Combined Insults of a MASH Diet and Alcohol Binges Activate Intercellular Communication and Neutrophil Recruitment via the NLRP3-IL-1ß Axis in the Liver.

Binge drinking in obese patients positively correlates with accelerated liver damage and liver-related death. However, the underlying mechanism and the effect of alcohol use on the progression of metabolic-dysfunction-associated steatotic liver disease (MASLD) remain unexplored. Here, we show that short-term feeding of a metabolic-dysfunction-associated steatohepatitis (MASH) diet plus daily acute alcohol binges for three days induce liver injury and activation of the NLRP3 inflammasome. We identify that a MASH diet plus acute alcohol binges promote liver inflammation via increased infiltration of monocyte-derived macrophages, neutrophil recruitment, and NET release in the liver. Our results suggest that both monocyte-derived macrophages and neutrophils are activated via NLRP3, while the administration of MCC950, an NLRP3 inhibitor, dampens these effects.In this study, we reveal important intercellular communication between hepatocytes and neutrophils. We discover that the MASH diet plus alcohol induces IL-1ß via NLRP3 activation and that IL-1ß acts on hepatocytes and promotes the production of CXCL1 and LCN2. In turn, the increase in these neutrophils recruits chemokines and causes further infiltration and activation of neutrophils in the liver. In vivo administration of the NLRP3 inhibitor, MCC950, improves the early phase of MetALD by preventing liver damage, steatosis, inflammation, and immune cells recruitment.

Mitochondrial Aldehyde Dehydrogenase 2 (ALDH2) Protects against Binge Alcohol-Mediated Gut and Brain Injury.

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) metabolizes acetaldehyde to acetate. People with ALDH2 deficiency and Aldh2-knockout (KO) mice are more susceptible to alcohol-induced tissue damage. However, the underlying mechanisms behind ALDH2-related gut-associated brain damage remain unclear. Age-matched young female Aldh2-KO and C57BL/6J wild-type (WT) mice were gavaged with binge alcohol (4 g/kg/dose, three doses) or dextrose (control) at 12 h intervals. Tissues and sera were collected 1 h after the last ethanol dose and evaluated by histological and biochemical analyses of the gut and hippocampus and their extracts. For the mechanistic study, mouse neuroblast Neuro2A cells were exposed to ethanol with or without an Aldh2 inhibitor (Daidzin). Binge alcohol decreased intestinal tight/adherens junction proteins but increased oxidative stress-mediated post-translational modifications (PTMs) and enterocyte apoptosis, leading to elevated gut leakiness and endotoxemia in Aldh2-KO mice compared to corresponding WT mice. Alcohol-exposed Aldh2-KO mice also showed higher levels of hippocampal brain injury, oxidative stress-related PTMs, and neuronal apoptosis than the WT mice. Additionally, alcohol exposure reduced Neuro2A cell viability with elevated oxidative stress-related PTMs and apoptosis, all of which were exacerbated by Aldh2 inhibition. Our results show for the first time that ALDH2 plays a protective role in binge alcohol-induced brain injury partly through the gut-brain axis, suggesting that ALDH2 is a potential target for attenuating alcohol-induced tissue injury.

Longitudinal alcohol-related brain changes in older adults: The Sydney Memory and Ageing Study.

Increases in harmful drinking among older adults indicate the need for a more thorough understanding of the relationship between later-life alcohol use and brain health. The current study investigated the relationships between alcohol use and progressive grey and white matter changes in older adults using longitudinal data. A total of 530 participants (aged 70 to 90 years; 46.0% male) were included. Brain outcomes assessed over 6 years included total grey and white matter volume, as well as volume of the hippocampus, thalamus, amygdala, corpus callosum, orbitofrontal cortex and insula. White matter integrity was also investigated. Average alcohol use across the study period was the main exposure of interest. Past-year binge drinking and reduction in drinking from pre-baseline were additional exposures of interest. Within the context of low-level average drinking (averaging 11.7 g per day), higher average amount of alcohol consumed was associated with less atrophy in the left (B = 7.50, pFDR = 0.010) and right (B = 5.98, pFDR = 0.004) thalamus. Past-year binge-drinking was associated with poorer white matter integrity (B = -0.013, pFDR = 0.024). Consuming alcohol more heavily in the past was associated with greater atrophy in anterior (B = -12.73, pFDR = 0.048) and posterior (B = -17.88, pFDR = 0.004) callosal volumes over time. Across alcohol exposures and neuroimaging markers, no other relationships were statistically significant. Within the context of low-level drinking, very few relationships between alcohol use and brain macrostructure were identified. Meanwhile, heavier drinking was negatively associated with white matter integrity.

