Neonatal binge alcohol exposure increases microglial activation in the developing rat hippocampus

Abstract

Aberrant activation of the developing immune system can have long-term negative consequences on cognition and behavior. Teratogens, such as alcohol, activate microglia, the brain's resident immune cells, which could contribute to the lifelong deficits in learning and memory observed in humans with Fetal Alcohol Spectrum Disorders (FASD) and in rodent models of FASD. The current study investigates the microglial response of the brain 24 h following neonatal alcohol exposure (postnatal days (PDs) 4-9, 5.25 g/kg/day). On PD10, microglial cell counts and area of cell territory were assessed using unbiased stereology in the hippocampal subfields CA1, CA3 and dentate gyrus (DG), and hippocampal expression of pro- and anti-inflammatory genes was analyzed. A significant decrease in microglial cell counts in CA1 and DG was found in alcohol-exposed and sham-intubated (SI) animals compared to undisturbed suckle controls (SCs), suggesting overlapping effects of alcohol exposure and intubation alone on the neuroimmune response. Cell territory was decreased in alcohol-exposed animals in CA1, CA3, and DG compared to controls, suggesting the microglia have shifted to a more activated state following alcohol treatment. Furthermore, both alcohol-exposed and SI animals had increased levels of pro-inflammatory cytokines IL-1β, TNF-α, CD11b, and CCL4; in addition, CCL4 was significantly increased in alcohol-exposed animals compared to SI as well. Alcohol-exposed animals also showed increased levels of anti-inflammatory cytokine TGF-β compared to both SI and SCs. In summary, the number and activation of microglia in the neonatal hippocampus are both affected in a rat model of FASD, along with increased gene expression of pro- and anti-inflammatory cytokines. This study shows that alcohol exposure during development induces a neuroimmune response, potentially contributing to long-term alcohol-related changes to cognition, behavior and immune function.

Keywords: FASD; cytokines; development; neuroimmune; pro-inflammatory.

Figure 1

Representative images of Iba-1+ microglia in the postnatal day 10 rat hippocampus. A) Quiescent microglia with small somas and long, thin processes. B) Fully activated amoeboid microglia with a large, round soma and very short processes. Both images taken with a 40x lens. C) Iba-1 immunostaining in the neonatal rat hippocampal CA1. Image taken with a 20x lens.

Figure 2

Microglial cell counts. Number of Iba-1+ microglia were significantly decreased in hippocampal CA1 (A) of AE and SI animals compared to the SC group. No effect of neonatal treatment was found in CA3 (B). In dentate gyrus (C), AE and SI animals had fewer microglia compared to the SC group, but the AE group also had significantly more microglia compared to the SI animals. AE = alcohol-exposed, SI = sham-intubated, SC = suckle control. ** = p < 0.01.

Figure 3

Microglia cell territory. A) In CA1, the average area covered by each microglia was decreased in the AE group compared to the SI and SC groups (p < 0.05 and 0.001, respectively). B) In CA3, cell territory was decreased in the AE group compared to SI and SC (p < 0.05 and 0.01, respectively). C) The AE group had significantly smaller microglia in the dentate gyrus (DG) compared to the SC group (p < 0.05), but was not significantly different from SI. Visualization of the size of the microglia measured in each region when categorized as small, medium or large. D) Representative drawings of small, medium, and large microglia. E) CA1, F) CA3, G) DG. AE = alcohol-exposed, SI = sham-intubated, SC = suckle control. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Figure 4

Pro- and anti-inflammatory gene expression in the neonatal hippocampus. Both AE and SI groups had elevated levels of IL-1β (A), TNF-α (B), and CD11b (C) compared to SC. For CCL4 (D), both AE and SI groups showed increased gene expression, however levels of CCL4 in the AE group were significantly higher than in the SI group (p < 0.05). E) Gene expression of anti-inflammatory cytokine TGF-β were significantly increased in the AE group over the SI and SC animals (p < 0.05). Data is expressed as a fold change from the suckle control (SC) group (shown as 1 on graphs). AE = alcohol-exposed, SI = sham-intubated, SC = suckle control. * = p < 0.05, ** = p < 0.01.