Sex differences in the association between systemic oxidative stress status and optic nerve head blood flow in normal-tension glaucoma

Masataka Sato; Masayuki Yasuda; Nana Takahashi; Kazuki Hashimoto; Noriko Himori; Toru Nakazawa

doi:10.1371/journal.pone.0282047

. 2023 Feb 24;18(2):e0282047. doi: 10.1371/journal.pone.0282047

Sex differences in the association between systemic oxidative stress status and optic nerve head blood flow in normal-tension glaucoma

Masataka Sato ^1,^#, Masayuki Yasuda ^1,^#, Nana Takahashi ^1,^#, Kazuki Hashimoto ^1,^#, Noriko Himori ^1,^2,^#, Toru Nakazawa ^1,^3,^4,^5,^*,^#

Editor: Demetrios G Vavvas⁶

PMCID: PMC9955941 PMID: 36827337

Abstract

Purpose

To investigate the association of systemic oxidative stress markers and optic nerve head (ONH) blood flow in normal-tension glaucoma (NTG) patients, as well as sex differences in this association.

Methods

This was a cross-sectional study of 235 eyes with NTG of 134 patients (56 male, 78 female; mean age, 60.9±14.1 years). Laser speckle flowgraphy (LSFG) was used to measure ONH blood flow (mean blur rate in the tissue area of the ONH; MBR-T) and LSFG pulse-waveform parameters, including flow acceleration index in the tissue area of the ONH (FAI-T). Oxidative stress markers, diacron-reactive oxygen metabolites (d-ROMs), and biological antioxidant potential (BAP) were measured with a free radical elective evaluator. Spearman’s rank correlation test and a multivariate linear mixed-effect model were used to investigate factors associated with ONH blood flow.

Results

MBR-T was significantly correlated with age (rs = -0.28, p < 0.001), mean arterial pressure (rs = -0.20, p = 0.002), intraocular pressure (rs = 0.24, p < 0.001), peripapillary retinal nerve fiber layer thickness (rs = 0.62, p < 0.001), and disc area (rs = -0.26, p < 0.001), but not with serum d-ROM level. Separate analyses of the subjects divided by sex showed that BAP was positively correlated to MBR-T (rs = 0.21, p = 0.036) and FAI-T (rs = 0.36, p < 0.001) only in male subjects. Similarly, BAP was significantly associated with MBR-T (β = 0.25, p = 0.026) and FAI-T (β = 0.37, p < 0.001) in male subjects in a multivariate linear mixed-effect model.

Conclusion

A lower serum antioxidant level, as indicated by BAP, was associated with reduced ONH blood flow only in male NTG patients. Our findings suggest that there are sex differences in the involvement of oxidative stress in the pathogenesis of reduced ocular blood flow in NTG.

Introduction

Glaucoma is the leading cause of blindness worldwide, with a prevalence that is expected to increase to 111.8 million by 2040 [1]. It is an ocular neurodegenerative disease characterized by progressive retinal ganglion cell death [2]. Elevated intraocular pressure (IOP) is the major risk factor for glaucoma, and the most common evidence-based treatment for glaucoma is lowering IOP [3]. However, we have often observed patients who have progressive glaucoma despite this treatment in daily clinical practice [4]. Normal-tension glaucoma (NTG) is defined as glaucoma with an open angle and no record of IOP 22 mmHg or higher. Epidemiological research has shown that the proportion of NTG cases among all cases of primary open-angle glaucoma was 50% in the US and 75 to 90% in China, Singapore, Japan, and Korea [5]. In Japan, NTG is the most common subtype of open-angle glaucoma, where it accounts for over 90% of cases [6]. Like other neurodegenerative diseases, it is considered that multiple factors are implicated in the pathogenesis of NTG [7]. However, IOP-independent risk factors for NTG remain unclear. In order to establish a novel treatment for glaucoma, there is a need to identify these risk factors.

Blood flow impairment is involved in various age-related diseases, such as Alzheimer’s disease [8] and major depressive disorder [9]. Many studies have suggested that changes in ocular blood flow (OBF) play a critical role in the development and progression of glaucoma. Blood flow in the peripapillary retina, which can be measured with laser Doppler flowmetry, has been found to be reduced in NTG [10]. Blood flow in the capillary area of the ONH, which can be measured with laser speckle flowgraphy (LSFG), has been shown to be reduced in the preperimetric stage of glaucoma [11] and the early stage of NTG [12]. In addition, the severity of glaucoma has been reported to be correlated to decreased ONH blood flow [13]. Furthermore, we recently demonstrated that decreased tissue blood flow in the ONH precedes glaucoma neurodegeneration in patients with risk factors for altered blood flow, such as old age and high pulse rate [14]. Treatment to inhibit reduced ONH blood flow may prevent the progression of glaucomatous optic neuropathy, but such treatment has not been established, nor have biomarkers for ONH blood flow in glaucoma.

Oxidative stress is defined as a state of an excess load of reactive oxygen species and/or reduction of antioxidants in cells and tissues. Chronic oxidative stress is involved in various age-related diseases, such as cancer [15] and atherosclerotic diseases [16]. Previous studies have suggested that systemic oxidative stress plays an important role in the pathogenesis of glaucoma. Himori et al. reported that urine levels of 8-hydroxy-2’-deoxyguanosine (8-OHdG), a systemic oxidative stress marker, were correlated to visual field defects in NTG [17]. In addition, Tanito et al. measured both systemic oxidative stress and the antioxidant capacity of blood samples using a free-radical analyzer system and found a positive correlation between antioxidant potential and mean deviation of the visual field [18]. Oxidative stress can induce decreased nitric oxide release and increased endothelin-1 production, which is involved in the blood flow impairment underlying various vascular diseases [19]. We thus hypothesized that systemic oxidative stress levels were associated with a reduction of ocular blood flow in NTG. In the current study, we aimed to investigate the relationship between markers of systemic oxidative stress and ONH blood flow, measured with LSFG, in NTG patients.

