NADPH oxidase and reactive oxygen species contribute to alcohol-induced microglial activation and neurodegeneration
© BioMed Central Ltd 2012
Received: 9 November 2011
Accepted: 12 January 2012
Published: 12 January 2012
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.
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.
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.
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.
KeywordsNADPH oxidase Reactive oxygen species neuroinflammation neurodegeneration
Alcohol can cause brain damage  and lead to neurodegeneration in some cases . Alcoholics, likely due to heavy alcohol consumption have reduced brain mass, cortical neuronal loss, other neuropathological changes as well as impaired cognitive functions and mild dementia [3–5]. Binge ethanol administration in adult rats is known to cause brain damage reduced by anti-oxidants [6–8]. However, the mechanism of ethanol induced neurodegeneration is uncertain. Previous work from our laboratory found 10 daily doses of ethanol treatment to male mice induced microglial activation, increased proinflammatory cytokines (TNFα, IL-1β, IL-6 etc.) and chemokines (MCP-1) and up-regulated NOX, resulting in production of ROS . We also found increased microglial markers and levels of the chemokine, MCP-1, in post-mortem human alcoholic brain . Others studying female mice following 5 months of ethanol drinking found chronic ethanol activation of nuclear factor kappa-B (NF-κB) 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 . In the present study, in male mice, we investigate NF-κB, NADPH oxidase (NOX) and ROS involvement in neuronal damage.
NF-κB is a transcription factor that in brain is involved in proinflammatory gene activation in glia as well as other gene regulation. Acute ethanol treatment of rats activates NF-κB in the brain . Chronic binge ethanol treatment of rats causes neuronal degeneration and increased NF-κB-DNA binding, with both being reversed by inhibition of NF-κB-DNA binding . Ethanol treatment of brain slice cultures has been found to increase multiple NF-κB proinflammatory target genes [14, 15]. However, the relationship between proinflammatory gene induction and neuronal death is not clearly understood. Activation of glial cells, especially microglia, that release pro-inflammatory factors and reactive oxygen species (ROS) have been implicated in several models of neurodegeneration [16, 17]. NADPH oxidase (NOX), an enzyme that produces ROS, is activated in brains from Alzheimer's disease (AD)  and Parkinson's disease (PD) . NOX is a multi-subunit enzyme complex that is activated and induced by inflammatory signals [20, 21]. The catalytic subunit of NOX, gp91phox, produces superoxide that can be toxic to neurons. In the present study, we find increased levels of NOX-gp91phox, and reactive oxygen species (ROS) following chronic ethanol treatment. Using transgenic mice that mark NF-κB transcription through induction of enhanced GFP mice (NF-κBEGFP) [22, 23] we find NF-κB transcription, NOX gp91phox activation and ROS production occur within the same cell. Further, increased levels of NOX-gp91phox and cell death markers within orbital frontal cortex (OFC) are found in both chronic ethanol treated mouse and human post-mortem alcoholic brain. Our data indicate ethanol activation of NF-κB transcription of proinflammatory genes and formation of NOX-ROS play a pivotal role in ethanol induced neurodegeneration.
Eight-week male (20-22g) C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, Maine). NF-κB enhanced GFP mice were gift from Dr. Christian Jobin's lab. All protocols in this study were approved by the Institutional Animal Care and Use Committee and were in accordance with the National Institute of Health regulations for the care and use of animals in research.
