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Retinal Cell Biology  |   February 2013
Green Tea Is Neuroprotective in Diabetic Retinopathy
Author Affiliations & Notes
  • Kamila C. Silva
    From the Renal Pathophysiology Laboratory, Investigation on Diabetes Complications, Faculty of Medical Sciences, State University of Campinas (Unicamp), Campinas, São Paulo, Brazil; and
  • Mariana A. B. Rosales
    From the Renal Pathophysiology Laboratory, Investigation on Diabetes Complications, Faculty of Medical Sciences, State University of Campinas (Unicamp), Campinas, São Paulo, Brazil; and
  • Dania E. Hamassaki
    Department of Histology and Embryology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Kelly C. Saito
    Department of Histology and Embryology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
  • Aline M. Faria
    From the Renal Pathophysiology Laboratory, Investigation on Diabetes Complications, Faculty of Medical Sciences, State University of Campinas (Unicamp), Campinas, São Paulo, Brazil; and
  • Patrícia A. O. Ribeiro
    From the Renal Pathophysiology Laboratory, Investigation on Diabetes Complications, Faculty of Medical Sciences, State University of Campinas (Unicamp), Campinas, São Paulo, Brazil; and
  • José B. Lopes de Faria
    From the Renal Pathophysiology Laboratory, Investigation on Diabetes Complications, Faculty of Medical Sciences, State University of Campinas (Unicamp), Campinas, São Paulo, Brazil; and
  • Jacqueline M. Lopes de Faria
    From the Renal Pathophysiology Laboratory, Investigation on Diabetes Complications, Faculty of Medical Sciences, State University of Campinas (Unicamp), Campinas, São Paulo, Brazil; and
  • Corresponding author: Jacqueline M. Lopes de Faria, Faculty of Medical Sciences, University of Campinas (Unicamp), Campinas, SP, Brazil; jmlfaria@fcm.unicamp.br
Investigative Ophthalmology & Visual Science February 2013, Vol.54, 1325-1336. doi:10.1167/iovs.12-10647
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      Kamila C. Silva, Mariana A. B. Rosales, Dania E. Hamassaki, Kelly C. Saito, Aline M. Faria, Patrícia A. O. Ribeiro, José B. Lopes de Faria, Jacqueline M. Lopes de Faria; Green Tea Is Neuroprotective in Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2013;54(2):1325-1336. doi: 10.1167/iovs.12-10647.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Green tea (GT), widely studied for its beneficial properties in protecting against brain ischemia, is a rich source of polyphenols, particularly (-)-epigallocatechin gallate (EGCG). The results presented here demonstrate the beneficial effects of GT in diabetic retinas and in retinal cells under diabetic conditions.

Methods.: Diabetes was induced in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats. Treatment animals received GT orally for 12 weeks. A vehicle was administered orally to the control animals. The protective effects of GT were also evaluated in Müller and in ARPE-19 cells.

Results.: In diabetic rats, there was an increase in the expression of glial fibrillary acidic protein (GFAP), oxidative retinal markers, and glutamine synthetase levels. In addition, there was a decrease in occludin and glutamate transporter and receptor. Diabetic SHR also demonstrated blood-retinal barrier breakdown and impaired electroretinography results. Müller cells exposed to high-glucose medium produced higher levels of reactive oxygen species (ROS) and glutamine synthetase but reduced levels of glutathione, glutamate transporter, and glutamate receptor. Similarly, ARPE-19 cells exhibited increased ROS production accompanied by decreased expression of claudin-1 and glutamate transporter. Treatment with GT fully restored all the above-mentioned alterations in diabetic animals as well as in retinal cells.

Conclusions.: GT protected the retina against glutamate toxicity via an antioxidant mechanism. These findings reveal a novel mechanism by which GT protects the retina against neurodegeneration in disorders such as diabetic retinopathy.

