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

Abstract

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. Adolescent, male rats were gavaged with ethanol (EtOH) or isocaloric control diet every 8hours for 4days and then sacrificed at 0, 2, 7, and 14days 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. 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. 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.