Association between the retinal vascular network and retinal nerve fiber layer in the elderly: The Montrachet study.

PURPOSE: To investigate the association between the characteristics of the retinal vascular network in the elderly and retinal nerve fiber layer (RNFL) thickness in a population-based study.
METHODS: We conducted a population-based study, the Montrachet study (Maculopathy Optic Nerve, nuTRition neurovAsCular, and HEarT disease), in participants aged ≥ 75 years. RNFL thickness was assessed with spectral-domain optical coherence tomography (SD-OCT). Analysis of the retinal vascular network was performed by means of the Singapore "I" Vessel Assessment (SIVA) software based on fundus photography.
RESULTS: Data from 970 participants were suitable for analysis. Patients with optic neuropathy were excluded. In multivariable analysis, each standard deviation (SD) decrease in the caliber of the six largest arterioles and veins in zone B and the six largest arterioles and veins in zone C was associated with a decrease in global RNFL thickness (β = -1.62 μm, P = 0.001; β = -2.39 μm, P < 0.001; β = -1.56 μm, P = 0.002; and β = -2.64 μm, P < 0.001, respectively).
CONCLUSIONS: Our study found that decreased retinal vessels caliber were associated with a decreased RNFL thickness in the elderly without optic neuropathy.

Introduction

There is growing interest in the quantitative description of the retinal microvascular network due to the fact that numerous studies have shown associations between retinal vascular characteristics and both ocular and systemic diseases [1-6]. Retinal vessels are easy to study, thus providing a noninvasive view of the human microcirculation network using fundus photography. In order to describe the geometric characteristics of arterial and velnous components of the retinal network, specific software programs have been developed that can automatically analyze and describe its geometric characteristics. Retinal Analysis (RA) was the pioneer software but required significant user input and manual tracing of vessels [7]. Automated software, such as the Singapore “I” Vessel Assessment (SIVA) was later developed. SIVA software enables quantitative analysis of vascular morphometry and was shown to be accurate and reproducible in assessing multiple in vivo architectural changes in the retinal vascular network [8, 9].

Today, the assessment of retinal nerve fiber layer (RNFL) thickness is investigated with spectral-domain optical coherence tomography (SD-OCT). Age, refraction, and axial length are consistently associated with RNFL thickness in large studies [10-13]. The association between narrower retinal vessel caliber and RNFL thinning has been described in individuals with glaucoma [14-19]. However, few studies have investigated the relationship between RNFL thickness and retinal vessels in non-glaucomatous populations [20, 21].

The purpose of this study was to investigate the association between quantitative retinal vascular characteristics and RNFL thickness in eyes without optic neuropathy in a population-based study focused on the elderly.

Methods

Study design and population

The Montrachet (Maculopathy Optic Nerve and nuTRition neurovAsCular and HEarT disease) study was an ancillary study of the Three-City (3C) study that started in 1999 and was designed to examine the relationship between vascular risk and dementia [22]. The methodology of the Montrachet study and the participant and nonparticipant characteristics have already been described [23]. Briefly, elderly urban inhabitants from three French cities: Montpellier, Bordeaux and Dijon were recruited between October 2009 and March 2013. This study aimed to investigate potential associations between age-related eye diseases with neurological and cardiovascular pathologies in elderly participants. The study was approved by the 3C study ethics committee. Participants gave their written consent to the study. It followed the tenets of the Declaration of Helsinki and it was registered as 2009-A00448-49. We followed the STROBE (STrengthening the Reporting of OBservational studies in Epidemiology) statement according to the EQUATOR (Enhancing the QUAlity and Transparency Of health Research) guidelines [24].

Cardiovascular history as well as medication and lifestyle were collected for each participants of the 3C study based on self-declaration at baseline and every 2 years thereafter for 10 years. Cardiovascular events and ischemic stroke were summarized in a single variable: major adverse cardiovascular or cerebrovascular events (MACCE). Blood samples were collected after fasting. The 10-year risk of fatal cardiovascular disease was estimated by means of Montrachet baseline information using the method proposed in the Systematic COronary Risk Evaluation (Heart SCORE) project [25].

SD-OCT RNFL thickness measurements

We performed SD-OCT (software version 5.4.7.0; Spectralis, Heidelberg Engineering Co., Heidelberg, Germany) of the optic disc after pupil dilation with tropicamide 0.5% (Théa, Clermont-Ferrand, France). Signal strength and the quality of segmentation were controlled by technicians based on their expertise. In the case of layer segmentation errors, manual correction was performed by the technician. RNFL thickness was measured around a 3.5-mm-diameter circle centered on the optic nerve head. The software calculated RNFL thickness and generated global and sector peripapillary RNFL thicknesses (nasal and temporal sectors).

Fundus photographs and vessel analysis

The methodology used to extract retinal vessel characteristics with the SIVA assessment software has already been extensively described in our previous study [26]. Briefly, we used fundoscopic retinal photographs (45 degrees) centered on the optic disc using a fundus camera (TRC-NW6S; Topcon, Tokyo, Japan). Photographs were sent to an external reading center at Yamagata University, Japan. We only retained one eye for analysis. Computerized analysis of the retinal microvascularization was based on the analysis of venules and arterioles from the center of the optic disc and then to three successive zones corresponding to a circular zone from the edge of the optic nerve head to 0.5 (zone A), 1 (zone B), and 2 (zone C) optic disc diameters. Retinal microvascular biomarkers were measured in zones B and C. The main retinal vascular parameters analyzed were vessel caliber, fractal dimension, and vascular tortuosity. Fractal dimension is a mathematical measure that quantifies complex geometric patterns in objects that are self-similar in their scaling patterns [27]. Retinal vascular fractal dimension represents a global measure of retinal vascular network complexity and density that summarizes the branching pattern of the retinal vasculature [9]. A higher fractal dimension value indicates a more complex vascular branching pattern and a lower fractal dimension indicates a sparser vascular network. Retinal vascular tortuosity reflects the straightness of the vessels; this measure is dimensionless [28]. A higher tortuosity value indicates more curved retinal vessels.

Exclusion criteria

Participants with glaucoma as defined by the International Society for Geographical and Epidemiological Ophthalmology were excluded [29]. Identified glaucomatous eyes were re-examined with Humphrey Swedish interactive thresholding algorithm 24–2 visual field (Carl Zeiss Meditec, Dublin, CA, USA) and a systematic gonioscopy. Optic disc photographs were interpreted by two trained ophthalmologists. In the case of discrepancy, the adjudication was made by a glaucoma specialist. To determine the three levels of evidence described by Foster et al., we used the 97.5th and 99.5th percentiles for the vertical cup-to-disk ratio found in our population-based study [29]. The 97.5th and 99.5th percentiles for the vertical cup-to-disk ratio and vertical cup-to-disk ratio asymmetry were 0.7, 0.2, and 0.8, 0.3, respectively. As previously reported, we also excluded participants presenting with epiretinal membrane from the analysis as they are known to modify vascular architecture [26]. Patients presenting with late age-related macular degeneration (AMD) and modification of the vascular architecture in zone B and C, attested by a trained grader, were not retained for the analysis. AMD was diagnosed on macular retinal photographs according to the modified international classification [30]. Participants with epiretinal membrane and AMD with modification of the vascular architecture were excluded because they present primary modifications of the retinal vascular profile without systemic involvement.