Fibroblast Growth Factor 19 Alters Bile Acids to Induce Dysbiosis in Mice With Alcohol-Induced Liver Disease.

BACKGROUND & AIMS: Excessive alcohol consumption can lead to alcohol-associated liver disease, a spectrum of conditions ranging from steatosis to fibrosis and cirrhosis. Bile acids regulate metabolic pathways by binding to cellular and nuclear receptors, and they also interact with the gut microbiome to control microbial overgrowth. Fibroblast growth factor 19 (FGF-19) is an ileum-derived hormone induced and released in response to bile acid activation of the nuclear receptor farnesoid X receptor. FGF-19 signaling is dysregulated with ethanol consumption and is increased in patients with alcoholic hepatitis. Here, we examined the effects of FGF-19 in a mouse model of chronic + binge ethanol feeding. METHODS: After injection of adeno-associated virus-green fluorescent protein or AAV-FGF-19, female C57BL/6J mice were pair-fed a Lieber DeCarli liquid diet (5% v/v) or control diet for 10 days and were given a bolus gavage of 5% ethanol or maltose control to represent a binge drinking episode. Tissues were collected for analysis 9 hours after the binge. RESULTS: Chronic + binge ethanol feeding induced steatosis regardless of FGF-19 expression. Interestingly, FGF-19 and ethanol resulted in significantly increased liver inflammation, as measured by Il6, Tgfß, and Tnfα, compared with ethanol alone. Both ethanol and FGF-19 decreased bile acid synthesis, and FGF-19 significantly reduced secondary bile acids, leading to overgrowth of specific pathogenic bacteria including Enterococcus faecalis, Escherichia coli, and Clostridium perfringens. CONCLUSIONS: Dysregulation of FGF-19 and consequent changes in bile acid synthesis and composition during alcohol consumption may be a contributing factor to alcohol-induced liver disease and dysbiosis.

Targeting Persistent Changes in Neuroimmune and Epigenetic Signaling in Adolescent Drinking to Treat Alcohol Use Disorder in Adulthood.

Studies universally find early age of drinking onset is linked to lifelong risks of alcohol problems and alcohol use disorder (AUD). Assessment of the lasting effect of drinking during adolescent development in humans is confounded by the diversity of environmental and genetic factors that affect adolescent development, including emerging personality disorders and progressive increases in drinking trajectories into adulthood. Preclinical studies using an adolescent intermittent ethanol (AIE) exposure rat model of underage binge drinking avoid the human confounds and support lifelong changes that increase risks. AIE increases adult alcohol drinking, risky decision-making, reward-seeking, and anxiety as well as reductions in executive function that all increase risks for the development of an AUD. AIE causes persistent increases in brain neuroimmune signaling high-mobility group box 1 (HMGB1), Toll-like receptor, receptor for advanced glycation end products, and innate immune genes that are also found to be increased in human AUD brain. HMGB1 is released from cells by ethanol, both free and within extracellular vesicles, that act on neurons and glia, shifting transcription and cellular phenotype. AIE-induced decreases in adult hippocampal neurogenesis and loss of basal forebrain cholinergic neurons are reviewed as examples of persistent AIE-induced pathology. Both are prevented and reversed by anti-inflammatory and epigenetic drugs. Findings suggest AIE-increased HMGB1 signaling induces the RE-1 silencing transcript blunting cholinergic gene expression, shifting neuronal phenotype. Inhibition of HMGB1 neuroimmune signaling, histone methylation enzymes, and galantamine, the cholinesterase inhibitor, both prevent and reverse AIE pathology. These findings provide new targets that may reverse AUD neuropathology as well as other brain diseases linked to neuroimmune signaling. SIGNIFICANCE STATEMENT: Adolescent underage binge drinking studies find that earlier adolescent drinking is associated with lifelong alcohol problems including high levels of lifetime alcohol use disorder (AUD). Preclinical studies find the underage binge drinking adolescent intermittent ethanol (AIE) model causes lasting changes in adults that increase risks of developing adult alcohol problems. Loss of hippocampal neurogenesis and loss of basal forebrain cholinergic neurons provide examples of how AIE-induced epigenetic and neuroimmune signaling provide novel therapeutic targets for adult AUD.

