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Intraocular Oxygen and Antioxidant Status: New Insights on the Effect of Vitrectomy and Glaucoma Pathogenesis

  • Carla J. Siegfried
    Correspondence
    Inquiries to Carla J. Siegfried, Washington University, Department of Ophthalmology and Visual Sciences, 660 South Euclid Avenue, Campus Box 8096, St. Louis, MO 63110, USA
    Affiliations
    Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri
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  • Ying-Bo Shui
    Affiliations
    Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri
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Open AccessPublished:February 14, 2019DOI:https://doi.org/10.1016/j.ajo.2019.02.008

      Purpose

      The purpose of this study was to investigate correlations of partial pressure of oxygen (pO2) in the ocular anterior segment of human eyes and aqueous humor antioxidant levels of ascorbate (AsA) and total reactive antioxidant potential (TRAP) with glaucoma and vitreous status.

      Methods

      This prospective, cross-sectional study stratified patients (n = 288 eyes) by lens and vitreous status and the presence of primary open-angle glaucoma for statistical analyses. Intraocular pO2 concentrations were measured using a fiberoptic probe in patients at the beginning of planned glaucoma and/or cataract surgery. Aqueous humor specimens were obtained for antioxidant analysis of AsA and TRAP.

      Results

      Following prior pars plana vitrectomy, pO2 levels were significantly higher than in the reference group of cataract surgery in the anterior chamber angle (16.2 ± 5.0 vs. 13.0 ± 3.9 mm Hg; P = .0171) and in the posterior chamber (7.6 ± 3.1 vs. 3.9 ± 2.7 mm Hg; P < .0001). AsA and TRAP levels were significantly lower (1.1 ± 0.4 vs. 1.4 ± 0.5 mM, respectively; 403.3 ±116.5 vs. 479.0 ± 146.7 Trolox units, respectively; P = .004 and P = .024, respectively) in patients after vitrectomy. In patients with an intact vitreous, neither pO2 nor antioxidant status correlated with lens status or glaucoma.

      Conclusions

      Increased pO2 and antioxidant depletion following vitrectomy suggests an alteration of the intraocular oxidant-antioxidant balance. Our study links physiologic factors such as increased pO2 in the anterior chamber angle and the posterior chamber to decreased antioxidant levels in aqueous humor following vitrectomy. Oxidative stress/damage to the trabecular meshwork in such post-vitrectomy cases may contribute to intraocular pressure elevation and increased risk of glaucoma. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.
      The precise pathogenesis of primary open angle glaucoma has not been fully elucidated. It likely represents a variety of different pathologies, genetic predispositions, and contributing environmental factors. Alterations in the local environment of the trabecular meshwork (TM), the main pathway for the conventional outflow of aqueous humor, may also affect its function, leading to increased intraocular pressure (IOP), an important risk factor for glaucoma. Understanding that ocular structures are “interconnected” is not a new idea. For example, feedback mechanisms of IOP regulation exist through nitric oxide synthesis in the TM,
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      found that vitreous gel, in comparison to liquefied vitreous due to myopia, aging, or surgical removal, has a higher concentration of ascorbate (AsA) and consumes pO2 at a faster rate. Previous studies indicated that antioxidant levels, specifically AsA and glutathione, are present in high concentrations in the vitreous humor.
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      Identification of increased pO2 levels within the vitreous cavity and at the posterior surface of the lens following vitrectomy led to the proposal that increased pO2 exposure leads to oxidative damage to the lens and nuclear cataract formation.
      • Holekamp N.M.
      • Shui Y.B.
      • Beebe D.C.
      Vitrectomy surgery increases oxygen exposure to the lens: a possible mechanism for nuclear cataract formation.
      Besides the lens, other ocular structures are continuously exposed to a broad spectrum of pO2 levels, ranging from hyperoxic to markedly hypoxic. Cells exposed to high pO2 levels as well as ultraviolet light (eg, corneal epithelium) contain nuclear ferritin,
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      AsA,
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      Ascorbic acid content of human corneal epithelium.
      glutathione,
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      Glutathione-related enzymes and the eye.
      superoxide dismutase, and catalase and other antioxidants.
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      Differences in activities of antioxidant superoxide dismutase, glutathione peroxidase and prooxidant xanthine oxidoreductase/xanthine oxidase in the normal corneal epithelium of various mammals.
      Ocular cells that function at low physiologic levels of pO2 are unlikely to adapt to altered (ie, higher) levels of pO2 exposure. Oxygen either is consumed by functioning cells and/or antioxidants, remains in its molecular form, or is transformed into other potentially unstable reactive oxygen species (ROS), capable of causing damage to RNA, DNA, and proteins. “Oxidative stress” is defined as an increase over physiologic values in the intracellular concentrations of ROS, which include superoxide anions, hydrogen peroxide (H2O2), hydroxyl radicals, peroxyl radicals, and singlet oxygen. Such ROS may be detrimental to cellular structures, destroying membrane lipids as well as structural and enzymatic proteins and DNA, contributing to cell senescence and potentially genetically programmed cell death or apoptosis. Cellular dysfunction results from decreased mitochondrial respiratory function and protein degradation.
      • Lee H.C.
      • Wei Y.H.
      Mitochondrial alterations, cellular response to oxidative stress and defective degradation of proteins in aging.
      Increases in intracellular ROS may be the result of increased endogenous production by mitochondrial respiration or decreased antioxidant capacity. Increased oxidative stress has been identified as a contributing factor to the pathogenesis of several age-related ocular diseases including glaucoma.
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      • Izzotti A.
      Oxidative stress and glaucoma: injury in the anterior segment of the eye.
      • Izzotti A.
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      The role of oxidative stress in glaucoma.
      Evidence supporting such oxidative damage to the TM was initially reported by Alvarado and colleagues
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      • Juster R.
      Age-related changes in trabecular meshwork cellularity.
      in 1981, as they first suggested that aging and oxidative stress underlay the degeneration of TM cells in patients with glaucoma. Cell senescence has been shown to increase generation of ROS, leading to reduced numbers and function of mitochondria.
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      As a result of this exposure to oxidative stress, changes occur in TM protein expression that affect extracellular matrix turnover. For example, in vivo perfusion of calf anterior segments with H2O2 following depletion of glutathione in the TM increases outflow resistance.
      • Kahn M.G.
      • Giblin F.J.
      • Epstein D.L.
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      TM tissue, compared to corneal and iris tissues, was found to be most sensitive to oxidative damage induced by exposure to H2O2.
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      Sensitivity of ocular anterior chamber tissues to oxidative damage and its relevance to the pathogenesis of glaucoma.
      Subsequent studies provide evidence of oxidative damage and reduced resistance to oxidative stress in the outflow pathway.
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      Oxidative damage to DNA is greater in TM cells in glaucoma patients than in controls
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      and correlates with IOP level and visual field loss.
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      • Izzotti A.
      Oxidative DNA damage in the human trabecular meshwork: clinical correlation in patients with primary open–angle glaucoma.
      Levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), an established biomarker of oxidative DNA damage, are significantly higher in both aqueous humor and sera in patients with glaucoma than in controls.
      • Sorkhabi R.
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      • Rashtchizadeh N.
      • Moharrery M.
      Oxidative DNA damage and total antioxidant status in glaucoma patients.
      Increased levels of 8-OHdG have also been found in TM specimens from primary open-angle glaucoma (POAG) patients.
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      • Capris P.
      • Izzotti A.
      Oxidative DNA damage in the human trabecular meshwork: clinical correlation in patients with primary open–angle glaucoma.
      Importantly, PPV has also been associated with increased pO2 exposure to the microenvironment of the TM and outflow pathways
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      • Tian B.
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      • Heatley G.A.
      • Kaufman P.L.
      Effects of vitrectomy and lensectomy on older rhesus macaques: oxygen distribution, antioxidant status, and aqueous humor dynamics.
      and increased risk of developing open-angle glaucoma in several retrospective clinical studies
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      LXII Edward Jackson lecture: open angle glaucoma after vitrectomy.
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      • Yoshida N.
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      • et al.
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      • Lam D.S.C.
      Presence of crystalline lens as a protective factor for the late development of open angle glaucoma after vitrectomy.
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      • Kimura H.
      • Matsumura M.
      • Kuroda S.
      Incidence of late-onset ocular hypertension following uncomplicated pars plana vitrectomy in pseudophakic eyes.
      • Wu L.
      • Berrocal M.H.
      • Rodriguez F.J.
      • et al.
      Intraocular pressure elevation after uncomplicated pars plana vitrectomy results of the Pan American Collaborative Retina Study Group.
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      and a recent population-based study.
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      • et al.
      The risk of primary open-angle glaucoma following vitreoretinal surgery-a population-based study.
      The present study was undertaken to provide further understanding of the impact of exposure to increased pO2 and/or its metabolites in the local environment of the aqueous outflow pathways following vitrectomy. We hypothesized that increased pO2 in these cases may contribute to alterations of oxidant-antioxidant balance leading to increased oxidative stress and damage of the TM. To assess these conditions in human subjects, we measured in vivo levels of pO2, total reactive antioxidant potential (TRAP) activity, and AsA levels in aqueous humor in eyes of patients undergoing glaucoma and/or cataract surgery to determine associations with glaucoma and vitreous status.