Materials and methods

Subjects

This was a cross-sectional study that examined 235 eyes of 134 NTG patients who visited Tohoku University Hospital, located in Miyagi, Japan, between October 2018 and July 2020. Written informed consent was obtained from the subjects at the time of sample collection. This study followed the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Tohoku University School of Medicine (Protocol number: 2021-1-430). NTG was diagnosed by a glaucoma specialist (T.N.) and was defined as an open angle in a gonioscopic examination, glaucomatous ONH changes, and corresponding visual field defects matching the Anderson–Patella criteria [20]. The exclusion criteria were as follows: (1) presence of ocular disease other than mild cataract, (2) a history of high IOP (22 mmHg or higher, as measured with Goldmann applanation tonometry) on the test day, and (3) high myopia, i.e., axial length (AL) longer than 26 mm.

Ophthalmic evaluation

Intraocular pressure (IOP) was measured with Goldmann applanation tonometry; AL was measured with the IOL Master (Zeiss Meditec, Dublin, CA, USA); circumpapillary retinal nerve fiber layer thickness (cpRNFLT) was measured with swept-source OCT (DRI OCT Triton, Topcon, Inc., Tokyo, Japan), and the visual field was measured with the 24–2 program of the Humphrey Field Analyzer (Carl Zeiss Meditec, Dublin, CA, USA). When both eyes of a patient met the inclusion criteria, both eyes were included in the statistical analysis.

Blood samples and oxidative stress measurement

The presence or absence of smoking history, diabetes (DM), hypertension, and dyslipidemia were recorded before the samples were collected. Blood samples were collected after at least 3 hours of fasting. Next, oxidative stress markers, including diacron-reactive oxygen metabolites (d-ROMs) and biological active potential (BAP), were measured with a free radical analyzer (Free Carpe Diem, Wismerll Co., Ltd., Tokyo, Japan). D-ROM level reflects oxidative stress and is an indicator of the serum activity of hydroperoxides. BAP reflects the antioxidant potential of a sample and represents its capacity to reduce ferric oxide to ferrous oxide. Details of these analysis procedures have been described previously [21].

Optic nerve head blood flow assessment with laser speckle flowgraphy

ONH blood flow was measured with LSFG (LSFG-NAVI, Softcare Co., Ltd., Fukutsu, Japan), as previously described [22]. Before the measurement, the pupils of the eyes of each subject were dilated with 0.5% tropicamide. Systolic and diastolic blood pressure (SBP and DBP) and pulse rate (PR) were measured after the subjects had rested for 10 min in a sitting position in a dark room. Mean arterial pressure (MAP) was calculated as follows: MAP = diastolic BP (DBP) + 1/3 (systolic BP [SBP]—DBP) [22]. Following these measurements, color maps of mean blur rate (MBR) in the ONH were obtained with LSFG, which is derived from the speckle pattern produced by the interference of a laser scattered by moving blood cells [23]. The MBR color maps were then automatically divided by the LSFG software (LSFG analyzer, version 3.2.12.0) into separate regions of the ONH, comprising the large vessel and tissue areas. MBR was assessed separately in the vessel and tissue areas and in the ONH overall. Our study focused on MBR in the tissue area (MBR-T) of the ONH, because it is considered to be a good indicator of blood flow in the deep ONH [24]. Pulse-waveform parameters were also measured with LSFG in the tissue area of the ONH, including flow acceleration index (FAI-T), blowout score (BOS-T), and blowout time (BOT-T). FAI-T indicates the instantaneous force with which blood flow increases in a short time; it is calculated as the maximum change among all frames (1/30 s) in a rising curve [25]. BOS-T is an indicator of the volume of blood flow maintained in a vessel between heartbeats [26]. BOT-T is defined as the ratio of the half width (i.e., the time that the waveform is higher than half of the mean of the minimum and maximum signals) in one heartbeat [27,28]. All statistical analyses of ONH blood flow were based on an average of three separate LSFG measurements.

Statistical analysis

All data are shown as the median (interquartile range [IQR]). Fisher’s exact test was used to analyze the significance of differences in the variables between male and female groups. Spearman’s correlation coefficient and a multivariate linear mixed-effect model were used to analyze the relationship of MBR-T and FAI-T to other variables. All statistical analyses were performed with R software version 4.1.1 (R Core Team 2021). The significance level was set at p < 0.05. All data were fully anonymized before analysis.

Results

The clinical and ophthalmological characteristics of the NTG patients enrolled in this study are shown in Tables 1 and 2, respectively. The male patients were older (p < 0.001), had higher SBP (p < 0.001), higher DBP (p = 0.016), higher MAP (p = 0.001), a lower d-ROM level (p = 0.014), lower IOP (p < 0.001), a wider disc area (p < 0.001), lower MBR-T (p < 0.001), and lower FAI-T (p = 0.004). A history of hypertension was more frequent in the male patients (p = 0.001). The number of topical anti-glaucoma drugs was not significantly different between the sexes (p = 0.912).

Table 1. Systemic characteristics of the normal-tension glaucoma patients.

Variable	Overall (N = 134)	Male (N = 56)	Female (N = 78)	p
Age (years)	62.0 (52.0–70.0)	67.5 (60.0–74.0)	57.5 (47.5–67.0)	<0.001***
SBP (mmHg)	124.0 (114.3–136.0)	129.0 (121.0–140.0)	119.0 (110.0–132.8)	<0.001***
DBP (mmHg)	73.0 (66.0–81.8)	74.5 (68.8–85.0)	71.5 (64.0–78.0)	0.016*
MAP (mmHg)	90.0 (83.4–100.6)	93.2 (88.6–103.0)	86.2 (80.0–96.0)	0.001**
HR (bpm)	69.0 (62.0–76.0)	68.0 (60.0–75.3)	70.0 (64.0–76.0)	0.209
d-ROM (U. Carr)	379.5 (344.0–416.8)	357.5 (333.8–404.5)	391.0 (351.0–427.0)	0.014*
BAP (μmol/L)	2208.0 (2035.0–2313.5)	2159.0 (2001.8–2301.3)	2219.0 (2085.0–2313.5)	0.419
HT (n)	39	25	14	0.001**
DM (n)	12	5	7	0.993
DL (n)	34	14	20	1
Smoking history (n)	12	8	4	0.127

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SBP: Systolic blood pressure; DBP: Diastolic blood pressure; MAP: Mean arterial pressure; HR: Heart rate; d-ROMs: Diacron reactive oxygen metabolites; U. Carr: Carrelli units; BAP: Biological antioxidant potential; HT: Hypertension; DM: Diabetes; DL: Dyslipidemia. Data are expressed as the median (IQR). The Mann-Whitney U test was used to compare groups. Categorical variables were analyzed with Fisher’s exact test. Asterisks indicate statistical significance (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

Table 2. Ocular characteristics of normal-tension glaucoma patients.

