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

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

IntroductionMuch 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.MethodsOcular 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.ResultsThe 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.ConclusionNiacin 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

Efficacia della combinazione di nutraceutici antiossidanti per il trattamento dello stress ossidativo nella degenerazione maculare senile e nella retinopatia diabetica

La degenerazione maculare senile (AMD) e la retinopatia diabetica (DR) rappresentano due tra le più comuni patologie degenerative della retina associate ad una progressiva perdita della vista. Entrambe le malattie sono caratterizzate da meccanismi patologici che determinano una degenerazione progressiva della neuroretina con seguenti alterazioni funzionali e morfologiche dell’intero organo. Tra i meccanismi molecolari caratterizzanti AMD e DR, lo stress ossidativo e l’infiammazione giocano un ruolo chiave per la loro progressione patologica. Nel caso dell’AMD, lo stress ossidativo e l’infiammazione sono principalmente causati sia dai processi di invecchiamento cellulare sia da fattori ambientali, quali eccessive esposizioni ad alte intensità luminose, alla base della degenerazione cellulare che coinvolge, perlopiù, la retina esterna e i fotorecettori. Nella DR, l’iperglicemia dovuta alla condizione diabetica, causa alterazioni metaboliche che contribuiscono all’incremento dei livelli di stress ossidativo alla base della degenerazione neuronale. La comunicazione tra le componenti neuronali e vascolari retiniche viene in questo modo compromessa, determinando una over-espressione di fattori pro-angiogenici responsabili della formazione di aberrazioni microvascolari tipiche delle fasi più avanzate della DR. L’incremento di stress ossidativo rappresenta quindi l’evento comune che determina la progressione delle due malattie. Pertanto, questo rappresenta un potenziale bersaglio di terapie antiossidanti che mirano a prevenire i meccanismi neurodegenerativi tipici di AMD e DR. In questo contesto, la somministrazione di antiossidanti di origine naturale con la dieta è emersa come un promettente approccio per la prevenzione di tali patologie, considerato la loro tipica bassa invasività e la bassa frequenza di effetti indesiderati. Il presente studio si propone quindi di valutare gli effetti derivanti dal trattamento combinato con una miscela di sostanze origine naturale (nutraceutici) riconosciute per il loro alto potere antiossidante (luteina, cyanidin-3-glucoside, zinco e verbascoside) volte alla prevenzione dei tipici danni retinici provocati dall’AMD e dalla DR. La condizione di AMD è stata riprodotta in ratti Sprague-Dawley mediante un danno da luce (LD) in seguito all’esposizione per 24h ad un’intensità di luce di 1000 lux. Il medesimo ceppo di ratti è invece stato utilizzato per l’induzione del diabete mediante streptozotocina (STZ) come modello di DR. Sulla base di questi modelli è stata testata l’efficacia preventiva di due dosi di miscela di nutraceutici antiossidanti somministrate per via orale. I ratti sottoposti a LD sono stati trattati per 7 giorni precedenti al danno da luce, mentre i ratti STZ hanno ricevuto un trattamento giornaliero per un mese consecutivo dopo l’induzione del diabete. L’efficacia del trattamento nel modello LD è stata valutata mediante l’analisi di marker di stress ossidativo, di reattività gliale, e analisi morfologiche della retina esterna. Nel modello STZ, l’efficacia del trattamento è stata valutata mediante analisi dei marker di stress ossidativo, della barriera emato-retinica (BRB) e analisi strutturale dei vasi della retina. Inoltre, analisi elettroretinografiche sono state effettuate per entrambi i modelli per valutare l’effetto del trattamento sulla funzionalità retinica. I dati confermano che sia nel modello LD sia nel modello STZ, l’attivazione di meccanismi di stress ossidativo e di infiammazione producono la perdita strutturale e funzionale delle componenti retiniche neuronali e vascolari, rispettivamente. Nel modello LD, il pretrattamento con la miscela antiossidante determina la diminuzione dello stress ossidativo e della reattività gliale con la conseguente prevenzione dei danni strutturali e funzionali della retina esterna. Nel modello STZ, il trattamento con la medesima miscela di nutraceutici antiossidanti contrasta l’insorgenza di stress ossidativo dovuto al diabete, prevenendo l’alterazione strutturale e funzionale della BRB e la relativa alterazione della funzionalità retinica. Age-related macular degeneration (AMD) and diabetic retinopathy (DR) represent two of the most common retinal degenerative diseases associated with a gradual loss of vision. Both are represented by pathological mechanism causing a progressive degeneration of neuroretina with functional and morphological damage to the whole organ. Among the molecular mechanisms that characterize AMD and DR, oxidative stress and inflammation play a key role in their pathological progression. In AMD, oxidative stress and inflammation are mainly caused by cellular aging/senescence processes and by environmental factors, including excessive exposure to high light intensity. These processes cause cellular degeneration events mostly occurring in the outer retina and photoreceptor layer. In DR, hyperglycemia due to the diabetic condition causes metabolic imbalances that contribute to the increase of oxidative stress underlying neuronal degeneration. This pathological event compromises the communication between the neuronal and vascular component of the retina, resulting an overexpression of pro-angiogenic factors responsible for the subsequent microvascular aberrations, typical of the several stages of DR. The increase of oxidative stress is defined as the common event underlying the progression of these diseases. Therefore, this represent a potential target of antioxidant therapies that aim to prevent the neurodegenerative mechanism typical of AMD and DR. In this context, the administration of antioxidants of natural origin as a promising approach for the prevention of these pathological processes, given their typical low invasiveness and low frequency of undesirable effects. This study aims to evaluate the effects deriving from the combined treatment with a mixture of natural substances with a high antioxidant power (formed by lutein, C3G, zinc and verbascoside) , aimed at preventing the typical retinal damage caused by AMD and DR. The AMD model was reproduced in Sprague-Dawley rats by 24 hours light damage with 100 lux of light intensity, and the animals were preventively treated in the 7 days preceding the light damage. The DR model was reproduced in the same strain of rats by intraperitoneal injection of streptozotocin (STZ), and the animals were treated for 30 days following the injection. To evaluate their antioxidant efficacy, some molecular techniques were used such as Western blot, immunohistochemistry and analysis of retinal function by electroretinogram (ERG). The results confirm that, both in the LD model and in the STZ model, the activation of oxidative stress and inflammation mechanisms produce the structural and functional loss of neuronal and vascular components of the retina. Pretreatment with the mixture of natural antioxidant in the LD model determines the reduction of oxidative stress and glial reactivity, preventing functional and structural damage of the retina. In the STZ model, the treatment with the same mixture counteracts the onset of oxidative stress caused by diabetes, preventing the structural and functional alterations of the BRB and related retinal function. Therefore, these results indicate that treatments based on the use of natural substances with powerful antioxidant action could prevent and/or counter the progression of degenerative retinal disease, such as AMD and DR