      Methods

       Study Design

      This prospective, cross-sectional study was approved by the Institutional Review Board of the Washington University School of Medicine, in compliance with the tenets of the Declaration of Helsinki and Health Insurance Portability and Accountability Act guidelines. Informed consent was obtained from subjects after the nature and possible consequences of the study were explained to them. This study was designed to measure pO2 distribution within the anterior segment of the eye and to collect aqueous humor for measurement of antioxidants in patients undergoing cataract and/or glaucoma surgery in an academic clinical practice. Patients were excluded from the study if there was evidence of corneal endothelial dysfunction; ischemic ocular disease, including diabetic retinopathy, anterior chamber angle closure, inflammatory or traumatic ocular disease, ocular neoplasia, requirement for general anesthesia; or monocular status.

       Patients and pO2 Measurements

      A complete general medical and ophthalmic history and comprehensive ophthalmic examinations, including Lens Opacities Classification System III (LOCS III) analysis for quantitative and qualitative assessment of lens opacities, were performed prior to surgical intervention. The use of topical glaucoma medications within 1 month of the surgical procedure was verified preoperatively. Patients with a diagnosis of POAG (based on optic nerve and visual field criteria) were classified by glaucoma severity as mild, moderate, or severe (using Hodapp Parrish Anderson criteria).
      • Hodapp E.P.R.I.
      • Anderson D.R.
      Clinical Decisions In Glaucoma.
      Central corneal thickness was measured by ultrasonography (DGH 55 Pachmate, DGH Technology Inc., Exton, Pennsylvania), and axial length measurements were recorded (IOLMaster 500, Carl Zeiss Meditec, Inc., Jena, Germany) for patients undergoing cataract extraction. Racial background was based on self-reports, as indicated on a standardized registration questionnaire.
      According to our routine surgical protocol, the patient was placed in a supine position, intravenous sedation was administered, the eye was prepared and draped, and a lid speculum was placed. Supplemental pO2 (21%–30%) was provided through nasal cannulation and separated from the ocular region by adhesive sterile drape to avoid any additional pO2 exposure. This technique did not impact intraocular pO2 measurements as previously reported.
      • Holekamp N.M.
      • Shui Y.B.
      • Beebe D.C.
      Vitrectomy surgery increases oxygen exposure to the lens: a possible mechanism for nuclear cataract formation.
      Blood oxygen saturation was monitored by continuous pulse oximetry and maintained between 95% and 100%. Topical lidocaine hydrochloride jelly, 2%, was placed on the ocular surface in the preoperative area. A sub-Tenons injection of 1 to 3 ml of 2% lidocaine and 0.375% bupivacaine mixture (50/50) was performed to provide additional local anesthesia as indicated. At the beginning of the planned surgical procedure, a 30-gauge needle was used for entry through the periphery of a clear cornea into the anterior chamber (AC) and an Oxylab pO2 optical oxygen sensor probe (Optode; Oxford Optronix, Oxford, United Kingdom) was then carefully introduced into the AC without aqueous humor leakage. The instrumentation was calibrated prior to each set of measurements. Under direct visualization with an operating microscope, the tip of the flexible fiberoptic probe was positioned for 3 measurements in all patients as described in our previous studies: 1) underneath the central corneal endothelium, 2) in the mid-AC, and 3) in the AC angle.
      • Siegfried C.J.
      • Shui Y.B.
      • Holekamp N.M.
      • Bai F.
      • Beebe D.C.
      Oxygen distribution in the human eye: relevance to the etiology of open-angle glaucoma after vitrectomy.
      • Siegfried C.J.
      • Shui Y.B.
      • Holekamp N.M.
      • Bai F.
      • Beebe D.C.
      Racial differences in ocular oxidative metabolism: implications for ocular disease.
      In pseudophakic patients or those scheduled to undergo cataract extraction, 2 additional measurements were obtained, 4) at the central anterior lens surface and 5) in the posterior chamber just behind the iris. These latter 2 measurements were not made in patients remaining phakic in order to avoid risk of lens damage. Approximately 46 seconds (total: <5 minutes) was required for each set of measurements. In order to confirm precise and consistent probe positioning and stabilization of the pO2 level, duplicate testing in the same locations were performed for verification.

       Aqueous Humor Specimen Collection

      After pO2 levels were measured, the needle entry site in the cornea was slightly enlarged with a 15-degree blade or side port instrument, and a sample of aqueous humor (50–100 μL) was drawn into a 1-mL tuberculin syringe through a 30-gauge blunt cannula, followed by re-inflation of the AC volume with balanced salt solution. Care was taken to avoid contamination of the specimen with blood. The aqueous humor specimen was immediately transferred to a sealed tube, placed on dry ice, and transported to storage in the gas phase of a liquid nitrogen tank until analysis. The scheduled surgical procedure was subsequently performed with standard postoperative management, and the patients were monitored for any complications.