Variable	Overall (N = 235)	Male (N = 100)	Female (N = 135)	p
IOP (mmHg)	12.00 (11.00–14.00)	12.00 (10.00–14.00)	13.00 (12.00–15.00)	<0.001***
Axial length (mm)	24.65 (23.87–25.40)	24.42 (23.98–25.03)	24.71 (23.80–25.51)	0.355
cpRNFLT (μm)	65.78 (53.55–78.66)	62.45 (47.00–77.08)	67.03 (56.50–81.12)	0.046*
Disc area (mm²)	1.96 (1.68–2.29)	2.12 (1.77–2.44)	1.87 (1.58–2.12)	<0.001***
MBR-T (AU)	9.07 (7.53–11.13)	8.32 (6.80–10.01)	9.73 (8.23–11.73)	<0.001***
FAI-T (AU)	0.97 (0.73–1.37)	0.88 (0.63–1.27)	1.07 (0.80–1.38)	0.003**
BOS-T	77.67 (73.87–81.62)	77.73 (73.60–81.77)	77.67 (73.92–81.58)	0.89
BOT-T	50.37 (47.30–53.58)	49.63 (46.41–53.20)	50.80 (47.52–53.68)	0.133
Topical anti-glaucoma drugs (n)	3.00 (2.00–4.00)	3.00 (2.00–4.00)	3.00 (2.00–4.00)	0.912

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IOP: Intraocular pressure; cpRNFLT: Circumpapillary retinal nerve fiber layer thickness; MBR-T: Mean blur rate in the tissue area of the optic nerve head; FAI-T: Flow acceleration index in the tissue area of the optic nerve head; AU: Arbitrary units; BOS-T: Blowout score in the tissue area of the optic nerve head; BOT-T: Blowout time in the tissue area of the optic nerve head. Data are expressed as the median (IQR). The Mann-Whitney U test was used to compare groups. Asterisks indicate statistical significance (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

Spearman’s rank correlation coefficient was calculated to evaluate the relationships between ONH blood flow parameters and their respective clinical parameters (Table 3). MBR-T was significantly correlated with age (rs = -0.28, p < 0.001), MAP (rs = -0.20, p = 0.002), IOP (rs = 0.24, p < 0.001), cpRNFLT (rs = 0.62, p < 0.001), and disc area (rs = -0.26, p < 0.001) in all patients. Separate analyses of patients grouped by sex showed that MBR-T was correlated to age (rs = -0.27, p = 0.002), MAP (rs = -0.21, p = 0.016), IOP (rs = 0.27, p = 0.002), and disc area (rs = -0.27, p = 0.002) in the female subjects, but not in the male subjects (age, rs = -0.10, p = 0.306; MAP, rs = 0.05, p = 0.602; IOP, rs = 0.006, p = 0.956; disc area, rs = -0.13, p = 0.193). We also investigated the correlation between the OBF parameters (i.e., MBR-T and FAI-T) and systemic oxidative stress markers (BAP and d-ROMs) (Table 3 and S1 Fig). MBR-T was significantly correlated to BAP only in the male patients (rs = 0.21, p = 0.036). There was no significant correlation between MBR-T and d-ROM level in any group. FAI-T was significantly correlated with age (rs = -0.13, p = 0.050), MAP (rs = -0.25, p < 0.001), IOP (rs = 0.21, p = 0.001), cpRNFLT (rs = 0.54, p < 0.001), BAP (rs = 0.16, p = 0.017), and disc area (rs = -0.21, p = 0.010). Analyses by sex showed that age (rs = -0.18, p = 0.041), MAP (rs = -0.30, p < 0.001), d-ROM level (rs = -0.18, p = 0.036), IOP (rs = 0.22, p = 0.010), and disc area (rs = -0.17, p = 0.045) were correlated to FAI-T only in the female subjects. CpRNFLT was significantly correlated to FAI-T in both sexes. FAI-T was correlated to BAP only in the male subjects (rs = 0.36, p < 0.001). BOS-T was significantly correlated with age (rs = -0.31, p < 0.001), MAP (rs = 0.17, p = 0.010), HR (rs = 0.28, p < 0.001), and cpRNFLT (rs = -0.24, p < 0.001). BOT-T was significantly correlated with age (rs = -0.44, p < 0.001) and HR (rs = 0.35, p < 0.001).

Table 3. Spearman’s correlation coefficient between MBR-T, pulse waveform parameters, and each variable.

LSFG parameter	Variable	Overall		Male		Female
LSFG parameter	Variable	rs	p	rs	p	rs	p
MBR-T (AU)
	Age (years)	-0.28	<0.001***	-0.1	0.306	-0.27	0.002**
	MAP (mmHg)	-0.2	0.002**	0.05	0.602	-0.21	0.016*
	HR (bpm)	0.11	0.1	-0.05	0.605	0.15	0.08
	BAP (μmol/L)	0.13	0.05	0.21	0.036*	0.01	0.882
	d-ROMs (U. Carr)	0.008	0.901	-0.03	0.766	-0.09	0.283
	IOP (mmHg)	0.24	<0.001***	0.006	0.956	0.27	0.002**
	cpRNFLT (μm)	0.62	<0.001***	0.6	<0.001***	0.65	<0.001***
	Disc area (mm²)	-0.26	<0.001***	-0.13	0.193	-0.27	0.002**
FAI-T (AU)
	Age (years)	-0.13	0.050*	0.06	0.548	-0.18	0.041*
	MAP (mmHg)	-0.25	<0.001***	-0.11	0.266	-0.3	<0.001***
	HR (bpm)	0.08	0.228	0.05	0.602	0.05	0.566
	BAP (μmol/L)	0.16	0.017*	0.36	<0.001***	-0.07	0.437
	d-ROMs (U. Carr)	-0.05	0.438	0.04	0.673	-0.18	0.036*
	IOP (mmHg)	0.21	0.001**	0.06	0.544	0.22	0.010**
	cpRNFLT (μm)	0.54	<0.001***	0.53	<0.001***	0.54	<0.001***
	Disc area (mm²)	-0.21	0.010*	-0.16	0.112	-0.17	0.045*
BOS-T
	Age (years)	-0.31	<0.001***	-0.43	<0.001***	-0.27	0.001**
	MAP (mmHg)	0.17	0.010*	0.09	0.388	0.24	0.005**
	HR (bpm)	0.28	<0.001***	0.2	0.044*	0.35	<0.001***
	BAP (μmol/L)	-0.01	0.82	-0.16	0.104	0.13	0.135
	d-ROMs (U. Carr)	0.03	0.646	-0.06	0.53	0.09	0.274
	IOP (mmHg)	-0.11	0.104	-0.11	0.262	-0.12	0.166
	cpRNFLT (μm)	-0.24	<0.001***	-0.24	0.017*	-0.25	0.004**
	Disc area (mm²)	0.06	0.388	0.06	0.523	0.03	0.691
BOT-T
	Age (years)	-0.44	<0.001***	-0.34	<0.001***	-0.51	<0.001***
	MAP (mmHg)	-0.11	0.087	-0.08	0.388	-0.09	0.296
	HR (bpm)	0.35	<0.001***	0.38	<0.001***	0.3	<0.001***
	BAP (μmol/L)	0.08	0.224	0.06	0.523	0.1	0.252
	d-ROMs (U. Carr)	-0.02	0.728	-0.06	0.885	-0.08	0.36
	IOP (mmHg)	-0.11	0.104	-0.11	0.268	-0.09	0.309
	cpRNFLT (μm)	-0.06	0.333	-0.18	0.081	-0.25	0.882
	Disc area (mm²)	-0.006	0.923	0.006	0.951	0.01	0.895

