3-nitrotyrosine and Retinal-Diseases

NCX 434 is a nitric oxide (NO)-donating triamcinolone acetonide (TA), shown to enhance optic nerve head (ONH) oxygen saturation in non-human primate eyes. Here, the effects of a single intravitreal (IVT) injection of TA were compared with those of NCX 434 on intraocular pressure (IOP), retinal function and retrobulbar haemodymamics in endothelin-1 (ET-1) induced ONH ischaemia/reperfusion in rabbits. Biochemical changes were also assessed in the aqueous humour and in retinal biopsies.. IOP and resistivity index of ophthalmic artery (RI-OA) were recorded using TonoPen and ecocolor Doppler, respectively. Retinal function was assessed using photopic electroretinography. Cytokine expression and oxidative stress markers were evaluated with immunoassay techniques.. At 4 weeks post IVT treatment, TA increased IOP and RI-OA while NCX 434 did not (IOP(Vehicle)=13.6±1.3, IOP(NCX 434)=16.9±2.2, IOP(TA)=20.9±1.9 mm Hg; p<0.05 vs vehicle; RI-OA(Vehicle)=0.44±0.03; RI-OA(NCX 434)=0.47±0.02; RI-OA(TA)=0.60±0.04). Both NCX 434 and TA reversed ET-1 induced decrease in electroretinography amplitude to similar extents. NCX 434 attenuated ET-1 induced oxidative stress markers and nitrotyrosine in retinal tissue, and interleukin-6 and tumour necrosis factor-α in aqueous humour more effectively than TA.. NCX 434 attenuates ET-1 induced ischaemia/reperfusion damage without increasing IOP, probably due to NO release. If data are confirmed in other species and models, this compound could represent an interesting new therapeutic option for retinal and ONH diseases, including diabetic retinopathy.

Topics: Animals; Caspase 3; Cytokines; Electroretinography; Endothelin-1; Glutathione; Hemodynamics; Intraocular Pressure; Intravitreal Injections; Laser-Doppler Flowmetry; Male; Nitrates; Nitric Oxide Donors; Ophthalmic Artery; Optic Disk; Rabbits; Reperfusion Injury; Retina; Retinal Diseases; Superoxide Dismutase; Tonometry, Ocular; Triamcinolone Acetonide; Tyrosine

Retinal ischemia/reperfusion (I/R) injury causes profound tissue damage, especially retinal ganglion cell (RGC) death. The aims of the study were to investigate whether catalase (CAT) has a neuroprotective effect on RGC after I/R injury in rats, and to determine the possible antioxidant mechanism. Wistar female rats were randonmized into four groups: normal control group (Control group), retinal I/R with vehicle group (I/R with vehicle group), retinal I/R with AAV-CAT group (I/R with AAV-CAT group), and normal retina with AAV-CAT group (normal with AAV-CAT group). One eye of each rat was pretreated with recombinant adeno-associated virus containing catalase gene (I/R with AAV-CAT group or normal with AAV-CAT group) and recombinant adeno-associated virus containing GFP gene (I/R with vehicle group) by intravitreal injection 21 days before initiation of I/R injury. Retinal I/R injury was induced by elevating intraocular pressure to 100mmHg for 1h. The number of RGC and inner plexiform layer (IPL) thickness were measured by fluorogold retrograde labeling and hematoxylin and eosin staining at 6h, 24h, 72 h and 5d after injury. Hydrogen peroxide (H(2)O(2)), the number of RGC, IPL thickness, malondialdehyde(MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), CAT activity and nitrotyrosine were measured by fluorescence staining, immunohistochemistry and enzyme-linked immunosorbent assay analysis at 5 days after injury. Electroretinographic (ERG) evaluation was also used. Pretreatment of AAV-CAT significantly decreased the levels of H(2)O(2), MDA, 8-OHdG and nitrotyrosine, increased the catalase activity, and prevented the reduction of a- and b- waves in the I/R with AAV-CAT group compare with the I/R with vehicle group (p<0.01). Catalase attenuated the I/R-induced damage of RGC and IPL and retinal function. Therefore, catalase can protect the rat retina from I/R-induced injury by enhancing the antioxidative ability and reducing oxidative stress, which suggests that catalase may be relevant for the neuroprotection of inner retina from I/R-related diseases.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antioxidants; Apoptosis; Catalase; Cell Count; Cell Survival; Deoxyguanosine; Dependovirus; Electroretinography; Enzyme-Linked Immunosorbent Assay; Female; Genetic Therapy; Genetic Vectors; Immunohistochemistry; Malondialdehyde; Neuroprotective Agents; Rats; Rats, Wistar; Reperfusion Injury; Retina; Retinal Diseases; Retinal Ganglion Cells; Tyrosine