       Aqueous Humor Analysis of Ascorbate

      AsA concentration was quantified in triplicate, based on its ability to reduce Fe3+ to Fe2+ and the resulting change in the A525 of complexes of Fe2+ with 2,2″-dipyridyl.
      • Kampfenkel K.
      • Van Montagu M.
      • Inze D.
      Extraction and determination of ascorbate and dehydroascorbate from plant tissue.
      Assay modifications enabled the analysis of 10-μL samples, and a standard curve was used for all measurements, as in our previous report.
      • Shui Y.B.
      • Holekamp N.M.
      • Kramer B.C.
      • et al.
      The gel state of the vitreous and ascorbate-dependent oxygen consumption: relationship to the etiology of nuclear cataracts.
      Gas chromatography-mass spectrometry studies confirmed the specificity of this colorimetric assay. Samples of aqueous humor were mixed with a known amount of carbon 13-labeled ascorbic acid (Omicron Biochemicals, South Bend, Indiana), dried, and then reacted with N,O-bis(trimethylsilyl)trifluoroacetamide. The sample was separated on a gas chromatograph (Varian Inc., Palo Alto, California) using a 30-m, 0.25-mm-internal gas chromatography column with a 0.25-μm film (DB-5 ms column; PJ Cobert Associates Inc., St. Louis, Missouri), maintained at 80°C for 1 minute and then eluted with a temperature gradient of 80°C to 300°C at 15°C/minute. The injection port and transfer line were at 250°C and the source temperature at 200°C of a mass spectrometer (Finnigan MS SSQ7000; Thermo Electron Corp, Waltham, Massachusetts) operated in the electron ionization mode at 70 eV. AsA concentration was calculated from the [13C]AsA-to-[12C]AsA ratio. In order to confirm the specificity of the AsA assay, 2 units of ascorbate oxidase (AO; A 0157, Sigma Chemical, St. Louis, Missouri) were added to each of the 10-μL samples and mixed well at room temperature, and AsA measurements were repeated.

       Total Reactive Antioxidant Potential

      The TRAP assay is a means to determine the ability of a sample to destroy chemically generated free radicals. A sample is added to a solution containing 2,2′-azobis(2-amidinopropane) (ABAP) (Sigma Aldrich, St. Louis, Missouri) and 40 μM luminol (3-aminophthalhydrazide; Sigma Aldrich). ABAP combines with pO2 to produce alkyl peroxyl radicals at a constant rate. In the absence of antioxidant, these radicals react with luminol to produce light, which is measured in a scintillation counter. Antioxidants quench luminescence by reacting with the peroxyl radicals. Because ABAP produces radicals at a constant rate, antioxidant activity is measured by the length of time required to quench luminescence. The assay is standardized using Trolox, a water-soluble vitamin E analog, and reported as “Trolox units,” with 1 unit equal to the amount of time required to quench luminescence by a sample containing 1 μM Trolox. TRAP was measured in the aqueous humor samples, and samples were treated with ascorbate oxidase to remove AsA as described above. Measurements were then repeated in order to differentiate AsA- from non-AsA-dependent effects on the composite TRAP value.

       Statistical Analysis

      Multivariate regression analyses were performed with adjustment for all potential confounding variables (P < .1) including age, sex, race, medications, and lens status by using SPSS version 24.0 software (Chicago, Illinois). The t-test, one-way ANOVA with multiple comparison analysis (Bonferroni correction), and Spearman correlation analyses were performed using Prism version 8.0 software (GraphPad, La Jolla, California). Results are expressed as means ± SD. P values less than .05 were defined as statistically significant.

      Results

       Patient Recruitment and Group Analysis

      A total of 288 eyes of 288 patients participated in the study between July 2007 and August 2015. Our initial cohort (July 2007-July 2010) of 112 eyes of 112 patients were included from a previously published study evaluating intraocular pO2 measurements.
      • Siegfried C.J.
      • Shui Y.B.
      • Holekamp N.M.
      • Bai F.
      • Beebe D.C.
      Oxygen distribution in the human eye: relevance to the etiology of open-angle glaucoma after vitrectomy.
      We extended that work to study a total 288 eyes for pO2, AsA, and TRAP measurements after exclusion of 24 eyes due to inadequate specimen collection. Patients with secondary open-angle glaucoma (ie, pseudoexfoliative or pigmentary) and low-tension glaucoma were excluded from this study. Patient characteristics (Table 1) indicate a greater number of females and caucasian patients in the study. The cataract group (CAT) had no history of ocular surgery, glaucoma, or exposure to ocular glaucoma medications. That group served as the reference or control group for select statistical analyses. Consistent with our previously published data,
      • Siegfried C.J.
      • Shui Y.B.
      • Holekamp N.M.
      • Bai F.
      • Beebe D.C.
      Oxygen distribution in the human eye: relevance to the etiology of open-angle glaucoma after vitrectomy.
      patients with a diagnosis of POAG (GL) were subdivided into patients undergoing glaucoma surgery or combined cataract and glaucoma surgery (GL/CAT) and pseudophakic patients undergoing glaucoma surgery (GL/IOL). Patients with a history of vitrectomy who had previously undergone PPV for vitreoretinal conditions including rhegmatogenous retinal detachment, epiretinal membrane, and macular hole but excluding those with proliferative retinopathy made up the VIT group. All these patients were either pseudophakic or were scheduled to undergo cataract extraction or glaucoma surgery. Patients in the GL/CAT and GL/IOL groups were older than those in the VIT group (P = .0005 and P = .0001, respectively). The GL/IOL patients were also older than the CAT reference group patients (P = .005). Subgroup analyses were performed to identify correlations with race, age, sex, lens, vitreous status, and ocular medications with pO2 levels and antioxidant status. We randomly selected 1 eye for the final data analysis in patients who had measurements and specimens taken from both eyes.
      Table 1Patient Demographic Information and Group Descriptions
      GroupsGroup DescriptionEyes (n)Age (y)F/MType of SurgeryRace
      CATGLCombinedAACCOther
      CATNo prior history of eye surgery or POAG (reference group)7268.0 ± 11.448/24720026442
      GL/CATPOAG undergoing glaucoma surgery or combined cataract/glaucoma surgery14370.4 ± 10.880/63353078391022
      GL/IOLPseudophakic POAG patients undergoing glaucoma surgery3973.8 ± 9.032/7039010290
      VITPatients who had undergone previous pars plana vitrectomy3463.1 ± 13.518/16241006271
      Total288178/1101317978812025
      AA = African American; CAT = cataract; CC = caucasian; F = female; GL = glaucoma; IOL = intraocular lens; M = male; POAG = primary open-angle glaucoma; VIT = prior vitrectomy.

       Intraocular pO2 Measurements

      Oxygen measurements at five intraocular locations were analyzed by multiple comparison analysis with a Bonferroni correction (Figure 1). Intraocular pO2 measurements were significantly higher after vitrectomy (VIT) than those in the reference (CAT) group in the AC angle (16.2 ± 5.0 mm Hg vs. 13.0 ± 3.9 mm Hg, respectively; P = .0171) and posterior chamber (7.6 ± 3.1 mm Hg vs. 3.9 ± 2.7 mm Hg, respectively; P < .0001). In the GL/IOL (pseudophakic) group, there were significantly higher levels of pO2 at the anterior lens surface than in the reference group (8.0 ± 4.1 mm Hg vs. 2.5 ± 2.4 mm Hg, respectively; P < .0001), in the mid-AC (11.1 ± 3.8 mm Hg vs. 8.4 ± 3.9 mm Hg, respectively; P = .007), and in the posterior chamber (5.7 ± 3.4 mm Hg vs. 3.9 ± 2.7 mm Hg, respectively; P = .0384).
      Figure thumbnail gr1
      Figure 1Intraocular pO2 measurements (mm Hg) were made at the indicated locations (green dots). (A) CAT groups were used as references for comparison with the other groups. Red numbers indicate P values < .05. (B) GL/CAT indicates diagnosis with cataract (C) GL/IOL shows glaucoma diagnosis with history of prior cataract surgery. (D) History of prior pars plana vitrectomy. (E) Comparison among pO2 levels at intraocular locations. Multiple comparisons were made using ANOVA and Bonferroni correction; P values < .05); error bar indicates mean mm Hg ± SD. ANOVA = analysis of variance; CAT = cataract; GL = glaucoma; IOL = intraocular lens; VIT = vitrectomy.