Statistical analysis

Categorical variables as expressed as number (n, %) and continuous variables as mean ± standard deviation (SD) or median (interquartile range [Q1–Q3]) according to their distribution. Participants and non-participants were compared with the chi-square test or Fisher’s exact test for categorical variables and the Student t test or ANOVA test for continuous variables where appropriate. Associations between axial length, Heart SCORE, and global RNFL were estimated with simple linear regression models. In order to estimate associations between retinal vascular parameters (caliber, fractal dimension, and vascular tortuosity) as independent variables and RNFL thickness (global, temporal, and nasal RNFL) as dependent variables, three individual multivariable linear regression models with one eye per individual as the unit of analysis were performed. All variables associated with global RNFL with P values less than 0.20 in bivariate analysis were included in the individual multivariable models except for Heart SCORE. First, models were systematically adjusted for age and sex. Second, final models were obtained after adjustment for age, sex, axial length, diabetes, and systemic hypertension. Associations were expressed as β (standard error, SE). For all analyses, the tests were two-tailed, and the results were considered significant when P-values were less than 0.05. Given the number of statistical tests in this study, we used the procedure of False Discovery Rate (FRD) correction to correct p values for multiple testing [31]. FDR Adjusted p values are presented in multivariable models for each independent variable. A separate analysis in participants without hypertension and diabetes was performed. Analyses were performed using SAS software (version 9.4; SAS Institute, Inc., Cary, NC, USA).

Results

Of the 1153 subjects included in the Montrachet study, 970 eyes had suitable data for analysis (Fig 1). The characteristics of the study population and a comparison between participants and non-participants are summarized in the supporting information (S1 Table). The group of non-participants was significantly older (P < 0.001) and had a longer axial length (P < 0.001). Two participants with diabetes and diabetic retinopathy were kept in the analysis. The mean RNFL of the study participants was 92.08 ± 13.29 μm and the mean age was 82.06 ± 3.64 years old.

Fig 1

Flow chart of the study.

SIVA, Singapore “I” Vessel Assessment; RNFL, retinal nerve fiber layer. Only patients with late age-related macular degeneration and modification of the vascular architecture in zone B and C were excluded.

Table 1 presents the demographics, lifestyle, and clinical characteristics of participants according to global RNFL thickness. A significantly thinner global RNFL thickness was found in males (P = 0.025) and patients with diabetes (P = 0.012). Participants with longer axial length also presented a thinner global RNFL thickness, β = −3.59 μm (P < 0.001).

Table 1

Associations between retinal nerve fiber layer thickness and demographics, lifestyle, clinical characteristics of participants.

Baseline characteristics	Participants, n = 970	Global RNFL, μm	P-value
Age, years
<80	351 (36.18)	92.25 ± 11.59	0.081
80–85	416 (42.89)	92.80 ± 14.86
>85	203 (20.93)	90.27 ± 12.49
Sex
Male	353 (36.39)	90.81 ± 14.87	0.025
Female	617 (63.61)	92.79 ± 12.25
Smoking status, self-declared
Non-smokers	512 (57.14)	92.18 ± 12.25	0.894
Smokers	384 (42.86)	92.30 ± 14.58
Diabetes, self-declared
No	761 (87.27)	92.61 ± 13.04	0.012
Yes	111 (12.73)	89.20 ± 15.05
Treatment for systemic hypertension
No	319 (34.52)	92.51 ± 12.79	0.063
Yes for < 10 years	256 (27.71)	93.27 ± 15.31
Yes for ≥ 10 years	349 (37.77)	90.51 ± 12.79
Cholesterol-lowering drug use
No	288 (30.90)	92.24 ± 14.34	0.964
Yes	644 (69.10)	92.19 ± 12.81
MACCE
No	811 (88.83)	92.21 ± 13.10	0.243
Yes	102 (11.17)	90.62 ± 12.10
Axial length, mm, per SD decrease, β (SE)	23.21 ± 1.21	-3.59 (0.37)	<0.001

The results are displayed as n (%) for categorical variables and mean ± standard deviation for continuous variables. RNFL, retinal nerve fiber layer; MACCE, major adverse cardiovascular or cerebrovascular events; SD, standard deviation; SE, standard error. Available data for each variable, smoking status n = 896; diabetes n = 872; treatment for systemic hypertension n = 924; cholesterol-lowering drug use n = 932; MACCE n = 913, axial length n = 798.

Table 2 presents the associations of retinal vascular parameters and global RNFL thickness. After adjustment for age and sex, a reduced caliber of arterioles and veins was significantly associated with a thinner global RNFL thickness (P < 0.001). Similar results were found with a reduced fractal dimension (P = 0.001 for total zone C, P = 0.005 for arteriole zone C, P < 0.001 for vein zone C) and tortuosity of vessels (P = 0.029). After further adjustment for age, sex, axial length, diabetes, and systemic hypertension, each SD decrease in the caliber of the six largest arterioles and veins in zone B and in the caliber of the six largest arterioles and veins in zone C was associated with a decrease in global RNFL thickness (β = −1.62 μm, P = 0.001; β = −2.39 μm, P < 0.001; β = −1.56 μm, P = 0.002; and β = −2.64 μm, P < 0.001, respectively). Finally, regarding fractal dimension, each SD decrease in the veins in zone C was associated with a 1.02-μm decrease in the global RNFL thickness (P = 0.028). In participants without hypertension and diabetes (n = 294), after adjustments for age, sex and axial length, each SD decrease in the caliber of the six largest arterioles and veins in zone B and in the caliber of the six largest arterioles and veins in zone C was associated with a decrease in global RNFL thickness (β = −1.88 μm, P = 0.0371; β = −3.28 μm, P < 0.001; β = −2.02 μm, P < 0.001; and β = −3.27 μm, P < 0.001, respectively) (Table 3).

Table 2

Associations between retinal vascular parameters and global retinal nerve fiber layer thickness.

