Identification of Prefrontal Cortex and Amygdala Expressed Genes Associated With Sevoflurane Anesthesia on Non-human Primate

Abstract

Clinical trials and animal studies have indicated that long-term use or multiple administrations of anesthesia may lead to fine motor impairment in the developing brain. Most studies on anesthesia-induced neurotoxicity have focused on the hippocampus and prefrontal cortex (PFC); however, the role of other vital encephalic regions, such as the amygdala, is still unclear. Herein, we focused on sevoflurane, the most commonly used volatile anesthetic in infants, and performed a transcriptional analysis of the PFC and amygdala of macaques after multiple exposures to the anesthetic by RNA sequencing. The overall, overlapping, and encephalic region-specific transcriptional patterns were separately analyzed to reveal their functions and differentially expressed gene sets that were influenced by sevoflurane. Specifically, functional, protein-protein interaction, neighbor gene network, and gene set enrichment analyses were performed. Further, we built the basic molecular feature of the amygdala by comparing it to the PFC. In comparison with the amygdala's changing pattern following sevoflurane exposure, functional annotations of the PFC were more enriched in glial cell-related biological functions than in neuron and synapsis development. Taken together, transcriptional studies and bioinformatics analyses allow for an improved understanding of the primate PFC and amygdala.

Keywords: RNA sequencing; amygdala; prefrontal cortex; primate; sevoflurane.

FIGURE 1

Differentially expressed genes (DEGs) in macaques’ amygdala and prefrontal cortex (PFC) after multiple sevoflurane anesthesia. (A) Experimental design. The rhesus macaques received 2.5–3% sevoflurane and 100% oxygen for 4 h on postnatal day (P) 7 and then on P21 and P35, respectively. The amygdala and PFC were harvested at the end of the third round of sevoflurane anesthesia on P35. Venn Diagram representation of 387 and 6,526 DEGs were identified from amygdala and PFC after multiple sevoflurane anesthesia, respectively. One hundred and eleven genes were overlapped. (B,C) Heatmap of significant DGEs in amygdala and PFC. (D) The levels of relative mRNA were assessed via qPCR as described. HOMER1, NF1, PLCB1, PPP3R1 mRNA expression increased in the macaques’ amygdala after multiple sevoflurane exposure (n = 5, p < 0.01, one-way ANOVA). Relative mRNA level of LRP6, DCC didn’t alter after sevoflurane exposure (n = 5, p = 0.7127 and p = 0.7065, respectively, one-way ANOVA). (** p < 0.01).

FIGURE 2

Functional annotation of DEGs in macaques’ amygdala and PFC after multiple sevoflurane anesthesia using Metascape. (A) The network plot of 898 DEGs in macaques’ amygdala after multiple sevoflurane anesthesia. (B) The network plot of 387 DEGs in macaques’ PFC after multiple sevoflurane anesthesia.

FIGURE 3

The overlapped 111 genes differently express in PFC and amygdala after multiple sevoflurane anesthesia. (A) Heatmap of the overlapped 111 genes. Samples in amygdala group, respectively, named Ctrl1, Ctrl2 and SEV1, SEV2. Samples in PFC group, respectively, named Ctrl1(P), Ctrl2(P) and SEV1(P), SEV2(P). (B) Significant enriched gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway terms of the overlapped DEGs in macaques’ amygdala and PFC after multiple sevoflurane anesthesia. (C) Functional annotation of transcription factors (TFs) in the overlapped 111 genes after multiple sevoflurane anesthesia using Metascape.

FIGURE 4

PFC-special function analysis. (A) The network of DEGs and related GO classes. (B) The network of PFC-specific DEGs and differentially expressed TFs.

FIGURE 5

Amygdala-special function analysis. (A) The network of DEGs and related GO classes. (B) The network of Amygdala-specific DEGs and differentially expressed TFs.