       Ascorbate Measurements

      AsA levels were significantly lower in the VIT group (1.1 ± 0.4 mM; P = .004) than in the CAT reference group (1.4 ± 0.5 mM). We further confirmed the fact that AsA is correlated with prior vitrectomy in a multivariate regression model (beta = −.198; P = .004). AsA levels were increased in phakic patients with POAG diagnosis (GL/CAT: 1.8 ± 0.7 mM; P = .002) as shown in Figures 2A and 2D. Multivariate regression analyses did not identify any correlations between pO2 and AsA following adjustment for race, age, sex, lens status, and presence of glaucoma.
      Figure thumbnail gr2
      Figure 2Comparison between aqueous humor antioxidant levels and reference groups (CAT black bars). (A) AsA; (B) TRAP; (C) Non-AsA TRAP; and (D) Mean value ± SD. P values were calculated from unpaired t-tests. Bars are standard deviations. AsA = ascorbate; CAT = cataract; GL = glaucoma; IOL = intraocular lens; REF = reference; TRAP = total reactive antioxidant potential; VIT = vitrectomy.

       TRAP and Non-AsA-Dependent TRAP

      TRAP and its component AsA are highly correlated in all human aqueous humor specimens, confirmed by the marked reduction of TRAP values in specimens treated with ascorbate oxidase. We designated the calculated remainder TRAP value non-AsA-dependent TRAP (non-AsA TRAP) in our subsequent analyses. As shown in Figures 2B, 2C and 2D, there were significantly lower levels of TRAP after vitrectomy (VIT: 403.3 ± 116.5 Trolox units; P = .024) than in the reference CAT group (479.0 ± 146.7 Trolox units). Multivariate regression analysis confirmed the correlation between TRAP and post-vitrectomy status (beta = −.186; P = .007). TRAP is significantly directly correlated with age (rs = .175; P = .01) as indicated in Figure 3. The non-AsA TRAP component percentage was significantly greater in the VIT group (149.9 ± 51.9 Trolox units [41.4%]) than in the CAT group (114.5 ± 61.9 Trolox units [24.3%]; P = .014). Multivariate regression also showed correlations between non-AsA TRAP and vitreous status (beta = .135; P = .05). There were no differences between the CAT group and the TRAP activity in both the GL/CAT and the GL/IOL groups. Figure 4 illustrates the comparative contributions of the components of TRAP in each group. AsA contributed 76% of TRAP in the CAT group whereas AsA in the VIT group contributed only 58%. Multivariate regression analyses did not indicate any correlations between pO2 and TRAP in the anterior segment following adjustment for race, age, sex, lens status, and medication use.
      Figure thumbnail gr3
      Figure 3Scatterplot diagrams show relationships between (A) AsA and TRAP; (B) TRAP and age; (C) non-AsA TRAP and age; and (D) AsA and age in all cases demonstrating the best linear fit to the data. Analysis used Spearman's rank-order correlations and confidence intervals. AsA = ascorbate; TRAP = total reactive antioxidant potential.
      Figure thumbnail gr4
      Figure 4Comparison between AsA and non-AsA components of TRAP. TRAP was designated as 100%, and the AsA component (light gray) and the non-AsA-dependent component (dark gray) are shown as percentages of TRAP values in each patient group. AsA = ascorbate; CAT = cataract; GL = glaucoma; IOL = intraocular lens; REF = reference; TRAP = total reactive antioxidant potential; VIT = vitrectomy.

       Topical Glaucoma Medications

      Medications were classified as beta-blockers (timolol, betaxolol), carbonic anhydrase inhibitors (dorzolamide, brinzolamide), alpha-2 agonist agents (brimonidine), or prostaglandin analogs (bimatoprost, latanoprost, travoprost). Fixed combination agents (brimonidine tartrate/timolol maleate ophthalmic solution [Combigan], dorzolamide hydrochloride-timolol maleate ophthalmic solution [Cosopt], and brinzolamide/brimonidine tartrate ophthalmic suspension [Simbrinza]) were categorized by their individual medication components. Because most patients were taking a combination of medications, each of the agents was analyzed individually. There was a significant correlation (beta 0.274; P = .004) between the use of topical carbonic anhydrase inhibitors (CAIs) and levels of AsA in the aqueous humor of all glaucoma patients (GL/CAT, GL/IOL). Notably, 69 of 146 patients (47.2%) in this group were taking topical CAIs as a component of their medical regimen. Four of 35 patients (11.4%) in the VIT group were taking CAI agents. After adjustments were made for race, age, sex, and lens status, no other medication class was correlated with AsA or TRAP levels. Multivariate regression analysis correcting for this variable resulted in demonstration of this drug's significant impact on AsA levels (Table 2).
      Table 2Results of Multivariate Regression Analyses Evaluating Effect of Topical Glaucoma Medications on Ascorbate (Asa) in Aqueous Humor
      Dependent VariableIndependent VariableβP value
      AsAAlpha0.009.927
      Beta0.111.306
      CAI0.274.004
      PG0.041.696
      Alpha = alpha-2 agonists; AsA = ascorbate; Beta = beta-blockers; CAI = carbonic anhydrase inhibitors; PG = prostaglandin analog.