	Crude associations			Age, sex-adjusted		Multivariable-adjusted*
Retinal vascular parameters (per SD decrease)	Mean ± SD	β (SE)	P-value	β (SE)	P-value	β (SE)	Unadjusted P-value	FDR Adjusted P-value
Caliber, μm
Six largest arterioles in zone B	139.14 ± 13.80	-3.16 (0.44)	<0.001	-3.04 (0.43)	<0.001	-1.62 (0.51)	0.001	0.004
Six largest veins in zone B	194.39 ± 21.60	-3.59 (0.43)	<0.001	-3.52 (0.43)	<0.001	-2.39 (0.49)	<0.001	<0.001
Six largest arterioles in zone C	143.33 ± 14.54	-3.00 (0.44)	<0.001	-2.88 (0.44)	<0.001	-1.56 (0.50)	0.002	0.005
Six largest veins in zone C	202.12 ± 21.29	-3.73 (0.43)	<0.001	-3.67 (0.43)	<0.001	-2.64 (0.49)	<0.001	<0.001
Fractal dimension
Total zone C	1.38 ± 0.07	-1.47 (0.42)	<0.001	-1.35 (0.42)	0.001	-0.75 (0.47)	0.106	0.159
Arterioles zone C	1.19 ± 0.07	-1.26 (0.41)	0.002	-1.17 (0.41)	0.005	-0.59 (0.45)	0.194	0.249
Veins zone C	1.16 ± 0.06	-1.51 (0.42)	<0.001	-1.43 (0.42)	<0.001	-1.02 (0.46)	0.028	0.042
Vascular tortuosity
Simple tortuosity, vessels, × 104	1.09 ± 0.02	-2.15 (0.94)	0.022	-2.05 (0.93)	0.029	-0.94 (1.02)	0.357	0.401
Curvature tortuosity, vessels, × 104	0.74 ± 0.17	-0.75 (0.41)	0.068	-0.68 (0.41)	0.099	-0.30 (0.44)	0.487	0.487

SD, standard deviation; SE, standard error; FDR, false discovery rate. 172 observations were deleted due to missing axial length variable.

*Adjusted for age, sex, axial length, diabetes, and systemic hypertension.

Table 3

Associations between retinal vascular parameters and global retinal nerve fiber layer thickness in participants without hypertension and diabetes.

Retinal vascular parameters (per SD decrease)	Mean ± SD	β (SE)1	P-value	β (SE)2	P-value	β (SE)3	Unajusted P-value4	FDR Adjusted P-value5
Caliber, μm
Six largest arterioles in zone B	139.73 ± 13.90	-4.01 (0.70)	< 0.001	-3.99 (0.71)	< 0.001	-1.88 (0.78)	0.0165	0.037
Six largest veins in zone B	193.86 ± 20.20	-5.53 (0.72)	< 0.001	-5.54 (0.73)	< 0.001	-3.28 (0.78)	< 0.001	< 0.001
Six largest arterioles in zone C	143.96 ± 13.67	-3.91 (0.72)	< 0.001	-3.89 (0.73)	< 0.001	-2.02 (0.77)	0.009	0.027
Six largest veins in zone C	201.47 ± 19.92	-5.33 (0.73)	< 0.001	-5.32 (0.73)	< 0.001	-3.27 (0.79)	< 0.001	< 0.001
Fractal dimension
Total zone C	1.39 ± 0.06	-2.43 (0.80)	0.005	-2.46 (0.81)	0.003	-1.07 (0.83)	0.198	0.251
Arterioles zone C	1.19 ± 0.06	-2.51 (0.76)	0.001	-2.54 (0.76)	< 0.001	-1.21 (0.77)	0.119	0.179
Veins zone C	1.16 ± 0.05	-1.78 (0.80)	0.027	-1.79 (0.81)	0.028	-0.51 (0.79)	0.523	0.523
Vascular Tortuosity
Simple tortuosity, vessels, × 104	1.09 ± 0.02	-2.04 (1.73)	0.242	-1.87 (1.74)	0.284	-2.15 (1.75)	0.223	0.251
Curvature tortuosity, vessels, × 104	0.73 ± 0.13	-1.17 (0.88)	0.186	-1.11 (0.88)	0.213	-1.49 (0.91)	0.102	0.179

SD, standard deviation; SE, standard error; FDR, false discovery rate.

1Crude associations.

2Adjusted for age and sex.

3Adjusted for age, sex and axial length.

4Unadjusted p-values.

5Corrected p-values for multiple testing.

The analyses of the association between retinal vascular parameters and sectorial temporal and nasal RNFL are shown in S2 and S3 Tables.

Discussion

In this population-based study of older adults, we found that thinner retinal vessels caliber and reduced fractal dimension of veins in zone C were associated with a thinner global RNFL thickness in non-glaucomatous participants. These results are in keeping with other published data. In a cross-sectional investigation of Asian participants, each SD decrease in retinal arteriolar caliber was associated with a 5.81 μm decrease in the mean global measured RNFL thickness (P < 0.001), and each SD decrease in retinal venular caliber was associated with an 8.37 μm decrease in mean global RNFL thickness (P < 0.001) [32]. In this study, participants’ mean age was 56.8 years. In a study derived from the Singapore Chinese Eye Study, each SD decrease in the retinal arteriolar caliber was associated with a 1.60 μm decrease in global RNFL thickness (P = 0.002), and each SD decrease in venular caliber was associated with a 1.97 μm decrease in average RNFL thickness (P < 0.001) (mean age was 53.6 ± 6.7 years) [21]. Finally, this association was also confirmed in a middle-aged Caucasian population, where each 10 μm increase in the retinal arteriolar and venular caliber was associated with, respectively, a 5.58 μm and a 3.79 μm increase in mean global RNFL thickness (P < 0.001) (mean age was 47.3 ± 0.9 years) [20]. Moreover, in a subgroup analysis (participants without hypertension or diabetes), we found similar results. Thus we confirmed in this study the association between RNFL thickness and retinal vessels caliber in an elderly population.

When investigating the RNFL nasal sector, the results were similar to those found for the global analysis, whereas an opposite association was found when investigating the temporal sector. A reduction in the caliber of vessels and a reduction in fractal dimension were associated with increased RNFL thickness.

It remains unknown whether the relationship between retinal vessel narrowing and reduced RNFL thickness is causal. Although it is impossible to provide a definite answer to this question, it is plausible that these changes are related to vascular autoregulation. Higher levels of oxidative factors have been found to lead to vasodilatation and elevated blood flow and could influence retinal vessel diameter [33]. It was also postulated that reduced venular caliber could lead to cytotoxic and vasogenic edema. This congestion would then cause arteriolar constriction and finally lead to early RNFL thinning. This vascular involvement seems to be reinforced, since vascular and neurovascular diseases such as stroke and dementia were shown to be associated with reduced RNFL thickness [34].

