The Influence of Arsenic Co-Exposure in a Model of Alcohol-Induced Neurodegeneration in C57BL/6J Mice.

Both excessive alcohol consumption and exposure to high levels of arsenic can lead to neurodegeneration, especially in the hippocampus. Co-exposure to arsenic and alcohol can occur because an individual with an Alcohol Use Disorder (AUD) is exposed to arsenic in their drinking water or food or because of arsenic found directly in alcoholic beverages. This study aims to determine if co-exposure to alcohol and arsenic leads to worse outcomes in neurodegeneration and associated mechanisms that could lead to cell death. To study this, mice were exposed to a 10-day gavage model of alcohol-induced neurodegeneration with varying doses of arsenic (0, 0.005, 2.5, or 10 mg/kg). The following were examined after the last dose of ethanol: (1) microglia activation assessed via immunohistochemical detection of Iba-1, (2) reactive oxygen and nitrogen species (ROS/RNS) using a colorimetric assay, (3) neurodegeneration using Fluoro-Jade® C staining (FJC), and 4) arsenic absorption using ICP-MS. After exposure, there was an additive effect of the highest dose of arsenic (10 mg/kg) in the dentate gyrus of alcohol-induced FJC+ cells. This additional cell loss may have been due to the observed increase in microglial reactivity or increased arsenic absorption following co-exposure to ethanol and arsenic. The data also showed that arsenic caused an increase in CYP2E1 expression and ROS/RNS production in the hippocampus which could have independently contributed to increased neurodegeneration. Altogether, these findings suggest a potential cyclical impact of co-exposure to arsenic and ethanol as ethanol increases arsenic absorption but arsenic also enhances alcohol's deleterious effects in the CNS.

Acute and Chronic Ethanol Effects during Adolescence on Neuroimmune Responses: Consequences and Potential Pharmacologic Interventions.

Heavy ethanol consumption during adolescence has been linked to neuroimmune response dysregulation and cognitive deficits in the developing adolescent brain. During adolescence, the brain is particularly susceptible to the pharmacological effects of ethanol that are induced by acute and chronic bouts of exposure. Numerous preclinical rodent model studies have used different ethanol administration techniques, such as intragastric gavage, self-administration, vapor, intraperitoneal, and free access, and while most models indicated proinflammatory neuroimmune responses in the adolescent brain, there are various factors that appear to influence this observation. This review synthesizes the most recent findings of the effects of adolescent alcohol use on toll-like receptors, cytokines, and chemokines, as well as the activation of astrocytes and microglia with an emphasis on differences associated with the duration of ethanol exposure (acute vs. chronic), the amount of exposure (e.g., dose or blood ethanol concentrations), sex differences, and the timing of the neuroimmune observation (immediate vs. persistent). Finally, this review discusses new therapeutics and interventions that may ameliorate the dysregulation of neuroimmune maladaptations after ethanol exposure.

The Influence of Sex on Hippocampal Neurogenesis and Neurotrophic Responses on the Persistent Effects of Adolescent Intermittent Ethanol Exposure into Adulthood.

In the United States, approximately 90% of alcohol consumed by adolescents is binge drinking. Binge-like ethanol exposure during adolescence promotes dysregulation of neurotrophic responses and neurogenesis in the hippocampus. These effects include changes in proliferation, regulation, differentiation, and maturation of neurons, and there is indication that such effects may be disproportionate between sexes. This study determined whether sex impacts neurotrophic responses and neurogenesis in adulthood after adolescent intermittent ethanol (AIE) exposure. To determine this, adolescent rats underwent AIE with ethanol (5 g/kg). In adulthood, animals were euthanized, and immunohistochemical techniques and ELISAs were utilized to determine AIE effects on sex-specific neurogenesis factors and neurotrophic markers, respectively. AIE exposure led to a significant decrease in neurogenesis in the dentate gyrus of the hippocampal formation indicated by reductions in the numbers of DCX+, SOX2+ and Ki-67+ cells in male and female AIE-exposed rats. Additionally, AIE increased markers for the pro-inflammatory cytokines, TNF-α and IL-1ß, in the hippocampus into adulthood in male AIE-exposed rats only. No significant AIE-induced differences were observed in the anti-inflammatory cytokines, IL-10 and TGF-ß, nor in the neurotrophic factors BDNF and GDNF. Altogether, our findings indicate that although AIE did not have a persistent effect on hippocampal neurotrophic levels, there was still a reduction in neurogenesis. The neurogenic impairment was not sex specific, but the neurogenic deficits in males may be attributed to an increase in pro-inflammatory cytokine expression. A persistent impairment in neurogenesis may have an impact on both behavioral maladaptations and neurodegeneration in adulthood.

Sex-specific effects of adolescent intermittent ethanol exposure-induced dysregulation of hippocampal glial cells in adulthood.