Case characteristics of Subjects used for immunohistochemical analyses - Alcohol Consumption
Age at Death
Clinical Cause of Death
Lifetime Ethanol (gm)
Ischaemic heart disease
Acute myocardial infarction
Ischaemic heart disease
Coronary heart disease
Ischaemic heart disease
Ischaemic heart disease
Ischaemic heart disease
Ischaemic heart disease
Coronary artery thrombosis
Ischaemic heart disease
Gastro intestinal haemorrhage
Atherosclerotic cardiovascular disease
Cleaved caspase-3 (Asp 175) antibody was from Cell Signaling Technology (Danvers, MA). Fluoro-Jade B and mouse Neu-N antibody were from Chemicon international (Temecula, CA). Rabbit anti-Iba1 antibody was purchased from Wako Pure Chemical Industries, Ltd. (1-2Doshomachi 3-Chome Chuo-ku Osaka 540-8605, Japan). Monoclonal anti-mouse gp91phox was from Transduction Laboratories (Lexington, KY). Rabbit polyclonal anti-gp91phox IgG was purchased from Upstate cell signaling solutions (Temecula, CA). Goat polyclonal gp91phox (C-15) antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal microtubule associated protein 2 (MAP2) antibody was purchased from Abcam (Cambridge, MA). Polyclonal Rabbit anti-Glial Fibrillary Acidic Protein was from DakoCytomation (Glostrup, Denmark). Hydroethidine was from Invitrogen Molecular Probes (Eugene, OR). All other reagents came from Sigma Chemical Co. (St. Louis, MO).
Sixty male C57BL/6 mice were randomly assigned to water control group (30 mice) and ethanol group (30 mice). The mice were treated intragastrically with water (control) or ethanol (5 g/kg, i.g., 25% ethanol w/v), with volumes matched, daily for 10 days. The average blood alcohol concentration at 1 hour after the first ethanol treatment and the last ethanol treatment was 302 mg/dl ± 12 (w/v, n = 10) and 297 mg/dl ± 11 (w/v, n = 10), respectively. The blood ethanol level is high and considered to model binge drinking . Twenty mice from each group were sacrificed at 24 hr after the last dose of ethanol for mRNA and histochemistry. Ten mice from each group were sacrificed at 1 week after the last dose of ethanol for NOX gp91phox immunostaining. For diphenyleneiodonium (DPI) treatment, forty male C57BL/6 mice were randomly assigned to 4 groups: control, EtOH, DPI and EtOH plus DPI (10 mice per group). The mice in EtOH and EtOH plus DPI groups were treated intragastrically with ethanol (5 g/kg, i.g., 25% ethanol w/v) daily for 10 days. The mice in control and DPI groups were gavaged with water daily for 10 days. DPI (3 mg/kg, i.p.) was given to mice at 0.5 hr and 24 hr after the last dose of ethanol. In both water and ethanol groups, mice were injected with saline, with volumes and time matched. Mice were sacrificed 3 hr after the last dose of DPI. For NF-κB transcription study, twenty male NF-κB enhanced GFP mice, a transgenic mouse expressing the enhanced GFP under the transcriptional control of NF-κB cis elements (cis-NF-κBEGFP) [22, 23], were treated intragastrically with water (control, 10 mice) or ethanol (5 g/kg, i.g., 25% ethanol w/v, 10 mice), daily for 10 days. The mice were sacrificed 24 hr after the last dose of ethanol. For ROS analysis, male C57BL/6 or NF-κB enhanced GFP mice (10 mice per group) were treated intragastrically with water (control) or ethanol (5 g/kg, i.g., 25% ethanol w/v), daily for 10 days. Mice were injected with dehydroethidium (10 mg/kg, i.p.) in 0.5% carboxymethyl cellulose at 23.5 hr after the last dose of ethanol. Brains were harvested 30 min later and frozen sections (15 μm) were examined for hydroethidine oxidation product, ethidium accumulation, by fluorescence microscopy. All experiments were repeated 2 to 3 times.
Real-time PCR analysis
Total RNA was extracted from the brain samples of C57BL/6 mice treated with ethanol or water, and reverse transcribed as described previously . The primer sequences used in this study were as follows: NF-κB p65 (essential modulator), 5'-GGC GGC ACG TTT TAC TCT TT-3' (forward) and 5'-CCG TCT CCA GGA GGT TAA TGC-3' (reverse); β-actin, 5'- GTA TGA CTC CAC TCA CGG CAA A-3' (forward) and 5'-GGT CTC GCT CCT GGA AGA TG-3' (reverse). The SYBR green PCR master mix (Applied Biosystems, Foster City, CA) was used for real-time PCR analysis. The relative differences in expression between groups were expressed using cycle time (Ct) values normalized with β-actin, and relative differences between control and treatment groups were calculated and expressed as relative increases setting control as 100%.