Adolescent intermittent ethanol exposure enhances adult stress effects in male rats.

Binge patterns of alcohol use, prevalent among adolescents, are associated with a higher probability of developing alcohol use disorders (AUD) and other psychiatric disorders, like anxiety and depression. Additionally, adverse life events strongly predict AUD and other psychiatric disorders. As such, the combined fields of stress and AUD have been well established, and animal models indicate that both binge-like alcohol exposure and stress exposure elevate anxiety-like behaviors. However, few have investigated the interaction of adolescent intermittent ethanol (AIE) and adult stressors. We hypothesized that AIE would increase vulnerability to restraint-induced stress (RS), manifested as increased anxiety-like behavior. After AIE exposure, in adulthood, animals were tested on forced swim (FST) and saccharin preference (SP) and then exposed to either RS (90 min/5 days) or home-cage control. Twenty-four hours after the last RS session, animals began testing on the elevated plus maze (EPM), and were re-tested on FST and SP. A separate group of animals were sacrificed in adulthood after AIE and RS, and brains were harvested for immunoblot analysis of dorsal and ventral hippocampus. Consistent with previous reports, AIE had no significant effect on closed arm time in the EPM (anxiety-like behavior). However, in male rats the interaction of AIE and adult RS increased time spent in the closed arms. No effect was observed among female animals. AIE and RS-specific alterations were found in glial and synaptic markers (GLT-1, FMRP and PSD-95) in male animals. These findings indicate AIE has sex-specific effects on both SP and the interaction of AIE and adult RS, which induces a propensity toward anxiety-like behavior in males. Also, AIE produces persistent hippocampal deficits that may interact with adult RS to cause increased anxiety-like behaviors. Understanding the mechanisms behind this AIE-induced increase in stress vulnerability may provide insight into treatment and prevention strategies for alcohol use disorders.

Activated and nonactivated MSCs increase survival in humanized mice after acute liver injury through alcohol binging.

The ability of the liver to regenerate after injury makes it an ideal organ to study for potential therapeutic interventions. Mesenchymal stem cells (MSCs) possess self-renewal and differentiation properties, as well as anti-inflammatory properties that make them an ideal candidate for therapy of acute liver injury. The primary aim of this study is to evaluate the potential for reversal of hepatic injury using human umbilical cord-derived MSCs. Secondary aims include comparison of various methods of administration as well as comparison of activated versus nonactivated human umbilical cord stem cells. To induce liver injury, humanized mice were fed high-cholesterol high-fat liquid diet with alcohol binge drinking. Mice were then treated with either umbilical cord MSCs, activated umbilical cord MSCs, or a placebo and followed for survival. Blood samples were obtained at the end of the binge drinking and at the time of death to measure alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Histology of all mouse livers was reported at time of death. Activated MSCs that were injected intravenously, intraperitoneally, or both routes had superior survival compared with nonactivated MSCs and with placebo-treated mice. AST and ALT levels were elevated in all mice before treatment and improved in the mice treated with stem cells. Conclusion: Activated stem cells resulted in marked improvement in survival and in recovery of hepatic chemistries. Activated umbilical cord MSCs should be considered an important area of investigation in acute liver injury.

