Retinal Oxidative Stress Research Articles

Sirt3 mediates the protective effect of hydrogen in inhibiting ROS-induced retinal senescence

Hydrogen possesses antioxidative effects and cures numerous types of ophthalmopathy, but the mechanism of hydrogen on ROS-induced retinal senescence remains elusive. In this study, retinal morphology revealed that hydrogen reduced the number and size of vitreous black deposits in Bruch's membrane in NaIO3 mice. Hydrogen also reduced ROS levels in the retina as assessed by DHE staining. Moreover, this result was consistent with the downregulation of expression of the oxidative stress hallmark OGG1. These findings suggested that hydrogen can reduce retinal oxidative stress induced by NaIO3, and this result was further verified using the antioxidant ALCAR. Mechanistic analysis revealed that hydrogen significantly inhibited the downregulation of Sirt3 expression, and this notion was confirmed using AICAR, which restores Sirt3 expression and activity. Moreover, hydrogen reduced the expression of p53, p21 and p16 and the number of blue-green precipitations in the retinas of NaIO3 mice as assessed by SA-β-gal staining. We also found that hydrogen decreased the expression of the DNA damage-related protein ATM, cyclinD1 and NF-κB but increased the expression of the DNA repair-related protein HMGB1, suggesting that hydrogen inhibits senescence in retinas of NaIO3 mice. Additionally, OCT examination revealed that hydrogen suppressed retinal high reflex formation significantly and prevented the retina from thinning. This result was supported by ERG assays that demonstrated that hydrogen prevented the reduction in a- and b-wave amplitude induced by NaIO3 in mice. Thus, our data suggest that hydrogen may inhibit retinal senescence by suppressing the downregulation of Sirt3 expression through reduced oxidative stress reactions.

Intraocular pressure-lowering effects of imidazo[1,2-a]- and pyrimido[1,2-a]benzimidazole compounds in rats with dexamethasone-induced ocular hypertension

Ocular hypertension is believed to be involved in the etiology of primary open-angle glaucoma. Although many pharmaceutical agents have been shown to be effective for the reduction of intraocular pressure (IOP), a significant opportunity to improve glaucoma treatments remains. Thus, the aims of the present study were: (1) to evaluate the IOP-lowering effect of four compounds RU-551, RU-555, RU-839 (pyrimido[1,2-a]benzimidazole), and RU-615 (imidazo[1,2-a]benzimidazole) on steroid-induced ocular hypertension in rats after single drop and chronic applications; and (2) to test in silico and in vitro conventional rho-associated kinase (ROCK) inhibitory activity of the selected compound. This study demonstrated that RU-551, RU-555, RU-839, and RU-615 significantly reduced IOP in Sprague Dawley rats with dexamethasone (DEXA) induced ocular hypertension after single drop administration (0.1%), however RU-615 showed the best IOP lowering effect as indicated by maximum IOP reduction of 22.32% from baseline. Repeated dose topical application of RU-615 caused sustained reduction of IOP from baseline throughout the 3 weeks of treatment with maximum IOP reduction of 30.31% on day 15. This study also showed that the steroid-induced increase in IOP is associated with increased retinal oxidative stress and significant retinal ganglion cells (RGCs) loss. Prolonged treatment with RU-615 over 3 weeks results in normalization of IOP in DEXA-treated rats with partial restoration of retinal antioxidant status (catalase, glutathione and superoxide dismutase) and subsequent protective effect against RGC loss. Thus, IOP lowering activity of RU-615 together with antioxidant properties might be the factors that contribute to prevention of further RGC loss. In vitro part of this study explored the ROCK inhibitory activity of RU-615 using dexamethasone-treated human trabecular meshwork cells as a possible mechanism of action of its IOP lowering activity. However, this study didn’t show conventional ROCK inhibition by RU-615 which was later confirmed by in silico consensus prediction. Therefore, in the future studies it is important to identify the upstream target receptors for RU-615 and then delineate the involved intracellular signalling pathways which are likely to be other than ROCK inhibition.

Taurine protects against NMDA-induced retinal damage by reducing retinal oxidative stress.

