Oxidative Stress-Associated Neuroretinal Dysfunction and Nitrosative Stress in Diabetic Retinopathy

Can J Diabetes 37 (2013) 401e407

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Canadian Journal of Diabetes journal homepage: www.canadianjournalofdiabetes.com

Original Research

Oxidative Stress-Associated Neuroretinal Dysfunction and Nitrosative Stress in Diabetic Retinopathy Lakshmi K. Mandal MS a, Subhadip Choudhuri PhD b, *, Deep Dutta MD c, Bhaskar Mitra MD d, Sunanda Kundu MSc e, Imran H. Chowdhury PhD b, Aditi Sen PhD b, Mitali Chatterjee PhD e, Basudev Bhattacharya MD b a

Regional Institute of Ophthalmology, Kolkata, India Department of Biochemistry, Dr. B C Roy Post Graduate Institute of Basic Medical Education and Research, Kolkata, India c Department of Endocrinology and Metabolism, B C Roy Post Graduate Institute of Basic Medical Education and Research and Seth Sukhlal Kanoria Memorial Hospital, Kolkata, India d Department of Pathology, Midnapore Medical College, Paschim Midnapore, India e Department of Pharmacology, Dr. B C Roy Post Graduate Institute of Basic Medical Education and Research, Kolkata, India b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 February 2013 Received in revised form 18 May 2013 Accepted 22 May 2013

Objective: The present study was intended to investigate whether oxidative stress is the key regulator to alter neuroretinal biochemical homeostasis and in turn aggravate the process of diabetic retinopathy by inducing nitrosative stress in the retinal neurovascular unit. Methods: Peripheral blood mononuclear cell reactive oxygen species level was measured by flow cytometry along with spectrophotometric detection of malondialdehyde (MDA) and glutamate from serum or plasma and a vitreous sample of study groups (i.e. subjects with proliferative diabetic retinopathy [PDR], type 2 diabetes without retinopathy [DNR] and healthy controls [HCs]). Further, nitrosative stress assessment was performed by spectrophotometric and enzyme-linked immunosorbent assay-based detection of serum and vitreous nitrite and nitrotyrosine concentrations, respectively. Results: The plasma glutamate level remains insignificant between subjects with PDR and DNR (p¼0.505) or in HC (p¼0.1344) individuals. However, serum MDA (p¼0.0004), nitrite (p¼0.0147) and nitrotyrosine (p¼0.0129) were found to be strikingly higher among PDR subjects compared with the DNR group. Significantly increased levels of peripheral blood mononuclear cell reactive oxygen species (p<0.0001), vitreous glutamate (p¼0.0009, p<0.0001), MDA (p¼0.0058, p¼0.0003), nitrite (p¼0.0014, p<0.0001) and nitrotyrosine (p¼0.0008, p<0.0001) were found in PDR subjects compared with DNR and HC subjects, respectively. Conclusions: Our observation suggests that oxidative stress is associated with impairment in neuroretinal biochemical homeostasis among PDR subjects, which further augments retinal nitrosative stress and thus worsens the pathogenic process of retinopathy among PDR subjects. Ó 2013 Canadian Diabetes Association

Keywords: diabetic retinopathy glutamate nitrosative stress oxidative stress reactive oxygen species (ROS)

r é s u m é Mots clés : rétinopathie diabétique glutamate stress nitrosatif stress oxydatif espèces réactives de l’oxygène (ERO)

Objectif : La présente étude avait pour but d’évaluer si le stress oxydatif est le régulateur principal de l’altération de l’homéostasie biochimique de la neurorétine et à son tour de l’aggravation du processus de rétinopathie diabétique par l’induction du stress nitrosatif à l’unité neurovasculaire de la rétine. Méthodes : La concentration des espèces réactives de l’oxygène (ERO) dans les cellules mononucléées du sang périphérique a été mesurée par la cytométrie de flux parallèlement à la détection spectrophotométrique de la malondialdéhyde (MDA) et du glutamate sérique et plasmatique, et d’un échantillon de corps vitré des groupes expérimentaux (c.-à-d. des sujets ayant une rétinopathie diabétique proliférante [RDP], le diabète de type 2 sans rétinopathie [DNR : diabetes with no retinopathy] et des témoins en santé [TS]). Par ailleurs, l’évaluation du stress nitrosatif a été réalisée par la détection spectrophotométrique et par l’immunoabsorption par enzyme liée des concentrations respectives de nitrite et de nitrotyrosine dans le sérum et le corps vitré.

