288 - Transcriptomic Reprogramming of Hippocampal Microglia Following Neonatal Hypoxic-Ischemic Brain Injury

Publication Number: 4283.288

JANA MIKE, University of California, San Francisco, School of Medicine, SAN FRANCISCO, CA, United States; Eesha Natarajan, University of California San Francisco, San Francisco, CA, United States; Emin Maltepe, UCSF Benioff Children's Hospital San Francisco, San Francisco, CA, United States; Donna Ferriero, University of California, San Francisco, School of Medicine, Stanford, CA, United States

Background: Neonatal hypoxic-ischemic (HI) brain injury causes lasting neurodevelopmental impairments, yet the cellular and molecular responses within the hippocampus remain incompletely understood. We investigated transcriptomic profiles of microglia to elucidate mechanisms of injury and repair.

Objective: To characterize the transcriptomic responses of microglial populations in the neonatal hippocampus following HI, focusing on shifts from the acute to subacute stages of tissue remodeling.

Design/Methods: Neonatal HI was induced at postnatal day 10 (P10) using the Vannucci model (left carotid occlusion followed by 50 minutes of 10% O₂). Tissues were collected at Day 1 (D1, acute) and Day 5 (D5, subacute). Sham mice served as controls. Sections from OCT-embedded frozen brains were processed on the Spatial Genomics Gene Positioning System (GenePS) using the Mouse Brain Mapper Panel supplemented with ARG1, ARG2, microglial (TMEM119, P2RY12), and fibrosis-related genes (TGFB1, COL1A2). Processed images were analyzed using Spatial Genomics Navigator, followed by clustering and differential expression analysis in MATLAB. Clustering identified 12 transcriptionally distinct clusters. Differential gene expression (DGE), gene set enrichment analysis (GSEA), and overrepresentation analysis (ORA) were applied to pseudobulk clusters (analyzed in Seurat) to assess cellular shifts and pathway enrichment across time points.

Results: Transcriptomic analysis revealed distinct temporal microglial phenotypes. D1 (acute response) microglia were enriched for pathways related to cell signaling, projection organization, and motility, with key genes such as Reln and Egr1 suggesting cytoskeletal remodeling and migration toward the injury site. By D5 (subacute response), microglia shifted toward extracellular matrix (ECM) remodeling and pro-fibrotic signaling. GSEA showed strong enrichment for connective tissue development, alongside significant upregulation of ECM-related DEGs including Bgn (log₂FC = +2.92), Col11a1 (log₂FC = +2.56), Cd44 (log₂FC = +3.23), and the pro-fibrotic mediator Tgfb1.

Conclusion(s): Neonatal HI in the hippocampus induces a microglial transcriptomic transition from early signaling and motility (D1) to active participation in tissue scarring and fibrotic ECM deposition (D5). Targeting ECM and fibrosis remodeling pathways may offer therapeutic potential for mitigating long-term structural damage following neonatal brain injury.