High-resolution transcriptome analysis on a mouse model of neonatal hypoxic-ischemic encephalopathy using single-nucleus RNA-seq

Abstract

Neonatal hypoxic-ischemic encephalopathy (HIE) encompasses brain injuries resulting from dysregulated oxygen or blood flow to the brain before, during, or immediately after birth. During the acute phase, neuronal damage is driven by excitotoxicity, with permanent injury manifesting over the subsequent hours. Treatment options have limited efficacy, requiring deeper insights into HIE pathogenesis. Recent advances in single-cell RNA sequencing have enabled molecular investigations of diverse diseases. However, the large size of certain cells, such as neurons, has posed challenges in studying conditions where neuronal damage is central. Thus, we employed single-nucleus RNA sequencing to evaluate damages in a mouse model of HIE and found pronounced changes in the hippocampus with significantly reduced neuronal populations. We observed the characteristic activation of hippocampal microglia, confirmed by immunostaining in the HIE model. These alterations were specific to combined hypoxic-ischemic conditions and were not observed with hypoxia or ischemia alone. These findings provide insights into the molecular and anatomical impact of HIE and highlight the hippocampus as a critical focus for understanding disease mechanisms and therapeutic development.

Keywords: Hippocampus; Interferon activation; Mouse model; Neonatal hypoxic-ischemic encephalopathy; Neuronal damage; Single-nucleus RNA sequencing.

Fig. 1

Procedures for creating an HIE neonatal mouse model (A) Under sedation with isoflurane, expanding the field of view of the left common carotid artery (CCA) of a P7 mouse. (B) Left CCA ligated with 6-0 silk. (C) Timeline for the experiments. Four experimental groups were established by combining ligation and hypoxia conditions. (D) Location of brain sections (at Bregma-1.5 mm) (E) Volume atrophy calculation ratios of each region. Each group contained four males and four females. (F)Hemisphere volume atrophy ratio. ∗∗p < 0.0001. Error bars show means + SD (G)Cerebral cortex volume atrophy ratio. ∗∗p < 0.0001. Error bars show means + SD (H)Hippocampal atrophy ratio. ∗∗p < 0.0001. Error bars show means + SD.

Fig. 2

Single-nucleus RNA-seq on brains including cerebral cortex and hippocampus. (A) UMAP plot of all snRNA-seq data containing cortex and hippocampus. (B) Marker genes for each cluster. Dot size represents the percentage of cells expressing a given gene within the cluster. Dot color intensity shows the mean expression level. (C) Each sample depth in terms of the number of genes. (D) Each sample depth in terms of the number of unique molecular indices (UMIs). (E) UMAP plot of cells from the hippocampus. (F) UMAP plot of cells from the cerebral cortex.

Fig. 3

Single-nucleus RNA-seq focused on neurons. (A) UMAP plot of cells from the hippocampus. (B) UMAP plot of cells from the cerebral cortex. (C) Differences in UMAP plots distribution of hippocampus neurons separated into four groups. (D) Differences in UMAP plots distribution of cerebral cortex neurons separated into four groups. (E) Difference in the percentage of neurons regarding the CA1-3 region of the hippocampus. (F) Difference in the percentage of neurons regarding layers of cerebral cortex.

Fig. 4

Single-nucleus RNA-seq focused on microglia. (A) Differences in UMAP plots distribution of separated hippocampus and cerebral cortex of four groups. (B) Number of differential expression genes (DEGs) in microglia compared with the Sham group. (C) Heat map focusing on genes that are upregulated in the hippocampus of the HIE group. (D) Gene Ontology (GO) analysis on the DEGs from the hippocampus microglia. (E) Samples of Iba-1 immunostained sections of the ipsilesional hippocampus. (F) Iba-1 positive area ratio of the hippocampus (ispi/contra). ∗p < 0.01. Error bars show means + SD. Each group contained four males and four females.