	0.130	0.194	0.100
• Inferior	0.099	0.105	0.061	0.131	0.216	0.106
• Temporal	0.056	0.410	0.068	0.221	0.009	0.085

μm = micrometer, SE = Standard Error, RNFL = Retinal Nerve Fiber Layer, GCL = Ganglion Cell Layer, IPL = Inner Plexiform Layer.

a values in change/year differed significantly at p<0.05 between no DR and DR.

Table 3

Multilevel analysis for change in retinal layer thickness overall and per quadrant in years for both absence or presence of Diabetic Retinopathy.

	No diabetic retinopathy			Diabetic retinopathy
	Change in μm/year	p-value	SE	Change in μm/year	p-value	SE
RNFL overall	-0.206	<0.001	0.023	-0.179	<0.001	0.035
• Superior	-0.079	0.336	0.082	-0.203	0.065	0.110
• Nasal	-0.294	<0.001	0.077	-0.391	0.002	0.124
• Inferior	-0.175	0.020	0.075	-0.098	0.454	0.131
• Temporal	-0.303a	<0.001	0.085	-0.035a	0.739	0.105
GCL overall	-0.105a	0.010	0.041	-0.294a	<0.001	0.062
• Superior	-0.130	0.131	0.086	-0.288	0.012	0.115
• Nasal	-0.154	0.054	0.080	-0.304	0.018	0.129
• Inferior	-0.124	0.112	0.078	-0.224	0.102	0.137
• Temporal	0.008a	0.927	0.088	-0.332a	0.003	0.110
IPL overall	0.081	0.021	0.035	0.163	<0.001	0.052
• Superior	0.101	0.126	0.066	0.123	0.167	0.089
• Nasal	0.084	0.175	0.062	0.128	0.201	0.100
• Inferior	0.097	0.112	0.061	0.127	0.231	0.106
• Temporal	0.053	0.436	0.068	0.219	0.010	0.085

Corrected for age, sex, HbA1C and time since diagnosis of Diabetes Mellitus. μm = micrometer, SE = Standard Error, RNFL = Retinal Nerve Fiber Layer, GCL = Ganglion Cell Layer, IPL = Inner Plexiform Layer.

a values in change/year differed significantly at p<0.05 between no DR and DR.

Fig 2

Bar charts for change in retinal layer thickness overall and per quadrant in years for both absence or presence of Diabetic Retinopathy (DR).

Note that per quadrant means that that particular quadrant did not have any DR during the follow-up period, but other quadrants within that same eye may have had DR. Means and 95% confidence intervals obtained with multilevel analyses, corrected for age, sex, HbA1C and time since diagnosis of Diabetes Mellitus. RNFL = Retinal Nerve Fiber Layer, GCL = Ganglion Cell Layer, IPL = Inner Plexiform Layer. * values in change/year differed significantly at p<0.05 between no DR and DR.

Discussion

This study, for the first time, shows that spatially, regional, structural DRN is associated with regional DR, confirming earlier studies that regional functional DRN is associated with regional DR. In addition, this study confirms earlier studies that DRN occurs irrespective of DR [8, 10, 11]. RNFL and GCL both showed a significant thinning over time, IPL showed a slight but significant thickening over time. For GCL and IPL, more pronounced changes were associated with the presence or development of DR in those eyes/quadrants.