Introduction
Diabetic retinopathy (DR) is a classic chronic microvascular complication of the retina caused by the deleterious metabolic effects of hyperglycemia, which results in extensive and early neurodegeneration. 1 Neuroretinal degeneration initiates several metabolic and signaling pathways that participate in the microvasculopathy process as well as in disturbances of the blood-retinal barrier (BRB), a key phenomenon in the pathogenesis of DR. 2  
The Müller cell is the predominant glial cell in the retina, interacting with most neurons in a symbiotic relationship. 3 Müller cells represent the anatomical and functional link between retinal neurons and also play a critical role in maintenance of the BRB 4 and its associated blood flow. 5 These cells modulate neuronal excitability and transmission through the release of gliotransmitters and other neuroactive substances, 6 neurotransmitter recycling, and the release of neurotransmitter precursors. Glutamate is the major retinal excitatory neurotransmitter in the photoreceptor-bipolar-ganglion cell circuit and is toxic when present in high concentrations, which ultimately results in neurodegeneration. 7 Previous studies have clarified the mechanisms by which glutamate is accumulated in the extracellular space in retinal tissue, including impaired glutamate uptake by glial cells 8 and a reduction in the capacity of the retina to oxidize glutamate to alpha-ketoglutarate. 9 The clearance of glutamate from the extracellular space is accomplished through the action of glutamate transporters. 10 GLAST, the high-affinity l-glutamate/L-aspartate transporter located in Müller cells, is the only glial-type glutamate transporter in the retina. In studies with GLAST-deficient mice, the electroretinogram is deeply depressed and retinal damage is exacerbated. These findings demonstrate that GLAST is required for normal signal transmission between photoreceptors and bipolar cells and plays a neuroprotective role during ischemia in the retina. 11 GLAST is essential not only to keep the extracellular glutamate concentration below a neurotoxic level but also to maintain glutathione levels by transporting glutamate, which is the substrate for glutathione synthesis, into Müller cells. This process represents a major antioxidant defense in the retina. This is the primary route for the uptake of cysteine, 12 the rate-limiting substrate for the synthesis of glutathione. 13 The reduced retinal concentrations of glutathione observed in GLAST-deficient mice suggest that glutamate neurotoxicity and oxidative stress are involved in retinal degenerative disease. 14  
The RPE is a specialized epithelium lying at the interface between the neural retina and the choriocapillaris. This layer of cells is an essential constituent of the outer BRB. It plays an important role in the proper function and maintenance of the neural retina, controlling the flow of solutes and fluid to prevent the accumulation of extracellular fluid in the subretinal space of the retina. 15 The human retinal epithelial cell line, ARPE-19, is a spontaneously immortalized cell line that has been commonly used as a model for the outer BRB because it has been demonstrated to have structural and functional properties characteristic of in vivo RPE cells. Notably, the high-affinity glutamate transporter EAAC1, an excitatory amino-acid transporter expressed by neurons in the central nervous system, 16 has been found in RPE cells. 17 Recent studies have shown that the EAAC1−/− mouse exhibits chronic mild neuron-specific oxidative stress. 12 It is believed that the EAAC1 receptor on RPE cells plays a role in regulation of the glutamate concentration in the subretinal space and also acts as a primary route for the uptake of cysteine. However, a possible role of the EAAC1 receptor in ARPE-19 cells under high-glucose conditions has not been investigated. 
Green tea (GT; Camellia sinensis ) is a rich source of polyphenols, particularly flavonoids. GT and (-)-epigallocatechin gallate (EGCG), the most active compound in GT, is reported to delay or prevent certain forms of cancer, arthritis, cardiovascular disease, and other disorders. These compounds also display strong antioxidant activity 1820 and anti-inflammatory effects. 21 In the context of diabetic retinal disease, it has been shown that GT not only reduces the level of anion production but also prevents the formation of acellular capillaries and pericyte ghosts. 20 EGCG also protects against brain ischemia. 22 Recently, it has been postulated that the neuroprotective effect of EGCG is mediated through reestablishment of the N-methyl-D-aspartate (NMDA) receptor-reactive oxygen species (ROS) system in an experimental model of Alzheimer disease. 23 To our knowledge, the effects of GT on an experimental model of DR have not been investigated thoroughly. 
In the present study, we sought to evaluate whether the oral administration of GT can protect the retina from the toxic effects of hyperglycemia. We also sought to identify the underlying mechanisms through in vitro studies. We showed that GT abrogated the retinal alterations presented in diabetic rats and that the neuroprotective effect observed in the retina was mediated by reestablishment of the glutamate cycle. 
Methods
Animal Experiments
Spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats were provided by Taconic (Germantown, NY). The study protocol complied with the care, use, and treatment of all animals in strict agreement with the guidelines of the Statement for the Use of Animals in Ophthalmic and Vision Research (ARVO) and was approved by the local Committee for Ethics in Animal Research (CEEA/IB/Unicamp). 
Diabetes was induced in 12-week-old male SHR and WKY rats with a single intravenous injection of streptozotocin (STZ, 50 mg/kg; Sigma, St. Louis, MO) or vehicle as a control. Blood glucose levels were measured by the glucose-oxidase method using a Labtest Kit (glucose PAP Liquiform, GOD-PAP colorimetric assay; Merck, Darmstadt, Germany) 72 hours after the injection and on the day before euthanasia. Values greater than or equal to 15 mmol/L were indicative of diabetes. The diabetic rats were then randomly assigned to receive oral treatment with GT (Midori Institute of the Tea, São Paulo, SP, Brazil) or water as the control. 24,25 HPLC (Waters, Milford, MA) chromatogram analyses were conducted in GT preparation to characterize it (see Supplementary Material and Supplementary Fig. S1). GT was prepared daily as described by the manufacturer: 10 g of dry tea was added to 750 mL of deionized boiled tap water cooled to 90°C and then brewed for 3 minutes, decanted, filtered, placed on ice, and protected from light with aluminum foil. 20 Fresh liquid was provided every day. The diabetic rats drank approximately 100 mL/d, which represents a dose of 5.7 g GT/kg/d. During the study, the diabetic rats received 2 units of long-acting insulin (human insulin HI-0310; Lilly, Indianapolis, IN) subcutaneously, three times per week. Systolic blood pressure (SBP) was obtained by tail-cuff plethysmography (Physiograph MK-III-S; Narco Bio-System, Houston, TX) as previously reported. 26 Twelve weeks after the induction of diabetes (DM), the rats were submitted to electroretinography and then euthanized, at which point the retinas were collected. We chose to use diabetic SHR because it has previously been shown that these rats displayed earlier and more severe retinal lesions than their normotensive counterparts, diabetic WKY, 27,28 and also because hypertension is frequently present in diabetic individuals with retinopathy. 29,30  
In Vitro Studies
Before the in vitro studies were initiated, a thiazolyl blue tetrazolium bromide (MTT) assay 31 was conducted in order to ensure that the concentrations of GT, EGCG, N-acetylcysteine (NAC), and tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) administered to retinal cells were safe, that is, below 10% of cell toxicity (data not shown). 
The GT concentrations used in the in vitro study were 1, 10, and 100 μg/mL. GT was prepared similarly for the in vitro studies as for the animal studies. 
Cell Culture
Primary Rat Retinal Müller Cells (PRRMC).
The enrichment of Müller cells was performed according to the protocol proposed by Hicks and Courtois. 32 The removal of cellular debris after 6 to 7 days yielded a purified flat cell preparation, which was maintained as a primary culture for 9 to 12 weeks. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin. All of the cells in these purified cultures stained positively with GS and GLAST monoclonal. 
Transformed Rat Retinal Müller Cell Line (rMC-1).
In this stage, rMC-1 cells, kindly donated by Vijay J. Sarthy, PhD (Northwestern University, Evanston, IL), were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin for 24, 48, and 72 hours. 
ARPE-19 Cell Line Culture.
ARPE-19 cells were obtained from the Federal University of Rio de Janeiro (RJCB Collection). Cells were cultured in DMEM and Ham's F12 (DMEM:F12) supplemented with 10% FBS and 1% penicillin/streptomycin. 
PRRMC, rMC-1, or ARPE-19 cell cultures at 70% of confluence were serum starved by reducing the FBS concentration to 2%, then exposed to 5.5-mM d-glucose (NG); 25-mM d-glucose (HG); HG plus 1, 10, or 100 μg/mL of GT (HG+GT); or with different treatments as specified; NG + 19.5 mM of mannitol was used as an osmotic control. 
Immunohistochemistry in Retinal Tissues and Immunofluorescence in Retinal Cells
The immunohistochemistry was performed as previously described. 26 The retinal sections were incubated with goat polyclonal antiglial fibrillary acidic protein (GFAP; Santa Cruz Biotechnologies, Santa Cruz, CA), rabbit polyclonal antioccludin (Invitrogen, Camarillo, CA), rabbit polyclonal antinitrotyrosine (NT; Upstate Cell Signaling Solutions, Lake Placid, NY), or rabbit polyclonal to phospho-neuronal nitric oxide synthase (nNOS, Ser847) (Abcam, Inc., Cambridge, MA) overnight at 4°C. The slides were then incubated with the appropriate secondary antibodies. The primary rat Müller cells and ARPE-19 cells were fixed in 2% paraformaldehyde, washed, and blocked. The primary Müller cells were stained with anti-GFAP antibody (Santa Cruz Biotechnologies), and ARPE-19 cells for claudin-1 (Invitrogen) followed by the appropriate secondary antibody. The analyses were performed by an observer with no knowledge of the groups studied using the Leica Application Suite (LAS Image Analysis; Leica Microsystems, Wetzlar, Germany) in nine nonconsecutive retinal sections divided among three slides per animal per group under high-powered microscopic viewing (×1000) or using confocal laser-scanning microscopy (Zeiss, Jena, Germany). 
Western Blotting
The protein obtained from each sample (retina or cell culture) was subjected to SDS-PAGE in a Bio-Rad slab gel apparatus (Mini-PROTEAN Tetra cell; Bio-Rad, Hercules, CA) and electrophoretically transferred to a nitrocellulose membrane. The membranes were incubated with primary antibodies for antiphospho-nNOS (Ser847) (Abcam, Inc.), antinNOS (Cell Signaling, Danvers, MA), antiphospho-endothelial nitric oxide synthase (eNOS, Ser1177) (Cell Signaling), anti-eNOS/NOS type III (BD Transduction Laboratories, San Diego, CA), anticopper/zinc superoxide dismutase (Cu/Zn-SOD; Upstate, Millipore, Billerica, MA), anti-GLAST (Alpha Diagnostic International, San Antonio, TX), anti-Nmdar1 (Millipore, Temecula, CA), anti-GS (Millipore, Temecula, CA), anticlaudin-1 (Invitrogen), anti-EAAC1 (Alpha Diagnostic International), antiphospho-Akt1/2/3 (Ser 473; Santa Cruz Biotechnology), and anti-Akt1/2/3 (Santa Cruz Biotechnology). 
Cyclic Guanosine Monophosphate ELISA
The retinal samples were prepared for the ELISA in accordance with the recommendations provided with commercial cyclic guanosine monophosphate (cGMP) ELISA assays (Cayman Chemical Company, Ann Arbor, MI). The protein concentrations were measured using the Bradford method. 33  
BRB Permeability in Whole-Mounted Retinas
The BRB was examined using the Evans blue method as previously reported. 34 For the evaluation of BRB breakdown sites, the retinal flat mounts were analyzed by confocal laser-scanning microscopy (CLSM, LSM510; Zeiss) using the appropriate emission filters. The digital images were captured using specific software (LSM; Zeiss) and then compared using a semiquantitative scale. 
Full-Flash Electroretinogram (ERG) Recording
Retinal function was measured in SHR animals at the end of the study using the UTAS-E3000 system (UTAS-E3000; LKG Technologies Inc., Gaithersburg, MD) as previously described with some modifications. 35 The pupils were dilated with tropicamide (Mydriacyl 0.5%; Allergan, Irvine, CA). General anesthesia was induced with ketamine and xylazine (75 and 7.5 mg/kg, respectively) under dim red illumination (λmax = 650 nm). The measurements were taken after overnight dark adaptation. A dark-adapted intensity-response series was recorded using a series of Ganzfeld flashes with intensities ranging from −3.60 to 2.40 log cd-s/m2 luminance. Recordings were amplified and digitized using a 24-bit A/D converter band-passed from 0.3 to 300 Hz with a 50-Hz notch filter. 
Measuring Intracellular ROS Production in Cell Culture
As previously described, 24 cells were grown on a 96-well plate and incubated for 30 minutes with 10 μM 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCF-DA) (Invitrogen) in Hank's buffer. The amount of fluorescence is measured using a fluorescence plate reader (SynergyMx; Biotek, Winooski, VT) at excitation and emission wavelengths of 485 and 528 nm, respectively. The qualitative assessment of ROS was carried out in the ARPE-19 cells after 24 hours of treatment. The cells were incubated for 30 minutes with 10 μM H2DCF-DA, washed, and imaged using a fluorescence microscope (Olympus IX-71; Olympus, Center Valley, PA). 
Calculating the Reduced Glutathione Expression
The reduced glutathione (GSH) levels in rMC-1 culture were measured using methods described previously. 26 The GSH concentration is expressed as μM GSH/μg protein. GSH was used for the preparation of a standard curve. 
Real-Time PCR in Müller Cells
Real-time PCR was performed as previously described. 36 Total RNA was isolated from Müller cells using Trizol reagent (Invitrogen). Primers for the GLAST (Glut1) and Nmdar1 (Grin1) genes were obtained from Applied Biosystems (Rn00570130_m1 and Rn01436034_m1; Applied Biosystems, Carlsbad, CA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression was used as a control (Applied Biosystems). 
Measurement of Glutamine Synthetase Activity
Glutamine synthetase (GS) activity was performed by colorimetric assay as previously described. 37  
Statistical Analysis
The results were expressed as the means ± SD. The groups were compared by one-way ANOVA, followed by the Fisher-protected least-significant difference test. For the ERG analyses, the area under the curve (AUC) was used to compare the ERG responses from each animal. The results were compared by ANOVA, followed by the Fisher-protected least-significant difference test. Statistical differences were considered significant at P less than 0.05. 
Results
Animal Experiments
GT treatment did not alter body weight or blood glucose levels. The final body weight was lower and blood glucose levels were higher in diabetic rats compared with nondiabetic groups (P = 0.002 and P < 0.0001, respectively). Systolic blood pressure was higher in SHR than in WKY rats (P < 0.0001) (Table). GT treatment did not alter any of the physiological parameters. 
Table. 
 
Physiological Characteristics of Studied Animals
Table. 
 