Mouse brains were fixed with 4% paraformidehide in Phosphate Buffered Saline (PBS) and processed for immunostaining as described previously . Human postmortem brains were processed to Paraffin sections for immunohistochemistry. Microglia were stained with rabbit anti-Iba1 antibody. Mouse NOX membrane subunit gp91phox was immunostained with monoclonal anti-mouse gp91phox or rabbit polyclonal anti-gp91phox IgG. Human gp91phox was immunostained with goat polyclonal gp91phox antibody. Caspase-3 was immunostained with polyclonal anti-cleaved caspase-3 antibody. Neurons were stained with Neu-N or MAP2 antibody. Astrocytes were labeled with GFAP antibody. Immunolabeling was visualized by using nickel-enhanced 3,3'-diaminobenzidinne (DAB) or Alexa Fluor 488 (green) or 555 (red) or 633 (blue) dye.
In situ visualization of O2 - and O2 - - derived oxidant production
In situ visualization of O2 - and O2 - - derived oxidant production was assessed by hydroethidine histochemistry [19, 30]. Mice were injected with dehydroethidium (10 mg/kg, i.p.) in 0.5% carboxymethyl cellulose at 23.5 hrs after the last dose of ethanol. Brains were harvested 30 min later and frozen sections (15 μm) were examined for hydroethidine oxidation product, ethidium accumulation, by fluorescence microscopy (excitation 510 nm; emission 580 nm).
Fluoro-Jade B staining with Neu-N labeling
Brain sections were immunostained with mouse Neu-N (a neuronal marker) antibody. Immunolabeling was visualized by using Alexa Fluor 555 dye. Sections were rinsed three times with PBS and one time with water before performing Fluoro-Jade B procedure. Sections stained with Neu-N were mounted on superfrost/plus microscope slides and air dried overnight. The sections were rinsed in distilled water for 2 min to rehydrate and transferred to a solution of 0.06% potassium permanganate for 10 min. The sections were then rinsed in distilled water for 2 min and placed in a 0.0004% Fluoro-Jade B solution made by adding 4 ml of a 0.01% stock solution of Fluoro-Jade B to 96 ml of 0.1% acetic acid. After 20 min in the Fluoro-Jade B staining solution, the stained slides were thoroughly washed in distilled water, dehydrated and coverslipped.
Where ∑Q is the sum of the caspase-3 or gp91phox + IR cells counted from each disector frame, ∑disector is the sum of the number of disector frames counted, A(fr) is the known area associated with each disector frame, and h is the known distance between two disector planes (we used 10 μm).
For colabeling study, double or triple stained sections were digitally photographed with Leica SP2-AOBS confocal microscope and analyzed with Leica SP2 LCS software.
The data are expressed as mean ± SEM and statistical significance was assessed with an ANOVA followed by Bonferroni's t-test using the StatView program (Abacus Concepts, Berkeley, CA). A value of P < 0.05 was considered statistically significant.
Chronic ethanol increases caspase-3 expression and Fluoro-Jade B staining
Chronic ethanol induces activation of microglia and astrocytes
Astrocyte activation was assessed by morphology using GFAP, an astrocyte-specific intermediate filament protein [39, 40]. Chronic ethanol treatment increased GFAP + IR in cortex and dentate gyrus (Figure 4B) 24 h after the last dose of ethanol. In addition to these two brain regions, astroglial activation was also notably observed in other brain areas, such as substantia nigra and forceps minor corpus callosum in the ethanol treated-mice (data not shown). Thus, the data together with increased cell death markers (caspase-3 and Fluoro-Jade B) by chronic ethanol treatment suggest that astroglial activation mediate ethanol-induced neurodegeneration.
Chronic ethanol enhances NF-κB mRNA and protein expression
Cell phenotype for NF-κB activation and ROS production was examined using histochemical markers. NF-κB enhanced GFP reporter mice showed green fluorescence. ROS were detected by red hydroethidine histochemistry and cell type markers, e.g. Iba1 microglial antibody, MAP2 neuronal antibody and GFAP astroglial antibody. Confocal microscopy found that NF-κB and ROS were triple-labeled with Iba1 or MAP2, but not with GFAP (Figure 5C), indicating ethanol-induced NF-κB activation and ROS production mostly occurred in microglia and neurons.