Earlier studies showed that retinal (layer) thinning occurs naturally over time, likely due to an aging effect. Thinning of 0.01–0.16μm per year of the macular RNFL, 0.05–0.10μm of the GCL of and 0.05μm of the IPL have been described, and this thinning was more prominent in individuals of higher age [23-25]. In our DM population, we found an overall thinning of 0.206μm per year for the macular RNFL and 0.105μm for the GCL, and a thickening of 0.081μm per year of the IPL, after correction for multiple confounders in the eyes not suffering from DR. These values were 0.179μm, 0.294μm and 0.163μm respectively for the eyes with the presence/development of DR (Table 3). The thinning of the RNFL and GCL both exceed what was reported by other studies as occurring with physiological degeneration through aging, especially considering the younger age (mean age 33 years at baseline) of our study population. This confirms many earlier studies claiming that DM induces increased DRN (i.e. thinning) of the inner retinal layers, even in the absence of DR [8–11, 18, 26, 27]. Thickening of the IPL, however, is not known to be a results of aging, nor could we find this in studies looking at the effect of DM on the retina. In fact, the opposite has been described: IPL thinning was also reported in the eyes of individuals with DM [8, 10, 18, 26, 27]. However, most of these studies considered the GCL and IPL as one layer, due to the difficulty of correctly separating these two layers, especially with the early lower resolution time domain OCTs [10, 18, 26]. By analyzing this GCIPL complex as a whole, a slight thickening of the IPL in diabetics may be masked by a more pronounced thinning of the GCL. The slight, but significant thickening of the IPL in this longitudinal study may be due to very early leakage, causing microscopic fluid assembly in the inner plexiform layer, leading to increased thickness of this layer. Subclinical macular edema has mostly been described to occur in the inner nuclear layer, but neighboring layers are also thought to be affected, supporting this theory [26]. When eliminating the studies looking at the GCIPL complex as a whole, only one study remains that found thinning of the IPL selectively, but only in a cross-sectional set-up when compared to healthy controls, and only in the eyes suffering from mild DR [27]. It could be that the IPL is thinner in DM patients with DR than controls, but IPL thickening over time may reflect a progress of leakage resulting in the build-up of subclinical edema. Furthermore, IPL thickening may also be caused by glial cell activation due to early inflammation, increasing the volume of these cells and therefore ultimately the volume of certain retinal layers. Lastly, the thickening of the IPL may be the result of an artifact from the segmentation algorithm, although this algorithm has been validated [21, 22].

In the overall values of both the GCL and the IPL, the eyes/quadrant with more pronounced changes were associated with the presence or development of DR during the follow-up period. For the GCL, this even reached a statistically significant difference. This suggests DR and DRN can develop in tandem. The finding that DR eyes show more severe DRN is one that is supported by many other studies [8, 9, 17, 18, 27]. However, the EUROCONDOR study did find that approximately 1 out of 3 diabetic patients (32%) do not express DRN despite the presence of minor DR, suggesting that while there seems to be a link in the pathogenesis of the two, this is not the same for all patients [15].

In this study, we also looked regionally in 4 different quadrants, to see if the finding of more pronounced DRN in areas with (development of) DR still holds true when analyzing these 4 quadrants within an eye. We found that the quadrants of the GCL showing increased DRN also suffered from or developed DR over the course of this study, with the temporal quadrant even reaching a statistically significant difference. This means that locally increased DRN is associated with an increased risk/presence of DR locally, i.e.: quadrants that suffer from increased DRN seem to suffer from DR more often, regardless of the overall DR status of that eye as a whole. This confirms an earlier functional DRN study using multifocal ERG, that the association between DR and neuroretinal degeneration is spatially limited and may fluctuate even within one eye [16]. In the case of the IPL, the quadrants with DR also had a higher increase in thickness over time, suggesting that the process of subclinical macular edema is also one that occurs very locally.

Both the overall RNFL changes and the RNFL changes per quadrant did not differ consistently between eyes/quadrants suffering from DR and those without, suggesting that RNFL is less influenced by the DR status in people with diabetes.

One of the main strengths of this study lies in its longitudinal set-up. This enables accurate estimations of retinal changes over time in diabetic patients, which are much less influenced by natural variation of retinal thickness or confounders such as age and sex. This is also illustrated by our use of both an uncorrected model and a model corrected for several confounders. The corrected model generally showed similar results as the uncorrected model, suggesting the confounders (age, sex, HbA1C and time since diagnosis of DM) to have little effect on the rate of change over time.

The lack of a control group in this longitudinal study is one of its limitations. Although several studies report normative data for retinal layer degeneration due to aging effects, a control group that underwent the exact same scanning protocol as our diabetic population would likely have given a more accurate insight in the added degeneration due to diabetes.

In conclusion, this study shows an association between spatially, regional, structural DRN and regional DR. We found a significant decrease of thickness over time of the RNFL and GCL, and an increase of thickness of the IPL in eyes of diabetic patients. The eyes/quadrants of the GCL and IPL with more pronounced DRN changes more often suffered from (development of) DR during the follow-up period, confirming DRN to develop even in eyes without DR, but once DR develops, this process is faster.