This study aimed to evaluate effect of TAU on NMDA-induced changes in retinal redox status, retinal cell apoptosis and retinal morphology in Sprague-Dawley rats. Taurine was injected intravitreally as pre-, co- or post-treatment with NMDA and 7days post-treatment retinae were processed for estimation of oxidative stress, retinal morphology using H&E staining and retinal cell apoptosis using TUNEL staining. Treatment with TAU, particularly pre-treatment, significantly increased retinal glutathione, superoxide dismutase and catalase levels compared to NMDA-treated rats; whereas, the levels of malondialdehyde reduced significantly. Reduction in retinal oxidative stress in TAU pre-treated group was associated with significantly greater fractional thickness of ganglion cell layer within inner retina and retinal cell density in inner retina. TUNEL staining showed significantly reduced apoptotic cell count in TAU pre-treated group compared to NMDA group. It could be concluded that TAU protects against NMDA-induced retinal injury in rats by reducing retinal oxidative stress.

Protective effects of sulforaphane on diabetic retinopathy: activation of the Nrf2 pathway and inhibition of NLRP3 inflammasome formation.

Sulforaphane (SFN) is abundant in cruciferous plants, providing significant protection against many chronic diseases. With the aim of clarifying the efficacy of sulforaphane in diabetic retinopathy (DR), a series of systematic studies were carried out in the present study. Male Sprague Dawley rats were intraperitoneally injected with streptozotocin (STZ, 65 mg/kg), and those with confirmed diabetes mellitus were given different doses of SFN (0.5 and 1 mg/kg/d) for 12 weeks. In vitro, Müller cells exposed to 25 mM glucose were treated with 2.5 µM SFN. The results indicated that SFN significantly reduced the generation of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) and enhanced the activity of antioxidant enzymes (GSH, SOD, and CAT) in the retina of STZ rats. Further, SFN enhanced the nuclear accumulation of Nrf2 and increased the expression of HO-1 and NQO1, two major antioxidants downstream to Nrf2, in the injured retina. In addition, retinal expression levels of NLRP3, cleaved caspase-1 p20, IL-1β p17, and ASC were dramatically increased in STZ-induced DR, and this was abolished by SFN intervention. In vitro, high glucose-induced inflammation and oxidative stress damage in Müller cells were attenuated by SFN. SFN also exerted antioxidant effects, activated the Nrf2 pathway, and inhibited the NLRP3 inflammasome in Müller cells. In conclusion, our work demonstrates that SFN attenuates retinal inflammation and oxidative stress induced by high glucose and activates the antioxidative Nrf2 pathway and inhibits the formation of the NLRP3 inflammasome in vivo and in vitro.

The protective effects of lycium barbarum polysaccharides on retinal neurons in diabetic rats and its mechanism

To clarify whether lycium barbarum polysaccharides (LBP) have protective effects on retina neuronal cells in diabetic rats and to identify the related mechanism involved in this process. Eighteen SD rats were randomly divided into 3 groups ( n= 6): normal control group (NC), diabetes mellitus group (DM) and LBP-treatment group (DM+LBP). The diabetic rat model was induced by single intraperitoneal injection of streptozotocin (STZ). The rats in DM+LBP group were treated with LBP at the dose of 1 mg/kg by gavage, once a day for 12 weeks. After the treatment, the weight and blood glucose, the generation of reactive oxygen species (ROS), the surviving retinal ganglion cells (RGCs) and amacrine cells and the protein expressions of nuclear factor E2-related factor 2 (Nrf2) and the heme oxygenase-1 (HO-1) were detected. The successful rate of diabetic model was 100%. Compared with NC group, the rats of DM group caused weight loss, elevated blood glucose, a marked increase of ROS generation and a significant decrease in the number of RGCs and amacrine cells (P＜0.01), and these effects were diminished or abolished by LBP treatment. Meanwhile, LBP significantly increased the expressions of Nrf2 and HO-1 in the retinas of diabetic rats (P＜0.01). LBP can improve retinal oxidative stress and exert beneficial neuroprotective effects in diabetic rats, and its mechanism may be associated with the activation of the Nrf2/HO-1 antioxidant pathway.