Chronic ethanol increases expression of NOX and production of ROS
The cellular localization of chronic ethanol-induced activation of NOX and production of ROS
DPI reduces chronic ethanol-induced microglial activation and ROS generation
Inhibition of NOX with DPI prevents ethanol-induced neurodegeneration
Chronic binge drinking can cause brain damage, cognitive dysfunction and neurodegeneration. Cerebral white matter atrophy and neuronal loss in the frontal cortex, the hypothalamus, and the thalamus are found in alcoholic brains [1, 45]. Binge ethanol treatment of adult rats induces neuronal damage . We have recently discovered that alcohol increases proinflammatory cytokines (TNFα, IL-1β), chemokine (MCP-1) and microglial activation in mouse brain that mimic increases found in post-mortem human alcoholic brain [9, 10]. Here, our data, for the first time, find that 10 daily binge doses of ethanol caused significant increases in the staining of cell death markers: cleaved caspase-3 and Fluoro-Jade B. Activated caspase-3+immunoreactivity (+IR) is a putative marker for dying cells . Fluoro-Jade B is an alternative marker selectively staining degenerating neurons in the central nervous system (CNS) . Our data found that chronic ethanol increased the number of activated caspase-3+IR cells 3.1 fold in cortex and 3.5 fold in dentate gyrus (Figure 1). Fluoro-Jade B positive cells was increased 10 fold in cortex and 7.6 fold in dentate gyrus (Figure 2). These results suggest that chronic ethanol can cause neurodegeneration in adult mice. We also studied human post-mortem alcoholic frontal cortex, the brain region most associated with alcoholic neurodegeneration [46–48]. We found that the orbitofrontal cortex (OFC) of human postmortem alcoholic brain has significantly more Fluoro-Jade B positive cells which are colocalized with Neu-N, a neuronal marker, compared to the OFC of human moderate drinking control brain. Together, these results indicate that alcohol can cause neurodegeneration in adult mice that mimics that found in human alcoholics.
The underlying mechanism of alcohol-induced brain damage is not well understood. Activation of glial cells is a critical event in many neuroinflammatory processes [49, 50]. Activation of microglia has been linked to neurodegeneration through the production of neurotoxic factors, such as proinflammatory cytokines and free radicals [51, 52]. Here we show that 10 doses of ethanol-treated mouse brain displayed the characteristics of activation of microglia: increased cell size, irregular shape, and intensified Iba-1 immunoreactivity (Figure 4A). We previously reported that chronic ethanol can activate microglia increasing proinflammatory factors (TNFα, IL-1β and MCP-1, etc.) . Astroglial activation we report here is also observed 24 hours after chronic ethanol treatment (Figure 4B). The activated astroglia were shown by a marked upregulation of GFAP immunoreactivity along with hypertrophic astrocytes in several brain regions, including cortex and dentate gyrus. These results are consistent with Guerri lab's findings that show hypertrophic astrocytes as well as increased caspase-3+IR cells in the mice treated with chronic ethanol administration (10% ethanol, v/v, for 5 months) . Reactive hypertrophic astrogliosis is a marker of neuroinflammation. Again, our data support that activation of microglia and astroglia contribute to chronic ethanol-induced neuroinflammation and neurodegeneration.
NF-κB is a family of transcription factors involved in regulating cell death/survival, differentiation, and inflammation. Acute ethanol administration has been demonstrated to activate the NF-κB system in the brain, and this in turn triggers the expression of TNFα as well as other proinflammatory cytokines and NF-κB-regulated genes . Increases in NF-κB DNA-binding activity during ethanol treatment correlate with the increased expression of proinflammatory genes in hippocampal-entorhinal cortex slice cultures . Blockade of NF-κB activation by p65 siRNA or the antioxidant butylated hydroxytoluene (BHT) reduces the induction of proinflammatory TNFα, IL-1β, MCP-1, protease TACE, tissue plasminogen activator (tPA) and inducible nitric oxide synthase by ethanol .
