Neurosteroid [3α,5α]3-hydroxypregnan-20-one inhibition of chemokine monocyte chemoattractant protein-1 in alcohol-preferring rat brain neurons, microglia, and astroglia

Background: Neuroimmune dysfunction in alcohol use disorder (AUD) is associated with activation of myeloid differentiation primary response 88 (MyD88)-dependent Toll-like receptors (TLR) resulting in overexpression of the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). MCP-1 overexpression in the brain is linked to anxiety, higher alcohol intake, neuronal death, and activation of microglia observed in AUD. The neurosteroid [3 alpha,5 alpha][3-hydroxypregnan-20-one (3 alpha,5 alpha-THP) has been reported as an inhibitor of MyD88-dependent TLR activation and MCP-1 overexpression in mouse and human macrophages and the brain of alcohol-preferring (P) rats. Methods: We investigated how 3 alpha,5 alpha-THP regulates MCP-1 expression at the cellular level in P rat nucleus accumbens (NAc) and central amygdala (CeA). We focused on neurons, microglia, and astrocytes, examining the individual voxel density of MCP-1, neuronal marker NeuN, microglial marker IBA1, astrocytic marker GFAP, and their shared voxel density, defined as intersection. Ethanol-na & iuml;ve male and female P rats were perfused 1 h after IP injections of 15 mg/kg of 3 alpha,5 alpha-THP, or vehicle. The NAc and CeA were imaged using confocal microscopy following double-immunofluorescence staining for MCP-1 with NeuN, IBA1, and GFAP, respectively. Results: MCP-1 intersected with NeuN predominantly and IBA1/GFAP negligibly. 3 alpha,5 alpha-THP reduced MCP-1 expression in NeuN-labeled cells by 38.27 +/- 28.09% in male and 56.11 +/- 21.46% in female NAc, also 37.99 +/- 19.53% in male and 54.96 +/- 30.58% in female CeA. In females, 3 alpha,5 alpha-THP reduced the MCP-1 within IBA1 and GFAP-labeled voxels in the NAc and CeA. Conversely, in males, 3 alpha,5 alpha-THP did not significantly alter the MCP-1 within IBA1 in NAc or with GFAP in the CeA. Furthermore, 3 alpha,5 alpha-THP decreased levels of IBA1 in both regions and sexes with no impact on GFAP or NeuN levels. Secondary analysis performed on data normalized to % control values indicated that no significant sex differences were present. Conclusions: These data suggest that 3 alpha,5 alpha-THP inhibits neuronal MCP-1 expression and decreases the proliferation of microglia in P rats. These results increase our understanding of potential mechanisms for 3 alpha,5 alpha-THP modulation of ethanol consumption.