Dataset used for analyses of the data.

(SAV)

Click here for additional data file.

31 Dec 2019

PONE-D-19-29938

The spatial relation of diabetic retinal neurodegeneration with diabetic retinopathy

PLOS ONE

Dear Mrs van de Kreeke,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Both reviewers found your paper interesting and convincing, but please address the concerns of the first reviewer concerning your multivalent modeling and please address the concerns of both reviewers about whether or not neurodegeneration can predict the appearance of microvascular abnormalities in the natural history of diabetic retinopathy.

We would appreciate receiving your revised manuscript by Feb 14 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Alfred S Lewin, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please disclose the author affiliation to IDx in your competing interest statement.

i)  Thank you for stating the following in the Competing Interests section:

[The authors have declared that no competing interests exist.].

We note that one or more of the authors are employed by a commercial company: "IDx".

Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

* Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors present an interesting analysis of structural changes in time series of OCT data from diabetic patients. The aim of their work is clearly expressed. In general, the statistical analysis and the methodology is correct.

I have some concerns regarding the multilevel modelling:

- From their description at lines 151-153 it would look like they have employed 4 nested factors (quadrants, eye, visits and patient). However, quadrants and visits are effectively fixed effects in the analysis (calculations per quadrants are clearly reported in the table and the visit is effectively the same as the time variable in the analysis). The only two random effects should therefore only be patient and eye (nested)

- Were random slopes included in the analysis? Since each eye represents a different time series, having random slopes specific for each eye would greatly improve the accuracy of the estimates

One of the stated aims (and claimed results in the discussion) is to understand whether structural changes are associated with the (local) development of DR. The authors seem to imply in their discussion that they have gathered evidence for thinning of the inner retina as a prognostic factor for the development of DR. This is evident when they draw the parallelism with similar studies investigating the prognostic ability of functional measurements. However, their analysis does not focus on an accurate assessment of the risk of developing DR given the structural parameters. Indeed, their results, as presented, would be more correctly interpreted as faster loss of inner retinal neurons as a consequence of DR. The authors are unclear about what interpretation they want to support and this should be clarified.

Also, there is no mention of how many subjects (sectors?) actually converted to DR during the study. If the aim is to show that neural loss is associated with an increased risk of developing DR, or simply micro-vascular changes in a given sector, an analysis with a logistic regression (or better yet, a survival analysis able to incorporate the temporal dimension) should be performed using the structural parameters as a predictor and the conversion to DR as the response variable.

Reviewer #2: In this paper the relationship between diabetes-induced retinal neurodegeneration and microvascular changes in fundus photography is examined in a prospective observational study. The main conclusion is that RNFL and GCL showed significant thinning over time and these changes were more pronounced with the presence or development of DR. Although a control group is lacking, this is a very interesting study which add new clinical data regarding the relationship between retinal neurodegeneration and the development microvascular abnormalities in the setting of diabetic retinopathy. There are, however, several issues that should be addressed:

In the introduction and/or in the discussion, the results of EUROCONDOR study in which two phenotypes of early stages of DR: one with diabetes induced retinal neurodegeneration + microvascular abnormalities, and another in which microvascular abnormalities were detected without any sign of neurodegeneration should be mentioned. In this regard, the authors could comment on the percentage of patients who developed microvascular abnormalities without significant thinning in RNFL and/or GCL in SD-OCT.

The progression of microvascular abnormalities during follow-up and their relationship with neurodegeneration should be better analyzed. A separate analysis between new incident microvascular lesions and those already present at the beginning of the study could clarify the role of neurodegeneration on new onset of vascular abnormalities (at least in a subset of patients). The question of whether or not neurodegeneration can predict the appearance of microvascular abnormalities in the natural history of DR should be addressed by the authors taken into account the prospective nature of this study.

The second sentence of the first paragraph of the discussion (lines 203-204) needs to be completed with appropriated references.