* Address for correspondence: Dr. Subhadip Choudhuri, PhD, Department of Biochemistry, Dr. B C Roy Post Graduate Institute of Basic Medical Education and Research, 244B A.J.C. Bose Road, Kolkata 700020, India. E-mail address: [email protected]. 1499-2671/$ e see front matter Ó 2013 Canadian Diabetes Association http://dx.doi.org/10.1016/j.jcjd.2013.05.004

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Résultats : La concentration plasmatique du glutamate demeure non significative entre les sujets ayant une RDP et un DNR (p ¼ 0,505) ou les TS (p ¼ 0,1344). Cependant, les concentrations sériques de la MDA (p ¼ 0,0004), du nitrite (p ¼ 0,0147) et de la nitrotyrosine (p ¼ 0,0129) ont été remarquablement plus élevées chez les sujets ayant une RDP comparativement au groupe ayant un DNR. Une augmentation significative des concentrations des ERO dans les cellules mononucléées du sang périphérique (p < 0,0001), du glutamate du corps vitré (p ¼ 0,0009, p <0,0001), de la MDA (p ¼ 0,0058, p ¼ 0,0003), du nitrite (p ¼ 0,0014, p < 0,0001) et de la nitrotyrosine (p ¼ 0,0008, p < 0,0001) a été observée de manière respective chez les sujets ayant une RDP comparativement aux sujets ayant un DNR et les TS. Conclusions : Notre observation montre que le stress oxydatif est associé à la détérioration de l’homéostasie biochimique de la neurorétine chez les sujets ayant une RDP, ce qui augmente par ailleurs le stress nitrosatif de la rétine, aggravant ainsi le processus pathogène de la rétinopathie chez les sujets ayant une RDP. Ó 2013 Canadian Diabetes Association

Introduction Retinal capillary damages are the classic hallmark of diabetic retinopathy (DR). DR is an incapacitating microvascular complication of diabetes mellitus, and hyperglycemia appears as a decisive factor in the etiology of retinopathy among subjects with type 2 diabetes (1). Hyperglycemic episodes are believed to have a close association with oxidative and nitrosative stress, which renders the retinal neurovascular unit and its associated glia susceptible to free radical injury (2). Muller cells are the principal glia of the retina, positioned critically between the microvasculature and the neurons of the retina. Muller cells express the high-affinity L glutamate/L aspartate transporter (GLAST), which plays an important role in the transport of extracellular glutamate from the synaptic spaces of the retina. Glutamate acts as a neurotransmitter at more than 90% of the synapses in the retina (3e5). The redox sensing elements present in the GLAST of Muller cells regulate the activity of the transporter through thiol disulfide interconversion. Considerable reduction in the activity of Muller cell GLASTs on exposure to oxidizing agents and its subsequent reversal by a chemical reductant has been reported (6,7). The calcium-permeable Nemethyl D aspartate (NMDA) receptor is an ionotrophic receptor for glutamate and is present in the neurons of the retina. The NMDA receptors have been associated with glutamate neurotoxicity and calcium influx into the neuronal cells. Influx of calcium in neuronal cells causes release of caspases from mitochondria, which are the key signalling molecule of apoptosis. Therefore, NMDA receptor overactivity during the hyperglycemic state is believed to play an important role in neurodegenerative changes of the retina (8e10). Nitric oxide (NO) is an ever-present intracellular messenger, modulates blood flow and regulates neural activity. Under chronic hyperglycemia and hypoxia, NO appears to be an important regulator of retinal vasculature autoregulation (11,12). The neurotoxic and detrimental role of NO appears once the molecule reacts with superoxide radicals to form powerful oxidant peroxynitrite (ONOO–). Peroxynitrite is a potent cytotoxin that attacks various biomolecules in the neurovascular unit of the retina and is a key element in resolving the contrasting roles of NO in microvascular pathology (13e15). However, the involvement of oxidative stress in retinal glutamate homeostasis and the association of subsequent nitrosative stress with DR pathology still remains inscrutable. To the best of our knowledge, no data have clarified this hypothesis in human beings. In this context, to scrutinize the role of oxidative stress on neuroretinal dysfunction and retinal glutamate homeostasis, we measured peripheral blood mononuclear cell (PBMC) reactive oxygen species (ROS) along with the plasma and vitreous glutamate and malondialdehyde (MDA) level in individuals from 3 study groups. We also measured nitrite and nitrotyrosine levels from

