Introduction: Neonatal hypoxic-ischemic (HI) brain injury is a major cause of mortality and long-term neurological disability, yet effective neuroprotective strategies remain limited. Microglia are central mediators of injury and repair, with arginase 1 (ARG1) marking anti-inflammatory, reparative states. However, the functional roles of ARG1+ microglia in tissue remodeling after HI are poorly understood. Methods: Neonatal mice (P10) underwent HI using the Vannucci procedure. ARG1 activity was inhibited pharmacologically using N-omega-hydroxy-nor-L-arginine (nor-NOHA). ARG1 expression, microglial morphology, efferocytosis, tissue scar, and injury volume were assessed via immunohistochemistry, Western blotting, and arginase activity assays at 1 and 5 days post-injury. Results: ARG1+ microglia rapidly engaged apoptotic neurons, exhibiting phagocytic activity confirmed by CD68 expression. Nor-NOHA treatment reduced ARG1 enzymatic activity, impaired microglial process extension, attenuated efferocytosis, and increased injury volume. ARG1+ microglia persisted in the glial scar and colocalized with collagen I alpha-1 (Col1a1), suggesting a role in extracellular matrix (ECM) deposition. Inhibition of ARG1 decreased Col1a1 expression, highlighting its contribution to tissue remodeling. Conclusions: ARG1+ microglia are pivotal in neonatal HI, mediating early efferocytosis and later ECM remodeling, thereby limiting injury and shaping scar architecture. Pharmacological blockade of ARG1 exacerbates injury, underscoring its reparative function. These findings establish ARG1 as a critical regulator of microglial-mediated neuroprotection and tissue repair, providing a potential therapeutic target for neonatal HI brain injury.

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.

Background: Hypoxic-ischemic encephalopathy (HIE) is the leading cause of neonatal morbidity and mortality globally. An early tool to diagnose and prognosticate disease could aid in timely intervention and reduction in disease burden. We aimed to identify biomarkers for HIE and define whether these biomarkers are associated with neurologic outcome severity in our ovine model. Methods: Study Cohort 1 ( n = 46) included lambs with HIE induced via umbilical cord occlusion (UCO) and healthy controls ( n = 19). Cohort 2 ( n = 25 UCO lambs) served as a validation cohort for identified biomarkers. Blood samples collected at multiple early time points were analyzed using untargeted liquid chromatography-mass spectrometry. Biomarkers were considered significant at p < 1e-6. Neurological outcome biomarkers were identified using ordinal logistic regression. Results: A total of 145 hypoxia biomarkers were identified, exhibiting a consistent and reproducible temporal pattern across both cohorts. Hypoxanthine level at 20 min of life showed a strong correlation with the severity of neurologic outcomes. Conclusions: Hypoxanthine emerged as a significant neurologic outcome biomarker in our study. Our findings support the potential utility of metabolomics for early diagnosis and prognostication in HIE. Further clinical validation is warranted to translate these biomarkers into accessible diagnostic and predictive tools, particularly for resource-limited settings. Impact: Our study identified plasma hypoxanthine as an early biomarker for hypoxic-ischemic encephalopathy (HIE) in an ovine model and uncovered a novel correlation between hypoxanthine levels and the severity of neurologic impairment in asphyxiated lambs. These findings highlight the potential of metabolomics in the early detection of HIE, paving the way for improved diagnostic and prognostic strategies. Translating these insights into clinical practice could enable the development of predictive tools for outcome assessment and resource allocation, ultimately enhancing early intervention and optimizing care for neonates at risk of HIE.