The interplay of Western diet and binge drinking on the onset, progression, and outlook of liver disease.

Non-alcoholic fatty liver disease and alcoholic liver disease, the two most prevalent liver diseases worldwide, share a common pathology but have largely been considered disparate diseases. Liver diseases are widely underestimated, but their prevalence is increasing worldwide. The Western diet (high-fat, high-sugar) and binge drinking (rapid consumption of alcohol in a short period of time) are two highly prevalent features of standard life in the United States, and both are linked to the development and progression of liver disease. Yet, few studies have been conducted to elucidate their potential interactions. Data shows binge drinking is on the rise in several age groups, and poor dietary trends continue to be prevalent. This review serves to summarize the sparse findings on the hepatic consequences of the combination of binge drinking and consuming a Western diet, while also drawing conclusions on potential future impacts. The data suggest the potential for a looming liver disease epidemic, indicating that more research on its progression as well as its prevention is needed on this critical topic.

Voluntary alcohol binge-drinking in adolescent C57Bl6 mice induces delayed appearance of behavioural defects in both males and females.

Adolescence is a developmental period characterized by significant changes in brain architecture and behaviour. The immaturity of the adolescent brain is associated with heightened vulnerability to exogenous agents, including alcohol. Alcohol is the most consumed drug among teenagers, and binge-drinking during adolescence is a major public health concern. Studies have suggested that adolescent alcohol exposure may interfere with the maturation of frontal brain regions and lead to long-lasting behavioural consequences. In this study, by using a slightly modified version of the Drinking in the Dark paradigm, adolescent C57Bl6 mice reach high blood alcohol concentration after voluntary binge-drinking. In order to assess short- and long-term consequences of adolescent alcohol exposure (AAE), a battery of behavioural tests was performed during late adolescence and during adulthood. We showed that AAE had no short-term effect on young mice behaviour but rather increased anxiety- and depressive-like behaviours, as well as alcohol consumption during adulthood. Moreover, alcohol binge-drinking during adolescence dramatically decreased recognition memory performances and behavioural flexibility in both adult males and females. Furthermore, we showed that voluntary consumption of alcohol during adolescence did not trigger any major activation of the innate immune system in the prefrontal cortex. Together, our data suggest that voluntary alcohol binge-drinking in adolescent mice induces a delayed appearance of behavioural impairments in adulthood.

Galantamine prevents and reverses neuroimmune induction and loss of adult hippocampal neurogenesis following adolescent alcohol exposure.

BACKGROUND: Binge ethanol exposure during adolescence reduces hippocampal neurogenesis, a reduction which persists throughout adulthood despite abstinence. This loss of neurogenesis, indicated by reduced doublecortin+ immunoreactivity (DCX+IR), is paralleled by an increase in hippocampal proinflammatory signaling cascades. As galantamine, a cholinesterase inhibitor, has anti-inflammatory actions, we tested the hypothesis that galantamine would prevent (study 1) or restore (study 2) AIE induction of proinflammatory signals within the hippocampus as well as AIE-induced loss of hippocampal neurogenesis. METHODS: Galantamine (4 mg/kg) or vehicle (saline) was administered to Wistar rats during adolescent intermittent ethanol (AIE; 5.0 g/kg ethanol, 2 days on/2 days off, postnatal day [P] 25-54) (study 1, prevention) or after AIE during abstinent maturation to adulthood (study 2, restoration). RESULTS: Results indicate AIE reduced DCX+IR and induced cleaved caspase3 (Casp3) in DCX-expressing immature neurons. Excitingly, AIE induction of activated Casp3 in DCX-expressing neurons is both prevented and reversed by galantamine treatment, which also resulted in prevention and restoration of neurogenesis (DCX+IR). Similarly, galantamine prevented and/or reversed AIE induction of proinflammatory markers, including the chemokine (C-C motif) ligand 2 (CCL2), cyclooxygenase-2 (COX-2), and high mobility group box 1 (HMGB1) protein, suggesting that AIE induction of proinflammatory signaling mediates both cell death cascades and hippocampal neurogenesis. Interestingly, galantamine treatment increased Ki67+IR generally as well as increased pan-Trk expression specifically in AIE-treated rats but failed to reverse AIE induction of NADPH-oxidase (gp91phox). CONCLUSIONS: Collectively, our studies suggest that (1) loss of neurogenesis after AIE is mediated by persistent induction of proinflammatory cascades which drive activation of cell death machinery in immature neurons, and (2) galantamine can prevent and restore AIE disruptions in the hippocampal environmental milieu to then prevent and restore AIE-mediated loss of neurogenesis.