serum and vitreous fluids to evaluate whether nitrosative stress is putatively associated with oxidative stress and subsequent DR pathology in proliferative diabetic retinopathy (PDR). Methods Study subjects Forty-five PDR subjects (mean age, 51.78.1 years) with type 2 diabetes mellitus, 32 subjects with type 2 diabetes without retinopathy (DNR) (mean age, 49.58.3 years) and 36 normal subjects without diabetes as healthy controls (HCs) (mean age, 48.67.8 years) who fulfilled all the inclusion and exclusion criteria and provided written informed consent (as per the declaration of Helsinki) were enrolled in this cross-sectional study. The presence of coronary artery disease or a strong family history of coronary artery disease, hypertension, peripheral vascular disease, recent infection, thromboembolitic event, microalbuminuria, prediabetes, ocular disorder (glaucoma, Eales disease, branch retinal venous occlusion and so forth) or any severe co-existing illness were considered exclusion criteria for this study. Patients with type 2 diabetes who received insulin were excluded. The study protocol was approved by the institute ethics committee. The samples were obtained from the retina clinic of the Regional Institute of Ophthalmology and the diabetic clinic of the Institute of Post Graduate Medical Education and Research in Kolkata. All subjects enrolled in this study were from the same geographic area (Gangetic Delta, Eastern India). Age, sex, blood pressure and body mass index were matched within the study groups. The glycemic status of all study subjects was assessed by measuring fasting and postprandial plasma glucose and glycosylated hemoglobin levels (A1C%). PDR cases were diagnosed by dilated fundus examination with slit lamp biomicroscopy by 90 diopter and 3 mirror lens, and 7-field digital fundus photography with fluorescence angiography. Grading of the retinopathy was performed according to a modified early treatment diabetic retinopathy study. Sample collection and processing Study subjects were advised to fast for 12 hours before blood sample collection. Fifteen milliliter venous blood samples were drawn and, from these, 10 mL blood samples were collected in a heparinized tube. From a 10 mL heparinized blood sample, 8 mL was collected in a 15 mL sterile centrifuge tube for PBMC isolation, and 2 mL samples were centrifuged at 3000 rpm for 10 minutes at 4 C to separate cellular components and plasma. Plasma samples were collected in cryocube vials for glucose and glutamate assay. The remaining 5 mL sample from the initial 15 mL was taken in a

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clot vial to obtain serum. Finally, serum samples were collected in cryocube vials for MDA, nitrite and nitrotyrosine assays. Mononuclear cells from peripheral whole blood were obtained from 8 mL heparinized blood by using Histopaque-1077 density gradient separating media (Sigma Aldrich, St. Louis, MO, USA) for 40 minutes at 1300 rpm and 20 C as previously described (16). PBMCs were further subjected to centrifugation at 1500 rpm for 10 minutes and washed with 1  phosphate-buffered saline (PBS) (pH 7.2) twice. Cells (5105) were pelleted and resuspended in 1 PBS (pH 7.2) for the estimation of ROS. Vitreous samples were drawn by 3-port pars plana vitrectomy from 45 PDR, 32 DNR and 36 HC subjects (DNR and normal vitreous were collected through parsplana vitrectomy during removal of a dropped nucleus, which occurred accidentally after blunt trauma and from a peroperative complication of phacoemulsification). MDA, glutamate, nitrite and nitrotyrosine levels also were measured from the vitreous fluid of study subjects. Undiluted vitreous gel (500 mL) was excised from the midvitreous by vitreous cutter and carefully aspirated into the hand-held sterile syringe attached to the suction port of the vitrectomy probe. Immediately after collection, the vitreous samples were kept in ice and centrifuged at 10 000 rpm for 15 minutes at 4 C. After centrifugation, the supernatant was aspirated and stored at 20 C for immediate use. Measurement of PBMC ROS Intracellular ROS generation in mononuclear cells was measured by ROS-sensitive cell permeable dye 2’7’ dihydro dichlorofluorescein diacetate (2’7’ H2DCF-DA), which in the presence of ROS was oxidized to highly fluorescent 2’7’DCF in the cell. The production of intracellular ROS is directly proportional to the oxidation of 2’7’ H2DCF-DA and thereby increases the cellular fluorescence level. Pelleted cells (5105) were washed twice with 1 PBS (pH 7.2) by centrifuging at 4000 rpm for 5 minutes and cells were resuspended in 500 mL of 1 PBS (pH 7.2). Thereafter, cells were incubated with 20 mm 2’7’ H2DCF-DA for 30 minutes at 37 C. Finally, the cells were washed again with 1 PBS (pH 7.2) and resuspended in 400 mL of 1 PBS. The mononuclear cells showing increased fluorescence of oxidized DCF was measured by flow cytometry (FACS Calibur; Becton Dickinson, San Jose, CA) equipped with an argon ion laser (15 mW) tuned to 488 nm (17). The fluorescence of DCF was collected in an FL1 channel, equipped with a 530/30 nm band pass filter. Fluorescence was measured in the long mode using Cell Quest Prosoftware (BD Bioscience, San Jose, CA) and expressed as the geometric mean fluorescence channel. Cells were gated on the basis of their characteristic morphology (i.e. forward scatter and side scatter of monocytes and lymphocytes). Acquisitions were performed on 10 000 gated events; data analysis was performed with Cell Quest Prosoftware (BD Bioscience). MDA measurement Lipid peroxidation in serum and vitreous fluid were measured by MDA estimation as described previously (18). Lipoproteins in serum and vitreous fluid were precipitated by adding 20% trichloro acetic acid and 8.1% sodium dodecyl sulfate. Thereafter, 0.8% aqueous solution of thiobarbituric acid was added to this precipitate, mixed well and finally was heated at 95 C for 1 hour for coupling of lipid peroxide with thio barbituric acid reagent. The resulting chromogen was extracted from the precipitate by adding a mixture of n-butanol and pyridine (15:1). The organic mixture was separated by centrifugation and the intensity of the organic layer was measured spectrophotometrically (Halo DB-20; Dynamica, SalzburgeMayrwise, Austria) by using a 530 nm filter against water blank. The concentration of MDA in serum and vitreous