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MBR-T: Mean blur rate in the tissue area of the optic nerve head; FAI-T: Flow acceleration index in the tissue area of the optic nerve head; AU: Arbitrary units; BOS-T: Blowout score in the tissue area of the optic nerve head; BOT-T: Blowout time in the tissue area of the optic nerve head; MAP: Mean arterial pressure; HR: Heart rate; BAP: Biological antioxidant potential; d-ROMs: Diacron reactive oxygen metabolites; U. Carr: Carrelli units; IOP: Intraocular pressure; cpRNFLT: Circumpapillary retinal nerve fiber layer thickness; rs: Spearman’s rank correlation coefficient. Asterisks indicate statistical significance (*: p< 0.05, **: p < 0.01, ***: p < 0.001).

Multivariate linear mixed-effect models were used to evaluate the association of BAP with MBR-T and FAI-T while adjusting for potential confounding factors, including sex, age, IOP, cpRNFLT, disc area, MAP, BAP, smoking history, HT, DM, and DL (Table 4). CpRNFLT was significantly associated with MBR-T in both sexes and in the group overall (overall, β = 0.45, p < 0.001; male, β = 0.51, p < 0.001; female, β = 0.44, p < 0.001). Disc area was also associated with MBR-T in both sexes and in the group overall (overall, β = -0.16, p = 0.003; male, β = -0.21, p = 0.021; female, β = -0.15, p = 0.028). This finding is compatible with a previous study conducted with healthy subjects [25]. Although IOP was associated with MBR-T in the overall group of subjects and in the female subjects (overall, β = 0.11, p = 0.036; male, β = 0.07, p = 0.433; female, β = 0.15, p = 0.042), the β value was very small and the p value was close to the significance threshold. On the other hand, BAP was significantly associated with MBR-T only in the male subjects (overall, β = 0.03, p = 0.579; male, β = 0.24, p = 0.026; female, β = -0.08, p = 0.368). FAI-T was also associated with cpRNFLT in all the groups (overall, β = 0.47, p < 0.001; male, β = 0.54, p < 0.001; female, β = 0.46, p < 0.001). Disc area was also significantly associated with FAI-T in the overall group and the male group (overall, β = -0.16, p = 0.009; male, β = -0.21, p = 0.021). Although the association of disc area with FAI-T did not reach statistical significance in the female subjects (β = -0.12, p = 0.125), the direction of β was similar to the overall group and the male group. IOP was associated with FAI-T in the overall group and the female group (overall, β = 0.17, p = 0.004; male, β = 0.17, p = 0.050; female, β = 0.22, p = 0.005). Unlike MBR-T, MAP showed a significant association with FAI-T in the overall group and the female group (overall, β = -0.21, p = 0.007; male, β = -0.01, p = 0.914; female, β = -0.26, p = 0.015). Interestingly, a positive association between FAI-T and BAP was also found in the male group (overall, β = 0.04, p = 0.512; male, β = 0.37, p < 0.001; female, β = -0.19, p = 0.040).

Table 4. The effect of clinical parameters on MBR-T and FAI analyzed with a linear mixed-effect model.

Response variable	Explanatory variable	Overall		Male		Female
Response variable	Explanatory variable	β	p	β	p	β	p
MBR-T (AU)
	Sex	-0.1	0.155	NA	NA	NA	NA
	Age (years)	-0.06	0.386	0.007	0.926	-0.08	0.446
	IOP (mmHg)	0.11	0.036*	0.07	0.433	0.15	0.042*
	cpRNFLT (μm)	0.45	<0.001***	0.51	<0.001***	0.44	<0.001***
	Disc area	-0.16	0.003**	-0.21	0.021*	-0.15	0.028*
	MAP (mmHg)	-0.08	0.219	0.06	0.612	-0.14	0.142
	BAP (μmol/L)	0.03	0.579	0.24	0.026*	-0.08	0.368
	Smoking history	0.07	0.252	0.04	0.693	0.06	0.505
	HT	-0.09	0.183	-0.21	0.046*	-0.01	0.905
	DM	-0.06	0.364	-0.05	0.621	-0.04	0.633
	DL	-0.006	0.925	-0.03	0.792	0.006	0.951
FAI-T (AU)
	Sex	-0.02	0.814	NA	NA	NA	NA
	Age (years)	0.15	0.057	0.19	0.067	0.12	0.234
	IOP (mmHg)	0.17	0.004**	0.17	0.05	0.22	0.005**
	cpRNFLT (μm)	0.47	<0.001***	0.54	<0.001***	0.46	<0.001***
	Disc area	-0.16	0.009**	-0.21	0.021*	-0.12	0.125
	MAP (mmHg)	-0.21	0.007**	-0.01	0.914	-0.26	0.015*
	BAP (μmol/L)	0.04	0.512	0.37	<0.001***	-0.19	0.040*
	Smoking history	0.12	0.101	0.08	0.477	0.08	0.365
	HT	-0.003	0.964	0.03	0.803	-0.07	0.517
	DM	0.004	0.958	0.05	0.656	0.03	0.768
	DL	-0.1	0.15	-0.11	0.294	-0.13	0.194