TRAIL Mediates Neuronal Death in AUD: A Link between Neuroinflammation and Neurodegeneration.

Although the cause of progressive neurodegeneration is often unclear, neuronal death can occur through several mechanisms. In conditions such as Alzheimer's or alcohol use disorder (AUD), Toll-like receptor (TLR) induction is observed with neurodegeneration. However, links between TLR activation and neurodegeneration are lacking. We report a role of apoptotic neuronal death in AUD through TLR7-mediated induction of death receptor signaling. In postmortem human cortex, a two-fold increase in apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in neurons was found in AUD versus controls. This occurred with the increased expression of TLR7 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors. Binge ethanol treatment in C57BL/6 mice increased TLR7 and induced neuronal apoptosis in cortical regions that was blocked by TLR7 antagonism. Mechanistic studies in primary organotypic brain slice culture (OBSC) found that the inhibition of TLR7 and its endogenous ligand let-7b blocked ethanol-induced neuronal cell death. Both IMQ and ethanol induced the expression of TRAIL and its death receptor. In addition, TRAIL-neutralizing monoclonal antibodies blocked both imiquimod (IMQ) and ethanol induced neuronal death. These findings implicate TRAIL as a mediator of neuronal apoptosis downstream of TLR7 activation. TLR7 and neuronal apoptosis are implicated in other neurodegenerative diseases, including Alzheimer's disease. Therefore, TRAIL may represent a therapeutic target to slow neurodegeneration in multiple diseases.

Recent Perspectives on Sex Differences in Compulsion-Like and Binge Alcohol Drinking.

Alcohol use disorder remains a substantial social, health, and economic problem and problem drinking levels in women have been increasing in recent years. Understanding whether and how the underlying mechanisms that drive drinking vary by sex is critical and could provide novel, more targeted therapeutic treatments. Here, we examine recent results from our laboratories and others which we believe provide useful insights into similarities and differences in alcohol drinking patterns across the sexes. Findings for binge intake and aversion-resistant, compulsion-like alcohol drinking are considered, since both are likely significant contributors to alcohol problems in humans. We also describe studies regarding mechanisms that may underlie sex differences in maladaptive alcohol drinking, with some focus on the importance of nucleus accumbens (NAcb) core and shell regions, several receptor types (dopamine, orexin, AMPA-type glutamate), and possible contributions of sex hormones. Finally, we discuss how stressors such as early life stress and anxiety-like states may interact with sex differences to contribute to alcohol drinking. Together, these findings underscore the importance and critical relevance of studying female and male mechanisms for alcohol and co-morbid conditions to gain a true and clinically useful understanding of addiction and neuropsychiatric mechanisms and treatment.

Alcohol stimulates the proliferation of mouse small intestinal epithelial cells via Wnt signaling.