403

samples was determined from a linear standard curve established by 1 to 8 nm of 1,1,3,3 tetra methoxy propane. Glutamate measurement Plasma and vitreous glutamate were measured by an enzymelinked spectrofluorimetric assay as described previously (19). In the presence of 1 U/mL glutamate dehydrogenase and 40 mmol/L beta-nicotinamide adenine dinucleotide phosphate (NADPþ), plasma and vitreous glutamate were oxidized to alpha keto glutarate with the fluorimetric production of NADPH in Krebs-RingerHEPES reaction buffer (145 mmol/L Nacl, 1.2 mmol/L Cacl2, 10 mmol/L dextrose, 15 mmol/L Kcl, 1.3 mmol/L Mgcl2 and 1.2 mmol/L Na2HPO4,H2O). After 30 minutes’ incubation of reaction mixture at 37 C, the fluorescence generated by the reduction of NADPþ to NADPH was monitored spectrofluorimetrically (model FP 6300; JASCO, Essex, UK) at excitation and emission wave lengths of 366 nm and 455 nm, respectively. The concentration of glutamate in plasma and vitreous samples was determined from a linear standard curve established by 25 to 150 mmol of L glutamate standard. Nitrite measurement We have measured serum and vitreous nitrite levels as an indicator of nitric oxide by the Griess reaction (20). Samples were deproteinized by methanol (sample: methanol¼1:2 volume/ volume). A sample with methanol mixture was centrifuged at 10 000 rpm for 10 minutes and the supernatant was collected for nitrite estimation. Equal amounts of sample and Griess reagent (0.5% sulfanilamide in 2.5% phosphoric acid and 0.05% N naphthyl ethylene diamine in 2.5% phosphoric acid) was mixed and vortexed. The mixture was allowed to incubate for 30 minutes at 37 C and, finally, the absorbance was read at 540 nm in a spectrophotometer (Halo DB- 20; Dynamica). The concentration of nitrite in serum and vitreous fluid was determined from a linear standard curve established by 15 to 140 mmol/L sodium nitrite. Nitrotyrosine measurement The nitrotyrosine present in the sample was measured by an enzyme-linked immunosorbent assay (ELISA) by using the Cell Biolabs kit (catalog STA-305; San Diego, CA). The nitrotyrosine present in the sample first was added to a nitrated bovine serum albumin (BSA) preabsorbed enzyme immunoassay plate. After a brief incubation, an antinitrotyrosine antibody was added, followed by a horseradish-peroxidase (HRP) conjugated secondary antibody. The nitrotyrosine content in the sample was determined by comparison with a standard curve that was prepared from nitrated bovine serum albumin standards in the nitrotyrosine concentration range from 1.95 to 8000 nm. The absorbance of the final color product was read at 450 nm as the primary wave length by a BioRad multiplate reader (model 680; Hercules, CA). Statistical analysis Data obtained from each sample group were expressed as the mean  standard deviation. The means obtained from different sample groups were compared by 1-way analysis of variance test and the nonparametric Mann-Whitney U test. To find out the correlation between the 2 variables, the Pearson product moment correlation coefficient was used. A p value of less than 0.05 was considered statistically significant. All statistical analyses were performed using Graph Pad prism software (version 5; San Diego, CA). Statistical analysis for sex distributions were evaluated by