Fractal dimension was also investigated in our study. This parameter reflects the complexity of the retinal microvascular tree as a geometric pattern including the degree of branching [27]. Optimal retinal vasculature with a higher fractal dimension is associated with greater efficiency in blood circulation [10, 35]. It has been reported to be associated with age, blood pressure, refractive errors, and lens opacity [3, 36]. Low fractal dimension was associated with cardiovascular mortality risk and advanced diabetic retinopathy [26, 37]. In our elderly population, a lower fractal dimension of veins in zone C was the only parameter to be associated with a reduction in mean RNFL thickness (P = 0.028). Tham et al. reported a similar finding, as each SD decrease in fractal dimension was associated with a 1.60 μm decrease in average RNFL thickness (P = 0.002) [21]. This low branching complexity could be a sign of microvascular alteration and neurodegeneration revealed by the decrease of RNFL thickness in our elderly population. However, in the subgroup analysis (Table 3), the association between a lower fractal dimension of veins in zone C and a reduction in mean RNFL thickness is no more significant. We could make the hypothesis that presence of hypertension or diabetes affect first and foremost retinal fractal dimension.

We also found that a thinner global RNFL was associated with sex, diabetes, and longer eyes. Regarding sex, we found in our population that male subjects had a significantly lower global RNFL thickness than females. The Beijing Eye Study showed women to have a higher RNFL thickness [38]. Mauschitz et al. found that sex was no longer associated with RNFL thickness after correcting for axial length, which was on average shorter in women and confounded the effect of sex on RNFL thickness [34]. Regarding diabetes, most studies report that patients with diabetes present with thinner RNFL thickness [39, 40]. One explanation could be neurodegeneration to be a consequence of microvascular damages and inflammation described in diabetic patients. A recent longitudinal study showed that diabetes was associated with accelerated RNFL loss regardless of the presence and progression of diabetic retinopathy or the intraocular pressure profile, suggesting that diabetic neurodegeneration may precede the microvascular abnormalities of diabetic retinopathy [41]. In this present study, we did not collect information about the duration of the diabetes and glycemic stability. These parameters should be taken into account in future studies. Regarding the association between greater axial length and thinner RNFL thickness, our findings are in line with several studies [42-45]. The mechanism remains unclear, but could be the result of longer axial length or measurement modifications due to magnification factors [46]. In contrast to Lee et al., in this study we did not compare RNFL thickness between participants with and without hypertension [47]. Nevertheless, hypertension was added to the adjusted-multivariable model. The association between hypertension history and RNFL thickness warranted further studies.

Strengths of our study include its large elderly population-based sample, extensive retinal vascular parameters phenotype (caliber, fractal dimension and vascular tortuosity) and a subgroup analysis in participants without hypertension or diabetes.

Potential limitations of this study should be mentioned. First, retinal vessel diameter has been reported to vary during the cardiac cycle [48-50]. Even though a validation study demonstrated that retinal parameters measured with SIVA are relatively stable, fundus photography analysis involves instantaneous images and cannot take into account vessel caliber variations [51]. Adaptive optics imaging could be an alternative, since this enables dynamic and high-resolution imaging and thereby integrates vessel caliber variations [52]. Second, our findings in an urban Caucasian European population cannot be extrapolated to different ethnic groups. Third, the causative relationship between retinal vascular geometry and RNFL thickness cannot be definitively assessed owing to the cross-sectional nature of our design. Fourth, we did not take into account the ocular magnification in the SIVA measurements. It could increase variation in RNFL thickness around the 3.5-mm-diameter measurements which may impact the analysis for extreme axial length eyes. Finally, the precision of retinal vasculature grading may be affected by measurement errors related to subjective grader input, variability in image, and contrast or brightness factor errors. There are no clear explanations as to why some parameters in zone B are associated with RNFL but not with zone C parameters. At present, measurement areas are limited owing to the time consumed to track vasculature. Future research using artificial intelligence technologies to aid segmenting vasculature will expand the potential of this analysis.

In conclusion, we found that a narrower retinal vasculature was associated with a thinner global RNFL in eyes without optic neuropathy. The clinical relevance of these findings warrants further studies.

Baseline characteristics between participants and non-participants in the Montrachet study.

(DOCX)

Click here for additional data file.

Associations between retinal vascular parameters and sectorial temporal retinal nerve fiber layer thickness.

(DOCX)

Click here for additional data file.

Associations between retinal vascular parameters and sectorial nasal retinal nerve fiber layer thickness.

(DOCX)

Click here for additional data file.

13 May 2020

PONE-D-20-00420

Association Between the Retinal Vascular Network and Retinal Nerve Fiber Layer in the Elderly: The Montrachet Study

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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: Partly

**********

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

Reviewer #1: I Don't Know

Reviewer #2: I Don't Know

**********

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: Yes

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: In this manuscript, Arnould, et al. report that in elderly study participants without optic neuropathy, thinner retinal nerve fiber layer (RNFL) thickness are associated with reduced caliber of retinal arteries and veins. They also report RNFL thickness is associated with several demographic features including sex, self-declared history of diabetes, and axial length.

Because several authors have published similar results, could the authors highlight or compare and contrast what is novel about their findings? Is it the age of the study participants? Is it the method of analyzing the retinal vessels?

Half of the study participants reported some degree of hypertension. Was there a significant difference in RNFL thickness or retinal vascular metrics for those with hypertension compared to those without?

The purpose of the manuscript defined in the introduction is the detailed analysis comparing retinal vasculature to RNFL thickness. I would recommend emphasizing these findings in the conclusion of the abstract which is vaguely written currently. Also, the discussion should be re-organized to focus on retinal vasculature metrics association with RNFL thickness and then followed by RNFL associations with demographic features.

What was the mean RNFL? How does this compare to other studies?

Could they authors explain their low rate (10 cases out of 1153) of age-related macular degeneration compared to reported prevalence data (Prevalence and incidence of age-related macular degeneration in Europe: a systematic review and meta-analysis. British Journal of Ophthalmology; 11 November 2019)?

Reviewer #2: Arnould et al. present a manuscript assessing the relationship of arterioles and venules to the RNFL thickness measurements. As noted by the authors, many studies have recently been published showing a link between the RNFL and ocular, systemic and neurodegenerative diseases. Their question, of how this layer is related in the elderly and with respect to the vasculature is an important component to this ongoing research.

This manuscript looks specifically at the relationship of different retinal vasculature morphology metrics (on a fundus image) to the thickness of the RNFL at 3.5mm from the optic disc. They find that arteriole and venous vessel caliber is related to RNFL thickness along with age. While this paper is interesting and normative data is needed to understand how aging affects the retina, this author has several concerns that need to be addressed.

