Transcriptome analysis of sevoflurane exposure effects at the different brain regions

Abstract

Backgrounds: Sevoflurane is a most frequently used volatile anesthetics, but its molecular mechanisms of action remain unclear. We hypothesized that specific genes play regulatory roles in brain exposed to sevoflurane. Thus, we aimed to evaluate the effects of sevoflurane inhalation and identify potential regulatory genes by RNA-seq analysis.

Methods: Eight-week old mice were exposed to sevoflurane. RNA from medial prefrontal cortex, striatum, hypothalamus, and hippocampus were analysed using RNA-seq. Differently expressed genes were extracted and their gene ontology terms were analysed using Metascape. These our anesthetized mouse data and the transcriptome array data of the cerebral cortex of sleeping mice were compared. Finally, the activities of transcription factors were evaluated using a weighted parametric gene set analysis (wPGSA). JASPAR was used to confirm the existence of binding motifs in the upstream sequences of the differently expressed genes.

Results: The gene ontology term enrichment analysis result suggests that sevoflurane inhalation upregulated angiogenesis and downregulated neural differentiation in each region of brain. The comparison with the brains of sleeping mice showed that the gene expression changes were specific to anesthetized mice. Focusing on individual genes, sevoflurane induced Klf4 upregulation in all sampled parts of brain. wPGSA supported the function of KLF4 as a transcription factor, and KLF4-binding motifs were present in many regulatory regions of the differentially expressed genes.

Conclusions: Klf4 was upregulated by sevoflurane inhalation in the mouse brain. The roles of KLF4 might be key to elucidating the mechanisms of sevoflurane induced functional modification in the brain.

Fig 1. RNA-seq analysis for brains exposed to sevoflurane.

(A)The workflow of the RNA-seq analysis for the anesthetized mice. After anesthetizing with 2.5% of sevoflurane and 40% oxygen for 3 hours, the brains were removed and sliced into 2 mm pieces. The striatum, medial prefrontal cortex (MPFC), hypothalamus and hippocampus were punched out. RNA was extracted from the punched out samples and RNA-seq was performed using NextSeq500.

Fig 2. Analysis of the upregulated Differently Expressed Genes (DEGs) in each part of the brain.

(A) Venn-diagram for the upregulated differently expressed genes (DEGs) in each part of brain. (B) The 13 genes commonly upregulated in the four parts of the brain and the fold changes (log2) for each gene. (C) Circos plot for the Metascape analysis of upregulated DEGs. The purple line links the same gene that is shared by multiple gene lists. The blue lines link the different genes where they fall into the same ontology term. (D) Heatmap for the gene ontology term analysis of the upregulated DEGs.

Fig 3. Analysis of the downregulated differently expressed genes for each part of the brain.

(A) Venn-diagram for the downregulated differently expressed genes (DEGs) in each part of brain. (B) Commonly downregulated gene in the four parts of the brain and its fold change (log2). (C) Circos plot for the Metascape analysis of the upregulated DEGs. The purple line links the same gene that are shared by multiple gene lists. The blue lines link the different genes where they fall into the same ontology term. (D) Heatmap for gene ontology terms analysis of the upregulated DEGs.

Fig 4. Estimation and comparison of the relative activities of the transcriptional factors.

(A)-(H) Weighted parametric gene set analysis (wPGSA) of the fold changes in each part of the brain. Transcription factors (TFs) with T-scores of > 2.0 or < -2.0 were identified and the distributions of the T-scores of the medial prefrontal cortex (MPFC) (A), striatum (C), hypothalamus (E), and hippocampus (G) were drawn. Furthermore, the transcription factors included in differently expressed genes (DEGs) were identified and the tables of the T-scores and expression fold changes for MPFC (B), striatum (D), hypothalamus (F), and hippocampus (H) were made.

Fig 5. Comparison of the activities of the transcription factors between the brains of the anesthetized and sleeping mice.

(A) Pie chart of the existence of KLF4-binding motifs in the 1000-bp upstream sequences of the genes annotated to the GO term “angiogenesis”. (B) Pie chart of the existence of KLF4-binding motifs in the 1000-bp upstream sequences of the genes annotated to the GO term “head development”. (C) Estimated mechanism of the effects of sevoflurane on the brain.