Physiological Characteristics of Studied Animals
Groups Initial Body Weight, g Final Body Weight, g Systolic Blood Pressure, mm Hg Glycemia, mmol/L
CT-WKY 322.3 ± 22 498.7 ± 48 143.7 ± 5 8.9 ± 0.4
DM-WKY 331.2 ± 16 384.6 ± 66* 139.8 ± 3 28.5 ± 5†
DM-WKY GT 325.6 ± 16 367.2 ± 42* 137.5 ± 8 27.4 ± 7†
CT-SHR 277.2 ± 17 345.5 ± 13 204.3 ± 10§ 8.4 ± 1
DM-SHR 277.6 ± 7 192.2 ± 38‡ 203.5 ± 8§ 29.5 ± 2‡
DM-SHR GT 269.7 ± 15 194.9 ± 25‡ 198.9 ± 10§ 30.5 ± 5‡
The Oral Administration of GT Ameliorated the Early Markers of DR.
The presence of diabetes markedly exacerbated GFAP staining throughout the retinal tissue in all cell types in both rat strains (P < 0.001) (Fig. 1A). The presence of diabetes also reduced the expression of occludin as compared with the levels observed in control rats (P < 0.03) (Fig. 1B). Occludin immunolabeling occurs mainly in the outer plexiform, inner nuclear, and ganglion cell layers. Treatment with oral GT prevented these alterations in diabetic rats, which suggests that GT exerts a protective effect in the early phases of DR. 
Figure 1. 
 
GT regulates the early markers of diabetic retinopathy, including GFAP and occludin levels. (A) A representative photomicrograph of glial reactivity as revealed by GFAP immunohistochemistry in rat retinas from WKY and SHR. In nondiabetic rat retinas, there was a light retinal glial reaction mainly in astrocytes. After 3 months of diabetes, marked staining can be observed throughout the retina; the oral administration of GT reduced this diabetes-mediated response. Magnification: ×100. The bars represent mean ± SD for the percentage of GFAP-positive retinal cells per mm2 of retina. *P = 0.02 and *P = 0.0003 for WKY and SHR, respectively. (B) Representative photomicrograph of the immunolocalization of occludin in WKY and SHR animals. In the control groups, occludin is present in the ganglion cell layer and in the inner nuclear layer around the vessels. In diabetic animals, there is a reduction in occludin expression; GT treatment reversed this effect. Magnification: ×100. The bars represent mean ± SD for the percentage of positivity of occludin/mm2 of retina. *P = 0.02 and *P = 0.01 for WKY and SHR, respectively. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; CT WKY, control WKY; DM WKY, diabetic WKY; DM WKY GT, diabetic WKY treated with GT; CT SHR, control SHR; DM SHR, diabetic SHR; DM SHR GT, diabetic SHR treated with GT.
Figure 1. 
 
GT regulates the early markers of diabetic retinopathy, including GFAP and occludin levels. (A) A representative photomicrograph of glial reactivity as revealed by GFAP immunohistochemistry in rat retinas from WKY and SHR. In nondiabetic rat retinas, there was a light retinal glial reaction mainly in astrocytes. After 3 months of diabetes, marked staining can be observed throughout the retina; the oral administration of GT reduced this diabetes-mediated response. Magnification: ×100. The bars represent mean ± SD for the percentage of GFAP-positive retinal cells per mm2 of retina. *P = 0.02 and *P = 0.0003 for WKY and SHR, respectively. (B) Representative photomicrograph of the immunolocalization of occludin in WKY and SHR animals. In the control groups, occludin is present in the ganglion cell layer and in the inner nuclear layer around the vessels. In diabetic animals, there is a reduction in occludin expression; GT treatment reversed this effect. Magnification: ×100. The bars represent mean ± SD for the percentage of positivity of occludin/mm2 of retina. *P = 0.02 and *P = 0.01 for WKY and SHR, respectively. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; CT WKY, control WKY; DM WKY, diabetic WKY; DM WKY GT, diabetic WKY treated with GT; CT SHR, control SHR; DM SHR, diabetic SHR; DM SHR GT, diabetic SHR treated with GT.
GT Treatment Prevented Oxidative Stress in Diabetic Rats Treated with GT.
NT expression serves as an index for the nitration of tyrosine, which is mediated by peroxynitrite. In diabetic as compared to control rats, NT staining was stronger throughout the layers of the retina (mainly in the inner and outer plexiform layers and outer segments of the photoreceptors) (P = 0.002 and P = 0.04, for WKY and SHR, respectively). GT completely restored retinal nitrosative status to nondiabetic rat levels (P < 0.04) (Fig. 2A). There was a tendency toward decreased expression of Cu/Zn-SOD, an important mediator of antioxidant defense, in diabetic SHR. In treated animals, we observed a marked elevation in Cu/Zn-SOD levels compared with those observed in nontreated groups (P = 0.0006) (Fig. 2B). 
Figure 2. 
 
Retinal oxidative stress was ameliorated by oral GT. (A) A representative photomicrograph of the immunolocalization of nitrotyrosine in WKY and SHR animals. Nitrotyrosine is a stable product formed from the reaction of peroxynitrate with tyrosine residues and is accepted as an index of nitrosative damage. In normal retinas, the signal of nitrotyrosine is faint and present in all retinal layers; in diabetic retinas, the staining is stronger and observed throughout the retinal tissue. This abnormality is abrogated by GT administration. The bars represent mean ± SD for the percentage of nitrotyrosine-positive retinal cells/mm2 of retina. *P = 0.04. (B) Western blot analysis of the Cu/Zn SOD enzyme in total retinal lysates of the SHR. The SOD family is a major antioxidant system, and a deficiency of Cu/Zn- SOD1 leads to features of ROS-mediated retinal degeneration. In this present study, there was no significant decrease in Cu/Zn SOD levels after 3 months of diabetes, but the administration of GT doubled SOD levels. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD for the band densities expressed in arbitrary densitometric units from at least three independent experiments. *P = 0.0006.
Figure 2. 
 
Retinal oxidative stress was ameliorated by oral GT. (A) A representative photomicrograph of the immunolocalization of nitrotyrosine in WKY and SHR animals. Nitrotyrosine is a stable product formed from the reaction of peroxynitrate with tyrosine residues and is accepted as an index of nitrosative damage. In normal retinas, the signal of nitrotyrosine is faint and present in all retinal layers; in diabetic retinas, the staining is stronger and observed throughout the retinal tissue. This abnormality is abrogated by GT administration. The bars represent mean ± SD for the percentage of nitrotyrosine-positive retinal cells/mm2 of retina. *P = 0.04. (B) Western blot analysis of the Cu/Zn SOD enzyme in total retinal lysates of the SHR. The SOD family is a major antioxidant system, and a deficiency of Cu/Zn- SOD1 leads to features of ROS-mediated retinal degeneration. In this present study, there was no significant decrease in Cu/Zn SOD levels after 3 months of diabetes, but the administration of GT doubled SOD levels. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD for the band densities expressed in arbitrary densitometric units from at least three independent experiments. *P = 0.0006.
Constitutive nNOS Expression Is Impaired in Diabetic SHR.
Synaptic activity regulates the production of nitric oxide (NO), which is catalyzed by phosphorylated nNOS. The phosphorylation of nNOS by calcium-calmodulin protein kinase II at Ser847 (phospho-nNOS Ser847) inhibits its activity. 38 There was no alteration in the expression of nNOS among the WKY rats studied (P > 0.05). In contrast, diabetic SHR presented a clear increase in the expression of inactive nNOS as compared with control animals (P < 0.0001). Treatment with GT restored expression to normal levels (P = 0.01) (Figs. 3A, 3B). These findings suggest that the changes in phospho-nNOS were dependent on the presence of hypertension. The exacerbation of retinopathy by hypertension likely involves nNOS inactivation. Further studies are necessary to clarify these results. 
Figure 3. 
 
The oral administration of GT re-establishes the constitutive neuronal nitric oxide synthase/cyclic GMP pathway in diabetic SHR. (A) The levels of constitutive neuronal NOS and its phosphorylated form at Ser847 (inactivated form) were examined in total retinal lysates using western blot analyses. In WKY rats, there was no change in the phospho-nNOS: total nNOS: β-actin ratio after 12 weeks of DM induction. This ratio was altered in diabetic SHR. Activation of nNOS was reduced in retinal tissue; GT reversed this effect. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities expressed in arbitrary densitometric units from at least three independent experiments. *P = 0.001. (B) A representative photomicrograph of the immunolocalization of phospho-nNOS in SHR animals. The phospho-nNOS staining exhibits a scattered pattern located mainly in the inner plexiform layer. In control rats, there was positivity mainly in the IPL; there is a marked increase in expression in the presence of DM. Treatment with GT reduced the magnitude of this increase. Magnification: ×100. (C) Cyclic guanosine monophosphate (cGMP) levels were analyzed by ELISA in retinas from SHR. The values represent mean ± SD of cGMP levels in pmol/mg protein from retinal tissue. *P < 0.05.
Figure 3. 
 