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Table 1 Clinical characteristics of HC, DNR and PDR subjects Parameters Sex Age (years) Duration of diabetes (years) Body mass index (kg/m2) Blood pressure (mm Hg) Blood glucose level (mg/dL) A1C%

HC (N¼36) Male Female

DNR (N¼32) PDR (N¼45) p value

22 (61.11%) 22 (68.75%) 14 (38.89%) 10 (31.25%) 48.67.8 49.58.3 —————— 15.26.45 24.13.9

Table 2 Level of different biochemical parameters among HC, DNR and PDR subjects

26.44.62

26 (57.77%) 19 (42.23%) 51.78.1 16.45.26

0.615

25.34.24

0.088

0.207 0.372

Systolic 126.46.65 128.27.7 Diastolic 79.85.9 826.2 Fasting 817.9 17222.9

1308.8 0.127 83.56 0.12 188.824.22 0.0001

PP

292.733.65 0.0001 10.41.4 0.0001

110.39.6 4.70.8

24629.4 8.91.2

PP, postprandial. A comparison of different groups enrolled in the present study shows no statistically significant differences for sex distribution, age, duration of disease, BMI and blood pressure. The A1C% and blood glucose values (fasting and postprandial) were increased in PDR groups compared with HC and DNR subjects, and statistical analysis showed a significant difference. Data were presented as mean  SD, and N indicates sample size. A 1-way analysis of variance test was performed and p<0.05 was considered the minimum level of significance.

chi-square test by using the statistical software Stata (version 8; Stata Corporation, College Station, TX). Results As shown in Table 1, different study groups enrolled in our present study showed no statistically significant differences for sex distribution, age, duration of diabetes, body mass index or blood pressure. Fasting and postprandial blood glucose levels were increased significantly among PDR subjects compared with DNR and HC subjects (p<0.0001). The A1C% was found to be higher in PDR subjects (10.4%1.4%) compared with the DNR group (8.9% 1.2%) and those who were considered HCs (4.7%0.8%). Further statistical analysis has shown a significant difference between the study groups (p<0.0001). The PBMC ROS level was expressed as the mean  SD of the geomean of DCF fluorescence per 5105 cells among the study subjects. The PBMC ROS level was increased significantly among PDR subjects compared with DNR and HC subjects (p<0.0001). The level was found to be significantly higher among DNR subjects compared with the HC group (p<0.0001) (Table 2). MDA level was increased remarkably in serum and vitreous fluid from PDR subjects compared with DNR (p¼0.0004, p¼0.0058, respectively) and HC individuals (p<0.0001, p¼0.0003, respectively). Yet again, serum MDA level was found to be significantly low in healthy individuals even compared with DNR subjects (p¼0.027) (Table 2). With regard to the plasma glutamate (p>0.05) level, no statistically significant difference was observed among PDR, DNR and HC individuals. However, the vitreous glutamate level was found to be noticeably high among PDR subjects compared with DNR (p¼0.0009) and HC (p<0.0001) subjects. Vitreous glutamate was found to be markedly low in HC subjects (p<0.0001) as compared with DNR subjects (Table 2). Serum and vitreous nitrite level was increased significantly among PDR subjects compared with the DNR (p¼0.0147, p¼0.0014) and HC groups (p<0.0001). Moreover, the level still remains significantly higher in serum and vitreous fluid of DNR subjects even compared with HC individuals (p<0.0001) (Table 2). However, a strikingly low level of nitrotyrosine was detected in serum and vitreous fluid of HC individuals compared with DNR (p<0.0001) and PDR subjects (p<0.0001). However, PDR subjects showed a significantly higher level of serum (p¼0.0129) and vitreous

Parameters

Sample type

ROS (geomean of DCF fluorescence/5105 cells) MDA (nmol/L)

PBMC

Serum Vitreous Glutamate (mmol/L) Plasma Vitreous Nitrite (NO) (mmol/L) Serum Vitreous Nitrotyrosine (nmol/L) Serum Vitreous

HC (n¼36), mean  SD 68.913.51

2.141.08 1.10.37 50.6820.6 10.674.18 19.477.04 9.493.24 12.446.27 4.622.61

DNR (n¼32), mean  SD

PDR (n¼45), mean  SD

98.621.35 155.3438.8

3.11.83 4.651.79 1.210.32 1.650.67 55.1320.8 57.9420.29 21.489.41 29.910.43 39.6516.16 54.0724.93 15.054.92 20.526.55 208.5669 277.2888.27 97.2529.12 119.6438.44