The intestinal epithelium is one of the fastest renewing tissues in mammals and is a barrier against toxic substances such as alcohol. Excessive alcohol can induce intestinal damage leading to intestinal bowel diseases. Thus, the control of small intestinal epithelial cell (IEC) regeneration is thought to be important for homeostasis in response to epithelium damage. However, reports on how epithelial cells respond to small intestinal damage are scarce. We investigated the effects of alcohol consumption on small intestinal epithelial cells of mice. To verify that alcohol altered the small intestinal epithelium, we used 8-10 weeks old male C57BL/6J mice for models of chronic and binge alcohol consumption (the NIAAA model) in addition to an organoid model. Alcohol promoted the proliferative activity of IECs and intestinal stem cells (ISCs) in intestinal crypts. Alcohol consumption increased expression of the proliferation marker cyclin D1 and activated the p44/42 MAPK (Erk1/2) signaling pathway in small intestinal epithelial cells. The Wnt target genes were markedly increased in IECs from alcohol-treated mice. In the small intestinal organoid model exposed to alcohol, the organoid area and numbers of buds increased with alcohol concentrations up to 0.5% similar to in vivo observations. These results suggest that alcohol consumption stimulates the proliferation of small intestinal epithelial cells via Wnt signaling.

Role of mTOR-regulated autophagy in spine pruning defects and memory impairments induced by binge-like ethanol treatment in adolescent mice.

Adolescence is a brain maturation developmental period during which remodeling and changes in synaptic plasticity and neural connectivity take place in some brain regions. Different mechanism participates in adolescent brain maturation, including autophagy that plays a role in synaptic development and plasticity. Alcohol is a neurotoxic compound and its abuse in adolescence induces neuroinflammation, synaptic and myelin alterations, neural damage and behavioral impairments. Changes in synaptic plasticity and its regulation by mTOR have also been suggested to play a role in the behavioral dysfunction of binge ethanol drinking in adolescence. Therefore, by considering the critical role of mTOR in both autophagy and synaptic plasticity in the developing brain, the present study aims to evaluate whether binge ethanol treatment in adolescence would induce dysfunctions in synaptic plasticity and cognitive functions and if mTOR inhibition with rapamycin is capable of restoring both effects. Using C57BL/6 adolescent female and male mice (PND30) treated with ethanol (3 g/kg) on two consecutive days at 48-hour intervals over 2 weeks, we show that binge ethanol treatment alters the density and morphology of dendritic spines, effects that are associated with learning and memory impairments and changes in the levels of both transcription factor CREB phosphorylation and miRNAs. Rapamycin administration (3 mg/kg) prior to ethanol administration restores ethanol-induced changes in both plasticity and behavior dysfunctions in adolescent mice. These results support the critical role of mTOR/autophagy dysfunctions in the dendritic spines alterations and cognitive alterations induced by binge alcohol in adolescence.

Neural Perturbations Associated With Recurrent Binge Alcohol in Male and Female Rats.

BACKGROUND: Binge drinking, characterized by brief periods of high intoxication interspersed with periods of abstinence, appears to be particularly damaging to the brain. Binge drinking is increasing among American women, yet few preclinical studies have assessed sex differences in the neurobehavioral effects of binge alcohol. METHODS: Adult Long-Evans rats were administered 4 g/kg ethanol (EtOH; or an isocaloric control dose) via intragastric gavage once-weekly. Brains were collected after 3 or 8 binge doses, and immunohistochemistry for mature neurons (NeuN), microglia (Iba1), neurogenesis (DCX), and reactive astrogliosis (vimentin) performed. Stereology was used to quantify target cell populations in the hippocampus and medial prefrontal cortex (mPFC). In a separate cohort of animals, cognition (spatial navigation and reversal learning), affect (tickling-evoked ultrasonic vocalizations), and task-induced c-fos activation were assessed after 3 or 8 binge doses. RESULTS: Blood EtOH concentration did not differ significantly between females (175 ± 3.6 mg/dl) and males (180 ± 3.7 mg/dl) and did not change significantly over time, indicating that tolerance did not develop. After 3 or 8 binge doses, the number of granule neurons in the hippocampal dentate gyrus of both sexes was significantly reduced in comparison with controls, although there was no binge effect on newly generated neurons. Moreover, 8 (but not 3) binge doses significantly increased the total number of microglia and the number of partially activated microglia in the hippocampus and mPFC in both sexes. There was no detectable reactive astrogliosis (vimentin) in either region at any timepoint. There was no effect of binge alcohol on behavior outcomes in either sex, but binged rats showed increased cellular activation in the mPFC following reversal learning. CONCLUSIONS: Our data indicate that recurrent binge alcohol results in similar neural damage and neuroimmune activation in alcohol-vulnerable corticolimbic brain regions in males and females.

