Developmental Stage-Dependent Transcriptomic Responses to Neonatal Intraventricular Hemorrhage

Neonatal intraventricular hemorrhage (IVH) is a major complication of preterm birth, yet how developmental stage influences the brain's response to injury remains unclear. We performed single-nucleus RNA sequencing on rat brains 24 hours after IVH at postnatal day 2 (PND2) or day 5 (PND5) to define transcriptional responses across cell types. We identified 42 distinct cell populations and found that PND5 brains exhibited a markedly stronger immune and inflammatory response to IVH, with a threefold increase in differentially expressed genes compared to PND2. Microglia were the most perturbed cell type at both stages, showing increased oxidative stress and polarization toward both pro- and anti-inflammatory phenotypes at PND5. Ligand-receptor and regulon analysis revealed a shift from reparative IGF2 and TGF-ß signaling at PND2 to proinflammatory Wnt signaling and activation of Runx1 and Stat5 at PND5. These findings highlight the importance of developmental timing in shaping the neuroimmune response to IVH and identify potential stage-specific therapeutic targets. Overall design: Using the single-nucleus RNA sequencing (snRNA-seq) platform from 10x Genomics, we obtained transcriptomic data from 161,933 nuclei from 32 rat brains. Rats underwent IVH at postnatal day 2 and postnatal day 5, and their brains were harvested for analysis 24 hours later. The experiment used 32 rats overall, with 16 for each timepoint. Of these 16, 8 were control and 8 underwent IVH at each timepoint. Four of each set were male and the other 4 were female.