      Discussion

       Oxygen Measurements and Homeostasis

      This prospective, cross-sectional study represents the largest reported cohort of patients undergoing cataract and/or glaucoma surgery in which assessments of both intraocular pO2 levels and aqueous humor antioxidant status were obtained. Precise in vivo measurements of pO2 made by our colleagues in rabbit and in human vitreous led to these studies of the anterior segment of the human eye, revealing consistent pO2 gradients.
      • Holekamp N.M.
      • Shui Y.B.
      • Beebe D.C.
      Vitrectomy surgery increases oxygen exposure to the lens: a possible mechanism for nuclear cataract formation.
      • Holekamp N.M.
      • Shui Y.B.
      • Beebe D.
      Lower intraocular oxygen tension in diabetic patients: possible contribution to decreased incidence of nuclear sclerotic cataract.
      • Shui Y.B.
      • Fu J.J.
      • Garcia C.
      • et al.
      Oxygen distribution in the rabbit eye and oxygen consumption by the lens.
      Our studies of how pO2 homeostasis is altered by surgical intervention, aging, and disease may reveal important insights of physiology and pathology. Because the ocular anterior segment represents an environment “protected” from direct blood flow, it provides an ideal site in which to study homeostatic mechanisms of oxygen metabolism in addition to oxidant-antioxidant balance. Additionally, by excluding patients with ischemic retinal disease and the use of general anesthesia, we separated effects of decreased retinal blood flow and hyperoxic conditions on intraocular pO2 levels, respectively.
      Increased pO2 in the AC angle of post-vitrectomy patients (VIT) compared to reference CAT patients may provide an important source of pro-oxidants leading to increased oxidative stress in the TM. Elevated pO2 levels in the TM region and in the posterior chamber may increase ROS in the aqueous outflow pathway by diffusion from the ciliary body stroma into the aqueous humor at the root of the iris. This movement is consistent with the mechanism of this pathway facilitating movement of plasma proteins through the TM, as described by Freddo and colleagues,
      • Freddo T.F.
      • Bartels S.P.
      • Barsotti M.F.
      • Kamm R.D.
      The source of proteins in the aqueous humor of the normal rabbit.
      and our previously published hypothesis regarding the correlation of pO2 levels in the AC and posterior chamber.
      • Siegfried C.J.
      • Shui Y.B.
      • Holekamp N.M.
      • Bai F.
      • Beebe D.C.
      Oxygen distribution in the human eye: relevance to the etiology of open-angle glaucoma after vitrectomy.
      Other body tissues exposed to excess levels of molecular pO2 have been shown to accumulate ROS. For example, pulmonary epithelial cells are adapted to much higher pO2 levels than other cells in the body (21% O2 or 160 mm Hg), but during prolonged exposure to levels as high at 40% O2 or greater, increased intracellular ROS leads to pulmonary pO2 toxicity.
      • Fridovich I.
      Oxygen toxicity: a radical explanation.
      • Jackson R.M.
      Pulmonary oxygen toxicity.
      Physiologic conditions for TM cells are relatively hypoxic as we discovered in the rabbit, monkey, and human.
      • Siegfried C.J.
      • Shui Y.B.
      • Tian B.
      • Nork T.M.
      • Heatley G.A.
      • Kaufman P.L.
      Effects of vitrectomy and lensectomy on older rhesus macaques: oxygen distribution, antioxidant status, and aqueous humor dynamics.
      • Siegfried C.J.
      • Shui Y.B.
      • Holekamp N.M.
      • Bai F.
      • Beebe D.C.
      Oxygen distribution in the human eye: relevance to the etiology of open-angle glaucoma after vitrectomy.
      • Shui Y.B.
      • Fu J.J.
      • Garcia C.
      • et al.
      Oxygen distribution in the rabbit eye and oxygen consumption by the lens.
      Exposure of these specialized cells to elevated pO2 may be “toxic,” leading to decreased TM cellularity, altered extracellular matrix formation, and ultimately decreased outflow facility and increased IOP. If the protective mechanisms of the aqueous humor are overwhelmed, then oxidative stress and damage may result. Notably, however, in POAG patients with an intact vitreous, we did not find increased intraocular pO2 in the TM region or posterior chamber, suggesting this may not be an important factor in all glaucoma subtypes.
      Adaptation of ocular structures to specific levels of pO2 is revealed in studies of oxidative damage and defense. For example, the basal layer of corneal epithelium is accustomed to high levels of pO2, essentially equivalent to air with pO2 of 160 mm Hg (21% pO2). In contrast, pO2 in inner retinal tissue and the vitreous adjacent to retinal blood vessels is approximately 20 mm Hg, consistent with other body tissues.
      • Holekamp N.M.
      • Shui Y.B.
      • Beebe D.C.
      Vitrectomy surgery increases oxygen exposure to the lens: a possible mechanism for nuclear cataract formation.
      • Alder V.A.
      • Cringle S.J.
      Vitreal and retinal oxygenation.
      • Sakaue H.
      • Tsukahara Y.
      • Negi A.
      • Ogino N.
      • Honda Y.
      Measurement of vitreous oxygen tension in human eyes.
      The environment surrounding the lens is notably hypoxic under normal conditions, measuring approximately 7 mm Hg at the posterior surface
      • Holekamp N.M.
      • Shui Y.B.
      • Beebe D.C.
      Vitrectomy surgery increases oxygen exposure to the lens: a possible mechanism for nuclear cataract formation.
      and 3 mm Hg at the anterior and lateral surfaces of the lens,
      • Siegfried C.J.
      • Shui Y.B.
      • Holekamp N.M.
      • Bai F.
      • Beebe D.C.
      Oxygen distribution in the human eye: relevance to the etiology of open-angle glaucoma after vitrectomy.
      with oxygen consumption by lens cells further decreasing pO2 within the lens nucleus.
      • Giblin F.J.
      • Quiram P.A.
      • Leverenz V.R.
      • Baker R.M.
      • Dang L.
      • Trese M.T.
      Enzyme-induced posterior vitreous detachment in the rat produces increased lens nuclear pO levels.
      • McNulty R.
      • Wang H.
      • Mathias R.T.
      • Ortwerth B.J.
      • Truscott R.J.W.
      • Bassnett S.
      Regulation of tissue oxygen levels in the mammalian lens.
      Extraction of the natural lens and replacement with an IOL removes the contributing factor of oxygen consumption by the lens epithelium, thereby increasing pO2 around the lens, including the posterior chamber. This pO2 elevation does not reach the pO2 levels following vitrectomy.

       Ocular Antioxidant Status: Ascorbate and TRAP

      We identified significantly decreased levels of both AsA and TRAP in aqueous humor of patients who had undergone vitrectomy (VIT) compared to those in the reference group (Figure 2). Vitrectomy, independent of lens status, results in decreased TRAP in comparison to all other groups analyzed. Vitrectomized eyes also displayed an increase in non-AsA TRAP compared to that in the reference group (Figure 4). Interestingly, we found that POAG patients (GL/CAT, GL/IOL) had higher levels of AsA and no differences in TRAP than in the reference group (Figure 2). Lee and colleagues
      • Lee P.
      • Lam K.W.
      • Lai M.
      Aqueous humor ascorbate concentration and open-angle glaucoma.
      also noted increased aqueous humor AsA in glaucoma patients compared to cataract surgery controls, whereas Ferreira and colleagues
      • Ferreira S.M.
      • Lerner S.F.
      • Brunzini R.
      • Evelson P.A.
      • Llesuy S.F.
      Antioxidant status in the aqueous humour of patients with glaucoma associated with exfoliation syndrome.
      found that AsA levels were decreased in patients with both POAG and exfoliation syndrome and that TRAP levels from glaucoma patients were significantly lower than those in the cataract group.
      • Ferreira S.M.
      • Lerner S.F.
      • Brunzini R.
      • Evelson P.A.
      • Llesuy S.F.
      Oxidative stress markers in aqueous humor of glaucoma patients.
      Our present study did not support these findings of decreased AsA in patients with glaucoma compared to those in cataract controls. Leite and coworkers
      • Leite M.T.
      • Prata T.S.
      • Kera C.Z.
      • Miranda D.V.
      • de Moraes Barros S.B.
      • Melo Jr., L.A.
      Ascorbic acid concentration is reduced in the secondary aqueous humour of glaucomatous patients.
      found that AsA levels were significantly lower in secondary aqueous obtained from patients with a history of previous intraocular surgery than in primary aqueous in patients with glaucoma and cataract. Our separate analysis of phakic glaucoma patients (GL/CAT) with a history of intraocular surgery confirmed this finding of decreased AsA compared to patients without history of surgery (P = .04; data not shown). Confounding variables such as frequent use of CAIs in the glaucoma subgroups significantly correlated with increased AsA and contributed to these contradictory findings. In addition, systemic ascorbate supplementation was not specifically documented in our medication review and might have also affected our results, especially in patients taking high doses of vitamin C (2 g/day).
      • Hah Y.S.
      • Chung H.J.
      • Sontakke S.B.
      • et al.
      Ascorbic acid concentrations in aqueous humor after systemic vitamin C supplementation in patients with cataract: pilot study.
      A recently published systematic review and meta-analysis of oxidant-antioxidant stress markers in glaucoma demonstrated decreased total antioxidant status in sera and aqueous humor in glaucoma patients, with the exception of 2 enzymatic antioxidants, superoxide dismutase and glutathione peroxidase.
      • Benoist d'Azy C.
      • Pereira B.
      • Chiambaretta F.
      • Dutheil F.
      Oxidative and anti-oxidative stress markers in chronic glaucoma: a systematic review and meta-analysis.
      These entities may represent a compensatory protective response to oxidative stress reflected in this study as non-AsA TRAP. A study of age-related changes in TRAP plasma levels showed that TRAP increased with age in both females and males.
      • Aejmelaeus R.T.
      • Holm P.
      • Kaukinen U.
      • et al.
      Age-related changes in the peroxyl radical scavenging capacity of human plasma.
      However, in males, levels increased only from 51 to 74 years of age, at which point they were noted to decline. Increases of antioxidant potential, especially in response to oxidative stress, were due to unidentified antioxidants which made up 35% of TRAP in both sexes.
      Huang and colleagues
      • Huang A.J.
      • Shui Y.B.
      • Han Y.P.
      • Bai F.
      • Siegfried C.J.
      • Beebe D.C.
      Impact of corneal endothelial dysfunctions on intraocular oxygen levels in human eyes.
      described “extreme” exposures to increased pO2 (42.7 ± 12.4 mm Hg at the corneal surface) in patients with Fuchs' dystrophy,
      • Huang A.J.
      • Shui Y.B.
      • Han Y.P.
      • Bai F.
      • Siegfried C.J.
      • Beebe D.C.
      Impact of corneal endothelial dysfunctions on intraocular oxygen levels in human eyes.
      another ocular condition associated with oxidative stress.
      • Buddi R.
      • Lin B.
      • Atilano S.R.
      • Zorapapel N.C.
      • Kenney M.C.
      • Brown D.J.
      Evidence of oxidative stress in human corneal diseases.
      Aqueous humor levels of AsA and TRAP were significantly lower than in the cataract reference group (P = .012 and P = .032, respectively [unpublished data]). In addition, we previously reported
      • Siegfried C.J.
      • Shui Y.B.
      • Holekamp N.M.
      • Bai F.
      • Beebe D.C.
      Racial differences in ocular oxidative metabolism: implications for ocular disease.
      increased pO2 in the anterior segment of patients with African American background compared to that in caucasians and confirmed in this expanded study cohort (data not shown). These increased levels of pO2 did not correlate with differences in antioxidant status, potentially suggesting alternative mechanisms for this racial group's increased risk and severity of POAG.
      Finally, AsA levels in aqueous humor of patients with cataract have been shown to decrease with age,
      • Canadananovic V.
      • Latinovic S.
      • Barisic S.
      • Babic N.
      • Jovanovic S.
      Age-related changes of vitamin C levels in aqueous humour.
      supporting the role of oxidative damage and accumulation of free radicals in cataract development. We did not identify correlations between AsA and age in this study (Figure 3). We acknowledge that the oxidant-antioxidant balance in the eye as reflected in the aqueous humor is undoubtedly highly complex and requires further study.

