The association between oxidative stress and corneal hysteresis in patients with glaucoma

Systemic antioxidative status has been implicated in glaucoma pathogenesis. Additionally, corneal hysteresis (CH) may contribute to glaucoma progression. Here, we evaluated the relationship between biological antioxidant potential (BAP) and CH. This study included 103 patients with open-angle glaucoma (OAG). We used a free radical analyzer to measure BAP, and an ocular response analyzer to measure CH and corneal resistance factor (CRF). We evaluated the relationship between systemic oxidative stress and other clinical parameters with Spearman’s rank correlation test and a multi-regression analysis. BAP was not correlated to either CH or CRF in the male or female OAG patients. BAP was correlated to both CH and CRF in the female OAG patients older than 57 years (r = 0.51, P = 0.003; r = 0.49, P = 0.004), but uncorrelated in the female OAG patients younger than 57 years. Multiple regression analysis revealed that BAP independently contributed to CH (P = 0.025) and CRF (P = 0.015) in the older female OAG patients. Systemic oxidative stress may significantly affect the viscoelasticity of the cornea in older female OAG patients. Future studies are needed to confirm that low systemic antioxidative status and low corneal hysteresis contribute to glaucoma pathogenesis.


Results
clinical characteristics of the oAG patients. This study included 103 Japanese glaucoma patients (male: 45; female: 58, NTG: 82, POAG: 21). In a previous study, we divided patients into two groups based on mean age 10 . In this study, we divided the patients into four groups based on sex and mean age in each sex group (male: 61 years, female: 57 years). Table 1 shows demographic characteristics. The younger female and older female groups differed significantly in dROMs, axial length (P = 0.019, P = 0.033), and the incidence of hypertension and hyperlipidemia (P = 0.007, P = 0.001). There were no statistical differences between younger and older male patients with OAG.
Association between cH or cRf and BAp. BAP was not correlated with either CH or CRF in the male or female patients ( Fig. 1A-D). We found that BAP was positively correlated to CH and CRF in the female OAG patients older than 57 years (r = 0.51, P corrected = 0.003; r = 0.49, P corrected = 0.004; Fig. 2C,D), but not correlated in the female OAG patients younger than 57 years (Fig. 3C,D). There were no significant relationships in the younger or older male patients (Figs. 2A,B and 3A,B).

Multiple regression analysis.
A univariable regression analysis revealed that age, BAP, GAT, CCT and hyperlipidemia were independent contributing factors to CH in female OAG patients older than 57 years (P = 0.007, P = 0.008, P = 0.001, P < 0.001, P = 0.010; Table 2A). There was a positive correlation between GAT and CCT. The regression coefficient (β) was higher in CCT than GAT, prompting us to choose CCT for further investigation. The regression coefficient (β) of hyperlipidemia was the lowest among the variables. We selected three variables: age, BAP, and CCT. We applied a multiple regression analysis, which showed that BAP, age and CCT remained independent contributing factors to CH (P = 0.025, P = 0.048, P < 0.001; Table 2A). Univariable regression analysis revealed that BAP, GAT and CCT were independent contributing factors to CRF in female OAG patients older than 57 years (P = 0.005, P < 0.001, P < 0.001; Table 2B).
Multiple regression analysis revealed that BAP and CCT remained independent contributing factors to CRF (P = 0.015, P < 0.001; Table 2B) in the older group of female OAG patients.

