Liposome-Mediated Delivery Improves the Efficacy of Lisosan G against Retinopathy in Diabetic Mice.

Nutraceuticals are natural substances whose anti-oxidant and anti-inflammatory properties may be used to treat retinal pathologies. Their efficacy is limited by poor bioavailability, which could be improved using nanocarriers. Lisosan G (LG), a fermented powder from whole grains, protects the retina from diabetic retinopathy (DR)-induced damage. For this study, we tested whether the encapsulation of LG in liposomes (LipoLG) may increase its protective effects. Diabetes was induced in mice via streptozotocin administration, and the mice were allowed to freely drink water or a water dispersion of two different doses of LG or of LipoLG. Electroretinographic recordings after 6 weeks showed that only the highest dose of LG could partially protect the retina from diabetes-induced functional deficits, while both doses of LipoLG were effective. An evaluation of molecular markers of oxidative stress, inflammation, apoptosis, vascular endothelial growth factor, and the blood-retinal barrier confirmed that the highest dose of LG only partially protected the retina from DR-induced changes, while virtually complete prevention was obtained with either dose of LipoLG. These data indicate that the efficacy of LG in contrasting DR is greatly enhanced by its encapsulation in liposomes and may lay the ground for new dietary supplements with improved therapeutic effects against DR.

Restored retinal physiology after administration of niacin with citicoline in a mouse model of hypertensive glaucoma.

Introduction: Much interest has been addressed to antioxidant dietary supplements that are known to lower the risk of developing glaucoma or delay its progression. Among them, niacin and citicoline protect retinal ganglion cells (RGCs) from degeneration by targeting mitochondria, though at different levels. A well-established mouse model of RGC degeneration induced by experimental intraocular pressure (IOP) elevation was used to investigate whether a novel combination of niacin/citicoline has better efficacy over each single component in preserving RGC health in response to IOP increase. Methods: Ocular hypertension was induced by an intracameral injection of methylcellulose that clogs the trabecular meshwork. Electroretinography and immunohistochemistry were used to evaluate RGC function and density. Oxidative, inflammatory and apoptotic markers were evaluated by Western blot analysis. Results: The present results support an optimal efficacy of niacin with citicoline at their best dosage in preventing RGC loss. In fact, about 50% of RGCs were spared from death leading to improved electroretinographic responses to flash and pattern stimulation. Upregulated levels of oxidative stress and inflammatory markers were also consistently reduced by almost 50% after niacin with citicoline thus providing a significant strength to the validity of their combination. Conclusion: Niacin combined with citicoline is highly effective in restoring RGC physiology but its therapeutic potential needs to be further explored. In fact, the translation of the present compound to humans is limited by several factors including the mouse modeling, the higher doses of the supplements that are necessary to demonstrate their efficacy over a short follow up period and the scarce knowledge of their transport to the bloodstream and to the eventual target tissues in the eye.

Increased efficacy of dietary supplement containing wax ester-rich marine oil and xanthophylls in a mouse model of dry macular degeneration.

Age-related macular degeneration (AMD) is nowadays considered among the retinal diseases whose clinical management lacks established treatment approaches, mainly for its atrophic (dry) form. In this respect, the use of dietary patterns enriched in omega-3 and antioxidant xanthophylls has emerged as a promising approach to counteract dry AMD progression although the prophylactic potential of omega-3 of fish origin has been discussed. Whether enriched availability of omega-3 and xanthophylls may increase the effectiveness of diet supplementation in preventing dry AMD remains to be fully established. The present study aims at comparing the efficacy of an existing orally administered formulation based on lutein and fish oil, as a source of omega-3, with a novel formulation providing the combination of lutein and astaxanthin with Calanus oil (COil), which contains omega-3 together with their precursors policosanols. Using a mouse model of dry AMD based on subretinal injection of polyethylene glycol (PEG)-400, we assessed the comparative efficacy of both formulations on PEG-induced major hallmarks including oxidative stress, inflammation, glial reactivity and outer retinal thickness. Dietary supplementation with both mixtures has been found to exert a significant antioxidant and anti-inflammatory activity as reflected by the overall amelioration of the PEG-induced pathological hallmarks. Noteworthy, the formulation based on COil appeared to be more protective than the one based on fish oil, presumably because of the higher bioavailability of omega-3 in COil. These results support the use of dietary supplements combining omega-3 and xanthophylls in the prevention and treatment of AMD and suggest that the source of omega-3 might contribute to treatment efficacy.

A Dietary Combination of Forskolin with Homotaurine, Spearmint and B Vitamins Protects Injured Retinal Ganglion Cells in a Rodent Model of Hypertensive Glaucoma.