In rats BHT blocked NF-κB-DNA binding and ethanol neurotoxicity . In this study, we find that 10 doses of ethanol significantly increase NF-κB -p65 gene expression (Figure 5A) in C57BL/6 mice. Consistent with the mRNA data, in ethanol treated group, NF-κB GFP reporter fluorescence was markedly increased in multiple brain regions, such as dentate gyrus in NF-κB enhanced GFP mice (Figure 5B). Increases occurred predominantly in microglia and neurons. There data support the hypothesis that ethanol-induced oxidative stress involves a neuroinflammatory mechanism under the regulation of NF-κB transcription.
Another novel discovery is that for the first time we show alcohol increases NADPH oxidase gp91phox (NOX2) in adult mouse brain that mimics that found in human post-mortem alcoholic brain. NOX gp91phox remained elevated 1 week after chronic ethanol treatment (Figure 6C). The orbitofrontal cortex (OFC) of human post-mortem alcoholic brain also had significant increases in the number of gp91phox + IR cells, compared to the OFC of human moderate drinking control brain (Figure 7A, B). Confocal microscopy of double IHC with markers specific for neurons, microglia and astrocytes indicated that human NOX gp91phox was expressed in all 3 cell types in alcoholics (Figure 7C). Previous studies have found increased NOX-proinflammatory responses in mice can persist for at least 10 months and longer . The persistence of NOX-proinflammatory responses suggests the elevated levels in human alcoholic brain may represent both recent alcohol drinking as well as heavy drinking periods earlier in the lifetime of the alcoholics studied. We previously reported increased microglial markers and the chemokine MCP1 in post-mortem human alcoholic brain . These findings are consistent with gene array studies in post-mortem human brain. One of the most prominent gene groups altered in frontal cortex and VTA of alcoholics are 'immune/stress response genes' [53, 54]. Similarly brain gene array studies in mice implicate pro-inflammatory genes in brain may as regulators of alcohol intake . Thus, our findings are consistent with others.
Activated NOX produces superoxide. Superoxide formation, assessed by ethidine, was increased by ethanol. Increased NOX gp91 expression, superoxide formation in neurons (Figure 9) and increased makers of neuronal death (Figure 1, 2) are consistent with neuroimmune activation and oxidative stress mediating the neuronal toxicity.
Diphenyleneiodonium (DPI) inhibits NADPH-dependent oxidase. Our data found that co-treatment of DPI and ethanol significantly reduced ethanol induced microglial activation and ROS production (Figure 10, 11). Also, DPI pretreatment reduced ethanol increased caspase-3 immunoreactivity and Fluoro-Jade B staining (Figure 12). These data link NOX-ROS to ethanol-induced microglial activation and neurodegeneration.
Chronic ethanol induces brain NADPH oxidase gp91phox (NOX2) up-regulation and neurodegeneration in adult C57BL/6 mice that mimics findings in human alcoholic brain. Activation of microglia and astrocytes, induction of NOX and production of ROS contribute to ethanol neurodegeneration. Inhibition of NOX, ROS and NF-κB may offer hope in prevention and treatment for alcoholics and other neurodegenerative diseases.
Reactive oxygen species
Polymerase chain reaction
Tumor necrosis factor-α
Monocyte chemotactic protein-1
Nuclear factor kappa-light-chain-enhancer of activated B cells
Enzyme-Linked ImmunoSorbent Assay
Institutional Animal Care and Use Committee
This work was supported by the National Institutes of Health, National Institute on Alcoholism and Alcohol Abuse [AA020023, AA020024, AA020022, AA019767, AA11605 and AA007573]. The authors wish to also acknowledge support from the Bowles Center for Alcohol Studies, The University of North Carolina at Chapel Hill, School of Medicine. The authors would also like to thank Donna Sheedy for providing human post-mortem brain sections and thank Dr. Christian Jobin, Dr. Kathy Sulik and Yan Dong for providing NF-κB reporter mice, and also thank Tonya Hurst, Michael Chua and Neal Kramarcy for their technical support, as well as Diana Lotito for assisting with the manuscript preparation.
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