Microglia Phenotypes Following the Induction of Alcohol Dependence in Adolescent Rats.

BACKGROUND: Activation of the innate immune system may play a role in the development of alcohol use disorders (AUDs), which often originate with adolescent alcohol abuse. A key player in the innate immune system is microglia, the activation of which occurs along a spectrum from proinflammatory, or M1-like, to anti-inflammatory, or M2-like, phenotypes. METHODS: Adolescent, male rats were gavaged with ethanol (EtOH) or isocaloric control diet every 8 hours for 4 days and then sacrificed at 0, 2, 7, and 14 days later. Microglia were isolated from the entorhinal cortex and hippocampus by Percoll gradient centrifugation, labeled with surface antigens for activation, and analyzed by flow cytometry. Polarization states of microglia, defined as CD11b+ CD45low cells, were determined by the expression of M1 surface markers, major histocompatibility complex (MHC) II, CD32, and CD86, and M2 surface marker, CD206 (mannose receptor). Cytokine gene expression was measured by reverse transcriptase polymerase chain reaction. RESULTS: Isolated cells were a highly enriched population (>95% pure) of microglia/macrophages according to CD11b immunoreactivity. EtOH rats showed the most dramatic increases in microglia activation markers CD11b and CD45, and M1 (MHC-II) and M2 (CD206) markers at T2, when additional M1 markers CD86 and CD32 were also increased. Surprisingly, proinflammatory gene expression of CCL2, IL-1ß, IL-6, and TNF-α generally was decreased at all time points in EtOH rats except for IL-6 which was increased at T0 and TNF-α which was not changed at T0 in either region. Simultaneously, BDNF expression was increased at T2 and T7, while IGF1 and TGF-ß gene expression was decreased. Arginase was also increased at T0 in hippocampus, but not changed by alcohol otherwise. CONCLUSIONS: These data show that microglia phenotype after alcohol dependence is not a simple M1 or M2 classification, though more indicators of an anti-inflammatory phenotype were observed. Determining microglia phenotype is critical for understanding their role in the development of AUDs.

Prospective Study Examining the Effects of Extreme Drinking on Brain Structure in Emerging Adults.

BACKGROUND: Emerging adulthood is a critical neurodevelopment period in which extreme drinking has a potentially pronounced neurotoxic effect. Therefore, extreme drinking, even a single episode, could be particularly harmful to the developing brain's structure. Relatedly, heavy alcohol use in emerging adults has been associated with structural brain damage, especially in the corpus callosum. However, it is unclear whether and how much a single extreme drinking episode would affect brain morphometry. METHODS: For the first time in the literature, the current study prospectively examined the impact of an extreme drinking episode (i.e., twenty-first birthday celebration) on the brain morphometry of emerging adults immediately following their birthday celebration (n = 50) and approximately 5 weeks post-birthday celebration (n = 29). RESULTS: We found evidence that a single extreme drinking episode was associated with structural changes immediately post-birthday celebration. Specifically, higher twenty-first birthday estimated blood-alcohol concentration was associated with decreased volume of the posterior and central corpus callosum immediately post-birthday celebration. This extreme drinking episode was not associated with further structural changes, or recovery, 5 weeks post-twenty-first birthday celebration. CONCLUSIONS: Overall, results suggest that a single episode of heavy drinking in emerging adulthood may be associated with immediate structural changes of the corpus callosum. Thus, emerging adulthood, which is characterized by high rates of extreme drinking, could be a critical period for targeted prevention and intervention.

Alcohol consumption during adolescence alters the hippocampal response to traumatic brain injury.