Neovascularization occurs in response to tissue ischemia and growth factor stimulation. In ischemic retinopathies, however, new vessels fail to restore the hypoxic tissue; instead, they infiltrate the transparent vitreous. In a model of oxygen-induced retinopathy (OIR), TNFalpha and iNOS, upregulated in response to tissue ischemia, are cytotoxic and inhibit vascular repair. The aim of this study was to investigate the mechanism for this effect.. Wild-type C57/BL6 (WT) and TNFalpha(-/-) mice were subjected to OIR by exposure to 75% oxygen (postnatal days 7-12). The retinas were removed during the hypoxic phase of the model. Retinal cell death was determined by TUNEL staining, and the microglial cells were quantified after Z-series capture with a confocal microscope. In situ peroxynitrite and superoxide were measured by using the fluorescent dyes DCF and DHE. iNOS, nitrotyrosine, and arginase were analyzed by real-time PCR, Western blot analysis, and activity determined by radiolabeled arginine conversion. Astrocyte coverage was examined after GFAP immunostaining.. The TNFalpha(-/-) animals displayed a significant reduction in TUNEL-positive apoptotic cells in the inner nuclear layer of the avascular retina compared with that in the WT control mice. The reduction coincided with enhanced astrocytic survival and an increase in microglial cells actively engaged in phagocytosing apoptotic debris that displayed low ROS, RNS, and NO production and high arginase activity.. Collectively, the results suggest that improved vascular recovery in the absence of TNFalpha is associated with enhanced astrocyte survival and that both phenomena are dependent on preservation of microglial cells that display an anti-inflammatory phenotype during the early ischemic phase of OIR.

Topics: Animals; Apoptosis; Arginase; Blotting, Western; Cell Count; Cell Death; Cell Survival; In Situ Nick-End Labeling; Ischemia; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Nitric Oxide Synthase Type II; Nitrosation; Oxidative Stress; Oxygen; Reactive Nitrogen Species; Reactive Oxygen Species; Retinal Diseases; Retinal Neurons; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; Tumor Necrosis Factor-alpha; Tyrosine

Retinal ischemia/reperfusion (I/R) occurs in many ocular diseases and leads to neuronal death. Lutein, a potent antioxidant, is used to prevent severe visual loss in patients with early age-related macular degeneration (AMD), but its effect on I/R insult is unclear. The objective of the present study is to investigate the neuroprotective effect of lutein on retinal neurons after acute I/R injury.. Unilateral retinal I/R was induced by the blockade of internal carotid artery using intraluminal method in mice. Ischemia was maintained for 2 hours followed by 22 hours of reperfusion, during which either lutein or vehicle was administered. The number of viable retinal ganglion cells (RGC) was quantified. Apoptosis was investigated using TUNEL assay. Oxidative stress was elucidated using markers such as nitrotyrosine (NT) and poly(ADP-ribose) (PAR).. In vehicle-treated I/R retina, severe cell loss in ganglion cell layer, increased apoptosis as well as increased NT and nuclear PAR immunoreactivity were observed. In lutein-treated I/R retina, significantly less cell loss, decreased number of apoptotic cells, and decreased NT and nuclear PAR immunoreactivity were seen.. The neuroprotective effect of lutein was associated with reduced oxidative stress. Lutein has been hitherto used principally for protection of outer retinal elements in AMD. Our study suggests that it may also be relevant for the protection of inner retina from acute ischemic damage.