Carteolol hydrochloride reduces visible light-induced retinal damage in vivo and BSO/glutamate-induced oxidative stress in vitro

The purpose of this study was to determine whether carteolol eye drops, a β-adrenoceptor antagonist used as an intraocular hypotensive agent, has protective effects against the light-induced oxidative stress in retina. Dark-adapted pigmented rats were pre-treated with topical carteolol ophthalmic solution or saline and then exposed to visible light. The effects on electroretinogram (ERG), morphology, oxidative stress, and expression of mRNAs in the retinas were determined. The l-buthionine-(S,R)-sulfoximine (BSO)/glutamate-induced oxidative stress in 661 W cells, a murine photoreceptor cell line, was evaluated by cell death assays, production of reactive oxygen species (ROS), and activation of caspase. In vivo studies showed that exposure to light caused a decrease in the amplitudes of ERGs and the outer nuclear layer (ONL) thickness and an increase of the 8-hydroxy-2′-deoxyguanosine (8-OHdG)-positive cells in the ONL. These changes were significantly reduced by pre-treatment with carteolol. Carteolol also significantly up-regulated the mRNA levels of thioredoxin 1 and glutathione peroxidase 1 compared to saline-treated group. Moreover, carteolol and timolol, another β-adrenoceptor antagonist, significantly inhibited BSO/glutamate-induced cell death and reduced caspase-3/7 activity and ROS production in vitro. Therefore, carteolol could protect retina from light-induced damage with multiple effects such as enhancing the antioxidative potential and decreasing the intracellular ROS production.

Cilostazol Attenuates Retinal Oxidative Stress and Inflammation in a Streptozotocin-Induced Diabetic Animal Model

ABSTRACTPurpose: To investigate the anti-oxidative and anti-inflammatory effects of cilostazol in the ocular tissues of streptozotocin (STZ)-induced diabetic rats.Materials and methods: Diabetes was induced in 6-week-old Wistar rats via peritoneal injections of STZ. The treatment group received cilostazol 18 mg/kg/day for 8 weeks (n = 10), and the diabetic group received phosphate buffer solution (n = 20). The expression of oxidative stress and inflammatory mediators in the ocular tissues was then assessed by reverse-transcription polymerase chain reactions, immunohistochemical (IHC) staining, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA).Results: Reverse-transcription polymerase chain reactions, IHC staining, Western blot analysis, and ELISA showed that cilostazol inhibited mRNA and protein expressions of intercellular adhesion molecule-1, monocyte chemoattractant protein-1 and fractalkine in the retina and aqueous humor (AqH). Consistent with these findings, cilostazol attenuated the activation of nuclear factor-κB (NF-κB) in the diabetic rats. The levels of oxidatively modified DNA (8-OHdG), nitrotyrosine and oxidative lipids (acrolein) were also diminished in the cilostazol-treated group. Chemiluminescence analysis showed that reactive oxygen species (ROS) levels in the AqH was significantly higher in the diabetic rats than in the non-diabetic rats. Treatment with cilostazol significantly reduced the ROS levels in the AqH compared to the diabetic rats.Conclusions: Our results indicated that cilostazol reduced inflammatory reactions and oxidative stress in diabetic eyes. The anti-inflammatory effects of cilostazol may be indirectly via reducing oxidative stress, inhibiting NF-κB activity, and subsequently decreasing inflammatory mediators. Cilostazol may be beneficial to prevent the progression of diabetic retinopathy.