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MBR-T: Mean blur rate in the tissue area of the optic nerve head; FAI-T: Flow acceleration index in the tissue area of the optic nerve head; AU: Arbitrary units; IOP: Intraocular pressure; cpRNFLT: Circumpapillary retinal nerve fiber layer thickness; MAP: Mean arterial pressure; BAP: Biological antioxidant potential; HT: Hypertension; DM: Diabetes; DL: Dyslipidemia; β: Standardized coefficient (linear mixed-effect model). Asterisks indicate statistical significance (*: p < 0.05, **: p < 0.01, ***: p < 0.001).

Discussion

In the current study, we investigated the relationship between OBF and oxidative stress markers and found that the serum BAP level was positively associated with MBR-T and FAI-T only in male NTG patients. Although cpRNFLT is strongly associated with OBF in the ONH, our findings suggest that antioxidant capacity might be implicated in OBF impairment in NTG. Furthermore, there might be a sex difference in the role of oxidative stress in NTG.

Oxidative stress and blood flow

Oxidative stress is defined as a disturbance in the balance between the production of reactive oxygen species (ROS) and antioxidants, and has been implicated in various diseases, including atherosclerotic cardiovascular disease (CVD) [16]. Although elevated IOP is the most significant risk factor for glaucoma, recent studies suggest that there is an association between oxidative stress and the pathophysiology of glaucoma. Oxidative stress can not only increase IOP via trabecular meshwork degeneration, but also directly damage the retinal ganglion cells through multiple cell death pathways [29,30] Furthermore, ROS can also induce vascular endothelial cell damage, which leads to blood flow impairment via decreased nitric oxide release [16,31]. Previously, we reported that increased urinary 8-OHdG levels were independently associated with decreased OBF in NTG [17], supporting the association between high oxidative stress and capillary damage in the ONH. However, we found only a poor correlation in the female subjects between OBF and the serum level of d-ROMs (rs = -0.18, p = 0.036), an indicator of systemic oxidative stress. Takayanagi et al. reported that serum d-ROMs were not correlated to the level of superoxide dismutase protein in the serum or aqueous humor or the level of serum sulfhydryl, and might not reflect the oxidative stress status of the eye [32]. Therefore, serum d-ROM levels might not directly reflect local ocular oxidative stress, which may explain the lack of an association between OBF parameters and d-ROMs in our study.

In the current study, we found that the serum BAP levels were positively associated with MBR-T in male patients with NTG. MBR represents a quantitative measurement of relative blood flow velocity. Reportedly, reduced MBR-T in the ONH precedes the loss of cpRFNLT and is independently associated with visual field loss in eyes with glaucoma, suggesting that MBR-T is a potential prognostic marker of glaucoma [13] [33]. Oxidative stress has been reported to enhance the inflammatory pathways responsible for the atherosclerotic process and to lower peripheral blood flow [34]. In addition, the acute oral intake of mitochondria-targeting antioxidants has been reported to contribute to improved endothelial function and peripheral circulation [35]. An animal study that used streptozotocin-induced diabetic rats showed that diabetic vascular dysfunction was accompanied by an accumulation of superoxides in arterioles, which normally provide blood flow to the sciatic nerve. That study also demonstrated that antioxidant treatment reduced vascular endothelial damage and improved peripheral nerve conduction [36]. Furthermore, decreased serum antioxidative capacity has been shown to increase oxidative stress in the aqueous humor of glaucoma patients [32]. Finally, oral antioxidant supplementation has been reported to increase ocular blood flow in the retinal and retrobulbar vascular beds in patients with open-angle glaucoma [37]. These previous studies support the findings of the current study, particularly that decreased antioxidant capacity was associated with decreased OBF, and suggest that the serum level of BAP is a useful biomarker of OBF impairment and underlying NTG pathogenesis. Thus, antioxidant supplements might be a promising treatment target for NTG patients with low serum BAP [38].

Sex differences

Although various studies have reported findings that suggest lower antioxidant capacity is implicated in blood flow impairment, we observed an association between serum BAP and ONH OBF only in male NTG patients. Such sex differences have been noted in other diseases, including CVD. Reportedly, women have higher mortality after acute cardiovascular events, though the incidence of CVD in women is usually lower than in men [39]. Yanagida et al. reported that women had better OBF than men among healthy volunteers, which is similar to our current findings. That study also suggested that the impact of age in decreased OBF was greater in women than men [40]. NTG has been reported to occur more frequently in women, and female sex has been reported to be a risk factor for the progression of NTG [41]. Vajaranant et al. reported that bilateral oophorectomy increased the risk of glaucoma [42], and Hulsman et al. found that reduced estrogen might be involved in blood flow impairment in women and that menopause was a risk factor for NTG [43]. Centofanti et al. reported that pre-menopausal women had significantly higher pulsatile ocular blood flow (POBF) and that estrogen replacement therapy in post-menopausal women improved POBF [44]. The secretion of estrogen varies among individuals, but it declines sharply during menopause at around age 50 [45,46]. Estrogen has been known to have anti-inflammatory and vasoprotective effects in addition to its role in stimulating follicles. Estrogen can modulate the expression of growth factors and oxidative stress, which is thought to partly explain its vasoprotective effects in injured arteries [47]. Moreover, the anti-inflammatory and vasoprotective effects of estrogen are lost in aging subjects. It has been also reported that blood total cholesterol, triglycerides, and low-density lipoprotein levels increase after menopause. Such unfavorable lipid profiles are involved in increased risk for CVD [48].

A recent systematic review and meta-analysis demonstrated that glaucoma patients had significantly higher total cholesterol, higher total cholesterol, and lower high-density lipoprotein levels than healthy controls [49]. Furthermore, hyperlipidemia can increase systematic oxidative stress in NTG patients [50]. Although we did not measure estrogen or cholesterol levels, these factors might affect OBF, resulting in sex differences in the association between antioxidant capacity and OBF in the ONH of NTG patients.