       Ascorbate Metabolism

      Ocular exposure to ultraviolet and visible light irradiation is greater than any other organ except skin. Consequently, this organ requires protective mechanisms against ROS generation. Because the TM represents the target tissue of glaucoma in the anterior segment, understanding the role of antioxidants in the trabecular tissue,
      • Freedman S.F.
      • Anderson P.J.
      • Epstein D.L.
      Superoxide dismutase and catalase of calf trabecular meshwork.
      • Ammar D.A.
      • Hamweyah K.M.
      • Kahook M.Y.
      Antioxidants protect trabecular meshwork cells from hydrogen peroxide-induced cell death.
      as well as in the aqueous humor in which it bathes, is critical to our comprehension of oxidant-antioxidant balance and its role in glaucoma development. The “pecking order” of aqueous humor antioxidants is affected by both the concentration and electrochemical activity of several low molecular weight water soluble species,
      • Buettner G.R.
      The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate.
      • Richer S.P.
      • Rose R.C.
      Water soluble antioxidants in mammalian aqueous humor: interaction with UV B and hydrogen peroxide.
      including AsA, l-tyrosine, l-cysteine, uric acid, and glutathione. Antioxidant enzymes such as superoxide dismutase, glutathione peroxidase,
      • Huang W.
      • Akesson B.
      Radioimmunoassay of glutathione peroxidase in human serum.
      and catalase have also been identified in aqueous humor. In contrast to nocturnal animals, the corneal epithelium,
      • Ringvold A.
      In vitro evidence for UV-protection of the eye by the corneal epithelium mediated by the cytoplasmic protein, RNA, and ascorbate.
      • Ringvold A.
      • Anderssen E.
      • Kjonniksen I.
      Distribution of ascorbate in the anterior bovine eye.
      lens,
      • Reddy V.N.
      • Giblin F.J.
      • Lin L.R.
      • Chakrapani B.
      The effect of aqueous humor ascorbate on ultraviolet-B-induced DNA damage in lens epithelium.
      vitreous,
      • Takano S.
      • Ishiwata S.
      • Nakazawa M.
      • Mizugaki M.
      • Tamai M.
      Determination of ascorbic acid in human vitreous humor by high-performance liquid chromatography with UV detection.
      and aqueous humor
      • De Berardinis E.
      • Tieri O.
      • Polzella A.
      • Iuglio N.
      The chemical composition of the human aqueous humour in normal and pathological conditions.
      • Kodama T.
      • Kabasawa I.
      • Tamura O.
      • Reddy V.N.
      Dynamics of ascorbate in the aqueous humor and tissues surrounding ocular chambers.
      of diurnal animals contain extremely high levels of AsA compared to that in plasma.
      • Duarte T.L.
      • Lunec J.
      Review: When is an antioxidant not an antioxidant? A review of novel actions and reactions of vitamin C.
      Shui and colleagues
      • Shui Y.B.
      • Holekamp N.M.
      • Kramer B.C.
      • et al.
      The gel state of the vitreous and ascorbate-dependent oxygen consumption: relationship to the etiology of nuclear cataracts.
      noted that metabolism of molecular oxygen in the vitreous gel occurs in an AsA-dependent manner, without an exogenous catalyst and independent of light, revealing the significance of AsA as a primary regulator of intraocular molecular oxygen.
      AsA is actively concentrated from the plasma through the sodium-dependent vitamin C (SVC2) transporter located in the pigmented epithelial cell layer of the ciliary body with uptake of dehydroascorbic acid (dAsA) through glucose transporter (GLUT1) receptors in the non-pigmented epithelial layers.
      • Ma N.
      • Siegfried C.
      • Kubota M.
      • et al.
      Expression profiling of ascorbic acid-related transporters in human and mouse eyes.
      AsA secretion into aqueous humor has been described in animals and humans with subsequent dAsA recycling back to AsA. However, neither the transporters implicated in the uptake of AsA and its metabolites nor other transporters of key antioxidants such as glutathione have been observed to provide specific information about antioxidant protection of the aqueous humor and TM.
      • Umapathy A.
      • Donaldson P.
      • Lim J.
      Antioxidant delivery pathways in the anterior eye.
      A review of published studies led us to propose that oxygen is consumed by AsA in aqueous humor through a 2-step reaction.
      • Matsuoka Y.
      • Yamato M.
      • Yamada K.
      Fluorescence probe for the convenient and sensitive detection of ascorbic acid.
      • Nimse S.B.
      • Pal D.
      Free radicals, natural antioxidants, and their reaction mechanisms.
      • Jiang D.
      • Li X.
      • Liu L.
      • Yagnik G.B.
      • Zhou F.
      Reaction rates and mechanism of the ascorbic acid oxidation by molecular oxygen facilitated by Cu(II)-containing amyloid-beta complexes and aggregates.
      • Traber M.G.
      • Stevens J.F.
      Vitamins C and E: beneficial effects from a mechanistic perspective.
      • Boatright W.L.
      Oxygen dependency of one-electron reactions generating ascorbate radicals and hydrogen peroxide from ascorbic acid.
      These reactions gradually decrease oxygen levels as AsA is converted to dAsA:
      2O2+2AsAFe/Cu2dAsA(dehydroascorbicacid)+2H2O2


