• Energy Research

Early ethanol consumption could be a risk factor for young brain integrity and its maturation, and also for the development of addictive behaviors in adulthood. Neuronal nitric oxide synthase (nNOS) expressing neurons are specifically located in the subgranular layer (SGL) of dentate gyrus and may be relevant for hippocampal neurogenesis. The focus of this work is aimed to determine local changes in the nNOS-like immunoreactive (nNOS-LIR) cell populations of the SGL after chronic ethanol exposure in young adult and mature adult rats.We used the nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase (NADPH-d) reaction as a qualitative marker of nNOS enzyme activity. We also analyzed the nNOS-LIR cell density by the nNOS immunocytochemistry in order to compare these two methods of labeling. Dorsal striatum (CPu) was also analyzed in order to compare two neural areas with high nNOS-LIR cell density.The young adult group showed less hippocampal NADPH-d(+) cell density than the mature adult group. Interestingly, the NADPH-d(+) cell density was increased in the SGL of the young adult ethanol-treated group, whereas it decreased in the mature adult ethanol-treated group, when compared with their respective controls. No change was observed in any of the groups for the hippocampal nNOS-LIR cell density and no differences could be established in CPu for nNOS-LIR and NADPH-d(+) cell densities in any of the groups studied.The NADPH-d expression is affected by chronic ethanol exposure in opposite ways between both age groups studied. Further studies are needed to evaluate the relative importance of these findings, especially when considering human subjects.

Naltrexone, an antagonist ofμ-opioid receptors, is clinically used as adjuvant therapy of alcohol dishabituation. The aim of the present work was to test the effect of 1 mg/kg body weight of naltrexone to revert oxidative stress-related biochemical alterations, in the hippocampus and serum of chronic alcoholic adult rats. Malondialdehyde concentration was increased and glutathione peroxidase activity was decreased in hippocampus and serum of alcohol-treated rats. Naltrexone treatment restored these alterations. The in vitro antioxidant ability of Ntx could not justify these effects considering the doses used. Thus this apparent protective effect of Ntx can only be attributed to its pharmacological effects, as herein discussed.

Ethanol consumption originates a wide spectrum of disorders, including alteration of visual function. Oxidative stress is included among the mechanisms by which alcohol predisposes nervous tissue to injury. Retina, which is the neurosensorial eye tissue, is particularly sensitive to oxidative stress.In this study we analyze the effect of long-term alcohol consumption on oxidative stress parameters of the rat retina, and its correlation to retinal function, as well as to the expression of the antiapoptotic protein Bcl-2. We also study the protective effect of ebselen, a synthetic selenoorganic antioxidant.Herein we show that ethanol has a toxic effect on rat retina associated with oxidative stress. Decreases in retina glutathione concentration and increases in malondialdehyde content in whole eye homogenate significantly correlate with ERG b-wave decrease and Bcl-2 overexpression. We also show how ebselen is able to prevent all the alterations observed.Chronic ethanol consumption induces oxidative stress in rat retina associated with an impairment of ERG and Bcl-2 overexpression, suggesting a role for glial cells. All these alterations in the rat allow the proposal of an alcoholic retinopathy in this species.

The aim of this study was to demonstrate the existence of alterations in glutathione and cholesterol homeostasis in brain mitochondria from alcoholic rats. Glutathione concentration decreased, whereas oxidized glutathione and cholesterol contents increased in these organelles, suggesting the ethanol-induced generation of reactive oxygen species, and the impairment of mitochondrial uptake of glutathione, possibly due to the increase in cholesterol deposition. The release of apoptogenic proteins was increased after stimulating mitochondria from the brain of alcoholic rats with atractyloside. As a conclusion, chronic alcohol consumption might sensitize brain mitochondria to apoptotic stimuli, and promote the subsequent release of apoptotic proteins.

