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

## TL;DR

This study maps how neonatal microglia change over time after brain injury, revealing early repair and later fibrotic phases that could guide new therapies.

## Contribution

The study reveals distinct temporal phases of microglial activation in neonatal hippocampal injury and identifies therapeutic targets linked to ARG1 signaling.

## Key findings

- Early microglia show efferocytic and inflammatory-resolving activity, while later microglia shift to extracellular matrix remodeling and fibrosis.
- HI injury leads to microglial and astrocytic expansion and loss of glutamatergic neurons by Day 5.
- ARG1-linked pathways, including TGF-β and PI3K–Akt, drive microglial transitions from repair to fibrotic states.

## Abstract

What are the main findings?
This study defines the temporal cellular architecture of the neonatal hippocampal response to hypoxic–ischemic injury by integrating cell-type-resolved transcriptomics and spatial mapping at Day 1 and Day 5.Early microglia display an efferocytic and inflammatory-resolving program, whereas subacute microglia shift toward extracellular matrix remodeling and fibrotic pathways.

This study defines the temporal cellular architecture of the neonatal hippocampal response to hypoxic–ischemic injury by integrating cell-type-resolved transcriptomics and spatial mapping at Day 1 and Day 5.

Early microglia display an efferocytic and inflammatory-resolving program, whereas subacute microglia shift toward extracellular matrix remodeling and fibrotic pathways.

What are the implications of the main findings?
These findings identify distinct phases of microglial activation and highlight therapeutic windows for modulating reparative microglial states.

These findings identify distinct phases of microglial activation and highlight therapeutic windows for modulating reparative microglial states.

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.