Abstract
BackgroundActivation 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.MethodsMale 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.ResultsEthanol 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.ConclusionsEthanol 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.
Highlights
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
Chronic ethanol increases caspase-3 expression and Fluoro-Jade B staining To determine the effect of ethanol exposure on neurodegeneration in mice, immunohistochemistry for cleaved caspase-3 [36] and Fluoro-Jade B histochemistry were performed on C57BL/6 mouse brain sections treated with water or ethanol (5 g/kg, i.g.) daily for 10 days
Confocal microscopy indicated that most activated caspase-3 +IR cells colocalize with Neu-N+IR cells (Figure 1C), suggesting chronic ethanol exposure causes neuronal cell death
Summary
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. Previous work from our laboratory found 10 daily doses of ethanol treatment to male mice induced microglial activation, increased proinflammatory cytokines (TNFa, IL-1b, IL-6 etc.) and chemokines (MCP-1) and up-regulated NOX, resulting in production of ROS [9]. Others studying female mice following 5 months of ethanol drinking found chronic ethanol activation of nuclear factor kappa-B (NF-B) pathways, markers of increased microglia and astrocyte activation, induction of the proinflammatory oxidases, inducible nitric oxide synthase, and cyclo-oxygenase COX-2, as well as increased cytokine levels in the cerebral cortex that were related to increased activated caspase-3, a marker of cell death [11]. In male mice, we investigate NF-B, NADPH oxidase (NOX) and ROS involvement in neuronal damage
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