We have explored the mechanisms underlying ethanol-induced mitochondrial dynamics disruption and mitophagy. Ethanol increases mitochondrial fission in a concentration-dependent manner through Drp1 mitochondrial translocation and OPA1 proteolytic cleavage. ARPE-19 (a human retinal pigment epithelial cell line) cells challenged with ethanol showed mitochondrial potential disruptions mediated by alterations in mitochondrial complex IV protein level and increases in mitochondrial reactive oxygen species production. In addition, ethanol activated the canonical autophagic pathway, as denoted by autophagosome formation and autophagy regulator elements including Beclin1, ATG5-ATG12 and P-S6 kinase. Likewise, autophagy inhibition dramatically increased mitochondrial fission and cell death, whereas autophagy stimulation rendered the opposite results, placing autophagy as a cytoprotective response aimed to remove damaged mitochondria. Interestingly, although ethanol induced mitochondrial Bax translocation, this episode was associated to cell death rather than mitochondrial fission or autophagy responses. Thus, Bax required 600 mM ethanol to migrate to mitochondria, a concentration that resulted in cell death. Furthermore, mouse embryonic fibroblasts lacking this protein respond to ethanol by undergoing mitochondrial fission and autophagy but not cytotoxicity. Finally, by using the specific mitochondrial-targeted scavenger MitoQ, we revealed mitochondria as the main source of reactive oxygen species that trigger autophagy activation. These findings suggest that cells respond to ethanol activating mitochondrial fission machinery by Drp1 and OPA1 rather than bax, in a manner that stimulates cytoprotective autophagy through mitochondrial ROS.

Background: Most of the previously published data suggest a role for oxidative or nitrosative stress in ethanol‐induced nervous system damage. Moreover, ethanol is able to impair learning abilities in adult mammalian brain, a process suggested to be directly related to hippocampal neurogenesis. Ebselen, a synthetic compound with antioxidant properties, is able to prevent ethanol‐induced impairment of neurogenesis in adult rats. The aim of the present work was to further demonstrate the ability of ebselen to prevent biochemical alterations, and preserve long‐term potentiation (LTP) and learning abilities, in the hippocampus of chronic alcoholic adult rats.Methods: Biochemical markers of oxidative stress (glutathione and malondialdehyde) were assayed in hippocampi of control rats and animals fed a liquid alcoholic diet (Lieber‐De Carli) supplemented or not with ebselen. Long‐term potentiation and hippocampal‐dependent tests were studied in all animal groups.Results: The hippocampal concentrations of glutathione and malondialdehyde were decreased and increased, respectively, in alcohol‐treated animals, and did not differ from those of the control and the alcohol+ebselen groups. Long‐term potentiation in hippocampal slices from ethanol‐treated animals was prevented, when compared with controls, and occurred with a similar profile in control animals and in the alcohol+ebselen groups. Learning ability was tested with the Morris water maze test. Escape latencies were higher in ethanol‐treated rats than in control animals or the ones treated with ethanol+ebselen.Conclusions: The results herein strongly suggest that oxidative mechanisms may underlie the hippocampal effects of ethanol in adult rats, in view of the protective effect of ebselen.

AbstractThe retinal pigment epithelium (RPE), a monolayer located between the photoreceptors and the choroid, is constantly damaged by oxidative stress, particularly because of reactive oxygen species (ROS). As the RPE, because of its physiological functions, is essential for the survival of the retina, any sustained damage may consequently lead to loss of vision. Exosomes are small membranous vesicles released into the extracellular medium by numerous cell types, including RPE cells. Their cargo includes genetic material and proteins, making these vesicles essential for cell‐to‐cell communication. Exosomes may fuse with neighbouring cells influencing their fate. It has been observed that RPE cells release higher amounts of exosomes when they are under oxidative stress. Exosomes derived from cultured RPE cells were isolated by ultracentrifugation and quantified by flow cytometry. VEGF receptors (VEGFR) were analysed by both flow cytometry and Western blot. RT‐PCR and qPCR were conducted to assess mRNA content of VEGFRs in exosomes. Neovascularization assays were performed after applying RPE exosomes into endothelial cell cultures. Our results showed that stressed RPE cells released a higher amount of exosomes than controls, with a higher expression of VEGFR in the membrane, and enclosed an extra cargo of VEGFR mRNA. Angiogenesis assays confirmed that endothelial cells increased their tube formation capacity when exposed to stressed RPE exosomes.