The oral administration of GT re-establishes the constitutive neuronal nitric oxide synthase/cyclic GMP pathway in diabetic SHR. (A) The levels of constitutive neuronal NOS and its phosphorylated form at Ser847 (inactivated form) were examined in total retinal lysates using western blot analyses. In WKY rats, there was no change in the phospho-nNOS: total nNOS: β-actin ratio after 12 weeks of DM induction. This ratio was altered in diabetic SHR. Activation of nNOS was reduced in retinal tissue; GT reversed this effect. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities expressed in arbitrary densitometric units from at least three independent experiments. *P = 0.001. (B) A representative photomicrograph of the immunolocalization of phospho-nNOS in SHR animals. The phospho-nNOS staining exhibits a scattered pattern located mainly in the inner plexiform layer. In control rats, there was positivity mainly in the IPL; there is a marked increase in expression in the presence of DM. Treatment with GT reduced the magnitude of this increase. Magnification: ×100. (C) Cyclic guanosine monophosphate (cGMP) levels were analyzed by ELISA in retinas from SHR. The values represent mean ± SD of cGMP levels in pmol/mg protein from retinal tissue. *P < 0.05.
The cGMP Levels in Diabetic Retinas Were Counteracted by the Administration of GT.
To assess a possible link between nNOS and diabetic neurotoxicity, we quantified the levels of cGMP in retinal tissue using an ELISA assay. We observed a reduction in cGMP levels in DM-SHR as compared with CT-SHR (P = 0.003). Treatment with GT prevented this increment (P < 0.05) (Fig. 3C). 
GT Prevents the DM-Induced Activation of Constitutive eNOS.
To understand the role of eNOS in BRB breakdown, the NOS system in retinal tissue was also assessed by measuring eNOS levels. It is known that the activation of eNOS along with inflammatory markers contributes strongly to BRB breakdown. 39 The NO produced by eNOS is essential for the increased vascular permeability induced by VEGF. 40 In DM-SHR, there was an increase in the levels of phospho-eNOS Ser1177 (P = 0.02). This upregulation was prevented by GT (P = 0.05, Fig. 4A). The phosphorylation of eNOS at Ser1177 activates eNOS. When comparing the retinas in diabetic rats to those in controls, the inner BRB in flat-mounted retinas displayed multiple and extensive sites of vascular leakage when examined using the Evans blue method. Treatment with GT significantly ameliorated this effect (Fig. 4B). 
Figure 4. 
 
GT prevented eNOS activation in diabetic SHR leading to a re-establishment of the BRB. (A) Western blot analysis of phospho-eNOS and total eNOS levels in total retinal lysates from SHR. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities expressed in arbitrary densitometric units from at least three independent experiments. *P < 0.05. (B) Representative images of vessel leakage in whole-mounted retinas. The fluorescein leakage in retinal tissue was classified based on the extent and sites of retinal leakage. Level 0: the absence of fluorescein leakage throughout the entire retina; Level 1: the presence of fluorescein leakage in only one quadrant; Level 1+: more than two sites of leakage in only one quadrant; Level 2: the presence of fluorescein leakage in two quadrants; Level 2+: more than two sites of leakage in one of two quadrants; Level 2++: more than two sites of leakage in both of two quadrants; Level 3: the presence of fluorescein leakage in three quadrants; Level 3+: more than two sites of leakage in one of three quadrants; Level 3++: more than two sites of leakage in two of three quadrants; Level 3+++: more than two sites of leakage in three quadrants; Level 4: the presence of fluorescein leakage in all four quadrants. In each control flat-mounted retina, the capillary bed was intact and minimal leakage was observed. After 12 weeks of DM, Evans blue dye leaked into retinal tissue at several sites, which indicated inner BRB breakdown. The oral administration of GT reduced the leakage to normal levels. (Scale bar: 200 μm for all groups. In DM-SHR, higher magnification was used for better visualization; scale bar: 100 μm.)
Figure 4. 
 
GT prevented eNOS activation in diabetic SHR leading to a re-establishment of the BRB. (A) Western blot analysis of phospho-eNOS and total eNOS levels in total retinal lysates from SHR. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities expressed in arbitrary densitometric units from at least three independent experiments. *P < 0.05. (B) Representative images of vessel leakage in whole-mounted retinas. The fluorescein leakage in retinal tissue was classified based on the extent and sites of retinal leakage. Level 0: the absence of fluorescein leakage throughout the entire retina; Level 1: the presence of fluorescein leakage in only one quadrant; Level 1+: more than two sites of leakage in only one quadrant; Level 2: the presence of fluorescein leakage in two quadrants; Level 2+: more than two sites of leakage in one of two quadrants; Level 2++: more than two sites of leakage in both of two quadrants; Level 3: the presence of fluorescein leakage in three quadrants; Level 3+: more than two sites of leakage in one of three quadrants; Level 3++: more than two sites of leakage in two of three quadrants; Level 3+++: more than two sites of leakage in three quadrants; Level 4: the presence of fluorescein leakage in all four quadrants. In each control flat-mounted retina, the capillary bed was intact and minimal leakage was observed. After 12 weeks of DM, Evans blue dye leaked into retinal tissue at several sites, which indicated inner BRB breakdown. The oral administration of GT reduced the leakage to normal levels. (Scale bar: 200 μm for all groups. In DM-SHR, higher magnification was used for better visualization; scale bar: 100 μm.)
Previous studies have demonstrated that serine/threonine protein kinase Akt/PKB mediates the activation of eNOS. The results of these studies demonstrate that activation of phosphatidylinositol 3-kinase and Akt signaling to eNOS represents a regulatory mechanism for activation of eNOS. 41,42 Based on this, we investigated whether the phosphorylation of eNOS in SHR animals was due to activation of Akt. We observed, by Western blot, that diabetes promotes a significant increase in Akt phosphorylation in retinal lysates from SHR rats compared with control rats (P = 0.001), and the treatment with GT completely restored this activation (P = 0.002) (see Supplementary Material and Supplementary Figs. S2A–C). 
In in vitro studies, we also observed that the rMC-1 exposed to high glucose had increased Akt phosphorylation accompanied by increased Ser 1177eNOS phosphorylation compared with normal glucose (P = 0.0007 and P = 0.007, respectively), and the treatment with GT prevented these phosphorylations (P = 0.005 and P = 0.0008, respectively) in a dose-dependent manner (see Supplementary Material and Supplementary Figs. S2D, S2E, S3). The treatment with Akt Inhibitor VIII (Calbiochem; Merck, Darmstadt, Germany, 10 uM) resulted in a similar response: significant reduction of p-Akt and p-eNOS to normal levels (see Supplementary Material and Supplementary Fig. S3). 
Alterations in the Retinal Glutamate/Glutamine Cycle Were Prevented by Oral Treatment with GT in the Diabetic SHR Group.
The glutamate cycle was investigated by measuring glutamate transporter, glutamate receptor, and glutamate-metabolizing enzyme (GS) expression in the retinas of SHR. Diabetic SHR animals exhibited a significant decrease in the retinal expression of GLAST (P = 0.04) and Nmdar1 (P < 0.05) proteins. This was associated with an increase in GS levels (P = 0.04). Notably, GT treatment prevented all of these alterations (P = 0.0008, P = 0.03, and P = 0.04, respectively) (Figs. 5A–C). 
Figure 5. 
 
GT exerts a neuroprotective effect in SHR. (A) Representative Western blots for GLAST in total retinal lysates from SHR animals. *P = 0.04. (B) Representative Western blots for Nmdar1 in total retinal lysates. *P < 0.05. (C) Representative Western blots for glutamine synthetase in total retinal lysates. *P = 0.04. Equal loading and transfer for all proteins were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities from at least three independent experiments in terms of the percentage of variation (GLAST and GS) or arbitrary densitometric units (Nmdar1). (D) Representative waveforms for a- and b-waves in the rats studied. The a-wave is the first negative deflection and represents the activity of the photoreceptors. The inner retinal function response, the b-wave, is a positive deflection generated in part by the Müller and mainly by the bipolar cell potentials. The figure shows representative full-flash ERG waveform for a CT-SHR (red), DM-SHR (blue), and DM-SHR GT (green). The bars represent the mean amplitude and latency in AUC of the a- and b-wave components and total latency of the ERG from the SHR animals. *P < 0.05 and #P < 0.03.
Figure 5. 
 
GT exerts a neuroprotective effect in SHR. (A) Representative Western blots for GLAST in total retinal lysates from SHR animals. *P = 0.04. (B) Representative Western blots for Nmdar1 in total retinal lysates. *P < 0.05. (C) Representative Western blots for glutamine synthetase in total retinal lysates. *P = 0.04. Equal loading and transfer for all proteins were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities from at least three independent experiments in terms of the percentage of variation (GLAST and GS) or arbitrary densitometric units (Nmdar1). (D) Representative waveforms for a- and b-waves in the rats studied. The a-wave is the first negative deflection and represents the activity of the photoreceptors. The inner retinal function response, the b-wave, is a positive deflection generated in part by the Müller and mainly by the bipolar cell potentials. The figure shows representative full-flash ERG waveform for a CT-SHR (red), DM-SHR (blue), and DM-SHR GT (green). The bars represent the mean amplitude and latency in AUC of the a- and b-wave components and total latency of the ERG from the SHR animals. *P < 0.05 and #P < 0.03.
Retinal Function Revealed an Important Protective Effect of GT in Diabetic Rats.
A significant decrease in the amplitude of the b-wave, an increase in the implicit time of the b-wave, and markedly reduced oscillatory potentials were observed in DM-SHR as compared with CT-SHR (P < 0.05). Treatment with oral GT alleviated these effects, which improved inner retinal function (P = 0.03) (Fig. 5D). 
In Vitro Studies
Primary Müller Cells.
Exposure to HG medium evoked a marked upregulation in the expression of GFAP, which is used to mark glial cells. Treatment with GT abolished this increase (Fig. 6A). The glutamate/glutamine cycle was investigated by measuring the expression of glutamate transporter, glutamate receptor, and GS in Müller cells. After 72 hours in HG conditions, GLAST and Nmdar1 protein levels were significantly decreased when compared to the levels observed in cells in the NG medium (P < 0.05). This effect was accompanied by a compensatory increase in GS expression levels (P = 0.01) and activity when compared with control Müller cells (P = 0.0006) (see Supplementary Material and Supplementary Fig. S4). GT treatment prevented these alterations (Figs. 6B, 6C, 6E). Similarly, quantitative real-time PCR for Nmdar1 mRNA showed decreased levels in the cells incubated in HG medium (P = 0.002). Treatment with GT increased Nmdar1 mRNA expression, but this trend failed to achieve statistical significance (Fig. 6D). 
Figure 6. 
 
