Temporal and Spatial Gene Expression Dynamics in Neonatal HI Hippocampus with Focus on Arginase.

BACKGROUND

Hypoxic-ischemic (HI) brain injury triggers a dynamic, multi-phase response involving early microglial efferocytosis followed by extracellular matrix (ECM) deposition and scar formation. Arginase-1 (ARG1), a key enzyme in tissue repair, is implicated in both processes, yet its role in neonatal microglia remains poorly defined. We characterize ARG1-linked pathways in neonatal microglia, identifying distinct efferocytic and fibrotic phases post-HI.

METHODS

HI was induced in P9 mice using the Vannucci model, and brains were collected at 24 h (D1) and 5 days (D5). Spatially resolved single-cell transcriptomics (seqFISH) was performed using a targeted panel enriched for microglial, ARG1-pathway, efferocytosis, and profibrotic genes. Cell segmentation, clustering, and spatial mapping were conducted using Navigator and Seurat. Differential expression, GSEA, and enrichment analyses were used to identify time- and injury-dependent pathways.

RESULTS

Spatial transcriptomics identified 12 transcriptionally distinct cell populations with preserved neuroanatomical organization. HI caused the expansion of microglia and astrocytes and the loss of glutamatergic neurons by D5. Microglia rapidly activated regenerative and profibrotic programs-including TGF-β, PI3K-Akt, cytoskeletal remodeling, and migration-driven by early DEGs such as Cd44, Reln, TGF-β1, and Col1a2. By D5, microglia adopted a collagen-rich fibrotic state with an upregulation of Bgn, Col11a1, Anxa5, and Npy.

CONCLUSION

Neonatal microglia transition from early efferocytic responses to later fibrotic remodeling after HI, driven by the persistent activation of PI3K-Akt, TGF-β, and Wnt/FZD4 pathways. These findings identify microglia as central regulators of neonatal scar formation and highlight therapeutic targets within ARG1-linked signaling.