Major Issues:

1. The inclusion of subjects with diabetes means that the relationship reported isn’t purely based on age although the authors do adjust for this by using diabetes (and hypertension as well) as a factor in one of their linear regressions. However, nothing is said about the prevalence of retinopathy (which can happen with both diabetes and hypertension). Additionally, there’s also a known relationship between diabetic retinopathy and the length of time someone has diabetes. Yet none of these things are taken into account leading this reviewer to wonder how the regression and these relationships would change. This reviewer would appreciate a deeper examination of subjects with diabetes and a better understanding of that population’s medical information. Additionally, the exclusion criteria does not mention how other ocular diseases were treated in terms of including or excluding those populations (i.e. sickle cell disease, Macular Telangiectasia etc.).

2. Was ocular magnification taken into account when measuring 3.5 mm away from the optic disc? While axial length itself does not impact thickness measurements (PMID: 30025118), it does impact the placement of where these measurements take place if from a set distance. With longer axial lengths, the circle, if ocular magnification is not taken into account, will cover a larger retinal distance than other subjects with short axial lengths. This leads to increased variation in RNFL thickness measurements which may impact the analysis. Please clarify if ocular magnification was corrected for. If not, it would be preferable for the authors to correct for ocular magnification so that 3.5 mm measurements were the same for each individual. If this is not possible, the lack of correction should be noted in the limitations paragraph.

3. Table 1: Stated both in the table and throughout the paper, it says that 970 subjects fit the criteria for the study yet there are additional n values in the same column as the baseline characteristic with no mention of what they mean. If only 878 subjects of the 970 subjects had axial length measurements, would this not also impact your findings for the regression? Given that some of these factors (such as axial length) are known to impact RNFL thickness, why include these subjects? Please clarify what the n values for the table mean. If subjects were included without a full set of data as presented, including them for the regression may be misleading and the authors should consider removing these subjects from their analysis.

4. This reviewer appreciates your transparency with the various regressions that you ran. However, with reading this paper, it is often difficult to remember the central point is to show that even with age, the relationship between retinal vasculature and RNFL thickness holds. If the authors could work to improve the clarity of their writing and work to focus the results in particular, this reviewer thinks it would read significantly easier and the main points would come across much stronger rather than the potential of readers to be caught in the details.

Minor Issues

1. Line 59: The sentence states “using fundus and other techniques”. Please expand on these other techniques and discuss the benefits of the fundus over these other techniques.

2. Lines 64-69: Multiple different software is cited but nothing is stated about why this goes into the introduction. I would expect a reason for why SIVA was used if this is brought up in the introduction over the others, or is the benefit of a fundus that there are these automated techniques?

3. Line 107-109: The sentence about the technicians controlling for image quality and segmentation is ambiguous. Was there an image quality metric with a cut off, were the technicians given any information or guidance or were these metrics purely based on their expertise?

4. Is it appropriate to keep p < 0.05 for all tests given the number of statistical tests done? This reviewer would suggest consulting with a biostatistician (if not already done) to make sure that the analysis provided is accurate and that the p-values do not need adjustment given the number of analyses this paper employs.

**********

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Reviewer #1: No

Reviewer #2: No

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21 Jul 2020

Manuscript ID PONE-D-20-00420

Association Between the Retinal Vascular Network and Retinal Nerve Fiber Layer in the Elderly: The Montrachet Study

Thank you for giving us the opportunity to revise this submission following peer review. After consultation with the other authors, we have responded to the reviewers’ constructive suggestions by revising the manuscript as attached. We would like to take the opportunity to respond to the individual points below.

Reviewer #1: In this manuscript, Arnould, et al. report that in elderly study participants without optic neuropathy, thinner retinal nerve fiber layer (RNFL) thickness are associated with reduced caliber of retinal arteries and veins. They also report RNFL thickness is associated with several demographic features including sex, self-declared history of diabetes, and axial length.

Because several authors have published similar results, could the authors highlight or compare and contrast what is novel about their findings? Is it the age of the study participants? Is it the method of analyzing the retinal vessels?

As suggested, we added these sentences in the revised version of our manuscript as follows (line 265-282), first paragraph of the discussion

“We found that thinner retinal vessels caliber and reduced fractal dimension of veins in zone C were associated with a thinner global RNFL thickness in non-glaucomatous participants. These results are in keeping with other published data. In a cross-sectional investigation of Asian participants, each SD decrease in retinal arteriolar caliber was associated with a 5.81 μm decrease in the mean global measured RNFL thickness (P < 0.001), and each SD decrease in retinal venular caliber was associated with an 8.37 μm decrease in mean global RNFL thickness (P < 0.001).43 In this study, participants’ mean age was 56.8 years. In a study derived from the Singapore Chinese Eye Study, each SD decrease in the retinal arteriolar caliber was associated with a 1.60 μm decrease in global RNFL thickness (P = 0.002), and each SD decrease in venular caliber was associated with a 1.97 μm decrease in average RNFL thickness (P < 0.001) (mean age was 53.6 ± 6.7 years).22 Finally, this association was also confirmed in a middle-aged Caucasian population, where each 10 μm increase in the retinal arteriolar and venular caliber was associated with a 5.58 μm and a 3.79 μm increase in mean global RNFL thickness, respectively (P < 0.001) (mean age was 47.3 ± 0.9 years).21 We confirmed in our study the association between RNFL thickness and retinal vessels caliber in an elderly population.”

Half of the study participants reported some degree of hypertension. Was there a significant difference in RNFL thickness or retinal vascular metrics for those with hypertension compared to those without?

We agree with the concern raised by the Reviewer. Indeed, Mauschitz et al. investigated pooled data from 8 European population-based studies (including the Montrachet Study) and showed reduced RNFL in hypertensive patients (Mauschitz et al. Ophthalmology 2018). Moreover, we fully agree with the Reviewer’s comment concerning hypertension and retinal vascular metrics. Ponto et al. showed that systemic hypertension was associated with lower values of arteriolar retinal caliber (Ponto et al. J Hypertension 2017). Our primary objective was to study the association between RNFL and retinal vascular parameters independently of cardiovascular risk factors. We did not perform in our analysis a comparison between participants with and without hypertension regarding their RNFL thickness as we decided to use this variable in our adjusted model. We added the following sentence in the revised version of our manuscript (line 343-347).

“In contrast to Lee et al., we did not compare RNFL thickness between participants with and without hypertension.47 Indeed, hypertension is known to be associated with reduced RNFL and decreased arteriolar retinal caliber.34,48 As a consequence, we added hypertension history to the adjusted-multivariable model. The association between hypertension history, RNFL thickness and retinal vascular metrics warrants further studies”

The purpose of the manuscript defined in the introduction is the detailed analysis comparing retinal vasculature to RNFL thickness. I would recommend emphasizing these findings in the conclusion of the abstract which is vaguely written currently.

We agree with the Reviewer and we modified the conclusion of the abstract as follows (line 46-47).