Discussion
Our findings suggest that systemic antioxidative status may significantly affect the viscoelasticity of the cornea in female OAG patients older than 57 years. In a previous study, we found that sex and age interacted with systemic antioxidative status to influence the pathogenesis of glaucoma and glaucomatous damage 10 . This prompted our decision to separately evaluate male and female groups, as well as older and younger, groups. Other studies have already demonstrated that low CH is associated with glaucoma progression 3,4 . Here, we evaluated the relationship between systemic antioxidative status and CH; to the best of our knowledge, this is the first study to do so. This study had two underlying hypotheses: first, that systemic antioxidative status may contribute to glaucoma pathogenesis by affecting CH; and second, that relatively older female patients with a low antioxidant level might be more susceptible to oxidative stress, resulting in glaucoma.
Collagen, keratocytes, and proteoglycans are the major components of the corneal stroma. Collagen is secreted by keratocytes, and eventually degraded by matrix metalloproteinases (MMPs). The collagen in the www.nature.com/scientificreports www.nature.com/scientificreports/ corneal stroma is continuous with that in the sclera and lamina cribrosa. Thus, it is likely that the cornea, sclera, and lamina cribrosa share similar biomechanical characteristics, and CH may represent an indirect measure of lamina cribrosa compliance 5 .
A variety of factors may modulate the balance between corneal collagen production and degradation. Oxidative stress accelerates the apoptosis of keratocytes, while hypoxia reduces collagen expression 11 . Moreover, oxidative stress and the nitric oxide pathway reduce the activity of tissue inhibitor of metalloproteinases (TIMP)-1 and MMP-2, resulting in destruction of the extracellular matrix 12 . Collagen cross-linking induced by oxidative stress increases corneal rigidity and collagen fiber thickness 13 . In this study, we found a significant difference between the younger and older female OAG patients in dROMs. High oxidative stress results in decreased collagen, which in turn enhances the compliance of the lamina cribrosa, yielding a worse environment for the ONH and leading to RGC death.
One of the distinguishing characteristics of relatively older female patients is low estrogen levels, a hormone known to have a neuroprotective effect in glaucoma. Importantly, studies have demonstrated that estrogen in the eye has a strong effect on the growth of collagen fibers, which are an important supportive tissue in the extracellular matrix of the lamina cribrosa. It has also been reported that CCT increases and decreases during the menstrual cycle with the estrogen level 14 . Zhou et al. investigated the effects of sex hormones on collagen degradation and MMP expression by corneal fibroblasts 15 . They found that 17β-estradiol inhibited collagen degradation by the corneal fibroblasts through down-regulation of the expression and activity of MMPs. Wickham et al. revealed the presence of estrogen receptors in the cornea 16 . These findings suggest that estrogen has a protective effect against glaucoma, acting by increasing collagen content and inducing an IOP decrease. The increased collagen also enhances the biomechanical properties of the lamina cribrosa, resulting in a better environment for the ONH connective tissues. Additionally, female hormones have an antioxidative effect. We previously found that in younger male patients, there was a relationship between reduced BAP and glaucoma severity, but that this relationship was absent in younger female patients 10 . However, after menopause, female subjects with low BAP are susceptible to changes in CH. Thus, abundant evidence suggests that estrogen has an influence on collagen fiber content in the eye and thereby contributes to many disease mechanisms.
Tanito et al. found that there was no significant difference in dROMs between control and glaucoma groups 17 . In the current study, there were no control subjects, but in a previous study, we found that there were no significant differences in dROMs between controls and any of several OAG subgroups 10 . In the current study, moreover, there was a significant association between dROMs and age in the female OAG patients, but not in the male OAG patients. Thus, oxidative stress, particularly dROM levels, may have depended on age in the female OAG patients in this study. As it is still difficult to evaluate systemic imbalances between ROS and antioxidant levels, we www.nature.com/scientificreports www.nature.com/scientificreports/ consider that BAP has great potential for future use as a predictive biomarker of oxidative stress-induced glaucomatous damage, particularly under circumstances when the production of ROS is normal.
Low systemic antioxidative capacity lowers the ability to defend against oxidative stress, which leads to oxidative damage and higher disease severity in glaucoma 10 . One of the most important antioxidants is glutathione. A recent clinical study of glaucoma showed that low glutathione levels were associated with higher susceptibility to glaucoma 18 . Nitric oxide synthase is also an important enzymatic antioxidant. Ingested nitrate is turned into nitric oxide, a vasodilator that improves ocular blood flow. Kang et al. showed that a diet high in dark green leafy vegetables, which are a source of nitrate, had a significant protective effect in OAG 19 . This suggests that oxidative stress may contribute to glaucoma by reducing the level of serum anti-oxidant proteins. The main addition of the current study to previous reports is the finding that there is a close association between systemic antioxidant levels and CH only in relatively older female patients with OAG. Thus, this subset of patients might benefit from anti-oxidant therapy.
The present study has several limitations. First, we did not measure the levels of female hormones. This might have provided useful data, because we found an association between BAP and CH in the female glaucoma patients older than 57 years, and the average age of menopause in Japan is 48.3 ± 4.7 YO 20 . Thus, the patients older than 57 years were likely postmenopausal and had a low level of female hormones. Second, somewhat paradoxically, we found that CH seemed to improve with age in the female glaucoma patients older than 57 years. It is possible that other, unknown pathways might compensate for changes in CH under low levels of female hormones. Third, BAP has been found to be associated with the level of uric acid, a marker of renal function 21,22 . Tanito et al. found that BAP was a good indicator of the functional effects of OAG, and reported an association between lower BAP and higher IOP in OAG 23 . Thus, measuring serum BAP may be a useful, reliable method of determining antioxidant capacity. We are planning to determine the association between glaucoma and directly measured antioxidant levels in a future study. Forth, there was a general association between dROMs and BAP. Along with an increased dROM level, BAP tended to increase in the female OAG patients older than 57 years compared to the younger female OAG patients. There were no other significant differences between the younger and older female OAG patients. Finally, the number of OAG patients was relatively small. Future, larger-scale investigations are thus required.
It is already known that alterations in IOP do not affect every eye in the same way. For example, the EMGT and the Collaborative Normal-Tension Study reported that progression still occurred in some eyes with NTG despite medically-lowered IOP 1 . Our study contributes to previous evidence that low CH is a risk factor for glaucomatous damage even when IOP is in the normal range 24 . Furthermore, CH might be a surrogate biomarker of the biomechanical properties of tissues located in the posterior of the eye, such as the lamina cribrosa 25 . Finally, we found www.nature.com/scientificreports www.nature.com/scientificreports/ that older female subjects were more susceptible to low CH and high-IOP-induced glaucomatous damage, likely because female hormone levels and systemic antioxidant potential are low in postmenopausal women. Thus, this study supports the view that low BAP and low CH are risk factors for glaucoma.

