Dietary Supplement Enriched in Antioxidants and Omega-3 Promotes Glutamine Synthesis in Müller Cells: A Key Process against Oxidative Stress in Retina.

To prevent ocular pathologies, new generation of dietary supplements have been commercially available. They consist of nutritional supplement mixing components known to provide antioxidative properties, such as unsaturated fatty acid, resveratrol or flavonoids. However, to date, only one preclinical study has evaluated the impact of a mixture mainly composed of those components (Nutrof Total®) on the retina and demonstrated that in vivo supplementation prevents the retina from structural and functional injuries induced by light. Considering the crucial role played by the glial Müller cells in the retina, particularly to regulate the glutamate cycle to prevent damage in oxidative stress conditions, we questioned the impact of this ocular supplement on the glutamate metabolic cycle. To this end, various molecular aspects associated with the glutamate/glutamine metabolism cycle in Müller cells were investigated on primary Müller cells cultures incubated, or not, with the commercially mix supplement before being subjected, or not, to oxidative conditions. Our results demonstrated that in vitro supplementation provides guidance of the glutamate/glutamine cycle in favor of glutamine synthesis. These results suggest that glutamine synthesis is a crucial cellular process of retinal protection against oxidative damages and could be a key step in the previous in vivo beneficial results provided by the dietary supplementation.

Cytoprotective Effects of Natural Highly Bio-Available Vegetable Derivatives on Human-Derived Retinal Cells.

Retinal pigment epithelial cells are crucial for retina maintenance, making their cytoprotection an excellent way to prevent or slow down retinal degeneration. In addition, oxidative stress, inflammation, apoptosis, neovascularization, and/or autophagy are key pathways involved in degenerative mechanisms. Therefore, here we studied the effects of curcumin, lutein, and/or resveratrol on human retinal pigment epithelial cells (ARPE-19). Cells were incubated with individual or combined agent(s) before induction of (a) H2O2-induced oxidative stress, (b) staurosporin-induced apoptosis, (c) CoCl2-induced hypoxia, or (d) a LED-autophagy perturbator. Metabolic activity, cellular survival, caspase 3/7 activity (casp3/7), cell morphology, VEGF levels, and autophagy process were assessed. H2O2 provoked a reduction in cell survival, whereas curcumin reduced metabolic activity which was not associated with cell death. Cell death induced by H2O2 was significantly reduced after pre-treatment with curcumin and lutein, but not resveratrol. Staurosporin increased caspase-3/7 activity (689%) and decreased cell survival by 32%. Curcumin or lutein protected cells from death induced by staurosporin. Curcumin, lutein, and resveratrol were ineffective on the increase of caspase 3/7 induced by staurosporin. Pre-treatment with curcumin or lutein prevented LED-induced blockage of autophagy flux. Basal-VEGF release was significantly reduced by lutein. Therefore, lutein and curcumin showed beneficial protective effects on human-derived retinal cells against several insults.

Dietary supplement enriched in antioxidants and omega-3 protects from progressive light-induced retinal degeneration.

In the present study, we have evaluated one of the dietary supplements enriched with antioxidants and fish oil used in clinical care for patient with age-related macular degeneration. Rats were orally fed by a gastric canula daily with 0.2 ml of water or dietary supplement until they were sacrificed. After one week of treatment, animals were either sacrificed for lipid analysis in plasma and retina, or used for evaluation of rod-response recovery by electroretinography (ERG) followed by their sacrifice to measure rhodopsin content, or used for progressive light-induced retinal degeneration (PLIRD). For PLIRD, animals were transferred to bright cyclic light for one week. Retinal damage was quantified by ERG, histology and detection of apoptotic nuclei. Animals kept in dim-cyclic-light were processed in parallel. PLIRD induced a thinning of the outer nuclear layer and a reduction of the b-wave amplitude of the ERG in the water group. Retinal structure and function were preserved in supplemented animals. Supplement induced a significant increase in omega-3 fatty acids in plasma by 168% for eicosapentaenoic acid (EPA), 142% for docosapentaenoic acid (DPA) and 19% for docosahexaenoic acid (DHA) and a decrease in the omega-6 fatty acids, DPA by 28%. In the retina, supplement induced significant reduction of linolenic acid by 67% and an increase in EPA and DPA by 80% and 72%, respectively, associated with significant decrease in omega-6 DPA by 42%. Supplement did not affect rhodopsin content or rod-response recovery. The present data indicate that supplement rapidly modified the fatty acid content and induced an accumulation of EPA in the retina without affecting rhodopsin content or recovery. In addition, it protected the retina from oxidative stress induced by light. Therefore, this supplement might be beneficial to slow down progression of certain retinal degeneration.