Natural History of Glaucoma Progression in the DBA/2J Model: Early Contribution of Müller Cell Gliosis

Glaucoma is a chronic optic neuropathy characterized by progressive degeneration of retinal ganglion cells (RGCs). Elevated intraocular pressure (IOP) and the resulting mechanical stress are classically considered the main causes of RGC death. However, RGC degeneration and ensuing vision loss often occur independent of IOP, indicating a multifactorial nature of glaucoma, with the likely contribution of glial and vascular function. The aim of the present study was to provide a comprehensive evaluation of the time course of neuro–glial–vascular changes associated with glaucoma progression. We used DBA/2J mice in the age range of 2–15 months as a spontaneous model of glaucoma with progressive IOP elevation and RGC loss typical of human open-angle glaucoma. We found that the onset of RGC degeneration at 10 months of age coincided with that of IOP elevation and vascular changes such as decreased density, increased lacunarity and decreased tight-junction protein zonula occludens (ZO)-1, while hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were already significantly upregulated at 6 months of age together with the onset of Müller cell gliosis. Astrocytes, however, underwent significant gliosis at 10 months. These results indicate that Müller cell activation occurs well before IOP elevation, with probable inflammatory consequences, and represents an early event in the glaucomatous process. Early upregulation of HIF-1α and VEGF is likely to contribute to blood retinal barrier failure, facilitating RGC loss. The different time courses of neuro–glial–vascular changes during glaucoma progression provide further insight into the nature of the disease and suggest potential targets for the development of efficient therapeutic intervention aside from IOP lowering

