七氟烷抑制新生小鼠前额叶皮质神经祖细胞向神经元分化发育

doi:10.3969/j.issn.1674-8115.2023.09.006

摘要/Abstract

摘要：

目的·基于单细胞转录组测序（single-cell RNA sequencing，scRNA-seq）探究单次和重复七氟烷麻醉对新生小鼠前额叶皮层（prefrontal cortex，PFC）神经元发育的损伤机制。方法·将新生小鼠分为重复麻醉暴露（Sev3）组、单次麻醉暴露（Sev1）组和对照组，每组3只。Sev3组于出生后第6、第7、第8日接受3%七氟烷和60%氧气的麻醉，Sev1组仅在出生后第6日接受麻醉，出生后第9日获取3组小鼠的PFC进行scRNA-seq。通过UMAP（uniform manifold approximation and projection）聚类、RNA速度分析、转录因子分析（SCENIC）获得七氟烷麻醉后新生小鼠PFC细胞图谱和神经元细分亚群图谱，并进行差异表达基因分析；通过基因本体论（Gene Ontology，GO）和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）数据库分析研究差异基因富集的生物学过程和通路；利用QuSAGE分析描述细胞周期及Hippo信号通路基因集的激活情况。通过CytoTRACE评分判断七氟烷麻醉后新生小鼠PFC神经元谱系细胞转录本丰富度和干性；利用拟时序分析（pseudo-time analysis）确定神经元分化轨迹，通过分支表达分析模型（BEAM）对发育节点进行解析，以确定决定细胞不同命运的关键基因。结果·3组新生小鼠PFC经scRNA-seq共获得40 061个细胞，10种细胞类型。单次七氟烷麻醉后PFC细胞下调的基因富集在细胞分化、前脑神经元分化、去甲肾上腺素能神经元分化和大脑皮层γ-氨基丁酸能中间神经元分化。重复七氟烷麻醉后下调基因富集在细胞分化的正向调节。单次七氟烷麻醉后下调的差异基因富集的KEGG通路有转化生长因子β信号通路。重复七氟烷麻醉后下调的差异基因富集的KEGG通路有Notch信号通路。SCENIC显示，早期生长反应因子1（early growth response 1，Egr1）、SRY-box转录因子7（SRY-box transcription factor 7，Sox7）在单次和重复七氟烷麻醉后均上调（均P<0.01），HES家族bHLH转录因子6（HES family bHLH transcription factor 6，Hes6）、NKX同源框-1基因（NK2 homeobox 1，Nkx2-1）仅在单次七氟烷麻醉后下调（均P<0.01）。七氟烷麻醉后放射状胶质细胞和神经元与细胞周期相关的基因集激活度增加，并且重复麻醉暴露后激活度增加更明显。重复七氟烷麻醉后神经元与Hippo信号通路相关的基因集由抑制变为激活。将8 224个神经元单独进行亚群分析获得8种神经元谱系细胞，CytoTRACE评分表明七氟烷麻醉后神经元干性增加，神经元发育迟滞。PFC神经元经拟时序分析区分为3个发育阶段，重复七氟烷麻醉使PFC神经元分化拟时间后退（P=0.000）。单次七氟烷麻醉暴露后新生小鼠PFC神经元下调的基因富集在细胞周期蛋白依赖性蛋白丝氨酸/苏氨酸激酶活性的调节、有丝分裂细胞周期相变、细胞分化、长期记忆、有丝分裂细胞周期G₁/S转换。重复七氟烷麻醉暴露后PFC神经元下调基因富集在细胞群增殖的负向调控、细胞分化的正向调节、前脑神经元的分化、典型Wnt信号通路的正向调控和细胞分化。结论·单次和重复七氟烷麻醉均促进新生小鼠PFC神经祖细胞增殖和神经元迁移，重复七氟烷麻醉抑制PFC的神经祖细胞向神经元分化，其潜在机制可能与细胞周期转换有关。

关键词: 七氟烷, 全身麻醉, 前额叶皮质, 神经元分化, 单细胞转录组测序

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% O₂on 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.

Key words: sevoflurane, general anesthesia, prefrontal cortex (PFC), neuron differentiation, single-cell RNA sequencing (scRNA-seq)

中图分类号:

R614.2

刘思雨, 张磊. 七氟烷抑制新生小鼠前额叶皮质神经祖细胞向神经元分化发育[J]. 上海交通大学学报（医学版）, 2023, 43(9): 1115-1130.

LIU Siyu, ZHANG Lei. Sevoflurane inhibits the differentiation and development of neural progenitor cells into neurons in the prefrontal cortex of newborn mice[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(9): 1115-1130.

图/表 9

图1 各组小鼠PFC细胞图谱、细胞类型分布及占比Note： A. Experimental procedures. The PFC cells acquired on postnatal day 9 (P9) from control (Ctrl) group, single anesthesia exposure (Sev1) group, and multiple anesthesia exposure (Sev3) group were prepared in single-cell suspension for 10x Genomics scRNA-seq. B. UMAP cluster showing PFC scRNA-seq results. C. Clustering visualization of RNA velocity analysis results of PFC cells. D. Bubble map of sorting gene markers in PFC cells. Bubble size represents the percentage of cells with the gene expressed in the population, and bubble colour represents the average value of the gene expressed in the population. E. Distribution of 3 groups of sample cells on the UMAP plot. F. Bar graphs of the percentages of cell types in 9 samples.