Pulse waveform in the ONH and antioxidant capacity in glaucoma

The current study is the first to investigate an association between waveform parameters and antioxidant capacity in NTG patients. We used LSFG to measure OBF parameters and found that MBR-T and FAI-T were positively associated with BAP in male NTG patients. We also found that IOP and MAP were associated with FAI-T in the overall group of patients and the female patients. Iwase et al. demonstrated that FAI in the ONH significantly increased with increasing IOP in healthy subjects [51]. In addition, Yanagida et al. showed that FAI in the overall ONH was negatively associated with MAP [40]. These previous reports support our results showing the association of FAI-T with IOP and MAP. Although the male patients did not show a significant association between IOP or MAP and FAI-T, the direction of the effect size was similar to that of the female patients. The sex differences in the median of MBR-T and FAI-T might be involved in these results, but the exact reason could not be clarified by this study.

As already mentioned, it has been suggested that MBR-T is a good indicator of structural and functional changes in glaucoma [13,33]. FAI-T is an indicator of the instantaneous force that can increase OBF in a short time [25]. A case-control study of patients with European ancestry has shown that NTG patients have lower FAI-T (i.e., a flattened pulse waveform) than healthy subjects [52]. Curiously, the association between FAI-T and BAP in the current study showed different directions between the sexes (male β = 0.37, female β = -0.19). Although the exact reason remains unknown, several differences in background characteristics (e.g., younger age, lower BP, and higher d-ROM level) as well as sex hormone levels might be involved in the negative association of FAI-T with BAP in the female NTG patients. Moreover, the β was low (β = -0.19) and the p-value was close to the threshold (p = 0.040), although it reached statistical significance. Therefore, the negative association of FAI-T with BAP in the female patients should be carefully interpreted. We previously reported that MBR decreases were milder in the ONH tissue of healthy subjects during IOP elevation, possibly because of increased FAI-T induced by the local autoregulatory system for OBF [53]. Therefore, antioxidant capacity might play an important role in OBF maintenance by preserving the autoregulatory system, as well as protecting retinal vessel structure.

Limitations

Our study has several limitations. First, it was limited to Japanese patients with NTG and was conducted at a single institution. Therefore, a follow-up study is required to determine whether the results apply to patients of other ethnicities. Second, our study used a cross-sectional design, and we were thus unable to determine whether the reduction in OBF was the effect of decreased antioxidant capacity or was related to structural changes in the ONH due to glaucoma progression. Third, although we found an association between MBR-T and BAP only in the male patients, we did not have any molecular data to support this finding of a sex difference. Multiple studies have reported that female hormones could contribute to the differences in OBF between men and women [40,54]. The effectiveness of postmenopausal hormone replacement therapy for glaucoma has been controversial [55], which might be due to a lack of detailed data on the time of menopause and estrogen levels. Therefore, future studies that include the time of menopause and estrogen level, as well as antioxidant capacity, are required to evaluate the association of sex differences with the oxidative stress-related reduction of OBF in NTG.

In conclusion, we found that a lower serum antioxidant level, as indicated by BAP, was associated with reduced MBR-T in ONH only in male NTG patients. In addition, BAP showed a different association with FAI-T between the sexes. This finding suggests that oxidative stress might be an IOP-independent risk factor implicated in the pathogenesis of reduced OBF in NTG. Furthermore, sex differences should be taken into consideration for the management of NTG patients. Further study is required to verify the causal relationships of oxidative stress and OBF in NTG and to elucidate the mechanisms underlying sex differences in these relationships.

Supporting information

S1 Fig. Correlation of BAP with MBR-T and FAI-T.

MBR-T was significantly correlated with BAP only in the male patients (rs = 0.21, p = 0.036). FAI-T was significantly correlated with BAP in the overall group (rs = 0.16, p = 0.017) and the male patients (rs = 0.36, p < 0.036).

(TIF)

Click here for additional data file.^{(653.1KB, tif)}

S1 Data. All relevant data.

All relevant data are available in S1 Data.

(CSV)

Click here for additional data file.^{(30.2KB, csv)}

Acknowledgments

Involved in design and conduct of the study were (M.S. and T.N.); data collection (M.S. and N.T.); management and analysis (M.S., K.H., and M.Y.); interpretation of the data (M.S., N.H., and M.Y.); drafting of the manuscript (M.S. and M.Y.); and review and approval of the manuscript (T.N.).

We thank Mr. Tim Hilts for reviewing and editing the language of the manuscript.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This paper was supported in part by JSPS KAKENHI Grants-in-Aid for Scientific Research (B) (T.N. JP20H03838), Grant-in-Aid for Challenging Exploratory Research (T.N. JP21K19548), and Grant-in-Aid for Early-Career Scientists (M.Y. JP21K16866).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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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: This work investigates the role of oxidative stress / antioxidant potential and optic nerve head perfusion parameters in normal tension glaucoma. The manuscript is well written, with interesting data for the scientific community but some revision is needed.

1) Lines 128-130: exclusion criteria “a history of high IOP (higher than 22 mmHg, as measured with Goldmann applanation tonometry) on the test day”. explain why patients with IOP of 22 mmHg were included, since this characterizes open angle glaucoma. Also, briefly explain why high myopia patients were excluded.

2) Line 154: “Measurements of ocular blood flow” This paragraph does not provide methods for measurement of ocular blood flow. Please review.

3) Lines 256-257 and 384-386: Authors state that ß value was very small / low. What criteria authors used to classify a ß value as very small/low? Other associations on the manuscript had similar ß value, but that information was not highlighted.

4) Lines 300-301: authors state no significant association between dROMs and OBF were found. However, authors also report a negative correlation between dROMs and FAI-T in females (rs=-0.18, p=0.036). Please review the statement.

5) Lines 305-306: If authors present previously published data showing that dROMs levels might not directly reflect local ocular oxidative stress, please explain why dROMs was used as oxidative stress marker in this study in the first place.

6) The main findings are related to BAP (antioxidant potential) and not dROMs (oxidative stress marker). BAP is not a direct oxidative stress marker, instead it reflects the systemic antioxidant potential by reducing ferric oxide to ferrous oxide. Therefore, the TITLE and ABSTRACT should be reworded to give less emphasis to “oxidative stress markers”.