There is indication that nutritional supplements protect retinal cells from degeneration. In a previous study, we demonstrated that dietary supplementation with an association of forskolin, homotaurine, spearmint extract and B vitamins efficiently counteracts retinal dysfunction associated with retinal ganglion cell (RGC) death caused by optic nerve crush. We extended our investigation on the efficacy of dietary supplementation with the use of a mouse model in which RGC degeneration depends as closely as possible on intraocular pressure (IOP) elevation. In this model, injecting the anterior chamber of the eye with methylcellulose (MCE) causes IOP elevation leading to RGC dysfunction. The MCE model was characterized in terms of IOP elevation, retinal dysfunction as determined by electrophysiological recordings, RGC loss as determined by brain-specific homeobox/POU domain protein 3A immunoreactivity and dysregulated levels of inflammatory and apoptotic markers. Except for IOP elevation, dysfunctional retinal parameters were all recovered by dietary supplementation indicating the involvement of non-IOP-related neuroprotective mechanisms of action. Our hypothesis is that the diet supplement may be used to counteract the inflammatory processes triggered by glial cell activation, thus leading to spared RGC loss and the preservation of visual dysfunction. In this respect, the present compound may be viewed as a potential remedy to be added to the currently approved drug therapies for improving RGC protection.

Protective Efficacy of a Dietary Supplement Based on Forskolin, Homotaurine, Spearmint Extract, and Group B Vitamins in a Mouse Model of Optic Nerve Injury.

Glaucoma is a multifactorial blinding disease with a major inflammatory component ultimately leading to apoptotic retinal ganglion cell (RGC) death. Pharmacological treatments lowering intraocular pressure can help slow or prevent vision loss although the damage caused by glaucoma cannot be reversed. Recently, nutritional approaches have been evaluated for their efficacy in preventing degenerative events in the retina although mechanisms underlying their effectiveness remain to be elucidated. Here, we evaluated the efficacy of a diet supplement consisting of forskolin, homotaurine, spearmint extract, and vitamins of the B group in counteracting retinal dysfunction in a mouse model of optic nerve crush (ONC) used as an in vivo model of glaucoma. After demonstrating that ONC did not affect retinal vasculature by fluorescein angiography, we determined the effect of the diet supplement on the photopic negative response (PhNR) whose amplitude is strictly related to RGC integrity and is therefore drastically reduced in concomitance with RGC death. We found that the diet supplementation prevents the reduction of PhNR amplitude (p < 0.001) and concomitantly counteracts RGC death, as in supplemented mice, RGC number assessed immunohistochemically is significantly higher than that in non-supplemented animals (p < 0.01). Major determinants of the protective efficacy of the compound are due to a reduction of ONC-associated cytokine secretion leading to decreased levels of apoptotic markers that in supplemented mice are significantly lower than in non-supplemented animals (p < 0.001), ultimately causing RGC survival and ameliorated visual dysfunction. Overall, our data suggest that the above association of compounds plays a neuroprotective role in this mouse model of glaucoma thus offering a new perspective in inflammation-associated neurodegenerative diseases of the inner retina.

Lisosan G Protects the Retina from Neurovascular Damage in Experimental Diabetic Retinopathy.

Lisosan G (LG), a fermented powder obtained from whole grains, is a recognized antioxidant compound that improves the bioactivity and survival of different cell types. The purpose of this study was to investigate whether LG ameliorates both the neural and the vascular damage characterizing early stages of diabetic retinopathy (DR). The effects of LG were studied in cultured explants of mouse retinas challenged with oxidative stress (OS) or in retinas of streptozotocin (STZ)-treated rats. Apoptosis, vascular endothelial growth factor (VEGF) expression, OS markers, blood-retinal barrier (BRB) integrity, and inflammation were assessed, while retinal function was evaluated with electroretinogram (ERG). LG extensively inhibited apoptosis, VEGF expression, and OS both in retinal explants and in STZ rats. In addition, STZ rats treated with LG displayed an almost total BRB integrity, reduced levels of inflammatory markers and a partially restored visual function as evaluated with ERG. In summary, we demonstrated that LG exhibits antioxidant and anti-inflammatory effects that exert powerful protective actions against neural and vascular defects characteristic of DR. Therefore, LG-containing foods or supplements may be considered to implement DR treatments.

Further Evidence on Efficacy of Diet Supplementation with Fatty Acids in Ocular Pathologies: Insights from the EAE Model of Optic Neuritis.