“In this study, we found that decreased retinal vessels caliber was associated with a decreased RNFL thickness in the elderly without optic neuropathy.”

Also, the discussion should be re-organized to focus on retinal vasculature metrics association with RNFL thickness and then followed by RNFL associations with demographic features.

We thank the Reviewer for emphasizing this crucial point. We re-organized the discussion as mentioned (line 265-282) and (line 324-347).

What was the mean RNFL? How does this compare to other studies?

The mean RNFL of the study participants was 92.08 ± 13.29 μm. This result was similar to other population-based studies. The mean RNFL thickness in the Beijing Eye Study was 103.2 ± 12.6 µm (66.2 ± 9.9 years) (YA Xing Wang et al. PlosOne 2013). The mean RNFL thickness in the Alienor study was 86.8 ± 13.7 µm (81.6 ± 4.2 years) (Juan Luis Méndez-Gómez et al. JAMA Netw Open 2018). We added the following sentence in the revised version of our manuscript (line 196)

“The mean RNFL of the study participants was 92.08 ± 13.29 μm”

Could they authors explain their low rate (10 cases out of 1153) of age-related macular degeneration compared to reported prevalence data (Prevalence and incidence of age-related macular degeneration in Europe: a systematic review and meta-analysis. British Journal of Ophthalmology; 11 November 2019)?

We thank the Reviewer for highlighting this point. Prevalence of age-related macular degeneration (AMD) in the Montrachet study was reported (Creuzot-Garcher et al. Acta Ophthalmologica 2015). The prevalence is lower compared to reported prevalence data because we investigated a white, urban, and generally healthy population with high economic status. Moreover, in this study, we only excluded participants with late AMD and modification of the vascular architecture in zone B and C attested by a trained grader. As suggested by the Reviewer, we added the following statement in the revised version of our manuscript (line 162-164) and we modified the caption of the Figure.

“Patients presenting with late age-related macular degeneration (AMD) and modification of the vascular architecture in zone B and C, attested by a trained grader, were not retained for the analysis.”

Reviewer #2: Arnould et al. present a manuscript assessing the relationship of arterioles and venules to the RNFL thickness measurements. As noted by the authors, many studies have recently been published showing a link between the RNFL and ocular, systemic and neurodegenerative diseases. Their question, of how this layer is related in the elderly and with respect to the vasculature is an important component to this ongoing research.

This manuscript looks specifically at the relationship of different retinal vasculature morphology metrics (on a fundus image) to the thickness of the RNFL at 3.5mm from the optic disc. They find that arteriole and venous vessel caliber is related to RNFL thickness along with age. While this paper is interesting and normative data is needed to understand how aging affects the retina, this author has several concerns that need to be addressed.

Major Issues:

1. The inclusion of subjects with diabetes means that the relationship reported isn’t purely based on age although the authors do adjust for this by using diabetes (and hypertension as well) as a factor in one of their linear regressions. However, nothing is said about the prevalence of retinopathy (which can happen with both diabetes and hypertension). Additionally, there’s also a known relationship between diabetic retinopathy and the length of time someone has diabetes. Yet none of these things are taken into account leading this reviewer to wonder how the regression and these relationships would change. This reviewer would appreciate a deeper examination of subjects with diabetes and a better understanding of that population’s medical information.

We fully understand this comment. As reported in our previous study (Creuzot-Garcher et al. Acta Ophthalmologica 2015), there were 9 participants of the Montrachet study with an history of diabetic retinopathy. In our study, we found in our database that only 2 diabetic patients presented with diabetic retinopathy. We decided not to exclude these patients because diabetes was chosen to be an adjustment variable in the linear regression. The primary objective of our study was to investigate the association between retinal nerve fiber layer thickness and retinal vascular architectural parameters independently of cardiovascular risk factors. Unfortunately, we did not collect information about the duration of the diabetes and the glycemic status. However, participants in the Montrachet study were healthy volunteers and their cardiovascular risk factors were well monitored.

We added the following statement in the revised version of our manuscript.

Line 195-196

“Two participants with diabetes and diabetic retinopathy were kept in the analysis.”

Line 337-338

“In the present study, we did not collect information about the duration of the diabetes and glycemic stability. These parameters should be taken into account in future studies.”

Additionally, the exclusion criteria does not mention how other ocular diseases were treated in terms of including or excluding those populations (i.e. sickle cell disease, Macular Telangiectasia etc.).

Participants ophthalmic history were collected in the same way for the entire population of the Montrachet study. In previous studies focusing on retinal history in the Montrachet study (Ben Ghezala et al. Retina 2020 and Gabrielle et al. IOVS 2019), we did not find other ocular vascular diseases.

2. Was ocular magnification taken into account when measuring 3.5 mm away from the optic disc? While axial length itself does not impact thickness measurements (PMID: 30025118), it does impact the placement of where these measurements take place if from a set distance. With longer axial lengths, the circle, if ocular magnification is not taken into account, will cover a larger retinal distance than other subjects with short axial lengths. This leads to increased variation in RNFL thickness measurements which may impact the analysis. Please clarify if ocular magnification was corrected for. If not, it would be preferable for the authors to correct for ocular magnification so that 3.5 mm measurements were the same for each individual. If this is not possible, the lack of correction should be noted in the limitations paragraph.

We agree with the concerns raised by the Reviewer. We added the following statement in the revised version of our manuscript (line 377-380).

“Fourth, we did not take into account the ocular magnification in the SIVA measurements. It could increase variation in RNFL thickness around the 3.5-mm-diameter measurements which may impact the analysis for extreme axial length eyes.”

3. Table 1: Stated both in the table and throughout the paper, it says that 970 subjects fit the criteria for the study yet there are additional n values in the same column as the baseline characteristic with no mention of what they mean.

We thank the Reviewer for highlighting this point. The n values in the first column of Table 1 and Table 2 correspond to the available data for each variable. We agree with the Reviewer that additional n values in the same column with no clear explanation is confusing. Therefore, we placed the n values in the notes under Table 1 and Table 2 to avoid any confusion.

If only 878 subjects of the 970 subjects had axial length measurements, would this not also impact your findings for the regression?

We fully agree with Reviewer’s comment. Missing data represent less than 10% of our dataset. As a consequence, we did not perform a multiple imputation method. However, it could affect the regression results and it could reduce the statistical power of our associations.

Given that some of these factors (such as axial length) are known to impact RNFL thickness, why include these subjects?

Axial length is a known confounder factor between all retinal vascular parameters and also for RNFL thickness. In this study, we kept the axial length as an adjustment variable in order to investigate the relationship between retinal vascular parameters and RNFL thickness irrespectively of the ametropia.

Please clarify what the n values for the table mean. If subjects were included without a full set of data as presented, including them for the regression may be misleading and the authors should consider removing these subjects from their analysis.