Etidronate protects chronic ocular hypertension induced retinal oxidative stress and promotes retinal ganglion cells growth through IGF-1 signaling pathway

Glaucoma is a common heterogeneous eye disorder that may lead to irreversible blindness. In the present study, we examined whether etidronate, a member of bisphosphonates, may have neuroprotective effects in in vivo and in vitro rat model of glaucoma. In an in vivo setting, chronic ocular hypertension (COH) was induced in adult rat retina. We discovered that systemic injection of etidronate reduced COH-induced retinal oxidative stress, including caspase-3 activity and MDA level, as well as promoted retinal ganglion cell survival. In an in vitro setting, neonatal retinal ganglion cell was incubated with etidronate. We found etidronate incubation promoted neurite growth, upregulated IGF-1 and p-IGF-1R protein expressions in retinal ganglion cell. In addition, application of a selective IGF-1R antagonist effectively blocked the pro-neuronal effect of etidronate on retinal ganglion cell growth, and reduced p-IGF-1R protein expression. Thus, our results demonstrated that etidronate might reduce retinal oxidative stress and promote retinal neuronal growth through IGF-1 signaling pathway. Future work may define its clinical feasibility to treating human patients with glaucoma.

Daily zeaxanthin supplementation prevents atrophy of the retinal pigment epithelium (RPE) in a mouse model of mitochondrial oxidative stress.

Oxidative damage is implicated in the pathogenesis of age-related macular degeneration (AMD). The dry form of AMD (geographic atrophy) is characterized by loss of RPE, photoreceptors, and macular pigments. The cumulative effects of oxidative stress impact mitochondrial function in RPE. In Sod2flox/floxVMD2-cre mice, the RPE specific deletion of Sod2, the gene for mitochondrial manganese superoxide dismutase (MnSOD), leads to elevated oxidative stress in retina and RPE, and causes changes in the RPE and underlying Bruch’s membrane that share some features of AMD. This study tested the hypothesis that zeaxanthin supplementation would reduce oxidative stress and preserve RPE structure and function in these mice. Zeaxanthin in retina/RPE/choroid and liver was quantified by LC/MS, retinal function and structure were evaluated by electroretinogram (ERG) and spectral domain optical coherence tomography (SD-OCT), and antioxidant gene expression was measured by RT-PCR. After one month of supplementation, zeaxanthin levels were 5-fold higher in the retina/RPE/choroid and 12-fold higher in liver than in unsupplemented control mice. After four months of supplementation, amplitudes of the ERG a-wave (function of rod photoreceptors) and b-wave (function of the inner retina) were not different in supplemented and control mice. In contrast, the c-wave amplitude (a measure of RPE function) was 28% higher in supplemented mice than in control mice. Higher RPE/choroid expression of antioxidant genes (Cat, Gstm1, Hmox1, Nqo1) and scaffolding protein Sqstm1 were found in supplemented mice than in unsupplemented controls. Reduced nitrotyrosine content in the RPE/choroid was demonstrated by ELISA. Preliminary assessment of retinal ultrastructure indicated that supplementation supported better preservation of RPE structure with more compact basal infoldings and intact mitochondria. We conclude that daily zeaxanthin supplementation protected RPE cells from mitochondrial oxidative stress associated with deficiency in the MnSOD and thereby improved RPE function early in the disease course.

Collagen peptide modified carboxymethyl cellulose as both antioxidant drug and carrier for drug delivery against retinal ischaemia/reperfusion injury.

Oxidative stress can cause injury in retinal endothelial cells. Carboxymethyl cellulose modified with collagen peptide (CMCC) is of a distinct antioxidant capacity and potentially a good drug carrier. In this study, the protective effects of CMCC against H2O2‐induced injury of primary retinal endothelial cells were investigated. In vitro, we demonstrated that CMCC significantly promoted viability of H2O2‐treated cells, efficiently restrained cellular reactive oxygen species (ROS) production and cell apoptosis. Then, the CMCC was employed as both drug and anti‐inflammatory drug carrier for treatment of retinal ischaemia/reperfusion (I/R) in rats. Animals were treated with CMCC or interleukin‐10‐loaded CMCC (IL‐10@CMCC), respectively. In comparisons, the IL‐10@CMCC treatment exhibited superior therapeutic effects, including better restoration of retinal structural thickness and less retinal apoptosis. Also, chemiluminescence demonstrated that transplantation of IL‐10@CMCC markedly reduced the retinal oxidative stress level compared with CMCC alone and potently recovered the activities of typical antioxidant enzymes, SOD and CAT. Therefore, it could be concluded that CMCC provides a promising platform to enhance the drug‐based therapy for I/R‐related retinal injury.