Topics: Acute Disease; Animals; Antioxidants; Apoptosis; Calbindin 2; Disease Models, Animal; In Situ Nick-End Labeling; Lutein; Male; Mice; Mice, Inbred C57BL; Neurons; Neuroprotective Agents; Oxidative Stress; Poly Adenosine Diphosphate Ribose; Reperfusion Injury; Retinal Diseases; Retinal Ganglion Cells; S100 Calcium Binding Protein G; Tyrosine

Thioredoxin (TRX) plays a variety of redox-related roles in organisms. To investigate its function as an endogenous redox regulator in NMDA-induced retinal neurotoxicity, we injected NMDA with TRX, mutant TRX or saline into the vitreous cavity of rat eyes. Retinal ganglion cells were rescued by TRX, compared with saline, when evaluated by retrograde labeling analysis at 7 days after NMDA injection. TRX, but not its mutant form, prevented NMDA-induced apoptosis in the retina, as measured by terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling. The induction of caspase 3 and 9, but not caspase 8, by NMDA was significantly lower in TRX-treated eyes than in saline-treated eyes. NMDA-induced activation of the MAPKs, p38 kinase and c-Jun N-terminal kinase after 6 h and of the MAPK kinases (MKKs) MKK3/6 and MKK4 after 3 h was markedly suppressed in retinal ganglion cells by TRX but not by the mutant form. NMDA-induced increases in protein carbonylation, nitrosylation and lipid peroxidation were also suppressed in TRX-treated eyes. We concluded that the intravitreous injection of TRX effectively attenuated NMDA-induced retinal cell damage and that suppression of oxidative stress and inhibition of apoptotic signaling pathways were involved in this neuroprotection.

Topics: Animals; Apoptosis; Blotting, Western; Caspase 3; Caspase 8; Caspase 9; Caspases; Excitatory Amino Acid Agonists; Immunoenzyme Techniques; Immunohistochemistry; In Situ Nick-End Labeling; Lipid Peroxidation; Male; Mitogen-Activated Protein Kinases; N-Methylaspartate; Neuroprotective Agents; Phosphorylation; Proto-Oncogene Proteins c-jun; Rats; Rats, Wistar; Retina; Retinal Diseases; Retinal Ganglion Cells; Thioredoxins; Tyrosine

Opposing effects have been ascribed to nitric oxide (NO) on retinal microvascular survival. We investigated whether changes in the redox state may contribute to explain apparent conflicting actions of NO in a model of oxygen-induced retinal vasoobliteration. Retinal microvascular obliteration was induced by exposing 7-day-old rat pups (P7) for 2 or 5 days to 80% O(2). The redox state of the retina was assessed by measuring reduced glutathione and oxidative and nitrosative products malondialdehyde and nitrotyrosine. The role of NO on vasoobliteration was evaluated by treating animals with nitric oxide synthase (NOS) inhibitors (N-nitro-l-arginine; L-NA) and by determining NOS isoform expression and activity; the contribution of nitrosative stress was also determined in animals treated with the degradation catalyst of peroxynitrite FeTPPS or with the superoxide dismutase mimetic CuDIPS. eNOS, but not nNOS or iNOS, expression and activity were increased throughout the exposure to hyperoxia. These changes were associated with an early (2 days hyperoxia) decrease in reduced glutathione and increases in malondialdehyde and nitrotyrosine. CuDIPS, FeTPPS, and L-NA treatments for these 2 days of hyperoxia nearly abolished the vasoobliteration. In contrast, during 5 days exposure to hyperoxia when the redox state rebalanced, L-NA treatment aggravated the vasoobliteration. Interestingly, VEGFR-2 expression was respectively increased by NOS inhibition after short-term (2 days) exposure to hyperoxia and decreased during the longer hyperoxia exposure. Data disclose that the dual effects of NO on newborn retinal microvascular integrity in response to hyperoxia in vivo depend on the redox state and seem mediated at least in part by VEGFR-2.