Reviewer #2: This is a very interesting study investigating and reporting on the association of ocular blood flow parameters with oxidative stress markers in patients with normal tension glaucoma. More specifically, the study focuses on differences in these correlations that are observed between genders. The authors managed to present a sound and thoroughly organized study and relate their findings very well to previous studies. However, the following points need to be taken into consideration.

Abstract: The investigation of sex differences regarding the association of ocular blood flow with oxidative stress should also be mentioned in the purpose of the study.

The methods and results presented in the abstract should precisely reflect the investigations and outcomes of the present study. In particular, the authors should additionally refer to the outcomes regarding the correlation of MBR-T to d-ROMs, the other oxidative stress marker. A possible association of pulse-waveform parameters with oxidative stress markers and other clinical parameters was also investigated in the present study and should be mentioned both in methods and results of the abstract.

Line 44: The authors had better replace “MBR-T” with “mean blur rate in the tissue area of the optic nerve head (MBR-T)”, since the abbreviation has not been explained earlier.

Line 52: MBR-T was significantly correlated with disc area, too. The text should be changed accordingly.

Line 132: It is advisable that the authors explain why high myopia is included in the exclusion criteria.

Line 154: The title should be changed in order to correspond to the text that follows.

Line 167: The authors should further explain in more detail what the parameter “mean blur rate” exactly indicates.

Lines 171-172: References should be provided.

Table 2: Explanations for the abbreviations “BOS-T”, “BOT-T” should also be included in the caption of the table.

Lines 206, 212: Statical should be changed to statistical.

Line 228: FAI-T was significantly correlated with disc area too. The text should be changed accordingly.

Line 230: cpRNFLT was significantly correlated to FAI-T both in males and females. The text should also be changed accordingly.

Tables 3 and 4: The statistical method that was used for the presented analysis should be mentioned at the title of each table, i.e. spearman’s correlation coefficient and multivariate linear-mixed effect models, respectively. Furthermore, explanation for the abbreviation “AU” should be included in the caption of the tables.

Figure S1: A caption should be provided.

I would like to look at a revised version of the manuscript.

**********

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Reviewer #2: No

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PLoS One. 2023 Feb 24;18(2):e0282047. doi: 10.1371/journal.pone.0282047.r002

Author response to Decision Letter 0

14 Dec 2022

Response letter:

We wish to express our appreciation to the reviewers for their insightful comments on our paper. We feel the comments have helped us significantly improve our paper.

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If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Author’s response: Thank you for your comments. In this study, the funders had no role. Thus, the statement below is correct:

“The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

We have added this statement to our cover letter, just in case.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Author’s response: All relevant data have been uploaded in the S1 Data file. We have added the following statement to the manuscript (line 691 in the revised version): “All relevant data are available in S1 Data.” We have also added this statement to the cover letter.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly.

Author’s response: We have added captions for “S1 Data” and “S1 Fig” at the end of our manuscript (lines 821-826).

5. Thank you for stating the following in the Acknowledgments Section of your manuscript:

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Author’s response:

We removed the funding-related text from the manuscript. The funding statement below is correct (we have also added this statement to our cover letter):

“The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Author’s response: We have made some modifications to the reference list, as follows:

We deleted one of the citations (No. 31 in the original manuscript) because it was a duplicate of No. 13. In addition, we have added references (Nos. 23 and 24) in response to the reviewers’ comments. We also updated the citation numbers in the revised manuscript.

Comments to the Author

Reviewer #1

Author’s response: Thank you for pointing out the error in the description of the exclusion criteria. The subjects of the current study were normal-tension glaucoma patients with an IOP of less than 22 mmHg. Therefore, “a history of high IOP (higher than 22 mmHg)” in the exclusion criteria was incorrect. We have revised “higher than 22 mmHg” to “22 mmHg or higher” (p.8, line 164).

The reason why we excluded high myopia patients was that the ocular circulation in myopic eyes has been reported to be different from normal eyes (PMID: 12084747, 12035987). In order to focus as closely as possible on ocular blood flow in normal-tension glaucoma, we excluded high myopia patients.

2) Line 154: “Measurements of ocular blood flow” This paragraph does not provide methods for measurement of ocular blood flow. Please review.

Author’s response: As the reviewer has pointed out, the title “Measurements of ocular blood flow” was inappropriate. We deleted this title and merged the paragraph with the next paragraph, “Optic nerve head blood flow assessment with laser speckle flowgraphy” (p 9-10, line 195-213).

Author’s response: We thank the reviewer for the comment. As pointed out, we mentioned no clear criteria for classifying the strength of ß values. However, an absolute correlation coefficient value of less than 0.2 has been judged to be a poor correlation in past reports, e.g., Acock et al, who defined |ß| < 0.2 as a weak effect size (Acock, A. C. 2014. A gentle introduction to Stata. College Station, Texas: Stata Press). Following this, we have described the ß value in line 433 as “very small” and the value in line 582 as “low” in the manuscript.

Author’s response: As the reviewer has pointed out, the description “we did not find any significant correlation between OBF and the serum level of d-ROMs” is incorrect. In the Spearman correlation analysis, d-ROM level was correlated with FAI-T in the female subjects (rs=-0.18, p=0.036). However, although the p-value reached the significance threshold, the rs value was less than 0.20, which is considered to be a poor correlation (Rea, L. M., & Parker, R. A. 2014. Designing and conducting survey research: A comprehensive guide. San Francisco: Jossey-Basspoor).

Moreover, we found a only poor association between d-ROM level and FAI-T in a multivariate linear mixed-effect model for the female subjects (data not shown). Thus, we revised “we did not find any significant correlation between OBF and the serum level of d-ROMs” to “we found only a poor correlation in the female subjects between OBF and the serum level of d-ROMs (rs = -0.18, p = 0.036)” (p 22, lines 492-493).

Author’s response:

The d-ROM level has advantages as a marker of oxidative stress for use in daily clinical practice, because it is easy to assess and can be measured simultaneously with BAP with a single analyzer. Therefore, we used d-ROMs as an oxidative stress marker. As the reviewer points out, however, d-ROM levels might not be a good indicator of local or ocular oxidative stress, as reported in previous papers (PMID: 33352680). We will investigate other oxidative stress markers for use in future analyses. We offer our thanks for the useful comment.

Author’s response: We agree with the reviewer’s comment. In response, we revised “oxidative stress markers” to “systemic oxidative stress status” in the title.