Fig 1 Cellular profiles, and cell type distribution and proportion in the PFCs of three groups of mice

图2 新生小鼠七氟烷麻醉后PFC细胞转录组变化和功能注释分析Note： A. Volcano map showing differentially expressed genes between Sev1 group and Ctrl group. B/C. Top 15 enrichment entries of down-regulated genes (B) and up-regulated genes (C) in the GO-BP database between Sev1 group and Ctrl group. D. Volcano map showing differentially expressed genes between Sev3 group and Ctrl group. E/F. Top 15 enrichment entries of down-regulated genes (E) and up-regulated genes (F) in the GO-BP database between Sev3 group and Ctrl group. Entries highlighted in red represent biological processes associated with PFC neurogenesis.

Fig 2 Analysis of transcriptome changes and functional annotation of PFC cells in neonatal mice after sevoflurane anesthesia

图3 新生小鼠七氟烷麻醉后PFC细胞差异表达基因的通路分析Note： A/B. Top 10 enrichment pathway entries of down-regulated genes (A) and up-regulated genes (B) in the KEGG database between Sev1 group and Ctrl group. C/D. Top 10 enrichment pathway entries of down-regulated genes (C) and up-regulated genes (D) in the KEGG database between Sev3 group and Ctrl group. Entries marked in red represent pathways associated with PFC neurogenesis.

Fig 3 Pathway analysis of differential expressed genes in PFC cells in neonatal mice after sevoflurane anesthesia

图4 新生小鼠七氟烷麻醉后PFC细胞转录因子变化Note： A. AUCell values of Egr1. B. AUCell values of Sox7. C. AUCell values of Hes6. D. AUCell values of Nkx2-1. ①P=0.000, ②P=0.009. ns—not significant.

Fig 4 Transcription factor changes in PFC cells in neonatal mice after sevoflurane anesthesia

图5 新生小鼠七氟烷麻醉后PFC细胞细胞周期和Hippo信号通路的基因集激活情况Note： QuSAGE analysis of differentially expressed genes after exposure to sevoflurane anesthesia. A. Cell cycle pathway gene set activation analysis. B. Hippo signaling pathway-multiple species gene set activation analysis.

Fig 5 Gene set activation of cell cycle pathways and Hippo signaling pathways in PFC cells in neonatal mice after sevoflurane anesthesia

图6 各组小鼠PFC神经元图谱、类型分布及干性分析Note： A. UMAP cluster showing the scRNA-seq results of PFC cells. B. Distribution of 3 groups of sample cells on the UMAP plot. C. Bar graphs of the percentages of neuron subdivisions in 9 samples. D. Clustering visualization of RNA velocity analysis results of PFC neurons. E. SCENIC analysis. F. Bubble map of sorting gene markers in PFC neurons. Bubble size represents the percentage of cells with the gene expressed in the population, and bubble colour represents the average value of the gene expressed in the population. G. CytoTRACE score. The color represents the score, and the higher the score, the higher the cell stemness. H. CytoTRACE box plot.

Fig 6 PFC neuron profiles, type distribution and stemness analysis of mice in 3 groups

图7 七氟烷麻醉后新生小鼠PFC神经元谱系细胞拟时序分析Note： A. Cell trajectory skeleton plots. B. Density profiles. The horizontal axis represents the proposed time, and the vertical axis represents the density of cell distribution. C. Boxplot of the pseudotime of PFC neuron development in 3 groups. ①P=0.000.

Fig 7 Pseudo‐time analysis of PFC neuronal lineage cells in neonatal mice after sevoflurane anesthesia

图8 七氟烷麻醉后新生小鼠PFC神经元谱系细胞BEAM分析热图Note： Left: heat map of top 1 000 genes that determine cell fate. Middle: top 15 enrichment entries of the 1 000 genes that determine cell fate in the GO-BP database. Right: heat map of Beam analysis of genes with significant differences in PFC neurons after sevoflurane anesthesia. The horizontal axis represents the proposed time, and the vertical axis represents the clustering of genes. The early developmental cluster included PIPC1?3, the mid-developmental cluster included IPC1?2, and the late developmental cluster included CRC, NRGN+ IN, and PyN.

Fig 8 BEAM analysis heat map of PFC neuronal lineage cells in neonatal mice after sevoflurane anesthesia

图9 新生小鼠七氟烷麻醉后PFC神经元的转录组变化和功能注释分析Note：A. Volcano map showing differentially expressed genes between Sev1 group and Ctrl group. B/C. Top 15 enrichment entries of down-regulated genes (B) and up-regulated genes (C) in the GO-BP database between Sev1 group and Ctrl group. D. Volcano map showing differentially expressed genes between Sev3 group and Ctrl group. E/F. Top 15 enrichment entries of down-regulated genes (E) and up-regulated genes (F) in the GO-BP database between Sev3 group and Ctrl group. Entries highlighted in red represent biological processes associated with PFC neurogenesis.

Fig 9 Analysis of transcriptome changes and functional annotation of PFC neurons in neonatal mice after sevoflurane anesthesia

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