Single-Cell Transcriptomic Profiling of Brain Cells in Newborn Rats Following Hypoxic Ischemic Encephalopathy

Abstract

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. In this study, we employed scRNA-seq to examine cellular heterogeneity during neonatal HIE. We analyzed a total of 87 580 high-quality brain cells to identify transcriptional changes associated with HIE. In the hyperacute phase, we observed astrocytes in response to tumor necrosis factors, 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 activated and involved in gliogenesis, while microglia proliferated. Neuroblasts were affected by metal ions, and oligodendrocytes decreased. In the subacute phase, astrocytes involved in inflammation and antigen presentation, 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. Spp1^high macrophages, T cells, and plasmacytoid dendritic cells increased during the acute and subacute phases, and B cells expanded during the subacute phase. 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.

Keywords: HIE; ScRNA‐seq; hypoxic–ischemic encephalopathy; newborn rats; single cell RNA sequencing.

FIGURE 1

Single‐cell profiling of the brain cells after HIE. (A) A UMAP plot identifying major cell types from brains isolated from Sham and HIE rat pups. (B) A dot plot of specific cell marker genes expressed in each cell type. (C) Proportion of each brain cell type among all cells in the Sham and HIE groups. OC, oligodendrocyte; OPC, oligodendrocyte precursor cell; MP, mononuclear phagocytes; CPC, choroid plexus cell; RBC, red blood cell; EC, endothelial cells.

FIGURE 2

Identification of astrocyte subpopulations after HIE. (A) Re‐clustering of astrocytes visualized in a UMAP plot. (B) A heatmap plot showing the top 10 DEGs expressed in each astrocyte subpopulation. (C‐E) Pathway enrichment analysis of DEGs of A8 (C), A3 (D), and A7 (E). (F) Venn diagrams showing overlaps of upregulated DEGs of A6 and A7. (G) Proportion of each group among all astrocyte subtypes.

FIGURE 3

Identification of microglial subpopulations after HIE. (A) Re‐clustering of microglia visualized in a UMAP plot. (B) A UMAP plot showing cell cycle analysis of microglia. (C) Pathway enrichment analysis of DEGs of MG10. (D) Proportion of each group among all microglial subtypes. (E) A UMAP plot showing the single cell trajectory of microglia using Monocle v3. Cells are ordered in pseudo time colored in a gradient from purple to yellow. (F) Flow cytometry showing MHC II positive microglia with or without incubating with myelin debris. (G) Statistical graph of F. (H and I) ELISA assays showing the secretion of IL‐1β (H) and IL‐6 (I) in microglia with or without incubating with myelin debris.

FIGURE 4

Identification of neuroblast and oligodendrocyte lineage subpopulations after HIE. (A) Re‐clustering of neuroblasts visualized in a UMAP plot. (B) Proportion of each group among all neuroblast subtypes. (C) Re‐clustering of oligodendrocyte lineage visualized in a UMAP plot. (D) Venn diagrams showing overlaps of DEGs of oligodendrocyte subtypes and OPC subtypes. (E) Proportion of each group among all oligodendrocyte lineage subtypes. (F) A dot plot showing the expression of Pdcd4, Cdkn1c, and Vgf in each oligodendrocyte lineage subpopulation.

FIGURE 5

Identification of endotheliocyte and fibroblast lineage subpopulations after HIE. (A) Re‐clustering of VEC visualized in a UMAP plot. (B) A dot plot showing specific cell marker genes expressed in each endotheliocyte subtype. (C) Proportion of each group among all endotheliocyte subtypes. (D) Re‐clustering of fibroblasts visualized in a UMAP plot. (E) Violin plots showing certain genes expressed in each fibroblast subtype. (F) Proportion of each group among all fibroblast subtypes.

FIGURE 6

Identification of immune cell subpopulations after HIE. (A) Re‐clustering of infiltrated immune cells visualized in a UMAP plot. (B) A dot plot showing specific cell marker genes expressed in each infiltrated immune cell subtype. (C) Proportion of each group among all infiltrated immune cell subtypes. (D) A dot plot showing pathway enrichment analysis of DEGs of Spp1 ^high macrophages. (E) Pathway enrichment analysis of DEGs of mast cells.