Lutein and Zeaxanthin Isomers Protect against Light-Induced Retinopathy via Decreasing Oxidative and Endoplasmic Reticulum Stress in BALB/cJ Mice.

Oxidative stress (OS) and endoplasmic reticulum stress (ERS) are the major factors underlying photoreceptor degeneration. Lutein, RR-zeaxanthin (3R,3’R-zeaxanthin) and RS (meso)-zeaxanthin (3R,3’S-RS- zeaxanthin) (L/Zi) could protect against cell damage by ameliorating OS in retina. In this study, we examined the effect of L/Zi supplementation in a mouse model of photoreceptor degeneration and investigated whether the treatment of L/Zi ameliorated OS and ERS. BALB/cJ mice after light exposure were used as the animal model. The protective effects of L/Zi were observed by electroretinography (ERG) and terminal deoxyuridine triphosphate nick-end labeling (TUNEL) analysis. The underlying mechanisms related to OS and ERS were explored by Western blotting. After L/Zi treatment, the ERG amplitudes were significantly higher, and the number of TUNEL-positive cells was significantly reduced compared to that of the vehicle group. Western blotting results revealed that OS was ameliorated according to the significant downregulation of phosphorylated c-Jun N-terminal kinase (p-JNK), and significant upregulation of nuclear factor erythroid 2-related factor 2 (Nrf2). In addition, ERS was reduced according to the significant downregulation of 78 kDa glucose-regulated protein (GRP78), phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (p-PERK), activating transcription factor 4 (ATF4) and activating transcription factor (ATF6). Our data shows that L/Zi provided functional and morphological preservation of photoreceptors against light damage, which is probably related to its mitigation of oxidative and endoplasmic reticulum stress.

D-cis-Diltiazem Can Produce Oxidative Stress in Healthy Depolarized Rods In Vivo.

PurposeNew perspectives are needed to understand decades of contradictory reports on the neuroprotective effects of the Cav1.2 L-type calcium channel blocker d-cis-diltiazem in retinitis pigmentosa (RP) models. Here, we address, in vivo, the following two knowledge gaps regarding d-cis-diltiazem's actions in the murine outer retina: (1) do normal mouse rods contain d-cis-diltiazem-insensitive Cav1.2 L-type calcium channels? (2) Can d-cis-diltiazem modify the normal rod redox environment?MethodsFirst, transretinal Cav1.2 L-type calcium channels were noninvasively mapped with manganese-enhanced magnetic resonance imaging (MRI) following agonist Bay K 8644 in C57BL/6 (B6) and in Cav1.2 L-type calcium channel BAY K 8644–insensitive mutant B6 mice. Second, d-cis-diltiazem–treated oxidative stress–vulnerable (B6) or –resistant [129S6 (S6)] mice were examined in vivo (QUEnch-assiSTed [QUEST] MRI) and in whole retina ex vivo (lucigenin). Retinal thickness was measured using MRI.ResultsThe following results were observed: (1) manganese uptake patterns in BAY K 8644–treated controls and mutant mice identified in vivo Cav1.2 L-type calcium channels in inner and outer retina; and (2) d-cis-diltiazem induced rod oxidative stress in dark-adapted B6 mice but not in light-adapted B6 mice or dark-adapted S6 mice (QUEST MRI). Oxidative stress in vivo was limited to inferior outer retina in dark-adapted B6 mice approximately 1-hour post d-cis-diltiazem. By approximately 4 hours post, only superior outer retina oxidative stress was observed and whole retinal superoxide production was supernormal. All groups had unremarkable retinal thicknesses.ConclusionsD-cis-diltiazem's unexpectedly complex spatiotemporal outer retinal oxidative stress pattern in vivo was dependent on genetic background and rod membrane depolarization, but not apparently dependent on Cav1.2 L-type calcium channels, providing a potential rationale for contradictory results in different RP models.