      2H2O2Catalase2H2O+O2


      Direct in vivo measurements of ROS are problematic due to their reactivity and transient nature.
      • Pryor W.A.
      • Godber S.S.
      Noninvasive measures of oxidative stress status in humans.
      As a result, quantification of antioxidants is frequently performed as a surrogate marker of oxidant-antioxidant balance. Using gas chromatography-mass spectrometry, we measured dAsA byproducts in ocular fluids and identified 2,3-diketogulonate and l-threonate (data not shown). The 2,3-diketogulonate reacts with H2O2, producing l-threonate. This provides indirect evidence that H2O2, an important ROS, exists in aqueous humor.
      • Linster C.L.
      • Van Schaftingen E.
      Vitamin C. Biosynthesis, recycling and degradation in mammals.
      Some investigators have suggested that elevated oxygen may produce enough H2O2 to exceed the ability of catalase to remove it,
      • Chang S.
      LXII Edward Jackson lecture: open angle glaucoma after vitrectomy.
      • Spector A.
      • Ma W.
      • Wang R.R.
      The aqueous humor is capable of generating and degrading H2O2.
      potentially increasing exposure of the aqueous outflow pathway to this toxic metabolite. Oxidative damage or death of TM endothelial cells could result as a consequence of this exposure, as observed in glaucoma patients with decreased cellularity of the TM.
      • Alvarado J.
      • Murphy C.
      • Polansky J.
      • Juster R.
      Age-related changes in trabecular meshwork cellularity.
      • Gabelt B.T.
      • Kaufman P.L.
      Changes in aqueous humor dynamics with age and glaucoma.
      • Grierson I.
      • Howes R.C.
      Age-related depletion of the cell population in the human trabecular meshwork.
      • Sacca S.C.
      • Izzotti A.
      • Rossi P.
      • Traverso C.
      Glaucomatous outflow pathway and oxidative stress.
      If increased oxygen oxidizes AsA and other antioxidants, one would expect antioxidant molecules to be depleted in the aqueous humor of patients with elevated pO2. Our findings of decreased AsA and TRAP levels in eyes after vitrectomy and IOL implantation support this theory.

       Vitrectomy and Risk of Open-Angle Glaucoma

      Increased pO2 in the AC angle and altered antioxidant status may be clinically significant. Alternative mechanisms of TM damage and physiologic responses may be represented in these vitrectomized patients compared to other forms of POAG. As indicated in several retrospective studies with varying inclusion/exclusion criteria and follow-up periods, the prevalence (2%–19.2%) of ocular hypertension and glaucoma following vitrectomy and subsequent lens extraction is inconsistent,
      • Koreen L.
      • Yoshida N.
      • Escariao P.
      • et al.
      Incidence of, risk factors for, and combined mechanism of late-onset open-angle glaucoma after vitrectomy.
      • Luk F.O.J.
      • Kwok A.K.H.
      • Lai T.Y.Y.
      • Lam D.S.C.
      Presence of crystalline lens as a protective factor for the late development of open angle glaucoma after vitrectomy.
      • Toyokawa N.
      • Kimura H.
      • Matsumura M.
      • Kuroda S.
      Incidence of late-onset ocular hypertension following uncomplicated pars plana vitrectomy in pseudophakic eyes.
      • Wu L.
      • Berrocal M.H.
      • Rodriguez F.J.
      • et al.
      Intraocular pressure elevation after uncomplicated pars plana vitrectomy results of the Pan American Collaborative Retina Study Group.
      with contrary conclusions in some studies.
      • Mi C.W.
      • Thompson J.T.
      Long–Term Follow–up of intraocular pressure after vitrectomy in eyes without preexisting glaucoma.
      • Yu A.L.
      • Brummeisl W.
      • Schaumberger M.
      • Kampik A.
      • Welge-Lussen U.
      Vitrectomy does not increase the risk of open-angle glaucoma or ocular hypertension: a 5-year follow-up.
      • Lalezary M.
      • Kim S.J.
      • Jiramongkolchai K.
      • Recchia F.M.
      • Agarwal A.
      • Sternberg Jr., P.
      Long-term trends in intraocular pressure after pars plana vitrectomy.
      The mechanisms of post-vitrectomy glaucoma likely represent a multifactorial process with various yet unidentified genetic and/or environmental risk factors.
      Lelazary and colleagues
      • Lalezary M.
      • Shah R.J.
      • Reddy R.K.
      • et al.
      Prospective Retinal and Optic Nerve Vitrectomy Evaluation (PROVE) study: twelve-month findings.
      recently completed the Prospective Retinal and Optic Nerve Vitrectomy Evaluation (PROVE) study. The 3-year data from Patel and colleagues
      • Patel S.K.S.
      • Lalezary M.
      • Shah R.J.
      • Kuchtey R.W.
      • Joos K.M.
      • Kammer J.A.
      • Cherney E.F.
      IOP changes: three-year findings of the Prospective Retinal and Optic Nerve Vitrectomy Evaluation (PROVE) study.
      revealed a significant increase in mean IOP in eyes having undergone both vitrectomy and cataract surgery with IOL implantation compared to baseline (P < .05) and compared to the fellow eye (P < .05), consistent with original reports by Chang.
      • Chang S.
      LXII Edward Jackson lecture: open angle glaucoma after vitrectomy.
      A recently published retrospective, population-based cohort study confirmed these findings of increased 10-year risk of POAG in post-vitrectomy eyes at 10.0% (95% confidence interval [CI]: 3.0–17.0%) and following vitrectomy combined with scleral buckle at 17.5% (95% CI: 0–34.9%) compared to the nonoperative group at 1% (95% CI: 1%).
      • Mansukhani S.A.
      • Barkmeier A.J.
      • Bakri S.J.
      • et al.
      The risk of primary open-angle glaucoma following vitreoretinal surgery-a population-based study.
      Of the patients in the VIT group who underwent glaucoma surgery, 5 of 10 (50%) had a history of controlled glaucoma prior to PPV surgery and subsequent lens extraction. The mean time from PPV to glaucoma surgery was 51.3 ± 39.4 months (range = 12–118 months). Delayed onset of elevated IOP and protective effects of the crystalline lens have been reported, consistent with our data.
      • Koreen L.
      • Yoshida N.
      • Escariao P.
      • et al.
      Incidence of, risk factors for, and combined mechanism of late-onset open-angle glaucoma after vitrectomy.
      • Luk F.O.J.
      • Kwok A.K.H.
      • Lai T.Y.Y.
      • Lam D.S.C.
      Presence of crystalline lens as a protective factor for the late development of open angle glaucoma after vitrectomy.
      Our findings of further increases of pO2 in the AC angle and posterior chamber in these cases following cataract extraction provides additional support for the theory of prolonged oxidative stress causing TM damage.
      Future recruitment of a subgroup of patients who have undergone PPV and lens extraction without a diagnosis of ocular hypertension or POAG may provide additional information. We performed longitudinal assessments of aqueous and vitreous humor oxidant-antioxidant balance in an older monkey model of PPV with subsequent lensectomy.
      • Siegfried C.J.
      • Shui Y.B.
      • Tian B.
      • Nork T.M.
      • Heatley G.A.
      • Kaufman P.L.
      Effects of vitrectomy and lensectomy on older rhesus macaques: oxygen distribution, antioxidant status, and aqueous humor dynamics.
      Our results indicated progressive decrease of both TRAP and AsA as well as increased 8-OHdG, a marker of oxidative damage, in both aqueous and vitreous specimens following each surgical procedure.