PBMC ROS was increased significantly among PDR subjects compared with DNR and HC subjects (p<0.0001). The level was significantly higher among DNR subjects even compared with the HC group (p<0.0001). A noticeable increase in serum and vitreous MDA level was observed among PDR subjects compared with DNR (p¼0.0004, p¼0.0058, respectively) and HC individuals (p<0.0001, p¼0.0003, respectively). Yet again, the serum MDA level was significantly low in healthy individuals even compared with DNR subjects (p¼0.027). No statistically significant difference was observed in plasma glutamate level among PDR, DNR and HC individuals (p>0.05). The vitreous glutamate level (p¼0.0009, p<0.0001, respectively) was noticeably high among PDR subjects compared with DNR and HC subjects; however, significantly lower vitreous glutamate was detected in HC subjects (p<0.0001) compared with DNR subjects. Serum and vitreous nitrite level was increased significantly among PDR subjects compared with the DNR (p¼0.0147, p¼0.0014, respectively) and HC groups (p<0.0001). Moreover, the level still remains significantly higher in serum and vitreous fluid of DNR subjects even compared with HC (p<0.0001). A significant low level of nitrotyrosine was detected in serum and vitreous fluid of HC individuals compared with DNR (p<0.0001) and PDR subjects (p<0.0001). PDR subjects showed a significantly higher level of serum (p¼0.0129) and vitreous (p¼0.0008) nitrotyrosine compared with the DNR group.

(p¼0.0008) nitrotyrosine even compared with the DNR group (Table 2). A significant correlation was observed between vitreous glutamate and PBMC ROS level among PDR (p<0.0001; r¼0.687) and DNR (p¼0.0122; r¼0.4375) subjects. However, no significant correlation was found in HC (p¼0.463; r¼0.126) individuals. Moreover, in vitreous fluid, the level of glutamate showed a significant correlation with MDA level among PDR (p¼0.0003; r¼0.5187) and DNR subjects (p¼0.0142; r¼0.4294), but not in control subjects (p¼0.2099; r¼0.2141) (Figure 1). Serum and vitreous nitrotyrosine levels showed a significant correlation with PBMC ROS in PDR subjects (p¼0.0023; r¼0.442, p<0.0001; r¼0.7397, respectively); however, in DNR serum (p¼0.071; r¼0.323) no significant correlation was observed between PBMC ROS and nitrotyrosine level. In vitreous fluid, a significant correlation was observed between both parameters (p¼0.0196; r¼0.41, respectively). In contrast, in HCs (p¼0.483, r¼0.1207; p¼0.0989, r¼ 0.279), no remarkable correlation was observed between PBMC ROS and serum or vitreous nitrotyrosine level, respectively (Figure 2). Discussion Over the past decade, there has been substantial interest on oxidative stress-mediated retinal neurodegeneration and subsequent progression of retinopathy in type 2 diabetes mellitus. Hyperglycemic conditions of cells are associated with increased ROS production, predominantly through mitochondrial electron transport chain and NADPH oxidase (21e23). ROS, the inhabitable component of oxidative stress, overwhelm the endogenous antioxidant system and act as a secondary messenger for several angiogenic and proinflammatory pathways associated with diabetic microvascular dysfunction (24,25). A retrospective casecontrol study on PDR and non-PDR patients showed an increased level of vitreous ROS in patients with PDR (26). Furthermore,

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Figure 1. (A) Correlation between vitreous glutamate and MDA level. XY scatter plot represents the correlation between vitreous glutamate and MDA level among PDR, DNR and HC individuals. In vitreous fluid, the level of glutamate showed significant correlation with MDA level among PDR (p¼0.0003; r¼0.5187) and DNR subjects (p¼0.0142; r¼0.4294), but not in control subjects (p¼0.2099; r¼0.2141). (B) Correlation between vitreous glutamate and PBMC ROS level. XY scatter plot represents correlation between vitreous glutamate level and PBMC ROS level among PDR, DNR and HC individuals. Significant correlation was observed between vitreous glutamate and PBMC ROS level among PDR (p<0.0001; r¼0.687) and DNR (p¼0.0122; r¼0.4375) subjects. But no significant correlation was found in HC (p¼0.463; r¼0.126) individuals.