7,8,3′-Trihydroxyflavone ameliorate oxidative stress in vivo and promotes neurite regeneration in vitro in rat retinal ganglion cells

In this study, we investigate the mechanism of 7,8,3′-Trihydroxyflavone (THF) on retinal oxidative stress and retinal ganglion cell (RGC) growth. Oxidative stress was induced by ocular hypertension (O.H.) in rat retina in vivo. Rats were then systemically injected with THF. We found THF ameliorated O.H.-induced oxidative stress in retina by reducing caspase-3 activity, downregulating MDA level and augmenting glutathione peroxidase expression. In addition, neonatal RGCs were cultured in vitro, and treated with THF. It was demonstrated that THF promoted neurite regeneration in RGC explant, and activated receptor kinase B (TrkB) signaling pathway through phosphorylation. In THF-treated RGC explant, application of TrkB antagonist, ANA12, effectively reversed the pro-neuronal effect of THF on promoting RGC neurite outgrowth, and inhibited TrkB signaling pathway through dephosphorylation. Thus, we concluded that THF could inhibit retinal oxidative stress and promote RGC growth through TrkB-dependent signaling pathway, thus providing a novel pharmaceutical candidate for glaucoma.

C-Reactive Protein as a Therapeutic Target in Age-Related Macular Degeneration

Age-related macular degeneration (AMD), a retinal degenerative disease, is the leading cause of central vision loss among the elderly population in developed countries and an increasing global burden. The major risk is aging, compounded by other environmental factors and association with genetic variants for risk of progression. Although the etiology of AMD is not yet clearly understood, several pathogenic pathways have been proposed, including dysfunction of the retinal pigment epithelium, inflammation, and oxidative stress. The identification of AMD susceptibility genes encoding complement factors and the presence of complement and other inflammatory mediators in drusen, the hallmark deposits of AMD, support the concept that local inflammation and immune-mediated processes play a key role in AMD pathogenesis that may be accelerated through systemic immune activation. In this regard, increased levels of circulating C-reactive protein (CRP) have been associated with higher risk of AMD. Besides being a risk marker for AMD, CRP may also play a role in the progression of the disease as it has been identified in drusen, and we have recently found that its monomeric form (mCRP) induces blood retinal barrier disruption in vitro. In this review, we will address recent evidence that links CRP and AMD pathogenesis, which may open new therapeutic opportunities to prevent the progression of AMD.

Dark Rearing Does Not Prevent Rod Oxidative Stress In Vivo in Pde6brd10 Mice

PurposeIn cyclic light-reared Pde6brd10 mice, rod cell oxidative stress contributes to the degenerative phenotype. Dark rearing Pde6brd10 mice slows but does not prevent atrophy. This suggests that outer retinal oxidative stress occurs in Pde6brd10 mice independent of light exposure, a hypothesis tested in this study.MethodsMouse strains Pde6brd10 and C57Bl/6 (wild type) were dark reared until postnatal day (P) 23 (P23) or P30. In subgroups of dark-reared mice, (1) layer-specific excessive free radical production (i.e., an oxidative stress biomarker) in vivo via QUEnch-assiSTed magnetic resonance imaging (QUEST MRI) was indicated by a significant reduction in the greater-than-normal spin-lattice relaxation rate R1 (1/T1) with methylene blue, (2) superoxide production was measured ex vivo in whole retina (lucigenin), and (3) retinal layer spacing and thickness were assessed in vivo (optical coherence tomography, MRI).ResultsIn P23 male Pde6brd10 mice, only the outer superior retina showed oxidative stress in vivo, as measured by QUEST MRI; a lucigenin assay confirmed supernormal superoxide production. In contrast, at P30, no evidence for retinal oxidative stress was observed. In P23 female Pde6brd10 mice, no retinal oxidative stress was apparent; however, at P30, oxidative stress was observed in superior inner and outer nuclear layers. Male and female Pde6brd10 mice at P23 had normal retinal thicknesses, whereas at P30, modest thinning was noted in inferior and superior retina.ConclusionsWe confirmed that outer retinal oxidative stress occurs in male and female dark-reared Pde6brd10 mice. Male and female Pde6brd10 mice demonstrated similar degrees of retinal thinning, but with unexpectedly distinct spatial and temporal retinal oxidative stress patterns.

