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 hours following neonatal alcohol exposure (postnatal days 4–9, 5.25 g/kg/day). On postnatal day 10, microglial cell counts and area of cell territory were assessed using unbiased stereology in the hippocampal subfields CA1, CA3 and dentate gyrus, and hippocampal expression of pro- and anti-inflammatory genes was analyzed. A significant decrease in microglial cell counts in CA1 and dentate gyrus was found in alcohol-exposed and sham-intubated animals compared to undisturbed suckle controls, 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 dentate gyrus compared to controls, suggesting the microglia have shifted to a more activated state following alcohol treatment. Furthermore, both alcohol-exposed and sham-intubated 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 sham-intubated as well. Alcohol-exposed animals also showed increased levels of anti-inflammatory cytokine TGF-β compared to both sham-intubated and suckle controls. 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.

Microglia are involved in various homeostatic processes in the brain, including phagocytosis, apoptosis, and synaptic pruning. Sex differences in microglia colonization of the developing brain have been reported, but have not been established following alcohol insult. Developmental alcohol exposure represents a neuroimmune challenge that may contribute to cognitive dysfunction prevalent in humans with Fetal Alcohol Spectrum Disorders (FASD) and in rodent models of FASD. Most studies have investigated neuroimmune activation following adult alcohol exposure or following multiple exposures. The current study uses a single day binge alcohol exposure model (postnatal day [PD] 4) to examine sex differences in the neuroimmune response in the developing rat hippocampus on PD5 and 8. The neuroimmune response was evaluated through measurement of microglial number and cytokine gene expression at both time points. Male pups had higher microglial number compared to females in many hippocampal subregions on PD5, but this difference disappeared by PD8, unless exposed to alcohol. Expression of pro-inflammatory marker CD11b was higher on PD5 in alcohol-exposed (AE) females compared to AE males. After alcohol exposure, C-C motif chemokine ligand 4 (CCL4) was significantly increased in female AE pups on PD5 and PD8. Tumor necrosis factor-α (TNF-α) levels were also upregulated by AE in males on PD8. The results demonstrate a clear difference between the male and female neuroimmune response to an AE challenge, which also occurs in a time-dependent manner. These findings are significant as they add to our knowledge of specific sex-dependent effects of alcohol exposure on microglia within the developing brain.