Analysis of Age-Dependent Transcriptomic Changes in Response to Intracerebral Hemorrhage in Mice

Abstract

Age is a well-known risk factor that is independently associated with poor outcomes after intracerebral hemorrhage (ICH). However, the interrelationship between age and poor outcomes after ICH is not well defined. In this study, we aimed to investigate this relationship based on collagenase-induced ICH mice models. After being assessed neurological deficit 24 h after ICH, mice were euthanized and brain perihematomal tissues were used for RNA-sequencing (RNA-seq). And then the functions of differentially expressed genes (DEGs) identified by RNA-seq were analyzed using Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, Ingenuity Pathway Analysis (IPA) and protein-protein interaction (PPI) analysis. In addition, we performed real-time quantitative polymerase chain reaction (RT-qPCR) for validation of candidate DEGs. In the behavioral tests, aged mice presented significantly worse neurological function than young mice and greater weight loss than aged sham controls 24 h after ICH. In DEGs analysis, ICH affected the expression of more genes in young mice (2,337 DEGs) compared with aged mice (2,005 DEGs). We found aged mice exhibited increased brain inflammatory responses compared with young animals and ICH induced significant activation of the interferon-β (IFN-β) and IFN signaling pathways exclusively in aged mice. Moreover, further analysis demonstrated that ICH resulted in the activation of cytosolic DNA-sensing pathway with the production of downstream molecule type I IFN, and the response to type I IFN was more significant in aged mice than in young mice. In agreement with the results of RNA-seq, RT-qPCR indicated that the expression of candidate genes of cyclic GMP-AMP synthase (cGAS), Z-DNA-binding protein 1 (ZBP1), and IFN-β was significantly altered in aged mice after ICH. Taken together, our study indicated that compared to young animals, aged mice exhibit increased vulnerability to ICH and that the differences in transcriptional response patterns to ICH between young and aged mice. We believe that these findings will facilitate our understanding of ICH pathology and help to translate the results of preclinical studies into a clinical setting.

Keywords: aging; inflammation; intracerebral hemorrhage; neurological deficit; transcriptomics; type I interferon.

FIGURE 1

Experimental protocols, sampling regions, the results of behavioral tests, quantification and quality control of RNA sequencing data. (A) Experimental flowchart. ICH, intracerebral hemorrhage; NDS, neurological deficit score; RNA-seq, RNA-sequencing; IPA, ingenuity pathway analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, gene ontology; RT-qPCR, real-time quantitative polymerase chain reaction. (B) Representative macrographs of coronal brain sections and sampling regions (marked with red traces) from the four groups. (C) Compared to sham surgery, ICH induced significant neurological dysfunction in both aged and young animals (n = 8); Compared to young mice, ICH induced more severe neurological deficits in aged mice. (D) The comparison of gait, climbing ability, and circling behavior scores between aged and young mice. (E) Body weight was significantly decreased in aged mice with ICH compared with aged sham mice (n = 8). (F) The box plot of fragments per kilobase of transcript sequence per millions mapped reads (FPKM) distribution per sample. (G) The principal component analysis plot of mRNA expression in the four groups (n = 3). Dim, dimension. The behavioral data were shown as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

FIGURE 2

Heatmaps of different pairwise comparisons. The criterion for identification DEGs is FC > 1.5 or FC < 0.67, P < 0.05. (A) Young sham vs. aged sham. (B) Young ICH vs. young sham. (C) Aged ICH vs aged sham. (D) Aged ICH vs. Young ICH. ICH, intracerebral hemorrhage.

FIGURE 3

Volcano plots of selected pairwise comparisons. The criterion for identification DEGs is FC > 1.5 or FC < 0.67, P < 0.05. (A) Young sham vs. aged sham. (B) Young ICH vs. young sham. (C) Aged ICH vs. aged sham. (D) Aged ICH vs. Young ICH. ICH, intracerebral hemorrhage.

FIGURE 4

Transcriptomic differences in the brain between young and aged mice. (A) Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis of significantly upregulated differentially expressed genes (DEGs) and (B) downregulated DEGs between young mice and aged mice after sham surgery was performed using Metascape. The significantly enriched GO terms and pathways (P < 0.01, z-score > 2) are presented in enrichment maps, and all of them are grouped into clusters based on membership similarities. The color of the nodes in the maps indicates the P value, and the size of the nodes reflects the number of genes in the hit list. Terms with a similarity > 0.3 are connected by lines. Circular nodes reflect GO terms, and diamond-shaped nodes reflect KEGG pathways. n = 3 per group.

FIGURE 5

Comparison of differentially expressed genes (DEGs) and functional clusters between young and aged mice with intracerebral hemorrhage (ICH). (A) Scatterplot showing the log₂ foldchange (FC) of genes between young and aged mice after ICH. Genes with an FC > 1.5 or FC < 0.67 are colored. (B) The Venn diagram showing overlap of up- or downregulated DEGs between selected pairwise groups. (C) Scatterplots of the log₂FC of genes between young and aged mice after ICH. The color of the scatters indicates the log₂FC of the genes in aged mice vs. young mice after sham surgery (D) or after ICH. (E) Bubble plots comparing ICH-induced alteration in functional clusters from several databases (including Metascape website and Ingenuity pathway analysis (IPA) software) in young mice (F) and aged mice. The Gene Ontology (GO) terms included biological process (BP), cellular component (CC) and molecular function (MF) terms. The size of the bubbles reflects the number of genes in each term, and the color of bubbles reflects the enriched category. (G) Protein-protein interaction (PPI) analysis and (H) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of 100 overlapping genes in the Venn diagram (marked with a red circle), overlapping genes among “upregulated in the young ICH group vs. the young sham group”, “upregulated in the aged ICH group vs. the aged sham group” and “upregulated in the aged ICH group vs. the young ICH group”. In the PPI network, the size of the nodes reflects the degree and the color reflects the FC value of aged ICH vs. sham. The most significant BP in GO enrichment analysis was marked in every module. (I) Pathways in the IPA system associated with genes that showed stronger upregulation after ICH in aged mice (“more upregulated in the aged ICH group”). n = 3 per group.

FIGURE 6

Functional annotation of differentially expressed genes (DEGs) between young and aged mice after intracerebral hemorrhage (ICH). (A) The scatterplot shows DEGs that were more strongly upregulated after ICH in aged mice (“more upregulated in the aged ICH group”), and the enrichment map shows significantly enriched Gene Ontology (GO) terms (circles) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (diamonds) for these genes (P < 0.01, z-score > 2). (B) The scatterplot and enrichment map for DEGs but for DEGs that were more strongly downregulated after ICH in aged mice (“more downregulated in the aged ICH group”). n = 3 per group.

FIGURE 7

Validation of candidate genes. (A) The mRNA expression levels of Cyclic GMP-AMP synthase (cGAS), (B) Z-DNA-binding protein 1 (ZBP1), (C) Interferon-β (IFN-β) and (D) interferon regulatory factor 7 (IRF7) were measured by real-time quantitative polymerase chain reaction (RT-qPCR). The mRNA expression is reported as the FC vs. young mice after sham surgery. The data are shown as the mean 2^–ΔΔCT value ± SEM; young sham: n = 5; young ICH: n = 4; aged sham: n = 4; aged ICH: n = 4; *P < 0.05.