Dataset Information

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

ABSTRACT:

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⁺ CD45^low 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-1β, 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-β 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.

SUBMITTER: Peng H

PROVIDER: S-EPMC8296648 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The objective of this study was to determine changes in gene expression within the extended amygdala following binge-like alcohol drinking by adolescent alcohol-preferring (P) rats. Starting at 28 days of age, P rats were given concurrent access to 15 and 30 % ethanol for 3 one-h sessions for 5 consecutive days each week until they were 49 days old. Rats were killed by decapitation 3 h after the first ethanol access session on the 15th day of drinking. RNA was prepared from micropunch samples of the nucleus accumbens shell (Acb-sh) and central nucleus of the amygdala (CeA). Ethanol intakes were 2.5 to 3.0 g/kg/session. There were 154 and 182 unique named genes that significantly differed (FDR = 0.2) between the water and ethanol group in the Acb-sh and CeA, respectively. Gene Ontology (GO) analyses indicated that adolescent binge drinking produced changes in the in biological processes involved in cell proliferation and regulation of cellular structure in the Acb-sh, and in neuron projection and positive regulation of cellular organization in the CeA. Ingenuity Pathway Analysis indicated that, in the Acb-sh, there were several major intracellular signaling pathways (e.g., cAMP-mediated and protein kinase A signaling pathways) altered by adolescent drinking, with 3-fold more genes up-regulated than down-regulated in the alcohol group. The cAMP-mediated signaling system was also up-regulated in the CeA of the alcohol group. Weighted gene co-expression network analysis (WGCNA) indicated significant G-protein coupled receptor signaling and transmembrane receptor protein kinase signaling categories in the Acb-sh and CeA, respectively. Overall, the results of this study indicated that binge-like alcohol drinking by adolescent P rats is differentially altering the expression of genes in the Acb-sh and CeA, some of which are involved in intracellular signaling pathways and may produce long-term changes in neuronal function. Differences in gene expression in the central nucleus of the amygdala (CeA) were compared in two groups of alcohol-preferring (P) rats, one given water only and the other given access to 15 & 30% ethanol during adolescence.

Project description:The objective of this study was to determine changes in gene expression within the extended amygdala following binge-like alcohol drinking by adolescent alcohol-preferring (P) rats. Starting at 28 days of age, P rats were given concurrent access to 15 and 30 % ethanol for 3 one-h sessions for 5 consecutive days each week until they were 49 days old. Rats were killed by decapitation 3 h after the first ethanol access session on the 15th day of drinking. RNA was prepared from micropunch samples of the nucleus accumbens shell (Acb-sh) and central nucleus of the amygdala (CeA). Ethanol intakes were 2.5 ‚Äì 3.0 g/kg/session. There were 154 and 182 unique named genes that significantly differed (FDR = 0.2) between the water and ethanol group in the Acb-sh and CeA, respectively. Gene Ontology (GO) analyses indicated that adolescent binge drinking produced changes in the in biological processes involved in cell proliferation and regulation of cellular structure in the Acb-sh, and in neuron projection and positive regulation of cellular organization in the CeA. Ingenuity Pathway Analysis indicated that, in the Acb-sh, there were several major intracellular signaling pathways (e.g., cAMP-mediated and protein kinase A signaling pathways) altered by adolescent drinking, with 3-fold more genes up-regulated than down-regulated in the alcohol group. The cAMP-mediated signaling system was also up-regulated in the CeA of the alcohol group. Weighted gene co-expression network analysis (WGCNA) indicated significant G-protein coupled receptor signaling and transmembrane receptor protein kinase signaling categories in the Acb-sh and CeA, respectively. Overall, the results of this study indicated that binge-like alcohol drinking by adolescent P rats is differentially altering the expression of genes in the Acb-sh and CeA, some of which are involved in intracellular signaling pathways and may produce long-term changes in neuronal function. Differences in gene expression in brain nucleus accumbens shell (Acb-sh) were compared in two groups of alcohol-preferring (P) rats, one given water only and the other given access to 15 & 30% ethanol during adolescence.