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
]. Binge ethanol treatment of adult rats induces neuronal damage [8
]. 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
]. 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 [36
]. Fluoro-Jade B is an alternative marker selectively staining degenerating neurons in the central nervous system (CNS) [37
]. 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 ). Fluoro-Jade B positive cells was increased 10 fold in cortex and 7.6 fold in dentate gyrus (Figure ). 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
]. 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
]. Activation of microglia has been linked to neurodegeneration through the production of neurotoxic factors, such as proinflammatory cytokines and free radicals [51
]. 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 ). We previously reported that chronic ethanol can activate microglia increasing proinflammatory factors (TNFα, IL-1β and MCP-1, etc.) [9
]. Astroglial activation we report here is also observed 24 hours after chronic ethanol treatment (Figure ). 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) [11
]. 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 [13
]. Increases in NF-κB DNA-binding activity during ethanol treatment correlate with the increased expression of proinflammatory genes in hippocampal-entorhinal cortex slice cultures [14
]. 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 [14
In rats BHT blocked NF-κB-DNA binding and ethanol neurotoxicity [13
]. In this study, we find that 10 doses of ethanol significantly increase NF-κB -p65 gene expression (Figure ) 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 ). 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 ). 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 ). 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 ). Previous studies have found increased NOX-proinflammatory responses in mice can persist for at least 10 months and longer [38
]. 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 [10
]. 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
]. Similarly brain gene array studies in mice implicate pro-inflammatory genes in brain may as regulators of alcohol intake [55
]. 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 ) and increased makers of neuronal death (Figure , ) 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 , ). Also, DPI pretreatment reduced ethanol increased caspase-3 immunoreactivity and Fluoro-Jade B staining (Figure ). These data link NOX-ROS to ethanol-induced microglial activation and neurodegeneration.
This study supports a role of NOX and ROS in chronic ethanol-induced neuroinflammation and neurodegeneration (Figure ). The present study and our previous report [9
] find that chronic ethanol induces microglial activation, increases proinflammatory cytokines (TNFα, IL-1β, IL-6 etc.) and chemokines (MCP-1) and up-regulates NOX, resulting in production of ROS. NF-κB transcription is activated and generates these proinflammatory factors (cytokines, chemokines, oxidases, ROS) that amplify NOX-ROS and NF-κB signaling cascades (Figure ). DPI, a NOX inhibitor, reduces microglial activation, ROS generation and neuronal death markers (activated caspase-3 and Fluoro-Jade B). Therefore, inhibition of NOX and ROS production may provide improved prevention and treatment for alcoholics and other neurodegenerative disorders.
Figure 13 NOX-ROS is a key signaling in alcohol neurodegeneration. Alcohol as a pro-inflammatory trigger activates microglia to release neurotoxic factors, such as TNFα, IL-1β, MCP-1, IL-6, ROS (O2-). Among these pro-inflammatory factors, ROS have (more ...)