Pre-treatment of adult rats with high doses of erythropoietin induces caspase-9 but prevents light-induced retinal injury.

Erythropoietin (Epo) had been shown to have a neuroprotective effect independent from its erythropoietic properties. In this study, we tested whether Epo could protect the retina from damage induced by a long period of moderate light insult and how it protected. First, rats were injected intraperitoneally (i.p.) by human recombinant Epo at 5000 or 30,000U/kg to assess Epo concentration in plasma and retina. Second, rats were untreated or injected i.p. with Epo at 30,000U/kg, 1 or 4h before being placed in constant light (24h; 2200lux). Electroretinograms (ERG) were recorded before treatment, 1day and 15days (D15) after light exposure. After the last ERG, eyes were taken for histology. In parallel, we tested Epo protection against oxidative stressors on isolated retinas and its effect on caspase-9 activity. Epo injected at 30,000U/kg body weight, 4h before exposure to the damaging light, protected retinal function and structure against light damage and induced an increase in caspase-9 activity and expression. Epo had no direct or indirect protective effect against free radicals-induced death on isolated retinas. Epo protected the retina from a long period of moderate light exposure through a mechanism independent from a free radical scavenging property or an antioxidant facilitating activity. The activation of caspase-9, 4h after Epo injection, corresponding to the start of light exposure, suggests that caspase-9 plays a role in neuroprotection.

P23H and S334ter opsin mutations: Increasing photoreceptor outer segment n-3 fatty acid content does not affect the course of retinal degeneration.

PURPOSE: The n-3 polyunsaturated fatty acids (PUFA) facilitate retinal development and function. Rats carrying transgenes with P23H and S334ter rhodopsin mutations lose their photoreceptors and have lower levels of 22:6n-3 in rod photoreceptor outer segments (ROS) than wild type (WT) animals. We tested the hypothesis that the rate of retinal degeneration in these mutant animals could be sensitive to the n-3 fatty acid content of retina. METHODS: Beginning embryonic day 15, WT and heterozygous transgenic rats with P23H and S344ter rhodopsin mutations were fed semi-synthetic diets enriched in n-6 (safflower oil, SO) or n-3 (flaxseed oil, FO) PUFA. At 35 and 55 days of age, electroretinographic (ERG) response, outer nuclear layer (ONL) thickness, and fatty acid composition of plasma and ROS were determined. Student's t-tests and multivariate analysis of variance with post hoc tests determined statistical differences. RESULTS: Rats fed FO or SO diets had different n-6/n-3 PUFA ratios in plasma (1.3 and 62) and ROS (0.2 and 1.1, respectively). Although there were profound effects of the diets on the plasma fatty acid composition, there were only minor differences between WT and transgenic animals within each dietary regime. The ROS of FO fed rats had 70% more 22:6n-3 than those fed SO, and the WT had higher concentrations of 22:6n-3 than the transgenic animals (WT>P23H>S334ter). In contrast, there was no difference in 22:6n-3 levels in ROS of WT and transgenic rats fed the SO diet. At P55, both transgenic lines had diminished ERGs and ONL thickness relative to the WT. There was no detectable effect of ROS fatty acid enrichment on the rate of retinal degeneration in the transgenic animals. However, the FO-diet provided a modest protection of function (b-wave) in S334ter animals. CONCLUSIONS: Feeding n-3 fatty acids to rats with mutant rhodopsin transgenes significantly increased the levels of 22:6n-3 in ROS membranes, but had no effect on the rate of retinal degeneration. Therefore, the degeneration is not the result of low (or high) 22:6n-3 in ROS and supplementation with 18:3n-3 will not rescue dying photoreceptor cells in these animal models of inherited retinal degenerations.