Apart from vascular leakage, glial activation could also contribute to the thinning of IPL. The authors perhaps could add a comment on this issue to the discussion section.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

15 Jan 2020

There is an uploaded file regarding the response to reviewers.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

3 Feb 2020

PONE-D-19-29938R1

The spatial relation of diabetic retinal neurodegeneration with diabetic retinopathy

PLOS ONE

Dear Mrs van de Kreeke,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The first reviewer indicates that that the sectors should not be treated as a random effect, and it is difficult to see how different sectors of the same eye can be treated as independent events.

We would appreciate receiving your revised manuscript by Mar 19 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Alfred S Lewin, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I appreciate that the authors provided explanation to some of my concerns. To my eyes, however, some sentences still read as confusing:

- Lines 245 - 246: "...thus making increased neurodegeneration a sign of potential worsening of retinal vascular status and vice versa in DM" should be removed

- Lines 255 - 258: "This means that locally increased DRN is associated with also constitutes an increased risk/presence for (development of) DR locally, i.e.: quadrants that suffer from increased DRN tend seem to develop or suffer from DR more often, regardless of the overall DR status of that eye as a whole." should be reworded to avoid suggesting a causative effect. The word "development" is especially misleading.

Finally, I am still convinced that the sector should not be a random effect. The author explicitly use the different sectors to make inference from direct comparisons, reporting p-values. This is exactly when a factor should be a fixed rather than a random effect. Moreover, having the same factor twice (as random and fixed effect) generates collinearity and is simply wrong. Numerical improvement of fitting should not replace good reasoning when building statistical models.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

20 Feb 2020

See included file

Submitted filename: Response to Reviewers 2.docx

Click here for additional data file.

11 Mar 2020

PONE-D-19-29938R2

The spatial relation of diabetic retinal neurodegeneration with diabetic retinopathy

PLOS ONE

Dear Mrs van de Kreeke,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The reviewer still does not believe that you have accounted for differences between quadrants in the same subject. Please see below.

We would appreciate receiving your revised manuscript by Apr 25 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Alfred S Lewin, Ph.D.

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have addressed all my concerns except the ones regarding the random effects in the mixed model. I appreciate the explanation, but what they have done does not account for differences between quadrants in the same subject/eye. If they wanted to model this, they should have used a random SLOPE for the effect of quadrants. This would model the INDIVIDUAL effect of quadrants within each eye/individual.

Allow me to elaborate with a simpler example (Quadrant is a discrete factor in the model):

y = B0 + B1*Quadrant + (1|Subject/Eye) #This models the quadrant as a fixed effect

y = B0 + B1*Quadrant + (1|Subject/Eye/Quadrant) #This models the quadrant as a fixed effect and random intercept,

nested within the eye. This model shows a bad use of random effects,

since the grouping factor is considered both as a random and a fixed

effect.

y = B0 + B1*Quadrant + (1|Quadrant/Subject/Eye) #This is useless

y = B0 + B1*Quadrant + (1|Subject/Eye) + (1|Quadrant) #This is equally useless

y = B0 + B1*Quadrant + (Quadrant|Subject/Eye) #THIS models individual effects of the quadrant within each eye/subject,

which is what the authors state as their goal for including the quadrant in

the random effects. In this case, Quadrant is a random slope on the fixed

effect.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

23 Mar 2020

Due to the concerns raised by the reviewer, we decided to remove the quadrant level. Tables 1 and 2, as well as figure 2, have been updated with the results obtained with the new analyses (i.e. analyses without the quadrant level). The methods section has also been adjusted accordingly (page 7, lines 153-154). As the results did not change drastically, the message our paper conveys did not change either, and therefore the discussion and conclusions required no alteration.

Submitted filename: Response to Reviewers 3.docx

Click here for additional data file.

26 Mar 2020

The spatial relation of diabetic retinal neurodegeneration with diabetic retinopathy

PONE-D-19-29938R3

Dear Dr. van de Kreeke,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Alfred S Lewin, Ph.D.

Section Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

2 Apr 2020

PONE-D-19-29938R3

The spatial relation of diabetic retinal neurodegeneration with diabetic retinopathy

Dear Dr. van de Kreeke:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Alfred S Lewin

Section Editor

PLOS ONE