Binge drinking is the consumption of large volumes of alcohol in short periods and exerts its effects on the central nervous system, including the hippocampus. We have previously shown that binge drinking alters mitochondrial dynamics and induces neuroinflammation in the hippocampus of adolescent rats. Mild traumatic brain injury (mTBI), is regularly linked to alcohol consumption and share mechanisms of brain damage. In this context, we hypothesized that adolescent binge drinking could prime the development of brain damage generated by mTBI. We found that alcohol binge drinking induced by the "drinking in the dark" (DID) paradigm increases oxidative damage and astrocyte activation in the hippocampus of adolescent mice. Interestingly, adolescent animals submitted to DID showed decreased levels of mitofusin 2 that controls mitochondrial dynamics. When mTBI was evaluated as a second challenge, hippocampi from animals previously submitted to DID showed a reduction in dendritic spine number and a different spine profile. Mitochondrial performance could be compromised by alterations in mitochondrial fission in DID-mTBI animals. These data suggest that adolescent alcohol consumption can modify the progression of mTBI pathophysiology. We propose that mitochondrial impairment and oxidative damage could act as priming factors, modifying predisposition against mTBI effects.

Chronic-plus-binge alcohol intake induces production of proinflammatory mtDNA-enriched extracellular vesicles and steatohepatitis via ASK1/p38MAPKα-dependent mechanisms.

Alcohol-associated liver disease is a spectrum of liver disorders with histopathological changes ranging from simple steatosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Recent data suggest that chronic-plus-binge ethanol intake induces steatohepatitis by promoting release by hepatocytes of proinflammatory mitochondrial DNA-enriched (mtDNA-enriched) extracellular vesicles (EVs). The aim of the present study was to investigate the role of the stress kinase apoptosis signal-regulating kinase 1 (ASK1) and p38 mitogen-activated protein kinase (p38) in chronic-plus-binge ethanol-induced steatohepatitis and mtDNA-enriched EV release. Microarray analysis revealed the greatest hepatic upregulation of metallothionein 1 and 2 (Mt1/2), which encode 2 of the most potent antioxidant proteins. Genetic deletion of the Mt1 and Mt2 genes aggravated ethanol-induced liver injury, as evidenced by elevation of serum ALT, neutrophil infiltration, oxidative stress, and ASK1/p38 activation in the liver. Inhibition or genetic deletion of Ask1 or p38 ameliorated ethanol-induced liver injury, inflammation, ROS levels, and expression of phagocytic oxidase and ER stress markers in the liver. In addition, inhibition of ASK1 or p38 also attenuated ethanol-induced mtDNA-enriched EV secretion from hepatocytes. Taken together, these findings indicate that induction of hepatic mtDNA-enriched EVs by ethanol is dependent on ASK1 and p38, thereby promoting alcoholic steatohepatitis.

Region-specific interneuron demyelination and heightened anxiety-like behavior induced by adolescent binge alcohol treatment.

Adolescent binge drinking represents a major public health challenge and can lead to persistent neurological and mental conditions, but the underlying pathogenic mechanisms remain poorly understood. Using a mouse model of adolescent binge ethanol treatment (ABET), we found that this treatment induced behavioral changes associated with demyelination in different brain regions. After ABET, adolescent mice exhibited anxiogenic behaviors with no change in locomotion on the elevated plus maze, and impaired spatial memory indicated by a significant reduction in spontaneous alternation in the Y maze test. Both effects persisted into adulthood. Anatomical studies further showed that ABET induced a significant reduction of parvalbumin-positive (PV+) GABAergic interneurons and myelin density in the hippocampus and medial prefrontal cortex (mPFC). While these deficits in PV+ interneurons and myelin persisted into early adulthood in the hippocampus, the myelin density recovered in the mPFC. Moreover, whereas ABET mainly damaged myelin of PV+ axons in the hippocampus, it primarily damaged myelin of PV-negative axons in the mPFC. Thus, our findings reveal that an adolescent binge alcohol treatment regimen disrupts spatial working memory, increases anxiety-like behaviors, and exerts unique temporal and spatial patterns of gray matter demyelination in the hippocampus and mPFC.