Reviewer #2

Abstract: The investigation of sex differences regarding the association of ocular blood flow with oxidative stress should also be mentioned in the purpose of the study.

Author’s response:

We thank the reviewer for the important suggestion. We have mentioned that a purpose of the study was to investigate sex differences in the association of ocular blood flow with oxidative stress.

As the reviewer has pointed out, the methods and results section in our abstract did not completely reflect the contents of the main manuscript. We revised the abstract to match the main manuscript as much as possible. However, we were unable to include all the results in the abstract because of the word-count limitation.

Line 44: The authors had better replace “MBR-T” with “mean blur rate in the tissue area of the optic nerve head (MBR-T)” since the abbreviation has not been explained earlier.

Author’s response: We thank the reviewer for pointing this out. We made the requested change (p 3, line 47).

Line 52: MBR-T was significantly correlated with disc area, too. The text should be changed accordingly.

Author’s response: We thank the reviewer for pointing this out. In response, we have added “and disc area (rs = -0.26, p < 0.001)” (p 3-4, lines 56-69).

Line 132: It is advisable that the authors explain why high myopia is included in the exclusion criteria.

Author’s response: As we also mentioned in our response to the comments of reviewer 1, the reason why we excluded high myopia patients was that the ocular circulation of myopic eyes has been reported to be different from normal eyes (PMID: 12084747, 12035987). In order to focus as closely as possible on ocular blood flow in normal-tension glaucoma, we excluded high myopia patients.

Line 154: The title should be changed in order to correspond to the text that follows.

Author’s response: Thank you for pointing this out. We removed the title “Measurements of ocular blood flow,” modified the relevant paragraph, and incorporated it into the next paragraph (p 9-10, lines 195-213).

Line 167: The authors should further explain in more detail what the parameter “mean blur rate” exactly indicates.

Author’s response: As the reviewer has suggested, we added an explanation of “mean blur rate” as follows (p 10, lines 215-216):

“...which is derived from the speckle pattern produced by the interference of a laser scattered by moving blood cells.”

We also have added a citation for this (No. 23).

Lines 171-172: References should be provided.

Author’s response: We have added a reference (No. 24).

Table 2: Explanations for the abbreviations “BOS-T”, “BOT-T” should also be included in the caption of the table.

Author’s response: We added expansions of the abbreviations “BOS-T” and “BOT-T” to the captions of Tables 2 and 3.

Lines 206, 212: Statical should be changed to statistical.

Author’s response: We have changed “statical” to “statistical” in all tables.

Line 228: FAI-T was significantly correlated with disc area too. The text should be changed accordingly.

Author’s response: We left out a note on the correlation between FAI-T and disc area. In response to this comment, we added the following text to the manuscript: “and disc area (rs = -0.21, p = 0.010)” (p 15, line 304).

Line 230: cpRNFLT was significantly correlated to FAI-T both in males and females. The text should also be changed accordingly.

Author’s response: We have changed “cpRNFLT was significantly correlated to FAI-T both in males and females” to “CpRNFLT was significantly correlated to FAI-T in both sexes” (p 15, lines 307-308).

Author’s response: We amended the title of Table 3 to “Spearman’s correlation coefficient between MBR-T, pulse waveform parameters, and each variable” and the title of Table 4 to “The effect of clinical parameters on MBR-T and FAI analyzed with a linear mixed effect model.”

We also added an expansion of the abbreviation “AU” to the captions of Tables 2, 3, and 4.

Figure S1: A caption should be provided.

Author’s response: We have provided a caption at the end of the manuscript, as follows:

“MBR-T was significantly correlated to BAP only in the male patients (rs = 0.21, p = 0.036). FAI-T was significantly correlated with BAP in the overall group (rs = 0.16, p = 0.017) and the male patients (rs = 0.36, p < 0.036).”

Additional change:

We deleted one of the citations (No. 31 in the original manuscript) because it was a duplicate of No. 13. In addition, we have added references (Nos. 23 and 24) in response to the reviewers’ comments. Accordingly, we updated the citation numbers in the revised manuscript.

In addition, we revised the expansions of the abbreviations at the end of each table.

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PLoS One. doi: 10.1371/journal.pone.0282047.r003

Decision Letter 1

Demetrios G Vavvas

7 Feb 2023

Sex differences in the association between systemic oxidative stress status and optic nerve head blood flow in normal-tension glaucoma

PONE-D-22-21223R1

Dear Dr. Nakazawa,

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.

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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: All comments have been addressed

**********

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

Reviewer #1: Yes

**********

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?

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: Authors have adequately addressed all reviewers comments. The manuscript has improved overall. I have no further comments.

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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

**********

PLoS One. doi: 10.1371/journal.pone.0282047.r004

Acceptance letter

Demetrios G Vavvas

15 Feb 2023

PONE-D-22-21223R1

Sex differences in the association between systemic oxidative stress status and optic nerve head blood flow in normal-tension glaucoma

Dear Dr. Nakazawa:

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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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Correlation of BAP with MBR-T and FAI-T.

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S1 Data. All relevant data.

All relevant data are available in S1 Data.

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Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

Sex differences in the association between systemic oxidative stress status and optic nerve head blood flow in normal-tension glaucoma

Masataka Sato

Masayuki Yasuda

Nana Takahashi

Kazuki Hashimoto

Noriko Himori

Toru Nakazawa

Roles

Abstract

Purpose

Methods

Results

Conclusion

Introduction

Materials and methods

Subjects

Ophthalmic evaluation

Blood samples and oxidative stress measurement

Optic nerve head blood flow assessment with laser speckle flowgraphy

Statistical analysis

Results

Table 1. Systemic characteristics of the normal-tension glaucoma patients.

Table 2. Ocular characteristics of normal-tension glaucoma patients.

Table 3. Spearman’s correlation coefficient between MBR-T, pulse waveform parameters, and each variable.

Table 4. The effect of clinical parameters on MBR-T and FAI analyzed with a linear mixed-effect model.

Discussion

Oxidative stress and blood flow

Sex differences

Pulse waveform in the ONH and antioxidant capacity in glaucoma

Limitations

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Demetrios G Vavvas

Roles

Author response to Decision Letter 0

Decision Letter 1

Demetrios G Vavvas

Roles

Acceptance letter

Demetrios G Vavvas

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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