title: NADPH oxidase and reactive oxygen species contribute to alcohol-induced microglial activation and neurodegeneration.
abstract: BACKGROUND
  Activation of microglia causes the production of proinflammatory factors and upregulation of NADPH oxidase (NOX) that form reactive oxygen species (ROS) that lead to neurodegeneration. Previously, we reported that 10 daily doses of ethanol treatment induced innate immune genes in brain. In the present study, we investigate the effects of chronic ethanol on activation of NOX and release of ROS, and their contribution to ethanol neurotoxicity.
  
  
  METHODS
  Male C57BL/6 and NF-κB enhanced GFP mice were treated intragastrically with water or ethanol (5 g/kg, i.g., 25% ethanol w/v) daily for 10 days. The effects of chronic ethanol on cell death markers (activated caspase-3 and Fluoro-Jade B), microglial morphology, NOX, ROS and NF-κB were examined using real-time PCR, immunohistochemistry and hydroethidine histochemistry. Also, Fluoro-Jade B staining and NOX gp91phox immunohistochemistry were performed in the orbitofrontal cortex (OFC) of human postmortem alcoholic brain and human moderate drinking control brain.
  
  
  RESULTS
  Ethanol treatment of C57BL/6 mice showed increased markers of neuronal death: activated caspase-3 and Fluoro-Jade B positive staining with Neu-N (a neuronal marker) labeling in cortex and dentate gyrus. The OFC of human post-mortem alcoholic brain also showed significantly more Fluoro-Jade B positive cells colocalized with Neu-N, a neuronal marker, compared to the OFC of human moderate drinking control brain, suggesting increased neuronal death in the OFC of human alcoholic brain. Iba1 and GFAP immunohistochemistry showed activated morphology of microglia and astrocytes in ethanol-treated mouse brain. Ethanol treatment increased NF-κB transcription and increased NOX gp91phox at 24 hr after the last ethanol treatment that remained elevated at 1 week. The OFC of human postmortem alcoholic brain also had significant increases in the number of gp91phox + immunoreactive (IR) cells that are colocalized with neuronal, microglial and astrocyte markers. In mouse brain ethanol increased gp91phox expression coincided with increased production of O2- and O2- - derived oxidants. Diphenyleneiodonium (DPI), a NOX inhibitor, reduced markers of neurodegeneration, ROS and microglial activation.
  
  
  CONCLUSIONS
  Ethanol activation of microglia and astrocytes, induction of NOX and production of ROS contribute to chronic ethanol-induced neurotoxicity. NOX-ROS and NF-κB signaling pathways play important roles in chronic ethanol-induced neuroinflammation and neurodegeneration.
authors.affiliation: Bowles Center for Alcohol Studies, School of Medicine, Chapel Hill, NC 27599, USA.
authors.forename: Liya
authors.initials: L
authors.lastname: Qin
authors.forename: Fulton T
authors.initials: FT
authors.lastname: Crews
mesh_terms: D000818:Animals; D001713:Biphenyl Compounds / Q000494:pharmacology; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D002492:Central Nervous System Depressants / Q000506:poisoning*; D016207:Cytokines / Q000378:metabolism; D000431:Ethanol / Q000506:poisoning*; D005452:Fluoresceins; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D017628:Microglia / Q000187:drug effects*; D019255:NADPH Oxidases / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D019636:Neurodegenerative Diseases / Q000139:chemically induced* / Q000209:etiology / Q000473:pathology; D009861:Onium Compounds / Q000494:pharmacology; D009930:Organic Chemicals; D018384:Oxidative Stress / Q000187:drug effects; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013997:Time Factors
chemical_list: D001713:Biphenyl Compounds; D002492:Central Nervous System Depressants; D016207:Cytokines; D005452:Fluoresceins; D016328:NF-kappa B; D009861:Onium Compounds; D009930:Organic Chemicals; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C485184:enhanced green fluorescent protein; C435731:fluoro jade; C031291:diphenyliodonium; D049452:Green Fluorescent Proteins; D000431:Ethanol; D019255:NADPH Oxidases; D053148:Caspase 3; D010751:Phosphopyruvate Hydratase