As suggested by the Reviewer we removed the participants with missing axial length variable in the multivariable models and a note has been written under the Tables in the revised version of our manuscript.

4. This reviewer appreciates your transparency with the various regressions that you ran. However, with reading this paper, it is often difficult to remember the central point is to show that even with age, the relationship between retinal vasculature and RNFL thickness holds. If the authors could work to improve the clarity of their writing and work to focus the results in particular, this reviewer thinks it would read significantly easier and the main points would come across much stronger rather than the potential of readers to be caught in the details.

Thank you to the Reviewer for emphasizing this crucial point. We re-organized the discussion as mentioned. (line 265-282) and (line 324-347).

Minor Issues

1. Line 59: The sentence states “using fundus and other techniques”. Please expand on these other techniques and discuss the benefits of the fundus over these other techniques.

We thank the Reviewer for highlighting this point. It is possible to investigate the human microcirculation network with noninvasive devices such as fundus photographs, capillaroscopy, renal doppler or brain imaging. In the Montrachet study, participants benefited from an exhaustive eye examination. Fundus photographs were performed for each participant. Compared to the other techniques, fundus photographs are less expensive, and it can be performed by a technician or a nurse. Moreover, semi-automated software based on fundus are available to thoroughly describe the retinal vascular network with quantitative metrics. Finally, it has been shown that retinal vascular network is reliable to assess the systemic vascular status (Arnould et al. PLoS One 2018). We deleted “other techniques” in the revised manuscript (line 65).

2. Lines 64-69: Multiple different software is cited but nothing is stated about why this goes into the introduction. I would expect a reason for why SIVA was used if this is brought up in the introduction over the others, or is the benefit of a fundus that there are these automated techniques?

We fully agree with the Reviewer’s comment. We used SIVA software because at the time of the data collection in the Montrachet study (2009-2013), it was the only software that was able to quantify complex vascular parameters such as fractal dimension and tortuosity. Other softwares were only focused on retinal caliber. We deleted information about other softwares in the introduction of the revised manuscript (line 62-70).

3. Line 107-109: The sentence about the technicians controlling for image quality and segmentation is ambiguous. Was there an image quality metric with a cut off, were the technicians given any information or guidance or were these metrics purely based on their expertise?

We thank the reviewer for highlighting this point. We modified the following statement in the revised version of our manuscript (line 121-122).

“Signal strength and the quality of segmentation were controlled by technicians based on their expertise and training”

4. Is it appropriate to keep p < 0.05 for all tests given the number of statistical tests done? This reviewer would suggest consulting with a biostatistician (if not already done) to make sure that the analysis provided is accurate and that the p-values do not need adjustment given the number of analyses this paper employs.

We agree with the Reviewer that, given the number of statistical tests in this study, keeping p < 0.05 for all tests is not appropriate. As suggested by the Reviewer, we used the procedure described by Benjamini and Hochberg (False Discovery Rate correction) (Benjamin Y et al . J R Stat Soc. 1995) to correct p values for multiple testing. FDR Adjusted p values are presented in multivariable models for each independent variable in Tables 3,4 and 5 in the revised version of our manuscript. Significant associations remained stable with this correction except the associations between fractal dimension (total zone C and arterioles zone C) and nasal RNFL thickness. We added the following statement in the revised version of our manuscript (line 183-185).

“Given the number of statistical tests in this study, we used the procedure of False Discovery Rate (FRD) correction to correct p values for multiple testing.31”

Submitted filename: Rebuttal letter.docx

Click here for additional data file.

21 Aug 2020

PONE-D-20-00420R1

Association Between the Retinal Vascular Network and Retinal Nerve Fiber Layer in the Elderly: The Montrachet Study

PLOS ONE

Dear Dr. Arnould,

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.

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We look forward to receiving your revised manuscript.

Kind regards,

Ireneusz Grulkowski, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Please, analyze the reviewers' comments and revise the paper again.

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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: (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

Reviewer #2: Yes

**********

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

Reviewer #1: I Don't Know

Reviewer #2: 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

Reviewer #2: Yes

**********

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: (No Response)

Reviewer #2: 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: Thank you for considering revisions. These analyses are difficult in elderly patients because of the confounding variables. For example, in this study nearly 70% of participants have a history of hypertension. Presence of hypertension, and diabetes, likely affects affect retinal vascular parameters, but this information is not presented in the manuscript.

Table 2 shows the association between RNFL to demographics, lifestyle and clinical characteristics. It would be useful to show associations between retinal vascular parameters to demographics, lifestyle and clinical characteristics as well. I would expect there to be significant associations with hypertension, diabetes and possibly axial length. Increased transparency with the multiple variables involved will help readers understand the complexity associated with these analyses.

Could the authors run a separate analysis evaluating for association of RNFL and retinal vasculature parameters after excluding hypertension, diabetes and extreme axial length differences? It is unclear if the numbers would support this evaluation.

For the RNFL analysis, this work is contrasted by others because it is an “elderly cohort” – could the authors clearly state the mean age in the results section?

Is Table 1 necessary? This is essentially comparing cases that were excluded to participants that were included. Why is this relevant to the comparison of RNFL to retinal vascular parameters? If important to include, perhaps these findings could be summarized in the text of the results.

I would exclude the sentence in the abstract describing thinner global RNFL thickness associated with males, patients with diabetes and longer eyes because these findings are not directly relevant to the purpose of the manuscript.

Why was axial length not included in Table 2? Axial length may confound other associations – such as gender differences.

Reviewer #2: In general, the authors have made considerable steps in improving their manuscript. The paper now reads clearer and the main points are not lost. However, there are still several points of concern about this study for this reviewer.

Major Concerns:

1. As previously mentioned by reviewer 1, several authors have published similar studies. While the discussion now clearly compares the various studies that have been published, it still remains unclear what is novel about this study or its contribution to the literature. Please highlight these points in your discussion.

2. The exclusion criteria state that subjects with epiretinal membranes and late-stage AMD were excluded due to known alterations these pathologies have on the vasculature architecture in the retina. Yet the same argument can be made for subjects with hypertension or with diabetes. Based on this, was the exclusions of the subjects based on the fact that those with ERMs or AMD had primarily ocular diseases and therefore the changes in the retinal vasculature had nothing to do with the cardio-vasculature system or was it truly because the vasculature in the retina is altered? If it’s the former, please update the section of the manuscript. If it’s the latter, then the argument for excluding those subjects but including subjects with diabetes and hypertension needs to be re-examined.

**********

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

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22 Sep 2020

Manuscript ID PONE-D-20-00420R1

Association Between the Retinal Vascular Network and Retinal Nerve Fiber Layer in the Elderly: The Montrachet Study

Thank you for giving us the opportunity to revise this submission following peer review. After consultation with the other authors, we have responded to the reviewers’ constructive suggestions by revising the manuscript as attached. We

would like to take the opportunity to respond to the individual points below.