High Glucose-Mediated Tyrosine Nitration of PI3-Kinase: A Molecular Switch of Survival and Apoptosis in Endothelial Cells.

Diabetes and hyperglycemia are associated with increased retinal oxidative and nitrative stress and vascular cell death. Paradoxically, high glucose stimulates expression of survival and angiogenic growth factors. Therefore, we examined the hypothesis that high glucose-mediated tyrosine nitration causes inhibition of the survival protein PI3-kinase, and in particular, its regulatory p85 subunit in retinal endothelial cell (EC) cultures. Retinal EC were cultured in high glucose (HG, 25 mM) for 3 days or peroxynitrite (PN, 100 µM) overnight in the presence or absence of a peroxynitrite decomposition catalyst (FeTPPs, 2.5 µM), or the selective nitration inhibitor epicatechin (100 µM). Apoptosis of ECs was assessed using TUNEL assay and caspase-3 activity. Immunoprecipitation and Western blot were used to assess protein expression and tyrosine nitration of p85 subunit and its interaction with the p110 subunit. HG or PN accelerated apoptosis of retinal ECs compared to normal glucose (NG, 5 mM) controls. HG- or PN-treated cells also showed significant increases in tyrosine nitration on the p85 subunit of PI3-kinase that inhibited its association with the catalytic p110 subunit and impaired PI3-kinase/Akt kinase activity. Decomposing peroxynitrite or blocking tyrosine nitration of p85 restored the activity of PI3-kinase, and prevented apoptosis and activation of p38 MAPK. Inhibiting p38 MAPK or overexpression of the constitutively activated Myr-Akt construct prevented HG- or peroxynitrite-mediated apoptosis. In conclusion, HG impairs pro-survival signals and causes accelerated EC apoptosis, at least in part via tyrosine nitration and inhibition of PI3-kinase. Inhibitors of nitration can be used in adjuvant therapy to delay diabetic retinopathy and microvascular complication.

Time- and dose-related effects of amyloid beta1-40 on retina and optic nerve morphology in rats

ABSTRACTPurpose: Amyloid beta (Aβ) is known to contribute to the pathophysiology of retinal neurodegenerative diseases such as glaucoma. Effects of intravitreal Aβ(1–42) on retinal and optic nerve morphology in animal models have widely been studied but not those of Aβ(1–40). Hence, we evaluated the time- and dose-related effects of intravitreal Aβ(1–40) on retinal and optic nerve morphology. Since oxidative stress and brain derived neurotrophic factor (BDNF) are associated with Aβ-induced neuronal damage, we also studied dose and time-related effects of Aβ(1–40) on retinal oxidative stress and BDNF levels.Materials and methods: Five groups of rats were intravitreally administered with vehicle or Aβ(1–40) in doses of 1.0, 2.5, 5 and 10 nmol. Animals were sacrificed and eyes were enucleated at weeks 1, 2 and 4 post-injection. The retinae were subjected to morphometric analysis and TUNEL staining. Optic nerve sections were stained with toluidine blue and were graded for neurodegenerative effects. The estimation of BDNF and markers of oxidative stress in retina were done using ELISA technique.Results and conclusions: It was observed that intravitreal Aβ(1–40) causes significant retinal and optic nerve damage up to day 14 post-injection and there was increasing damage with increase in dose. However, on day 30 post-injection both the retinal and optic nerve morphology showed a trend towards normalization. The observations made for retinal cell apoptosis, retinal glutathione, superoxide dismutase activity and BDNF were in accordance with those of morphological changes with deterioration till day 14 and recovery by day 30 post-injection. The findings of this study may provide a guide for selection of appropriate experimental conditions for future studies.

Alterations in System xc- Expression in the Retina of Type 1 Diabetic Rats and the Role of Nrf2.