GT exerts a neuroprotective effect in primary rat Müller cells and restores the oxidative balance in rMC-1. (A) Immunofluorescence for GFAP in primary Müller cells cultured for 72 hours. Scale bar: 20 μm. (B) Representative Western blots for GLAST in total cell lysates. *P < 0.03. (C) Representative Western blots for NMDAR1 in total cell lysates. *P < 0.04. (D) The Nr1 gene expression profile normalized to GAPDH in primary Müller cell mRNA. *P = 0.002. (E) Representative Western blots for glutamine synthetase in total cell lysates. *P < 0.01. Equal loading and transfer for all proteins were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities expressed as the percentage of variation from at least three independent experiments. (F) The quantification of total intracellular ROS levels in rMC-1 cultured for 24 hours in normal d-glucose (NG, 5 mM) and high d-glucose (HG, 25 mM) in the presence or absence of GT (HG + GT: 1, 10, and 100 μg/mL). Mannitol (MAN) was used as an osmotic control. The values are means ± SD and expressed as percentages of fluorescence units. Values were corrected by the number of cells at the end of each treatment. *P < 0.01 versus NG; #P < 0.01 versus HG; ¶P < 0.04 versus HG+GT 1 μg/mL; §P < 0.0007 versus HG+GT 10 μg/mL. (G) Concentrations of reduced glutathione (GSH) from rMC-1 cells cultured for 24 hours (μM glutathione/μg of protein). Treatment with GT (10–100 μg/mL) or NAC (1 mM) prevented the decrease in GSH levels in cells in HG. The bars represent mean ± SD. *P < 0.001. (H) The GLAST gene expression profiles normalized with GAPDH in rMC-1. *P = 0.02 versus NG; #P = 0.02 versus HG; ¶P < 0.05 versus HG. (I) The Nr1 gene expression profile normalized with GAPDH in rMC-1. *P < 0.04 versus NG; #P = 0.03 versus HG. NG, normal d-glucose; HG, high d-glucose; HG+GT, high d-glucose + 100 μg/mL of green tea; MAN, mannitol.
Figure 6. 
 
GT exerts a neuroprotective effect in primary rat Müller cells and restores the oxidative balance in rMC-1. (A) Immunofluorescence for GFAP in primary Müller cells cultured for 72 hours. Scale bar: 20 μm. (B) Representative Western blots for GLAST in total cell lysates. *P < 0.03. (C) Representative Western blots for NMDAR1 in total cell lysates. *P < 0.04. (D) The Nr1 gene expression profile normalized to GAPDH in primary Müller cell mRNA. *P = 0.002. (E) Representative Western blots for glutamine synthetase in total cell lysates. *P < 0.01. Equal loading and transfer for all proteins were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities expressed as the percentage of variation from at least three independent experiments. (F) The quantification of total intracellular ROS levels in rMC-1 cultured for 24 hours in normal d-glucose (NG, 5 mM) and high d-glucose (HG, 25 mM) in the presence or absence of GT (HG + GT: 1, 10, and 100 μg/mL). Mannitol (MAN) was used as an osmotic control. The values are means ± SD and expressed as percentages of fluorescence units. Values were corrected by the number of cells at the end of each treatment. *P < 0.01 versus NG; #P < 0.01 versus HG; ¶P < 0.04 versus HG+GT 1 μg/mL; §P < 0.0007 versus HG+GT 10 μg/mL. (G) Concentrations of reduced glutathione (GSH) from rMC-1 cells cultured for 24 hours (μM glutathione/μg of protein). Treatment with GT (10–100 μg/mL) or NAC (1 mM) prevented the decrease in GSH levels in cells in HG. The bars represent mean ± SD. *P < 0.001. (H) The GLAST gene expression profiles normalized with GAPDH in rMC-1. *P = 0.02 versus NG; #P = 0.02 versus HG; ¶P < 0.05 versus HG. (I) The Nr1 gene expression profile normalized with GAPDH in rMC-1. *P < 0.04 versus NG; #P = 0.03 versus HG. NG, normal d-glucose; HG, high d-glucose; HG+GT, high d-glucose + 100 μg/mL of green tea; MAN, mannitol.
Rat MC-1 (rMC-1) Cells.
To address the relationship between oxidative stress and neurodegeneration, we evaluated the oxidative balance and glutamate pathway in Müller cells. We evaluated the time course effect of HG medium in the production of ROS. After 48 and 72 hours of treatment, the increase in ROS production was not significant (see Supplementary Material and Supplementary Fig. S5). Therefore, the experiments were conducted at 24 hours of treatment, when a significant difference in ROS production between NG and HG was detected (P < 0.05). 
The rMC-1 cells exposed to the HG medium displayed an elevation in ROS production as compared to those exposed to the NG medium (P = 0.01). The presence of GT at 10 and 100 μg/mL prevented this increase (P < 0.001) (Fig. 6F). GSH levels decreased in the HG as compared to the NG condition (P = 0.001). Similarly, GT treatment at 10 to 100 μg/mL restored GSH levels to those observed in the NG condition (P = 0.006) (Fig. 6G). To verify whether impairment of the GSH system is due to the compromised uptake of cysteine in HG conditions, we treated the cells with NAC (1 mM), a direct intracellular cysteine donor. As expected, the supplementation of cysteine fully counteracted the GSH depletion (P = 0.1) In contrast, treatment with tempol (1 mM), a superoxide dismutase mimetic, did not affect GSH levels (P = 0.5 versus HG). These findings indicate that in HG conditions, rMC-1 cells present markedly inadequate oxidative defense. This is probably due to a cysteine deficiency. As observed with the markers of oxidative stress, the gene expression of GLAST and Nmdar1 decreased dramatically. GT counteracted this effect (10 and 100 μg/mL for GLAST and 100 μg/mL for Nmdar1, P < 0.05) (Figs. 6H, 6I). It appears that impaired glutamate transporter and receptor function as well as the compensatory upregulation of a metabolizing enzyme after 24 hours (as observed under HG conditions) limited cysteine uptake. This reduced GSH levels and increased ROS production. Treatment with GT counteracted the functional impairment of both the glutamate receptor and the glutamate transporter. These measures protect the glial cells against HG-induced oxidative stress. 
ARPE-19 Cells.
Because ARPE-19 cells express glutamate transporter (EAAC1) and represent an in vitro model of the outer BRB, we evaluated the possible effects of GT and EGCG treatments on ROS production, EAAC1 protein expression, and tight-junction integrity. As observed in Müller cells, the presence of HG increased total intracellular levels of ROS (P < 0.0001). Treatment with GT or EGCG counteracted this effect (P < 0.0001) (Fig. 7A). At the same time, EAAC1 protein expression decreased, but this trend failed to achieve statistical significance. The presence of GT reversed this effect (P = 0.03) (Fig. 7B). The integrity of the intercellular junction, as represented by claudin-1 expression, decreased markedly in the HG medium (P = 0.02). GT treatment reversed this effect (P = 0.03) (Figs. 7C, 7D). These observations indicate that GT treatment protected ARPE-19 cells from the oxidative stress induced in HG conditions and improved the function of glutamate receptor EAAC1 by restoring the original structure of the intercellular junction. 
Figure 7. 
 
GT prevented outer BRB dysfunction, ROS production and decreased glutamate transport levels in ARPE-19 cells. (A) Representative fluorescent microscopic images of H2DCF-DA in ARPE-19 cells. The fluorescence intensity of H2DCF-DA indicates the level of ROS production. The bars represent the quantification of total intracellular ROS levels in ARPE-19 cells cultured for 24 hours. The values are means ± SD and expressed as percentages of fluorescence units. The values were corrected by the number of cells at the end of each treatment. *P < 0.0001. (B) Representative Western blots for claudin-1 in ARPE-19 cells. *P < 0.02. (C) Immunofluorescence analysis for claudin-1 in ARPE-19 cells. Scale bar: 20 μm. (D) Representative Western blots for EAAC1 in ARPE-19 cells. *P < 0.03. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD for the band densities expressed as a percentage of variation from at least three independent experiments. NG, normal d-glucose; HG, high d-glucose, HG+GT, high d-glucose + 100 μg/mL of GT; HG+EGCG (10, 25, and 50 μM), high d-glucose plus (-)-epigallocatechin gallate; MAN, mannitol.
Figure 7. 
 
