Sevoflurane inhibits the differentiation and development of neural progenitor cells into neurons in the prefrontal cortex of newborn mice

Abstract

Objective·To investigate the mechanism of sevoflurane damaging neuron development in the prefrontal cortex (PFC) of the neonatal mice after single or multiple sevoflurane anesthesia based on the single-cell RNA sequencing (scRNA-seq).Methods·The neonatal mice were divided into multiple anesthesia exposure (Sev3) group, single anesthesia exposure (Sev1) group, and control group with 3 mice each. The Sev3 group received anesthesia with 3% sevoflurane and 60% O2 on postnatal day 6, 7, and 8, and the Sev1 group received anesthesia only on postnatal day 6. The PFC from mice in the 3 groups was harvested on postnatal day 9 for scRNA-seq. PFC cell profiles and neuronal subpopulation profiles of newborn mice after sevoflurane anesthesia were obtained by UMAP (uniform manifold approximation and projection) clustering, RNA velocity analysis, and transcription factor analysis (SCENIC). Differential expression gene analysis was performed. The biological processes and pathways of the differential genes were investigated through Gene Ontology (GO) database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database; QuSAGE analysis was used to describe the activation of the cell cycle and Hippo signaling pathway gene sets. Transcript enrichment and stemness of PFC neuronal lineage cells of neonatal mice after sevoflurane anesthesia was determined by CytoTRACE score. The differentiation trajectory of PFC neurons was determined by using pseudo-time analysis, and the developmental nodes were resolved by BEAM analysis to identify key genes that determine different cell fates.Results·A total of 40 061 cells with 10 cell types were obtained from the PFC of newborn mice in the 3 groups by scRNA-seq. The down-regulated genes in the PFC cells after single sevoflurane anesthesia were enriched in cell differentiation, forebrain neuron differentiation, noradrenergic neuron differentiation, and cerebral cortex GABAergic interneuron differentiation. The down-regulated genes after multiple sevoflurane anesthesia were enriched in positive regulation of cell differentiation. KEGG analysis showed that the down-regulated genes after single sevoflurane anesthesia were enriched in transforming growth factor-β signaling pathway, and the down-regulated genes after multiple sevoflurane anesthesia were enriched in the Notch signaling pathway. SCENIC analysis showed that early growth response 1 (Egr1) and SRY-box transcription factor 7 (Sox7) were up-regulated after both single and multiple sevoflurane anesthesia (both P<0.01), and HES family bHLH transcription factor 6 (Hes6) and NK2 homeobox 1 (Nkx2-1) were down-regulated only after single sevoflurane anesthesia (P<0.01). Activation of the gene set of the cell cycle in radial glial cells and neurons increased after sevoflurane anesthesia, and the increase in activation was more pronounced after multiple sevoflurane anesthesia. The gene set of the Hippo signaling pathway in neurons changed from inhibition to activation after multiple sevoflurane anesthesia. Subpopulation analysis of 8 224 neurons identified 8 neuronal lineage cells, and CytoTRACE scores indicated increased neuron stemness and delayed neuron development after sevoflurane anesthesia. The PFC neurons were divided into 3 developmental stages by pseudo-time analysis, and multiple sevoflurane anesthesia receded the differentiation of PFC neurons in pseudo-time (P=0.000). The down-regulated genes in PFC neurons of newborn mice after single sevoflurane anesthesia were enriched in the regulation of cyclin-dependent protein serine/ threonine kinase activity, mitotic cell cycle phase transition, cell differentiation, long-term memory, and G1/S transition of the mitotic cell cycle. The down-regulated genes in PFC neurons after multiple sevoflurane anesthesia were enriched in the negative regulation of cell population proliferation, positive regulation of cell differentiation, forebrain neuron differentiation, positive regulation of canonical Wnt signaling pathway, and cell differentiation.Conclusion·Both single and multiple sevoflurane anesthesia promote PFC neuron proliferation and migration, and multiple sevoflurane anesthesia inhibits the differentiation of neural progenitor cell into neuron in PFC. The underlying mechanism might be related to cell cycle transitions.

Keywords

• sevoflurane
• general anesthesia
• prefrontal cortex (pfc)
• neuron differentiation
• single-cell rna sequencing (scrna-seq)