Single-cell Transcriptomic Profiling of Brains in Newborn Rats Following Hypoxic Ischemic Encephalopathy

Abstract Background Neonatal hypoxic-ischemic encephalopathy (HIE) is a severe neurological condition associated with high rates of mortality or long-term disability. Despite its clinical significance, the detailed cellular mechanisms underlying HIE remain unclear. Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for investigating cellular heterogeneity across development, aging, and disease processes. However, no scRNA-seq studies have yet addressed neonatal HIE. Methods We employed scRNA-seq to examine cellular heterogeneity during the hyperacute (3 hours), acute (2 days), and subacute (7 days) phases of neonatal HIE. Uniform Manifold Approximation and Projection (UMAP) was used to visualize the cell clustering. Differentially expressed genes (DEGs) were calculated and identified using the Seurat’s FindAllMarkers function, which was enriched for pathway analysis (GO, KEGG pathway, WikiPathways, and Reactome Gene Sets). CytoTRACE v2 was used to identify the maturity state of each cell type and pseudotime analysis was performed using Monocle v3. Results We analyzed a total of 87,580 high-quality brain cells to identify transcriptional changes associated with HIE. In the hyperacute phase, we observed activation of astrocytes in response to reactive oxygen species, involvement of microglia in phagocytosis, Stat3-mediated ischemic responses in oligodendrocyte precursor cells, and an increase in senescent lymphatic endothelial cells. In the acute phase, astrocytes were found to exacerbate inflammation and impede brain development, while microglia proliferated. Neuroblasts were affected by metal ions, and oligodendrocytes decreased. In the subacute phase, astrocytes facilitated tissue repair, while inflammatory microglia highly expressing MHC II were induced by the IL27 and type I interferon pathways and expanded. Additionally, peripheral immune cells played vital roles in HIE. Specifically, neutrophils infiltrated and expanded throughout all phases post-HIE. Spp1high macrophages, T cells, and plasmacytoid dendritic cells increased during the acute and subacute phases, and B cells expanded during the subacute phase. Conclusion This study offers deep insights into the molecular alterations of key cell types following HIE, elucidating the pathological processes involved. These findings have significant implications for developing effective clinical strategies for managing HIE.