GT prevented outer BRB dysfunction, ROS production and decreased glutamate transport levels in ARPE-19 cells. (A) Representative fluorescent microscopic images of H2DCF-DA in ARPE-19 cells. The fluorescence intensity of H2DCF-DA indicates the level of ROS production. The bars represent the quantification of total intracellular ROS levels in ARPE-19 cells cultured for 24 hours. The values are means ± SD and expressed as percentages of fluorescence units. The values were corrected by the number of cells at the end of each treatment. *P < 0.0001. (B) Representative Western blots for claudin-1 in ARPE-19 cells. *P < 0.02. (C) Immunofluorescence analysis for claudin-1 in ARPE-19 cells. Scale bar: 20 μm. (D) Representative Western blots for EAAC1 in ARPE-19 cells. *P < 0.03. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD for the band densities expressed as a percentage of variation from at least three independent experiments. NG, normal d-glucose; HG, high d-glucose, HG+GT, high d-glucose + 100 μg/mL of GT; HG+EGCG (10, 25, and 50 μM), high d-glucose plus (-)-epigallocatechin gallate; MAN, mannitol.
Discussion
The present study aimed to evaluate the potential protective effect of GT when used as an oral antioxidant in diabetic retinas. In animal experiments, GT was able to ameliorate the structural lesions present in DR and restore the glutamate transporter, glutamate receptor and GS. This maintained a level of retinal function similar to that observed in nondiabetic animals. To identify the major pathways involved in retinal protection, in vitro studies were carried out using retinal cells. GT played a pivotal role by counteracting glutamine/glutamate impairment in Müller cells. GT induced this effect by restoring the cysteine route, which prevented the oxidative stress-related excitotoxicity present under HG conditions. In ARPE-19 cells, GT prevented the decrease in EAAC1 transporter expression and reduced ROS production, thus reestablishing the intercellular junction and regulating the subretinal environment. These results provide novel evidence that GT acts as a potent neuroprotector in the diabetic retina. These effects of GT were independent of glycemic or blood pressure controls. Our study reveals a number of important findings that have not been described before, that is, the mechanisms by which improvement of oxidative stress restores the glutamine/glutamate cycle, which leads to the neuroprotection of diabetic retina and the preservation of tight junction proteins and EAAC1 expressions at RPE levels, thus maintaining the subretinal environment. 
The treatment regimen adopted in this study was the same as in a previous study by Mustata et al., 20 in which after 12 months the authors could demonstrate effective protection from retinal vascular lesions in treated diabetic rats. A more recent study investigating the possible antioxidative stress and anti-inflammatory effects for diabetic rats for 16 weeks could demonstrate the improvement of antioxidant defenses and diminishment of inflammatory markers, preventing the thickening of basement membrane 21 with a much lower dose of GT (200 mg/kg/d). Assuming equivalency on the basis of mg/m2, 43 the appropriate dose in an adult subject would vary from 0.7 to 0.03 g/kg/d of GT, based on data from the current study or from recently a published article, 21 respectively. From that, an adult subject should ingest from 2 to 50 g of GT per day. These calculations are important to demonstrate that the treatment is feasible and may be beneficial in the prevention and/or treatment of diabetic retinopathy in human subjects. 
Previous studies have suggested mechanisms by which hypertension exacerbates retinopathy in diabetic models, such as in vitro studies in which cyclic stretch upregulated VEGF and its receptor in retinal endothelial cells. 44,45 Previous data from our groups have already shown that experimentally, diabetic SHR exhibit more severe neurodegeneration, inflammation, and oxidative stress as compared with normotensive WKY rats. 27,28 After 12 weeks of induced DM, levels of the inactive form of nNOS were observed only in hypertensive rats. This could be attributed to the duration of DM, which might not be sufficient to produce this effect in normotensive WKY rats. For this reason, subsequent experiments (investigating eNOS expression, BRB permeability in flat-mounted retinas, the glutamate cycle, and retinal function) were conducted only in SHR rats. 
The NMDA receptor, which is localized to excitatory synapses, regulates nNOS activity. 46 NO derived from the NOS system activates soluble guanylyl cyclase and increases cGMP production, which results in retinal transduction. 47 Levels of cGMP can be taken as an index of neuroprotection, because neurons expressing nNOS are dependent on NO for survival. The survival of these neurons requires cGMP-dependent activation of the Akt survival pathway. 48 In this study, the increase in phospho-nNOS accompanied by a decrease in cGMP levels in diabetic animals was reversed by the oral administration of GT. To our knowledge, this is the first report demonstrating that oral GT improves the efficiency of the NO/cyclic GMP pathway in models of diabetic retinal disease. In support of our findings, Zhang et al. 49 demonstrated that orally administered EGCG attenuated the injuries to the retina caused by ischemia/reperfusion in a model of retinal ischemia. This suggests that oral GT exerts a neuroprotective effect in a rat model of glaucoma. 
To investigate the role of oxidative stress in glutamate/glutamine cycle imbalance, we evaluated ROS production and glutathione levels in GT-treated Müller cells under HG conditions. The presence of GT at 10 to 100 μg/mL reduced ROS formation and restored glutathione expression to baseline levels, which prevented the oxidative stress induced by HG conditions. This piece of information indicates that in hyperglycemic conditions, glutamate transporter/receptor impairment leads to oxidative stress. In the presence of GT, glutamate uptake is restored, which prevents oxidative damage. Concordant with our findings, Zeng et al. 37 verified that Müller cells treated with high levels of glucose displayed decreased GLAST mRNA expression. Treatment with taurine, a retinal amino acid, prevented degradation under diabetic conditions by exerting antioxidant effects. Studies by Siu et al. 50 involving mass spectrometry have shown that in retinal homogenates, treatment with glutamate induced lipid peroxidation. The presence of catechin, a polyphenol present in GT, significantly reversed the changes in levels of thioredoxin peroxidase, 5-hydroxytryptamine receptor, peroxiredoxin 6, and pyruvate, thus protecting the retinal tissue from glutamate-induced lipoperoxidation. Our results suggest that in ARPE-19 cells, the upregulation of claudin-1 by GT may be involved in sealing the outer BRB. The functional consequences and clinical applicability of these observations require further study. More recently, the role of outer BRB breakdown in the pathogenesis of DR has garnered increasing attention. Data from Villarroel et al. have demonstrated the complexity of this barrier and that occludin, ZO-1, and claudin-1 might play different roles in the outer BRB barrier. 51  
The results presented here demonstrate the beneficial effects of GT in the diabetic retina and in retinal cells exposed to high glucose conditions. Although the design of this study was one of primary intervention (concomitant with the induction of diabetes or in the presence of high glucose), we demonstrated considerable protective effects in neuro-glial and vascular sites of the retina under high-glucose conditions. The results from the current study are novel and describe the neuroprotective effects of GT in an animal model of DR and in in vitro studies mimicking diabetic conditions. Previous studies by Lee et al. 52 and Renouf et al. 53 reveal that the plasmatic levels of the main catechins present in GT were in nM levels among healthy volunteers. This present translational study can be used as a rationale for future clinical trials to test dose and efficiency of GT as a low-cost co-adjuvant therapy for patients with diabetic retinal disease. 
Supplementary Materials
Acknowledgments
We thank Priscilla Sayami (Department of Cell & Developmental Biology, University of São Paulo, São Paulo, Brazil) for her work culturing primary rat Müller cells. The authors are very grateful to the personnel from the Laboratory of Renal Pathophysiology (Investigation on Diabetes Complications, Faculty of Medical Sciences [FCM], State University of Campinas [Unicamp], Campinas, São Paulo, Brazil) for supporting this work and to Sandra R. Brambilla for technical assistance. 
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Footnotes
 Supported by São Paulo Research Foundation (FAPESP) Grants 2008/57560-0 and 2010/11514-7 and a scholarship from FAPESP (KCSI).
Footnotes
 Disclosure: K.C. Silva, None; M.A.B. Rosales, None; D.E. Hamassaki, None; K.C. Saito, None; A.M. Faria, None; P.A.O. Ribeiro, None; J.B. Lopes de Faria, None; J.M. Lopes de Faria, None
Figure 1. 
 
GT regulates the early markers of diabetic retinopathy, including GFAP and occludin levels. (A) A representative photomicrograph of glial reactivity as revealed by GFAP immunohistochemistry in rat retinas from WKY and SHR. In nondiabetic rat retinas, there was a light retinal glial reaction mainly in astrocytes. After 3 months of diabetes, marked staining can be observed throughout the retina; the oral administration of GT reduced this diabetes-mediated response. Magnification: ×100. The bars represent mean ± SD for the percentage of GFAP-positive retinal cells per mm2 of retina. *P = 0.02 and *P = 0.0003 for WKY and SHR, respectively. (B) Representative photomicrograph of the immunolocalization of occludin in WKY and SHR animals. In the control groups, occludin is present in the ganglion cell layer and in the inner nuclear layer around the vessels. In diabetic animals, there is a reduction in occludin expression; GT treatment reversed this effect. Magnification: ×100. The bars represent mean ± SD for the percentage of positivity of occludin/mm2 of retina. *P = 0.02 and *P = 0.01 for WKY and SHR, respectively. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; CT WKY, control WKY; DM WKY, diabetic WKY; DM WKY GT, diabetic WKY treated with GT; CT SHR, control SHR; DM SHR, diabetic SHR; DM SHR GT, diabetic SHR treated with GT.
Figure 1. 
 