In the experimental autoimmune encephalomyelitis (EAE) mouse model of optic neuritis, we recently demonstrated that diet supplementation with a balanced mixture of fatty acids (FAs), including omega 3 and omega 6, efficiently limited inflammatory events in the retina and prevented retinal ganglion cell (RGC) death, although mechanisms underlying the efficacy of FAs were to be elucidated. Whether FAs effectiveness was accompanied by efficient rescue of demyelinating events in the optic nerve was also unresolved. Finally, the possibility that RGC rescue might result in ameliorated visual performance remained to be investigated. Here, the EAE model of optic neuritis was used to investigate mechanisms underlying the anti-inflammatory effects of FAs, including their potential efficacy on macrophage polarization. In addition, we determined how FAs-induced rescue of RGC degeneration was related to optic nerve histopathology by performing ultrastructural morphometric analysis with transmission electron microscopy. Finally, RGC rescue was correlated with visual performance by recording photopic electroretinogram, an efficient methodology to unravel the role of RGCs in the generation of electroretinographic waves. We conclude that the ameliorative effects of FAs were dependent on a predominant anti-inflammatory action including a role on promoting the shift of macrophages from the inflammatory M1 phenotype towards the anti-inflammatory M2 phenotype. This would finally result in restored optic nerve histopathology and ameliorated visual performance. These findings can now offer new perspectives for implementing our knowledge on the effectiveness of diet supplementation in counteracting optic neuritis and suggest the importance of FAs as possible adjuvants in therapies against inflammatory diseases of the eye.

Fatty Acids Dietary Supplements Exert Anti-Inflammatory Action and Limit Ganglion Cell Degeneration in the Retina of the EAE Mouse Model of Multiple Sclerosis.

Optic neuritis is an acute inflammatory demyelinating disorder of the optic nerve (ON) and is an initial symptom of multiple sclerosis (MS). Optic neuritis is characterized by ON degeneration and retinal ganglion cell (RGC) loss that contributes to permanent visual disability and lacks a reliable treatment. Here, we used the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, a well-established model also for optic neuritis. In this model, C57BL6 mice, intraperitoneally injected with a fragment of the myelin oligodendrocyte glycoprotein (MOG), were found to develop inflammation, Müller cell gliosis, and infiltration of macrophages with increased production of oncomodulin (OCM), a calcium binding protein that acts as an atypical trophic factor for neurons enabling RGC axon regeneration. Immunolabeling of retinal whole mounts with a Brn3a antibody demonstrated drastic RGC loss. Dietary supplementation with Neuro-FAG (nFAG®), a balanced mixture of fatty acids (FAs), counteracted inflammatory and gliotic processes in the retina. In contrast, infiltration of macrophages and their production of OCM remained at elevated levels thus eventually preserving OCM trophic activity. In addition, the diet supplement with nFAG exerted a neuroprotective effect preventing MOG-induced RGC death. In conclusion, these data suggest that the balanced mixture of FAs may represent a useful form of diet supplementation to limit inflammatory events and death of RGCs associated to optic neuritis. This would occur without affecting macrophage infiltration and the release of OCM thus favoring the maintenance of OCM neuroprotective role.

Efficacy of a Fatty Acids Dietary Supplement in a Polyethylene Glycol-Induced Mouse Model of Retinal Degeneration.

Current knowledge of the benefits of nutrition supplements for eye pathologies is based largely on the use of appropriate animal models, together with defined dietary supplementation. Here, C57BL6 mice were subretinally injected with polyethylene glycol (PEG)-400, an established model of retinal degeneration with a dry age-related macular degeneration (AMD)-like phenotype, an eye pathology that lacks treatment. In response to PEG-400, markers of the complement system, angiogenesis, inflammation, gliosis, and macrophage infiltration were upregulated in both retinas and retinal pigment epithelium (RPE)/choroids, whereas dietary supplementation with a mixture based on fatty acids counteracted their upregulation. Major effects include a reduction of inflammation, in both retinas and RPE/choroids, and an inhibition of macrophage infiltration in the choroid, yet not in the retina, suggesting a targeted action through the choroidal vasculature. Histological analysis revealed a thinning of the outer nuclear layer (ONL), together with dysregulation of the epithelium layer in response to PEG-400. In addition, immunohistofluorescence demonstrated Müller cell gliosis and macrophage infiltration into subretinal tissues supporting the molecular findings. Reduced ONL thickness, gliosis, and macrophage infiltration were counteracted by the diet supplement. The present data suggest that fatty acids may represent a useful form of diet supplementation to prevent or limit the progression of dry AMD.