Reviewer #1: Thank you for considering revisions. These analyses are difficult in elderly patients because of the confounding variables. For example, in this study nearly 70% of participants have a history of hypertension. Presence of hypertension, and diabetes, likely affects affect retinal vascular parameters, but this information is not presented in the manuscript.

Table 2 shows the association between RNFL to demographics, lifestyle and clinical characteristics. It would be useful to show associations between retinal vascular parameters to demographics, lifestyle and clinical characteristics as well. I would expect there to be significant associations with hypertension, diabetes and possibly axial length. Increased transparency with the multiple variables involved will help readers understand the complexity associated with these analyses.

We thank the Reviewer for highlighting this point. We previously presented associations between retinal vascular parameters and demographics, lifestyle and clinical characteristics in our manuscript : Association between the retinal vascular network with Singapore “I” Vessel Assessment (SIVA) software, cardiovascular history and risk factors in the elderly: The Montrachet study. (Arnould L et al. PlosOne April 2018). As suggested by the Reviewer, we found significant associations between retinal vascular parameters and hypoglycemic treatment, history of systemic vascular events and increased Heart Score risk. For greater clarity, we did not repeat these results.

Could the authors run a separate analysis evaluating for association of RNFL and retinal vasculature parameters after excluding hypertension, diabetes and extreme axial length differences? It is unclear if the numbers would support this evaluation.

Thank you to the Reviewer for emphasizing this crucial point. We ran a separate analysis in participants without hypertension and diabetes. This analysis is presented in Table 3. We added these sentences in the revised version of our manuscript as follows

Line 220-225

“In participants without hypertension and diabetes (n = 294), after adjustment for age, sex and axial length, each SD decrease in the caliber of the six largest arterioles and veins in zone B and in the caliber of the six largest arterioles and veins in zone C was associated with a decrease in global RNFL thickness (β = −1.88 μm, P = 0.0371; β = −3.28 μm, P < 0.001; β = −2.02 μm, P < 0.001; and β = −3.27 μm, P < 0.001, respectively) (Table 3).”

Line 271-272

“Moreover, in a subgroup analysis (participants without hypertension or diabetes), we found similar results.”

Line 306-310

“However, in the subgroup analysis (Table 3), the association between a lower fractal dimension of veins in zone C and a reduction in mean RNFL thickness is no more significant. We could make the hypothesis that presence of hypertension or diabetes affect first and foremost retinal fractal dimension.”

For the RNFL analysis, this work is contrasted by others because it is an “elderly cohort” – could the authors clearly state the mean age in the results section?

The mean age in this study was 82.06 (± 3.64) years old. We added this information in the results section as follows (Line 184)

“The mean RNFL of the study participants was 92.08 ± 13.29 μm and the mean age was 82.06 ± 3.64 years old.”

Is Table 1 necessary? This is essentially comparing cases that were excluded to participants that were included. Why is this relevant to the comparison of RNFL to retinal vascular parameters? If important to include, perhaps these findings could be summarized in the text of the results.

We agree with the concern raised by the Reviewer. Table 1 is now part of the supporting information. In the revised version of our manuscript, it is reported as S1 Table. We added these sentences in the revised version of our manuscript as follows (Line 180-181)

“The characteristics of the study population and a comparison between participants and non-participants are summarized in the supporting information (S1 Table).”

Moreover, we transferred Table 4 and 5 in the supporting information (S2 and S3 Table). We added a caption section as requested in the revised version of our manuscript.

I would exclude the sentence in the abstract describing thinner global RNFL thickness associated with males, patients with diabetes and longer eyes because these findings are not directly relevant to the purpose of the manuscript.

We fully agree with the reviewer’s comment. We deleted this sentence in the abstract of the revised manuscript.

Why was axial length not included in Table 2? Axial length may confound other associations – such as gender differences.

We agree with the concerns raised by the Reviewer. We added this information in the revised version of our manuscript (Table 1).

Reviewer #2: In general, the authors have made considerable steps in improving their manuscript. The paper now reads clearer and the main points are not lost. However, there are still several points of concern about this study for this reviewer.

Major Concerns:

1. As previously mentioned by reviewer 1, several authors have published similar studies. While the discussion now clearly compares the various studies that have been published, it still remains unclear what is novel about this study or its contribution to the literature. Please highlight these points in your discussion.

We thank the reviewer for highlighting this point. We added this sentence in the discussion of the revised manuscript (Line 334-337).

“Strengths of our study include its large elderly population-based sample, extensive retinal vascular parameters phenotype (caliber, fractal dimension and vascular tortuosity) and a subgroup analysis in participants without hypertension or diabetes.”

2. The exclusion criteria state that subjects with epiretinal membranes and late-stage AMD were excluded due to known alterations these pathologies have on the vasculature architecture in the retina. Yet the same argument can be made for subjects with hypertension or with diabetes. Based on this, was the exclusions of the subjects based on the fact that those with ERMs or AMD had primarily ocular diseases and therefore the changes in the retinal vasculature had nothing to do with the cardio-vasculature system or was it truly because the vasculature in the retina is altered? If it’s the former, please update the section of the manuscript. If it’s the latter, then the argument for excluding those subjects but including subjects with diabetes and hypertension needs to be re-examined.

We agree with the concerns raised by the Reviewer. We performed a subgroup analysis in participants without hypertension or diabetes (Table 3) and we added the following statement in the revised version of our manuscript (Line 149-152).

“Participants with epiretinal membrane and AMD with modification of the vascular architecture were excluded because they present primary modifications of the retinal vascular profile without systemic involvement.”

Submitted filename: Rebuttal letter R2.docx

Click here for additional data file.

8 Oct 2020

Association Between the Retinal Vascular Network and Retinal Nerve Fiber Layer in the Elderly: The Montrachet Study

PONE-D-20-00420R2

Dear Dr. Arnould,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Ireneusz Grulkowski, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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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)

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Reviewer #1: Yes

Reviewer #2: (No Response)

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Reviewer #1: Yes

Reviewer #2: (No Response)

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Reviewer #1: Yes

Reviewer #2: (No Response)

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Reviewer #1: I would like to thank the authors for making the recommended changes to the manuscript over the past two revisions. Nice work.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

12 Oct 2020

PONE-D-20-00420R2

Association Between the Retinal Vascular Network and Retinal Nerve Fiber Layer in the Elderly: The Montrachet Study

Dear Dr. Arnould:

I'm 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 let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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.

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Thank you for submitting your work to PLOS ONE and supporting open access.

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on behalf of

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Academic Editor

PLOS ONE