GT regulates the early markers of diabetic retinopathy, including GFAP and occludin levels. (A) A representative photomicrograph of glial reactivity as revealed by GFAP immunohistochemistry in rat retinas from WKY and SHR. In nondiabetic rat retinas, there was a light retinal glial reaction mainly in astrocytes. After 3 months of diabetes, marked staining can be observed throughout the retina; the oral administration of GT reduced this diabetes-mediated response. Magnification: ×100. The bars represent mean ± SD for the percentage of GFAP-positive retinal cells per mm2 of retina. *P = 0.02 and *P = 0.0003 for WKY and SHR, respectively. (B) Representative photomicrograph of the immunolocalization of occludin in WKY and SHR animals. In the control groups, occludin is present in the ganglion cell layer and in the inner nuclear layer around the vessels. In diabetic animals, there is a reduction in occludin expression; GT treatment reversed this effect. Magnification: ×100. The bars represent mean ± SD for the percentage of positivity of occludin/mm2 of retina. *P = 0.02 and *P = 0.01 for WKY and SHR, respectively. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; CT WKY, control WKY; DM WKY, diabetic WKY; DM WKY GT, diabetic WKY treated with GT; CT SHR, control SHR; DM SHR, diabetic SHR; DM SHR GT, diabetic SHR treated with GT.
Figure 2. 
 
Retinal oxidative stress was ameliorated by oral GT. (A) A representative photomicrograph of the immunolocalization of nitrotyrosine in WKY and SHR animals. Nitrotyrosine is a stable product formed from the reaction of peroxynitrate with tyrosine residues and is accepted as an index of nitrosative damage. In normal retinas, the signal of nitrotyrosine is faint and present in all retinal layers; in diabetic retinas, the staining is stronger and observed throughout the retinal tissue. This abnormality is abrogated by GT administration. The bars represent mean ± SD for the percentage of nitrotyrosine-positive retinal cells/mm2 of retina. *P = 0.04. (B) Western blot analysis of the Cu/Zn SOD enzyme in total retinal lysates of the SHR. The SOD family is a major antioxidant system, and a deficiency of Cu/Zn- SOD1 leads to features of ROS-mediated retinal degeneration. In this present study, there was no significant decrease in Cu/Zn SOD levels after 3 months of diabetes, but the administration of GT doubled SOD levels. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD for the band densities expressed in arbitrary densitometric units from at least three independent experiments. *P = 0.0006.
Figure 2. 
 
Retinal oxidative stress was ameliorated by oral GT. (A) A representative photomicrograph of the immunolocalization of nitrotyrosine in WKY and SHR animals. Nitrotyrosine is a stable product formed from the reaction of peroxynitrate with tyrosine residues and is accepted as an index of nitrosative damage. In normal retinas, the signal of nitrotyrosine is faint and present in all retinal layers; in diabetic retinas, the staining is stronger and observed throughout the retinal tissue. This abnormality is abrogated by GT administration. The bars represent mean ± SD for the percentage of nitrotyrosine-positive retinal cells/mm2 of retina. *P = 0.04. (B) Western blot analysis of the Cu/Zn SOD enzyme in total retinal lysates of the SHR. The SOD family is a major antioxidant system, and a deficiency of Cu/Zn- SOD1 leads to features of ROS-mediated retinal degeneration. In this present study, there was no significant decrease in Cu/Zn SOD levels after 3 months of diabetes, but the administration of GT doubled SOD levels. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD for the band densities expressed in arbitrary densitometric units from at least three independent experiments. *P = 0.0006.
Figure 3. 
 
The oral administration of GT re-establishes the constitutive neuronal nitric oxide synthase/cyclic GMP pathway in diabetic SHR. (A) The levels of constitutive neuronal NOS and its phosphorylated form at Ser847 (inactivated form) were examined in total retinal lysates using western blot analyses. In WKY rats, there was no change in the phospho-nNOS: total nNOS: β-actin ratio after 12 weeks of DM induction. This ratio was altered in diabetic SHR. Activation of nNOS was reduced in retinal tissue; GT reversed this effect. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities expressed in arbitrary densitometric units from at least three independent experiments. *P = 0.001. (B) A representative photomicrograph of the immunolocalization of phospho-nNOS in SHR animals. The phospho-nNOS staining exhibits a scattered pattern located mainly in the inner plexiform layer. In control rats, there was positivity mainly in the IPL; there is a marked increase in expression in the presence of DM. Treatment with GT reduced the magnitude of this increase. Magnification: ×100. (C) Cyclic guanosine monophosphate (cGMP) levels were analyzed by ELISA in retinas from SHR. The values represent mean ± SD of cGMP levels in pmol/mg protein from retinal tissue. *P < 0.05.
Figure 3. 
 
The oral administration of GT re-establishes the constitutive neuronal nitric oxide synthase/cyclic GMP pathway in diabetic SHR. (A) The levels of constitutive neuronal NOS and its phosphorylated form at Ser847 (inactivated form) were examined in total retinal lysates using western blot analyses. In WKY rats, there was no change in the phospho-nNOS: total nNOS: β-actin ratio after 12 weeks of DM induction. This ratio was altered in diabetic SHR. Activation of nNOS was reduced in retinal tissue; GT reversed this effect. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities expressed in arbitrary densitometric units from at least three independent experiments. *P = 0.001. (B) A representative photomicrograph of the immunolocalization of phospho-nNOS in SHR animals. The phospho-nNOS staining exhibits a scattered pattern located mainly in the inner plexiform layer. In control rats, there was positivity mainly in the IPL; there is a marked increase in expression in the presence of DM. Treatment with GT reduced the magnitude of this increase. Magnification: ×100. (C) Cyclic guanosine monophosphate (cGMP) levels were analyzed by ELISA in retinas from SHR. The values represent mean ± SD of cGMP levels in pmol/mg protein from retinal tissue. *P < 0.05.
Figure 4. 
 
GT prevented eNOS activation in diabetic SHR leading to a re-establishment of the BRB. (A) Western blot analysis of phospho-eNOS and total eNOS levels in total retinal lysates from SHR. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities expressed in arbitrary densitometric units from at least three independent experiments. *P < 0.05. (B) Representative images of vessel leakage in whole-mounted retinas. The fluorescein leakage in retinal tissue was classified based on the extent and sites of retinal leakage. Level 0: the absence of fluorescein leakage throughout the entire retina; Level 1: the presence of fluorescein leakage in only one quadrant; Level 1+: more than two sites of leakage in only one quadrant; Level 2: the presence of fluorescein leakage in two quadrants; Level 2+: more than two sites of leakage in one of two quadrants; Level 2++: more than two sites of leakage in both of two quadrants; Level 3: the presence of fluorescein leakage in three quadrants; Level 3+: more than two sites of leakage in one of three quadrants; Level 3++: more than two sites of leakage in two of three quadrants; Level 3+++: more than two sites of leakage in three quadrants; Level 4: the presence of fluorescein leakage in all four quadrants. In each control flat-mounted retina, the capillary bed was intact and minimal leakage was observed. After 12 weeks of DM, Evans blue dye leaked into retinal tissue at several sites, which indicated inner BRB breakdown. The oral administration of GT reduced the leakage to normal levels. (Scale bar: 200 μm for all groups. In DM-SHR, higher magnification was used for better visualization; scale bar: 100 μm.)
Figure 4. 
 
GT prevented eNOS activation in diabetic SHR leading to a re-establishment of the BRB. (A) Western blot analysis of phospho-eNOS and total eNOS levels in total retinal lysates from SHR. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities expressed in arbitrary densitometric units from at least three independent experiments. *P < 0.05. (B) Representative images of vessel leakage in whole-mounted retinas. The fluorescein leakage in retinal tissue was classified based on the extent and sites of retinal leakage. Level 0: the absence of fluorescein leakage throughout the entire retina; Level 1: the presence of fluorescein leakage in only one quadrant; Level 1+: more than two sites of leakage in only one quadrant; Level 2: the presence of fluorescein leakage in two quadrants; Level 2+: more than two sites of leakage in one of two quadrants; Level 2++: more than two sites of leakage in both of two quadrants; Level 3: the presence of fluorescein leakage in three quadrants; Level 3+: more than two sites of leakage in one of three quadrants; Level 3++: more than two sites of leakage in two of three quadrants; Level 3+++: more than two sites of leakage in three quadrants; Level 4: the presence of fluorescein leakage in all four quadrants. In each control flat-mounted retina, the capillary bed was intact and minimal leakage was observed. After 12 weeks of DM, Evans blue dye leaked into retinal tissue at several sites, which indicated inner BRB breakdown. The oral administration of GT reduced the leakage to normal levels. (Scale bar: 200 μm for all groups. In DM-SHR, higher magnification was used for better visualization; scale bar: 100 μm.)
Figure 5. 
 