Nrf2 (nuclear factor erythroid 2-related factor 2), a transcription factor that controls expression of several proteins that are related to cellular antioxidant capacity, such as the subunit xCT of the system xc-, is dysregulated in diabetes. Recently, it was described that system xc- is decreased in the retina after 3weeks of diabetes. So, in the present work, the temporal relationship between xCT and Nrf2 in the retina of diabetic animals was investigated. Diabetes was induced in male Wistar rats (200g) by a single injection of streptozotocin, and retinas were collected after 1, 2, and 6months of diabetes induction. Expression of xCT, Nrf2 activity, and binding to antioxidant-responsive element (ARE) sequence were evaluated. Glutathione and reactive oxygen species (ROS) levels were also assessed. After 1month of diabetes, Nrf2 activity, xCT expression, and glutathione levels were reduced whereas ROS were increased. Although glutathione and ROS levels remain unchanged until later stages, Nrf2 activity and xCT expression returned to normal levels after 2months. However, they were decreased again at 6months of diabetes. Accordingly, Nrf2 binding to xCT ARE sequence followed the same pattern of Nrf2 activity and xCT expression. These data showed that retinal xCT expression is regulated by Nrf2 in diabetic condition. The results also demonstrated a temporal relationship between Nrf2 and system xc- which could be implicated in the initiation of oxidative stress in retina in diabetes.

Alterations of Ocular Hemodynamics Impair Ophthalmic Vascular and Neuroretinal Function

Hypertension is associated with numerous diseases, but its direct impact on the ocular circulation and neuroretinal function remains unclear. Herein, mouse eyes were challenged with different levels of hemodynamic insult via transverse aortic coarctation, which increased blood pressure and flow velocity by 50% and 40%, respectively, in the right common carotid artery, and reduced those parameters by 30% and 40%, respectively, in the left common carotid artery. Blood velocity in the right central retinal artery gradually increased up to 40% at 4 weeks of transverse aortic coarctation, and the velocity in the left central retinal artery gradually decreased by 20%. The fundus and retinal architecture were unaltered by hemodynamic changes. Endothelium-dependent vasodilations to acetylcholine and adenosine were reduced only in right (hypertensive) ophthalmic arteries. Increased cellularity in the nerve fiber/ganglion cell layers, enhanced glial fibrillary acidic protein expression, and elevated superoxide level were found only in hypertensive retinas. The electroretinogram showed decreased scotopic b-waves in the hypertensive eyes and decreased scotopic oscillatory potentials in both hypertensive and hypotensive eyes. In conclusion, hypertension sustained for 4 weeks causes ophthalmic vascular dysfunction, retinal glial cell activation, oxidative stress, and neuroretinal impairment. Although ophthalmic vasoregulation is insensitive to hypotensive insult, the ocular hypoperfusion causes neuroretinal dysfunction.

Taurine protects against retinal and optic nerve damage induced by endothelin-1 in rats via antioxidant effects.

Endothelin-1 (ET-1), a potent vasoconstrictor, is involved in retinal vascular dysregulation and oxidative stress in glaucomatous eyes. Taurine (TAU), a naturally occurring free amino acid, is known for its neuroprotective and antioxidant properties. Hence, we evaluated its neuroprotective properties against ET-1 induced retinal and optic nerve damage. ET-1 was administered intravitreally to Sprague-Dawley rats and TAU was injected as pre-, co- or post-treatment. Animals were euthanized seven days post TAU injection. Retinae and optic nerve were examined for morphology, and were also processed for caspase-3 immunostaining. Retinal redox status was estimated by measuring retinal superoxide dismutase, catalase, glutathione, and malondialdehyde levels using enzyme-linked immuosorbent assay. Histopathological examination showed significantly improved retinal and optic nerve morphology in TAU-treated groups. Morphometric examination showed that TAU pre-treatment provided marked protection against ET-1 induced damage to retina and optic nerve. In accordance with the morphological observations, immunostaining for caspase showed a significantly lesser number of apoptotic retinal cells in the TAU pre-treatment group. The retinal oxidative stress was reduced in all TAU-treated groups, and particularly in the pre-treatment group. The findings suggest that treatment with TAU, particularly pre-treatment, prevents apoptosis of retinal cells induced by ET-1 and hence prevents the changes in the morphology of retina and optic nerve. The protective effect of TAU against ET-1 induced retinal and optic nerve damage is associated with reduced retinal oxidative stress.