Abstract
BackgroundRecent studies have implicated microglia—the resident immune cells of the brain—in the pathophysiology of alcoholism. Indeed, post-mortem alcoholic brains show increased microglial markers and increased immune gene expression; however, the effects of ethanol on microglial functioning and how this impacts the brain remain unclear. In this present study, we investigate the effects of acute binge ethanol on microglia and how microglial depletion changes the brain neuroimmune response to acute binge ethanol withdrawal.MethodsC57BL/6J mice were treated intragastrically with acute binge ethanol for time course and dose-response studies. Cultured mouse BV2 microglia-like cells were treated with ethanol in vitro for time course studies. Mice were also administered the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia from the brain. These mice were subsequently treated with acute binge ethanol and sacrificed during withdrawal. Brain and BV2 mRNA were isolated and assessed using RT-PCR to examine expression of microglial and neuroimmune genes.ResultsAcute binge ethanol biphasically changed microglial (e.g., Iba1, CD68) gene expression, with initial decreases during intoxication and subsequent increases during withdrawal. Acute ethanol withdrawal dose dependently increased neuroimmune gene (e.g., TNFα, Ccl2, IL-1ra, IL-4) expression beginning at high doses. BV2 cells showed biphasic changes in pro-inflammatory (e.g., TNFα, Ccl2) gene expression following ethanol treatment in vitro. Administration of PLX5622 depleted microglia from the brains of mice. Although some neuroimmune genes were reduced by microglial depletion, many others were unchanged. Microglial depletion blunted pro-inflammatory (e.g., TNFα, Ccl2) gene expression and enhanced anti-inflammatory (e.g., IL-1ra, IL-4) gene expression during acute binge ethanol withdrawal.ConclusionsThese studies find acute binge ethanol withdrawal increases microglial and neuroimmune gene expression. Ethanol exposure also increases microglial pro-inflammatory gene expression in vitro. Furthermore, microglial depletion decreases expression of microglia-specific genes but has little effect on expression of many other neuroimmune signaling genes. Microglial depletion blunted the acute binge ethanol withdrawal induction of pro-inflammatory genes and enhanced induction of anti-inflammatory genes. These findings indicate microglia impact the brain response to acute binge ethanol withdrawal.
Highlights
Recent studies have implicated microglia—the resident immune cells of the brain—in the pathophysiology of alcoholism
TNFα protein increased 345%, while Ccl2 increased from less than 1 pg/ mL to approximately 17 pg/mL as determined by enzyme-linked immunosorbent assay (ELISA) (Additional file 5: Figure S5). These results find that acute binge ethanol withdrawal increases brain TNFα and Ccl2 mRNA and protein during withdrawal, similar to the microglial markers
TNFα and Ccl2 expression increased exponentially to 672% (p < 0.05) and 873% (p < 0.05), respectively, with the 6 g/kg dose (Fig. 2c, d). These results find that acute binge ethanol dose dependently increases brain pro-inflammatory cytokine expression during withdrawal beginning at high doses
Summary
Recent studies have implicated microglia—the resident immune cells of the brain—in the pathophysiology of alcoholism. Post-mortem alcoholic brains show increased microglial markers and increased immune gene expression; the effects of ethanol on microglial functioning and how this impacts the brain remain unclear. In this present study, we investigate the effects of acute binge ethanol on microglia and how microglial depletion changes the brain neuroimmune response to acute binge ethanol withdrawal. Alcohol use disorders (AUDs) are a common mental health disease in which individuals drink excessive amounts of alcohol despite negative consequences. Much remains unknown regarding the mechanisms by which excessive alcohol use negatively impacts the brain
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