Methods
Subjects. This retrospective study comprised a consecutive series of 103 Japanese patients who had previously been diagnosed with OAG. All patients were already undergoing treatment with antiglaucoma drugs. All subjects were examined at the ophthalmology clinic of Tohoku University Hospital during a period starting in April 2017 and ending in April 2018. Study protocols followed the Declaration of Helsinki and were approved by the Tohoku University School of Medicine Ethics Committee (study 2017-1-290). All participants provided informed consent. Data on the presence or absence of hyperlipidemia, hypertension, diabetes, and smoking habits were obtained. clinical parameters. All patients received an ophthalmic evaluation that included best-corrected visual acuity (logMAR: logarithm of the minimal angle of resolution) and axial length. Intraocular pressure (IOP) was measured using Goldmann applanation tonometry (GAT). Mean deviation (MD) was measured with the Humphrey field analyzer (HFA; Carl Zeiss Meditec, Dublin, CA; Swedish interactive threshold algorithm (SITA)-standard strategy of the 24-2 program. Anterior-segment optical coherence tomography (AS-OCT; Casia, Tomey Co., Nagoya, Japan) was used to measure central corneal thickness (CCT). The subjects also underwent cpRNFLT measurement with conventional OCT (3D-OCT 2000; Topcon Co., Tokyo, Japan). A glaucoma specialist evaluated the optic disc in each patient using a 90-diopter lens. Exclusion criteria were the presence of other eye disease, including trauma, pigment dispersion glaucoma, angle-closure glaucoma, exfoliative glaucoma, secondary glaucoma, or strong myopia (i.e., axial length above 26.5 mm). The statistical analysis used the eye with worse MD. corneal hysteresis measurement. ORA (Reichert Ophthalmic Instruments Inc, Depew, New York, USA) was used to measure CH, corneal-compensated IOP (IOPcc), corneal resistance factor (CRF), and Goldmann-correlated IOP (IOPg). Trained examiners measured each eye 3 times. Three measurements were averaged for use in the analysis. The quality of ORA measurements is determined based on a waveform score produced by the device. Measurements with a score of at least 6.5 were included. Details of these techniques have been reported previously 4,26 . Statistical Analysis. This study used Spearman's rank correlation test to determine single correlations between variables in each of the four groups (male/female, younger/older). Mann-Whitney U test or Fisher's exact test were used to compare groups. We performed simple and multiple regression analyses to investigate variables independently affecting CH or CRF. Data are the mean ± standard deviation. We defined statistical significance as P < 0.05. Bonferroni corrected P values were used, to compensate for the effect of multiple testing (P corrected ). Differences were considered significant at P corrected < 0.015 (0.05/4 groups) for Spearman's rank correlation test. JMP Pro 11 software was used for all statistical analyses (SAS Institute Japan, Inc., Tokyo, Japan).  Table 2. Factors contributing to CH and CRF in female OAG patients older than 57 years. VA = visual acuity, logMAR = logarithm of minimum angle of resolution, dROM = diacron reactive oxygen metabolites, BAP = biological antioxidant potential, U. Carr = Carrelli units, GAT = Goldmann applanation tonometry, CCT = central corneal thickness, MD = mean deviation, cpRNFLT = circumpapillary retinal nerve fiber layer thickness, β = standard partial regression coefficient.