Single-cell reanalysis characterizes an Osmr+ astrocyte state and predicts midkine signaling to Cox6b1+ glutamatergic neurons at 24 h after traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) remains a leading cause of mortality and long-term neurological disability worldwide. The cellular heterogeneity and intercellular communication in the injured brain remain incompletely defined, particularly the astrocyte-neuron crosstalk that could drive potential interventions. METHODS: We reanalyzed the publicly available single-cell RNA-sequencing dataset GSE290150, comprising 60,962 high-quality cells from the ipsilateral cortex of mice at 24 h after TBI or sham surgery. Integrated bioinformatic analyses, including unsupervised clustering, gene-set activity scoring, pseudotime inference, transcriptional regulatory network analysis using SCENIC, and cell-cell communication inference using CellChat, were performed to characterize the early post-TBI cellular landscape. RESULTS: We identified five astrocyte subpopulations. Descriptive analysis of the pooled cells showed a greater relative proportion of C3 Osmr+ astrocytes in the TBI group than in the Sham group. This subpopulation exhibited a highly reactive transcriptional state with concurrent protection-associated and neurotoxicity-associated features, together with relatively high oxidative-phosphorylation- and glutamate-metabolism-related activity scores and elevated inferred Tfe3 regulon activity. Among ten neuronal subpopulations, C0 Cox6b1+ glutamatergic neurons displayed oxidative-phosphorylation- and aerobic-respiration-related features. CellChat analysis prioritized Mdk-Ncl as a candidate ligand-receptor interaction contributing to inferred communication from C3 Osmr+ astrocytes to C0 Cox6b1+ neurons, suggesting a potential astrocyte-to-neuron communication pattern after TBI. CONCLUSIONS: This study identifies a TBI-associated C3 Osmr+ astrocyte subpopulation characterized by the highest pan-reactive signature together with protection-associated, neurotoxicity-associated, and metabolic gene expression features, and identifies C0 Cox6b1+ glutamatergic neurons as a candidate recipient population of astrocyte-derived MK signaling. Tfe3 was further prioritized as a candidate transcriptional regulator associated with the C3 Osmr+ astrocyte state. These findings provide a valuable framework for advancing experimental studies of astrocyte-neuron communication after TBI.