Preventive Efficacy of an Antioxidant Compound on Blood Retinal Barrier Breakdown and Visual Dysfunction in Streptozotocin-Induced Diabetic Rats

: In diabetic retinopathy (DR), high blood glucose drives chronic oxidative stress and inflammation that trigger alterations of the neurovascular balance finally resulting in vascular abnormalities and retinal cell death, which converge towards altered electroretinogram (ERG). In the last years, a growing body of preclinical evidence has suggested that nutrients with anti-inflammatory/antioxidant properties can be able to hamper DR progression since its very early stages. In the present study, we used a streptozotocin-induced rat model of DR, which mimics most aspects of the early stages of human DR, to test the preventive efficacy of a novel compound containing cyanidin-3-glucoside (C3G), verbascoside and zinc as nutrients with antioxidant and anti-inflammatory properties. Western blot, immunofluorescence and electroretinographic analyses demonstrated a dose-dependent inhibition of oxidative stress- and inflammation-related mechanisms, with a significant counterpart in preventing molecular mechanisms leading to DR-associated vasculopathy and its related retinal damage. Preventive efficacy of the compound on dysfunctional a- and b-waves was also demonstrated by electroretinography. The present demonstration that natural compounds, possibly as a consequence of vascular rescue following ameliorated oxidative stress and inflammation, may prevent the apoptotic cascade leading to ERG dysfunction, adds further relevance to the potential application of antioxidants as a preventive therapy to counteract DR progression

Dietary Supplementation of Antioxidant Compounds Prevents Light‐Induced Retinal Damage in a Rat Model

Light‐induced retinal damage (LD) is characterized by the accumulation of reactive oxy‐ gen species leading to oxidative stress and photoreceptor cell death. The use of natural antioxidants has emerged as promising approach for the prevention of LD. Among them, lutein and cyanidin‐3‐ glucoside (C3G) have been shown to be particularly effective due to their antioxidant and anti‐in‐ flammatory activity. However, less is known about the possible efficacy of combining them in a multicomponent mixture. In a rat model of LD, Western blot analysis, immunohistochemistry and electroretinography were used to demonstrate that lutein and C3G in combination or in a multi‐ component mixture can prevent oxidative stress, inflammation, gliotic and apoptotic responses thus protecting photoreceptor cells from death with higher efficacy than each component alone. Com‐ bined efficacy on dysfunctional electroretinogram was also demonstrated by ameliorated rod and cone photoreceptor responses. These findings suggest the rationale to formulate multicomponent blends which may optimize the partnering compounds bioactivity and bioavailability

Meldonium Inhibits Cell Motility and Wound-Healing in Trabecular Meshwork Cells and Scleral Fibroblasts: Possible Applications in Glaucoma

Meldonium (MID) is a synthetic drug designed to decrease the availability of L-carnitine—a main player in mitochondrial energy generation—thus modulating the cell pathways of energy metabolism. Its clinical effects are mostly evident in blood vessels during ischemic events, when the hyperproduction of endogenous carnitine enhances cell metabolic activities, leading to increased oxidative stress and apoptosis. MID has shown vaso-protective effects in model systems of endothelial dysfunction induced by high glucose or by hypertension. By stimulating the endothelial nitric oxide synthetase (eNOS) via PI3 and Akt kinase, it has shown beneficial effects on the microcirculation and blood perfusion. Elevated intraocular pressure (IOP) and endothelial dysfunction are major risk factors for glaucoma development and progression, and IOP remains the main target for its pharmacological treatment. IOP is maintained through the filtration efficiency of the trabecular meshwork (TM), a porous tissue derived from the neuroectoderm. Therefore, given the effects of MID on blood vessels and endothelial cells, we investigated the effects of the topical instillation of MID eye drops on the IOP of normotensive rats and on the cell metabolism and motility of human TM cells in vitro. Results show a significant dose-dependent decrease in the IOP upon topic treatment and a decrease in TM cell motility in the wound-healing assay, correlating with an enhanced expression of vinculin localized in focal adhesion plaques. Motility inhibition was also evident on scleral fibroblasts in vitro. These results may encourage a further exploration of MID eye drops in glaucoma treatment