Topics: Animals; Animals, Newborn; Antioxidants; Glutathione; Isoenzymes; Malondialdehyde; Metalloporphyrins; Microcirculation; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxidation-Reduction; Oxidative Stress; Oxygen; Rats; Rats, Sprague-Dawley; Retina; Retinal Diseases; Retinal Vessels; Salicylates; Tyrosine; Vascular Endothelial Growth Factor Receptor-2

To investigate the effect of termination of galactose feeding after a very short duration of experimental galactosemia on the biochemical abnormalities that are postulated to contribute to the development of retinopathy.. Experimentally galactosemic rats (normal rats fed a 30% galactose-rich diet for 2 months) were fed a galactose-free diet for an additional 1 month. At the end of 3 months, retinas were removed to measure oxidative stress, nitric oxides (NOs), activity of PKC, and levels of nitrotyrosine. Data were compared between rats in the control group (fed a normal diet) and those in the experimentally galactosemic group (30% galactose diet for the entire 3 months).. Interruption of 2 months of galactose feeding by withdrawal of galactose from the diet for 1 additional month had partially beneficial effects on retinal lipid peroxides, but the levels of an endogenous antioxidant, reduced glutathione (GSH), remained subnormal in the retina of galactose-withdrawal rats (P < 0.05 vs. normal and P > 0.05 vs. galactose group). Cessation of the galactose-rich diet had partially beneficial effects on NO levels in the retina, but the levels of nitrotyrosine, an indicator of the formation of peroxynitrite, and activation of PKC were not affected.. The results show that retinal dysmetabolism continues to progress after experimental galactosemia is terminated in rats: Particularly, antioxidant levels remain subnormal, and nitrotyrosine levels are elevated for at least 1 month. Identification of metabolic abnormalities associated with the progression of incipient retinopathy after hyperglycemia is normalized may help in the search for the cause of retinopathy.

Topics: Animals; Disease Progression; Drug Administration Schedule; Galactose; Galactosemias; Glutathione; Hexoses; Lipid Peroxides; Male; Metabolic Diseases; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidative Stress; Protein Kinase C; Rats; Rats, Sprague-Dawley; Retina; Retinal Diseases; Tyrosine

Ischemic proliferative retinopathy (e.g., diabetes mellitus, retinopathy of prematurity, or retinal vein occlusion) is a major cause of blindness worldwide. Apart from neovascularization, ischemic proliferative retinopathy leads to retinal degeneration. Apoptosis has been ascribed to be the leading mechanism in ischemic retinal degeneration. We showed recently that inducible nitric oxide synthase (iNOS) is expressed in the avascular retina in proliferative retinopathy in vivo and that iNOS expression in retinal glial cells is responsible for retinal neuronal cell death in vitro. Here we show that retinal apoptosis and subsequent degeneration occur in the murine model of ischemic proliferative retinopathy. Furthermore, because NO can have beneficial or detrimental effects in the retina, we analyzed the role of iNOS on retinal apoptosis in ischemic proliferative retinopathy. Using iNOS knock-out mice and iNOS inhibitor 1400W, we demonstrate in vivo that iNOS expression induces apoptosis locally in the inner nuclear layer of the avascular retina and that protein nitration may be involved in this process. These findings are the first evidence for retinal apoptosis in an animal model of ischemic proliferative retinopathy, demonstrating that iNOS plays a crucial role not only in retinal neovascular disease but also in retinal degeneration. We show that it is an ideal target to protect the hypoxic retina from degeneration and to improve its vascularization.

Topics: Aging; Amidines; Animals; Animals, Newborn; Apoptosis; Benzylamines; Cell Count; Crosses, Genetic; Diabetic Retinopathy; Disease Models, Animal; Drug Administration Routes; Enzyme Inhibitors; Immunohistochemistry; In Situ Nick-End Labeling; Ischemia; Mice; Mice, Inbred Strains; Mice, Knockout; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxygen; Proteins; Retina; Retinal Diseases; Retinal Vein Occlusion; Tyrosine