Adolescent alcohol abuse is a significant public health concern, with approximately 4.3 million U.S. adolescents reporting monthly binge drinking. Excessive ethanol consumption during adolescence has been linked to dysregulation of the neuroimmune system, particularly in the hippocampus. Because there are sex differences in both neuroimmune responses and ethanol's pharmacologic actions, this study tested whether there were disparate effects based on sex in glial cells and neurodegeneration in adulthood after the adolescent intermittent ethanol (AIE) model. Male and female adolescent Sprague-Dawley rats underwent AIE. In adulthood, immunohistochemical techniques were utilized to determine the effects of AIE on astrocytes and microglia, and Fluoro-Jade C (FJC) was used to assess neurodegeneration in the hippocampus. AIE exposure significantly increased astrocyte activation in the cornu ammonis 1 (CA1), CA2/3, and dentate gyrus (DG) in both male and female rats with no discernible sex differences in immunoreactivity. Likewise, the number of GFAP + cells was significantly increased by AIE across the hippocampus. In our microglial assessment, AIE only led to increased Iba1 immunoreactivity in the CA1 but not CA2/3 or DG regions. However, the number of Iba1+ cells was increased by AIE in both the CA1 and DG subregions. In the DG, the ethanol effect was observed in both sexes, but in the CA1, AIE-induced increased Iba1 cells were only observed in females. In regard to neurodegeneration, there were no persisting AIE effects on FJC + cells. These findings indicate that AIE alters hippocampal glial cells in adulthood, in the absence of active neurodegeneration. However, while AIE induced long-term elevation of astroglial measures in both males and females, persisting AIE-induced microglial activation was more sparse and sex-dependent. While the majority of these findings suggest that AIE has similar effects on glial morphology and number between males and females, additional work should determine whether there are molecular differences as well as innate sex differences in glial interaction with AIE's influence on glial functions in behavior.

Chemogenetic manipulation of astrocytic signaling in the basolateral amygdala reduces binge-like alcohol consumption in male mice.

Binge drinking is a common occurrence in the United States, but a high concentration of alcohol in the blood has been shown to have reinforcing and reciprocal effects on the neuroimmune system in both dependent and non-dependent scenarios. The first part of this study examined alcohol's effects on the astrocytic response in the central amygdala and basolateral amygdala (BLA) in a non-dependent model. C57BL/6J mice were given access to either ethanol, water, or sucrose during a "drinking in the dark" paradigm, and astrocyte number and astrogliosis were measured using immunohistochemistry. Results indicate that non-dependent consumption increased glial fibrillary acidic protein (GFAP) density but not the number of GFAP+ cells, suggesting that non-dependent ethanol is sufficient to elicit astrocyte activation. The second part of this study examined how astrocytes impacted behaviors and the neurochemistry related to alcohol using the chemogenetic tool, DREADDs (designer receptors exclusively activated by designer drugs). Transgenic GFAP-hM3Dq mice were administered clozapine N-oxide both peripherally, affecting the entire central nervous system (CNS), or directly into the BLA. In both instances, GFAP-Gq-signaling activation significantly reduced ethanol consumption and correlating blood ethanol concentrations. However, GFAP-Gq-DREADD activation throughout the CNS had more broad effects resulting in decreased locomotor activity and sucrose consumption. More targeted GFAP-Gq-signaling activation in the BLA only impacted ethanol consumption. Finally, a glutamate assay revealed that after GFAP-Gq-signaling activation glutamate concentrations in the amygdala were partially normalized to control levels. Altogether, these studies support the theory that astrocytes represent a viable target for alcohol use disorder therapies.

Repetitive binge-like consumption based on the Drinking-in-the-Dark model alters the microglial population in the mouse hippocampus.

Alcoholism causes various maladaptations in the central nervous system, including the neuroimmune system. Studies of alcohol-induced dysregulation of the neuroimmune system generally focus on alcohol dependence and brain damage, but our previous research indicates that repetitive binge-like consumption perturbs cytokines independent of cell death. This paper extends that research by examining the impact of binge-like consumption on microglia in the hippocampus and the amygdala. Microglia were assessed using immunohistochemistry following binge-like ethanol consumption based on Drinking-in-the-Dark model. Immunohistochemistry results showed that binge-like ethanol consumption caused an increase in Iba-1 immunoreactivity and the number of Iba-1+ cells after one Drinking-in-the-Dark cycle. However, after three Drinking-in-the-Dark cycles, the number of microglia decreased in the hippocampus. We showed that in the dentate gyrus, the average immunoreactivity/cell was increased following ethanol exposure despite the decrease in number after three cycles. Likewise, Ox-42, an indicator of microglia activation, was upregulated after ethanol consumption. No significant effects on microglia number or immunoreactivity (Iba-1 nor Ox-42) were observed in the amygdala. Finally, ethanol caused an increase in the expression of the microglial gene Aif-1 during intoxication and ten days into abstinence, suggesting persistence of ethanol-induced upregulation of microglial genes. Altogether, these findings indicate that repetitive binge-like ethanol is sufficient to elicit changes in microglial reactivity. This altered neuroimmune state may contribute to the development of alcohol use disorders.