increased PBMC ROS production among subjects with type 2 diabetes compared with healthy individuals is in agreement with an earlier study (27). In our present study, we found that the PBMC ROS level was increased remarkably among PDR subjects as compared with DNR and healthy individuals. It has been postulated that an increase in ROS production contributes to the initiation of lipid peroxidation. Furthermore, lipid peroxides undergoes complex chemical reactions with protein to yield advanced lipoxidation end products, which has pro-atherogenic and proinflammatory effects on microvascular cells of the retina (28). In this context we have measured MDA as an indicator of lipid peroxidation and have evaluated the increased level of serum and vitreous MDA among PDR subjects as compared with DNR and HC subjects. In addition, Mancino et al (29) reported that increased blood and vitreous MDA level is associated with oxidative stress along with poor antioxidant defense, which further promotes the progression of DR to its proliferative form. Therefore, based on this observation and previous studies it may be said that an increased level of ROS is intimately associated with the pathogenesis of PDR. Moreover, an increased level of vitreous and serum MDA among PDR subjects generates speculation that retinal microvascular complication is closely associated with the severity of oxidative stress.

Retinal Muller cells optimize the extracellular ionic environment to facilitate proper electrophysiologic function of retinal neurons, and GLAST transporter molecules expressed by this glia are implicated in glutamate uptake to keep its extracellular concentration below neurotoxic levels (4,5,30). Glutamate excitotoxicity disrupts retinal neurons and has been proposed as a mechanism of neurodegeneration in glaucoma (31). A previous study reported that under high-glucose concentrations, glutamate uptake and GLAST expression in Muller cells decreased significantly along with increased generation of ROS in Muller cells (32). Another study performed on an animal model suggested that in early stages of DR the function of glutamate transporter in retinal Muller cells is decreased by a mechanism that is likely to involve oxidation (33). In the present study, we speculate that the vitreous level of glutamate was significantly high among PDR subjects compared with DNR and HC subjects. The level also was increased significantly among DNR subjects compared with HC individuals. However, plasma glutamate level was not increased significantly among PDR subjects as compared with DNR and HC individuals. Our finding explores the theory that increased vitreous glutamate in PDR and DNR subjects may be caused by failure of glutamate transporter buffering in retina. Further significant correlation between vitreous

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Figure 2. Correlation between PBMC ROS and serum or vitreous nitrotyrosine, respectively. XY scatter plot represents the correlation between PBMC ROS and serum or vitreous nitrotyrosine level, respectively, among PDR, DNR and HC individuals. Serum and vitreous nitrotyrosine level showed a significant correlation with PBMC ROS in PDR subjects (p¼0.0023, r¼0.442; p<0.0001, r¼0.7397, respectively). Although in DNR serum (p¼0.071; r¼0.323) no significant correlation was observed between PBMC ROS and nitrotyrosine level, in vitreous fluid a significant correlation was observed between both parameters (p¼0.0196; r¼0.41, respectively). In contrast, in HC individuals (p¼0.483, r¼0.1207; p¼0.0989, r¼0.279, respectively), no remarkable correlation was observed between PBMC ROS and serum or vitreous nitrotyrosine level.

glutamate and PBMC ROS or vitreous MDA was observed in DNR subjects. The strongest correlation was evaluated between vitreous glutamate and PBMC ROS or vitreous MDA in PDR subjects. These findings suggest that increased oxidative stress may be involved in retinal glutamate homeostasis derangement, which was shown by the increased level of glutamate in PDR vitreous fluid. Our finding of altered glutamate homeostasis in PDR vitreous fluid suggests the close association of this biochemical disarray with DR manifestation. NO plays an important role in homeostatic vasodilatation and regulation of blood flow. Formation of NO in retinal nerve cells involves a constitutively active calcium/calmodulin-dependent neuronal NO synthase and endothelial NO synthase enzymes (14,34). In the present study, we measured nitrite level as an indicator of NO production and found an increased level of vitreous and serum nitrite among PDR subjects as compared with DNR and HC subjects. Our findings are consistent with the cross-sectional study performed on Japanese people, which elucidated a significant association of NO with the pathogenesis of DR (35). Peroxynitrite, a potent reactive nitrogen species (RNS), induces DNA single-strand breakage and subsequent activation of nuclear