GT exerts a neuroprotective effect in SHR. (A) Representative Western blots for GLAST in total retinal lysates from SHR animals. *P = 0.04. (B) Representative Western blots for Nmdar1 in total retinal lysates. *P < 0.05. (C) Representative Western blots for glutamine synthetase in total retinal lysates. *P = 0.04. Equal loading and transfer for all proteins were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities from at least three independent experiments in terms of the percentage of variation (GLAST and GS) or arbitrary densitometric units (Nmdar1). (D) Representative waveforms for a- and b-waves in the rats studied. The a-wave is the first negative deflection and represents the activity of the photoreceptors. The inner retinal function response, the b-wave, is a positive deflection generated in part by the Müller and mainly by the bipolar cell potentials. The figure shows representative full-flash ERG waveform for a CT-SHR (red), DM-SHR (blue), and DM-SHR GT (green). The bars represent the mean amplitude and latency in AUC of the a- and b-wave components and total latency of the ERG from the SHR animals. *P < 0.05 and #P < 0.03.
Figure 5. 
 
GT exerts a neuroprotective effect in SHR. (A) Representative Western blots for GLAST in total retinal lysates from SHR animals. *P = 0.04. (B) Representative Western blots for Nmdar1 in total retinal lysates. *P < 0.05. (C) Representative Western blots for glutamine synthetase in total retinal lysates. *P = 0.04. Equal loading and transfer for all proteins were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities from at least three independent experiments in terms of the percentage of variation (GLAST and GS) or arbitrary densitometric units (Nmdar1). (D) Representative waveforms for a- and b-waves in the rats studied. The a-wave is the first negative deflection and represents the activity of the photoreceptors. The inner retinal function response, the b-wave, is a positive deflection generated in part by the Müller and mainly by the bipolar cell potentials. The figure shows representative full-flash ERG waveform for a CT-SHR (red), DM-SHR (blue), and DM-SHR GT (green). The bars represent the mean amplitude and latency in AUC of the a- and b-wave components and total latency of the ERG from the SHR animals. *P < 0.05 and #P < 0.03.
Figure 6. 
 
GT exerts a neuroprotective effect in primary rat Müller cells and restores the oxidative balance in rMC-1. (A) Immunofluorescence for GFAP in primary Müller cells cultured for 72 hours. Scale bar: 20 μm. (B) Representative Western blots for GLAST in total cell lysates. *P < 0.03. (C) Representative Western blots for NMDAR1 in total cell lysates. *P < 0.04. (D) The Nr1 gene expression profile normalized to GAPDH in primary Müller cell mRNA. *P = 0.002. (E) Representative Western blots for glutamine synthetase in total cell lysates. *P < 0.01. Equal loading and transfer for all proteins were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities expressed as the percentage of variation from at least three independent experiments. (F) The quantification of total intracellular ROS levels in rMC-1 cultured for 24 hours in normal d-glucose (NG, 5 mM) and high d-glucose (HG, 25 mM) in the presence or absence of GT (HG + GT: 1, 10, and 100 μg/mL). Mannitol (MAN) was used as an osmotic control. The values are means ± SD and expressed as percentages of fluorescence units. Values were corrected by the number of cells at the end of each treatment. *P < 0.01 versus NG; #P < 0.01 versus HG; ¶P < 0.04 versus HG+GT 1 μg/mL; §P < 0.0007 versus HG+GT 10 μg/mL. (G) Concentrations of reduced glutathione (GSH) from rMC-1 cells cultured for 24 hours (μM glutathione/μg of protein). Treatment with GT (10–100 μg/mL) or NAC (1 mM) prevented the decrease in GSH levels in cells in HG. The bars represent mean ± SD. *P < 0.001. (H) The GLAST gene expression profiles normalized with GAPDH in rMC-1. *P = 0.02 versus NG; #P = 0.02 versus HG; ¶P < 0.05 versus HG. (I) The Nr1 gene expression profile normalized with GAPDH in rMC-1. *P < 0.04 versus NG; #P = 0.03 versus HG. NG, normal d-glucose; HG, high d-glucose; HG+GT, high d-glucose + 100 μg/mL of green tea; MAN, mannitol.
Figure 6. 
 
GT exerts a neuroprotective effect in primary rat Müller cells and restores the oxidative balance in rMC-1. (A) Immunofluorescence for GFAP in primary Müller cells cultured for 72 hours. Scale bar: 20 μm. (B) Representative Western blots for GLAST in total cell lysates. *P < 0.03. (C) Representative Western blots for NMDAR1 in total cell lysates. *P < 0.04. (D) The Nr1 gene expression profile normalized to GAPDH in primary Müller cell mRNA. *P = 0.002. (E) Representative Western blots for glutamine synthetase in total cell lysates. *P < 0.01. Equal loading and transfer for all proteins were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD of band densities expressed as the percentage of variation from at least three independent experiments. (F) The quantification of total intracellular ROS levels in rMC-1 cultured for 24 hours in normal d-glucose (NG, 5 mM) and high d-glucose (HG, 25 mM) in the presence or absence of GT (HG + GT: 1, 10, and 100 μg/mL). Mannitol (MAN) was used as an osmotic control. The values are means ± SD and expressed as percentages of fluorescence units. Values were corrected by the number of cells at the end of each treatment. *P < 0.01 versus NG; #P < 0.01 versus HG; ¶P < 0.04 versus HG+GT 1 μg/mL; §P < 0.0007 versus HG+GT 10 μg/mL. (G) Concentrations of reduced glutathione (GSH) from rMC-1 cells cultured for 24 hours (μM glutathione/μg of protein). Treatment with GT (10–100 μg/mL) or NAC (1 mM) prevented the decrease in GSH levels in cells in HG. The bars represent mean ± SD. *P < 0.001. (H) The GLAST gene expression profiles normalized with GAPDH in rMC-1. *P = 0.02 versus NG; #P = 0.02 versus HG; ¶P < 0.05 versus HG. (I) The Nr1 gene expression profile normalized with GAPDH in rMC-1. *P < 0.04 versus NG; #P = 0.03 versus HG. NG, normal d-glucose; HG, high d-glucose; HG+GT, high d-glucose + 100 μg/mL of green tea; MAN, mannitol.
Figure 7. 
 
GT prevented outer BRB dysfunction, ROS production and decreased glutamate transport levels in ARPE-19 cells. (A) Representative fluorescent microscopic images of H2DCF-DA in ARPE-19 cells. The fluorescence intensity of H2DCF-DA indicates the level of ROS production. The bars represent the quantification of total intracellular ROS levels in ARPE-19 cells cultured for 24 hours. The values are means ± SD and expressed as percentages of fluorescence units. The values were corrected by the number of cells at the end of each treatment. *P < 0.0001. (B) Representative Western blots for claudin-1 in ARPE-19 cells. *P < 0.02. (C) Immunofluorescence analysis for claudin-1 in ARPE-19 cells. Scale bar: 20 μm. (D) Representative Western blots for EAAC1 in ARPE-19 cells. *P < 0.03. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD for the band densities expressed as a percentage of variation from at least three independent experiments. NG, normal d-glucose; HG, high d-glucose, HG+GT, high d-glucose + 100 μg/mL of GT; HG+EGCG (10, 25, and 50 μM), high d-glucose plus (-)-epigallocatechin gallate; MAN, mannitol.
Figure 7. 
 
GT prevented outer BRB dysfunction, ROS production and decreased glutamate transport levels in ARPE-19 cells. (A) Representative fluorescent microscopic images of H2DCF-DA in ARPE-19 cells. The fluorescence intensity of H2DCF-DA indicates the level of ROS production. The bars represent the quantification of total intracellular ROS levels in ARPE-19 cells cultured for 24 hours. The values are means ± SD and expressed as percentages of fluorescence units. The values were corrected by the number of cells at the end of each treatment. *P < 0.0001. (B) Representative Western blots for claudin-1 in ARPE-19 cells. *P < 0.02. (C) Immunofluorescence analysis for claudin-1 in ARPE-19 cells. Scale bar: 20 μm. (D) Representative Western blots for EAAC1 in ARPE-19 cells. *P < 0.03. Equal loading and transfer were ascertained by reprobing the membranes for β-actin. The bars represent mean ± SD for the band densities expressed as a percentage of variation from at least three independent experiments. NG, normal d-glucose; HG, high d-glucose, HG+GT, high d-glucose + 100 μg/mL of GT; HG+EGCG (10, 25, and 50 μM), high d-glucose plus (-)-epigallocatechin gallate; MAN, mannitol.
Table. 
 
Physiological Characteristics of Studied Animals
Table. 
 
Physiological Characteristics of Studied Animals
Groups Initial Body Weight, g Final Body Weight, g Systolic Blood Pressure, mm Hg Glycemia, mmol/L
CT-WKY 322.3 ± 22 498.7 ± 48 143.7 ± 5 8.9 ± 0.4
DM-WKY 331.2 ± 16 384.6 ± 66* 139.8 ± 3 28.5 ± 5†
DM-WKY GT 325.6 ± 16 367.2 ± 42* 137.5 ± 8 27.4 ± 7†
CT-SHR 277.2 ± 17 345.5 ± 13 204.3 ± 10§ 8.4 ± 1
DM-SHR 277.6 ± 7 192.2 ± 38‡ 203.5 ± 8§ 29.5 ± 2‡
DM-SHR GT 269.7 ± 15 194.9 ± 25‡ 198.9 ± 10§ 30.5 ± 5‡
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