       Antioxidant Properties of Topical Glaucoma Medications

      An interesting finding in this study was the correlation of topical CAIs with AsA levels in aqueous humor collected from human patients in vivo (Table 2). CAIs administered topically or systemically to rabbits resulted in increased concentrations of AsA in the aqueous of the posterior chamber but not the AC.
      • Bar-Ilan A.
      • Pessah N.I.
      • Maren T.H.
      The effects of carbonic anhydrase inhibitors on aqueous humor chemistry and dynamics.
      These findings, confirming prior studies by Becker,
      • Becker B.
      The effects of the carbonic anhydrase inhibitor, acetazoleamide, on the composition of the aqueous humor.
      were noted to be a reflection of decreased aqueous production and flow in this region. However, these measurements were based on acute therapy with systemic carbonic anhydrase inhibitors, and may not be reproduced with chronic topical use, a common component of glaucoma therapy.
      Our findings of significantly higher AsA concentrations in patients taking topical CAIs may represent a potential secondary mechanism of action, as revealed in the reduction of free radical formation in glaucoma patients taking topical dorzolamide.
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      Antioxidant status modifications by topical administration of dorzolamide in primary open-angle glaucoma.
      Timolol, a beta-blocker, also has been shown to exert direct antioxidant protection of human endothelial cells in culture.
      • Izzotti A.
      • Sacca S.C.
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      • Penco S.
      • Bassi A.M.
      Antioxidant activity of timolol on endothelial cells and its relevance for glaucoma course.
      Metipranolol, in addition to its active metabolite desacetylmetipranolol, also exhibits antioxidant properties in vitro.
      • Melena J.
      • Osborne N.N.
      Metipranolol attenuates lipid peroxidation in rat brain: a comparative study with other antiglaucoma drugs.
      Brimonidine, an alpha-2 adrenergic agonist, has been shown to exert a neuroprotective effect on rat retinal ganglion cells in the presence of glutamate, oxidative and hypoxic stress,
      • Lee K.Y.
      • Nakayama M.
      • Aihara M.
      • Chen Y.N.
      • Araie M.
      Brimonidine is neuroprotective against glutamate-induced neurotoxicity, oxidative stress, and hypoxia in purified rat retinal ganglion cells.
      but no changes in antioxidant levels of the anterior segment. Pre-incubation of cultured human TM cells with prostaglandin analogs followed by exposure to H2O2 has been shown to reduce glaucomatous TM changes in these cells.
      • Yu A.L.
      • Fuchshofer R.
      • Kampik A.
      • Welge-Lussen U.
      Effects of oxidative stress in trabecular meshwork cells are reduced by prostaglandin analogues.
      Further studies of potential antioxidant effects of glaucoma therapies may be warranted.

       Study Limitations

      The cross-sectional design of this study and others identified in our review of published studies limits our understanding of how responses to oxidative stress occur over time in a given patient. Future longitudinal analyses may help to understand questions surrounding progressive TM damage and glaucoma development. As in any human study, individual patient variation may affect group mean data analysis. Dependence on a patient's historical information regarding medication (eg, antioxidant supplements) and social history (eg, tobacco use) may significantly alter results, especially with limited sample size within each of the study groups. Our results did not confirm previously published findings of decreased antioxidant protection in glaucoma versus cataract patients, despite similar protocol techniques. Although we designated patients undergoing cataract surgery as references/controls for our comparisons, it is important to note that these were not “normal controls,” as they did have a condition(s) associated with oxidative damage (cataract and aging). Differences in cataract type and glaucoma severity may have had an impact on the results as well as our observation of the effect of specific glaucoma medications on AsA in aqueous humor. Given our limited number of VIT subjects, recruitment of additional post-vitrectomy subjects (with and without glaucoma) could be informative as vitreoretinal pathology may independently influence antioxidant levels. Because both patients and specimen quantities were limited, assays of multiple antioxidants could not be performed for all patients, depending on volumes required. We identified AsA and TRAP as the most promising agents, given their biochemical reactions with oxygen as the dominant measurements of antioxidant potential, but other molecules may play a significant role in antioxidant defense (eg, non-AsA TRAP).

      Conclusions

      Our observation of increased pO2 levels in the anterior segment and decreased levels of AsA and TRAP in the group of patients who had undergone PPV compared to the reference group may provide important insights into how this surgery may increase oxidative stress and glaucoma risk in select patients. We propose increased intraocular pO2 levels in these patients could be a potential source of pro-oxidants for generation of ROS, decreasing antioxidant defenses in the ocular anterior segment (Figure 5). Further understanding of this surgical intervention's impact on oxygen homeostatic mechanisms, antioxidant balance, and oxidative stress is vital, and these investigations may lead to future therapies targeted to this specific population as well as to other individuals afflicted with this leading cause of irreversible blindness.
      Figure thumbnail gr5
      Figure 5Proposed mechanism for development of post-vitrectomy glaucoma. A: Low levels of pO2 in vitreous cavity, surrounding clear lens and in anterior chamber (AC) angle. Oxygen diffuses across cornea into the AC and from retinal blood vessels into the vitreous cavity. B: Following pars plana vitrectomy, pO2 increases in the vitreous cavity, around lens, in AC angle and posterior chamber with development of nuclear sclerotic cataract. C: Following cataract extraction and intraocular lens implantation (IOL), pO2 increases in the AC angle, posterior chamber and mid-A(C) D: Expanded view of AC angle illustrating alterations in aqueous humor including increased pO2 surrounding lens implant and in AC angle leading to increased reactive oxygen species formation (brown; ROS) and decreased antioxidants (green). E: Proposed cascade of events following vitrectomy and IOL implantation ultimately leading to increased intraocular pressure (IOP) and risk of glaucoma. AsA = ascorbate; GSH = glutathione; H2O2 = hydrogen peroxide; O2 = superoxide anion; O2−2 = peroxide; OH = hydroxyl ion; SOD = superoxide dismutase; TM = trabecular meshwork; TRAP = total reactive antioxidant potential.
      Funding/support: Supported by grants NEI EY021515 , NEI EY015863 , and NEI P30EY002687 , a Grace Nelson Lacy Glaucoma Research Grant, an American Health Assistance Foundation /Brightfocus Foundation-National Glaucoma Research Grant, a Glaucoma Research Foundation grant (Shaffer grant), and an unrestricted grant from Research to Prevent Blindness (New York, New York) to the Washington University Department of Ophthalmology And Visual Sciences. The funding organizations had no role in the design or conduct of this research.
      Financial disclosures: Dr. Siegfried has received lecture fees from Allergan Inc. Dr. Shui has no financial disclosures. All authors attest that they meet the current ICMJE criteria for authorship.
      Contributions of Authors: Design of the study (C.J.S., Y.B.S.); collection, management, analysis, and interpretation of the data (C.J.S., Y.B.S.), preparation, review and approval of the manuscript (C.J.S., Y.B.S.)
      Acknowledgments: The authors thank David C. Beebe, PhD (deceased), for inspiration and passion to bring this scientific investigation to life; Andrew Huang, MD, for guidance and contribution of patients to the study; and Fang Bai, MD, for assistance with the aqueous humor assays.

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