enzyme poly-ADP ribose polymerase. Recent studies have reported an important role of poly-ADP ribose polymerase activation in the pathogenesis of diabetic vascular and neural dysfunction in retinopathy (13,15,36). In the present study, we measured nitrotyrosine level, which is a footprint of peroxynitrite-induced injury and recently was considered a collective index of RNS, rather than a specific indicator of peroxynitrite formation (37,38). Our study showed a significantly increased level of vitreous and serum nitrotyrosine among PDR subjects. In addition, the level was increased noticeably in DNR subjects compared with HCs. A crosssectional study on type 2 subjects with diabetes showed increased nitrotyrosine immunoreactivity in skin tissues (39). Another study showed increased monocyte nitrosylated protein expression as a biomarker of metabolic control and inflammation in subjects with diabetes with macroangiopathy (40). Yet again, we observed a significant correlation between PBMC ROS and serum or vitreous nitrotyrosine level among PDR subjects. Therefore, our observation suggests that increased generation of ROS and NO are the fundamental factors that may contribute to an increase in RNS generation among PDR subjects, which was shown by the increased level of nitrotyrosine in PDR vitreous and serum. Therefore, based on

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earlier study reports and our present findings, it may be said that oxidative stress-associated increased production of retinal RNS is intimately associated with PDR occurrence. In contrast, relatively less significant correlation with PBMC ROS and vitreous nitrotyrosine level in DNR vitreous fluid suggests that hyperglycemia is the key mediator to initiate reactive oxygen and nitrogen-sensing mechanisms in nerve and the endothelial cell lining of the retinal neurovascular unit. This speculation provides an explanation for the increased susceptibility of subjects with poorly controlled type 2 diabetes to develop microangiopathy. Although we have found increased levels of vitreous MDA, glutamate and nitrite levels among PDR and DNR individuals, pharmacologic experiments performed in the same field reported an increased level of glutamate, thiobarbituric acid-reactive substances and NO in their study using rats after 2 months of diabetes (13). Furthermore, the study evaluated that inhibition of glutamate results in decreased production of NO in rat retina. Based on our present observation type and findings, we could not explore the exact mechanism involved in a simultaneous increase of nitrite and glutamate level in PDR and DNR vitreous fluid. However, vitreous samples were evaluated in our study because vitreous gel is a potent bio-indicator of the retinal vascular and neural microenvironment status in chronic oxidative tissue injury or in acute inflammatory disease states (24,41). Therefore, we believe that an increased level of vitreous MDA with a concomitant increase of vitreous glutamate and nitrotyrosine level might explain the fact that oxidative stress is the key regulator, which is associated significantly with neuroretinal metabolic disparity, and thus initiates the process of diabetic microangiopathy characterized by vascular endothelial and pericyte cell loss (21,22). In summary, based on all of our findings and previous study reports it may be reasonable to hypothesize that increased ROS production might be associated with an increased level of vitreous glutamate and furthermore an increased neurotoxic glutamate level may worsen neuroretinal biochemical homeostasis among subjects with PDR. Still, increased oxidative stress augments retinal nitrosative stress and their sustained increased levels are closely associated with the pathogenic process of retinopathy and might aggravate the course of retinopathy among PDR subjects. Author Disclosures This work was supported by an intramural grant from the Indian Council of Medical Research, Government of India. No potential conflicts of interest relevant to this article were reported. References 1. Cusick M, Chew EY, Chan CC, et al. Histopathology and regression of retinal hard exudates in diabetic retinopathy after reduction of elevated serum lipid levels. Ophthalmology 2003;110:2126e33. 2. Pacher P, Obrosova IG, Mabley JG, Szabo C. Role of nitrosative stress and peroxynitrite in the pathogenesis of diabetic complications. Emerging new therapeutical strategies. Curr Med Chem 2005;12:267e75. 3. Newman E, Reichenbach A. The Muller cell: a functional element of the retina. Trends Neurosci 1996;19:307e11. 4. Ng YK, Zeng XX, Ling EA. Expression of glutamate receptors and calcium binding proteins in the retina of streptozotocin induced diabetic rats. Brain Res 2004;1018:66e72. 5. Ambati J, Chalam KV, Chawla DK, et al. Elevated g-aminobutyric acid, glutamate, and vascular endothelial growth factor levels in the vitreous of patients with proliferative diabetic retinopathy. Arch Ophthalmol 1997;115:1161e6. 6. Trott D, Rizzini BL, Rossi D, et al. Neuronal and glial glutamate transporters possess and SH based redox regulatory mechanism. Eur J Neurosci 1997;9: 1236e43. 7. Trott D, Danbolt NC, Volterra A. Glutamate transporters are oxidant vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration? Trends Pharmacol Sci 1998;19:328e34. 8. DeVries SH. Bipolar cells use kainate and AMPA receptors to filter visual information into separate channels. Neuron 2000;28:847e56.

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