Single-cell transcriptomic profiling reveals sex-specific microglial responses to simulated galactic cosmic radiation without classical inflammatory activation

Long-duration deep space missions expose astronauts to galactic cosmic radiation (GCR), a complex mixture of high- and low-LET ions that can compromise central nervous system (CNS) health. Microglia, the resident immune cells of the brain, are highly sensitive to radiation, yet their cell-state adaptations to space-relevant mixed-field exposure remain poorly defined. Here, we performed single-cell RNA sequencing (scRNA-seq) of hippocampal microglia isolated from male and female C57BL/6J mice 53 days after acute 100 cGy 5-ion GCR simulation. Behavioral testing showed no overt deficits, but scRNA-seq revealed subtle, sex-specific transcriptional remodeling. Male microglia engaged a stress-adaptive program characterized by upregulation of Fkbp5, Rtp4, Creb5, and metabolic regulators, with concurrent downregulation of ER chaperone and ribosomal genes. Female microglia displayed a transcriptionally blunted phenotype with selective induction of Fkbp5, Ccnd3, and Tnfaip3, and broad downregulation of inflammatory and NOD/TNF pathways. Canonical inflammatory or disease-associated microglia signatures were absent and homeostatic markers (P2ry12, Cx3cr1) were preserved. These findings define a non-inflammatory, sex-specific microglial response to space-relevant GCR and provide a cellular framework for sex-informed CNS risk assessment and countermeasure development for deep space travel. Overall design: Six-month-old male and female C57BL/6J mice were randomly assigned to sham irradiation or exposure to 100 cGy simplified 5-ion galactic cosmic radiation simulation (GCRsim) at the NASA Space Radiation Laboratory (Brookhaven National Laboratory). Behavioral testing (open field, novel object recognition, contextual fear conditioning) was performed 48–51 days post-irradiation. At 53 days post-irradiation, mice were euthanized by injection with a mixture of xylazine (10 mg/kg, i.p.) and ketamine (100 mg/kg, i.p.) and perfused intracardially with 0.15 M PBS containing 0.5% sodium nitrite and 2 IU heparin/mL. Hippocampus and overlying cortex were dissected and CD11b+ microglia were isolated by fluorescence-activated cell sorting. Samples were multiplexed using TotalSeq-B anti-CD45 antibody hashing and profiled by 10x Genomics single-cell 3' gene expression sequencing. Single-cell transcriptomes were analyzed to quantify sex- and radiation-dependent changes in microglial cell states, differential gene expression, and pathway enrichment.