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

doi:10.3969/j.issn.1674-8115.2023.09.006

Abstract

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)

CLC Number:

R614.2

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.

Figures/Tables 9

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

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

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

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

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

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

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

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

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

TrendMD

References 50

1	SHI Y W, WANG G, LI J Y, et al. Hydrogen gas attenuates sevoflurane neurotoxicity through inhibiting nuclear factor κ-light-chain-enhancer of activated B cells signaling and proinflammatory cytokine release in neonatal rats[J]. Neuroreport, 2017, 28(17): 1170-1175.
2	TIAN Y, CHEN K Y, LIU L D, et al. Sevoflurane exacerbates cognitive impairment induced by Aβ_1-40 in rats through initiating neurotoxicity, neuroinflammation, and neuronal apoptosis in rat hippocampus[J]. Mediators Inflamm, 2018, 2018: 3802324.
3	WU L Z, ZHAO H L, WENG H, et al. Lasting effects of general anesthetics on the brain in the young and elderly: mixed picture of neurotoxicity, neuroprotection and cognitive impairment[J]. J Anesth, 2019, 33(2): 321-335.
4	ZHAO S, FAN Z Q, HU J, et al. The differential effects of isoflurane and sevoflurane on neonatal mice[J]. Sci Rep, 2020, 10(1): 19345.
5	OLUTOYE O A, BAKER B W, BELFORT M A, et al. Food and Drug Administration warning on anesthesia and brain development: implications for obstetric and fetal surgery[J]. Am J Obstet Gynecol, 2018, 218(1): 98-102.
6	CHEN Q C, CHU W, SHENG R, et al. Maternal anesthesia with sevoflurane during the mid-gestation induces social interaction deficits in offspring C57BL/6 mice[J]. Biochem Biophys Res Commun, 2021, 553: 65-71.
7	FANG F, SONG R X, LING X M, et al. Multiple sevoflurane anesthesia in pregnant mice inhibits neurogenesis of fetal hippocampus via repressing transcription factor PAX6[J]. Life Sci, 2017, 175: 16-22.
8	LIANG L R, ZENG T, ZHAO Y Y, et al. Melatonin pretreatment alleviates the long-term synaptic toxicity and dysmyelination induced by neonatal sevoflurane exposure via MT1 receptor-mediated Wnt signaling modulation[J]. J Pineal Res, 2021, 71(4): e12771.
9	RAPER J, DE BIASIO J C, MURPHY K L, et al. Persistent alteration in behavioural reactivity to a mild social stressor in rhesus monkeys repeatedly exposed to sevoflurane in infancy[J]. Br J Anaesth, 2018, 120(4): 761-767.
10	YIN J, ZHAO X, WANG L J, et al. Sevoflurane-induced inflammation development: involvement of cholinergic anti-inflammatory pathway[J]. Behav Pharmacol, 2019, 30(8): 730-737.
11	SUN M Y, XIE Z C, ZHANG J Q, et al. Mechanistic insight into sevoflurane-associated developmental neurotoxicity[J]. Cell Biol Toxicol, 2022, 38(6): 927-943.
12	XIE L H, LIU Y, HU Y H, et al. Neonatal sevoflurane exposure induces impulsive behavioral deficit through disrupting excitatory neurons in the medial prefrontal cortex in mice[J]. Transl Psychiatry, 2020, 10(1): 202.
13	XU X Y, TIAN X, WANG G L. Sevoflurane reduced functional connectivity of excitatory neurons in prefrontal cortex during working memory performance of aged rats[J]. Biomed Pharmacother, 2018, 106: 1258-1266.
14	ZHAO T Y, CHEN Y X, SUN Z X, et al. Prenatal sevoflurane exposure causes neuronal excitatory/inhibitory imbalance in the prefrontal cortex and neurofunctional abnormality in rats[J]. Neurobiol Dis, 2020, 146: 105121.
15	ZHANG L, CHENG Y Y, XUE Z Y, et al. Sevoflurane impairs m⁶A-mediated mRNA translation and leads to fine motor and cognitive deficits[J]. Cell Biol Toxicol, 2022, 38(2): 347-369.
16	JIANG J L, LI S S, WANG Y Q, et al. Potential neurotoxicity of prenatal exposure to sevoflurane on offspring: metabolomics investigation on neurodevelopment and underlying mechanism[J]. Int J Dev Neurosci, 2017, 62: 46-53.
17	WANG C Y, LIU F, FRISCH-DAIELLO J L, et al. Lipidomics reveals a systemic energy deficient state that precedes neurotoxicity in neonatal monkeys after sevoflurane exposure[J]. Anal Chimica Acta, 2018, 1037: 87-96.
18	CHENG Y Y, LIU S Y, ZHANG L, et al. Identification of prefrontal cortex and amygdala expressed genes associated with sevoflurane anesthesia on non-human primate[J]. Front Integr Neurosci, 2022, 16: 857349.
19	XU G, LU H, DONG Y, et al. Coenzyme Q₁₀ reduces sevoflurane-induced cognitive deficiency in young mice[J]. Br J Anaesth, 2017, 119(3): 481-491.
20	ZHANG J, DONG Y L, ZHOU C, et al. Anesthetic sevoflurane reduces levels of hippocalcin and postsynaptic density protein 95[J]. Mol Neurobiol, 2015, 51(3): 853-863.
21	ZHANG L, XUE Z Y, LIU Q D, et al. Disrupted folate metabolism with anesthesia leads to myelination deficits mediated by epigenetic regulation of ERMN[J]. EBioMedicine, 2019, 43: 473-486.
22	CHEN S F, ZHOU Y Q, CHEN Y R, et al. Fastp: an ultra-fast all-in-one FASTQ preprocessor[J]. Bioinformatics, 2018, 34(17): i884-i890.
23	LIM L, MI D, LLORCA A, et al. Development and functional diversification of cortical interneurons[J]. Neuron, 2018, 100(2): 294-313.
24	HAMMOND T R, DUFORT C, DISSING-OLESEN L, et al. Single-cell RNA sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes[J]. Immunity, 2019, 50(1): 253-271.e6.
25	JOGLEKAR A, PRJIBELSKI A, MAHFOUZ A, et al. A spatially resolved brain region- and cell type-specific isoform atlas of the postnatal mouse brain[J]. Nat Commun, 2021, 12(1): 463.
26	LI X S, LIU G P, YANG L, et al. Decoding cortical glial cell development[J]. Neurosci Bull, 2021, 37(4): 440-460.
27	MARQUES S, ZEISEL A, CODELUPPI S, et al. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system[J]. Science, 2016, 352(6291): 1326-1329.
28	WOLF F A, ANGERER P, THEIS F J. SCANPY: large-scale single-cell gene expression data analysis[J]. Genome Biol, 2018, 19(1): 15.
29	AIBAR S, GONZÁLEZ-BLAS C B, MOERMAN T, et al. SCENIC: single-cell regulatory network inference and clustering[J]. Nat Methods, 2017, 14(11): 1083-1086.
30	ASHBURNER M, BALL C A, BLAKE J A, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium[J]. Nat Genet, 2000, 25(1): 25-29.
31	DRAGHICI S, KHATRI P, TARCA A L, et al. A systems biology approach for pathway level analysis[J]. Genome Res, 2007, 17(10): 1537-1545.
32	YAARI G, BOLEN C R, THAKAR J, et al. Quantitative set analysis for gene expression: a method to quantify gene set differential expression including gene-gene correlations[J]. Nucleic Acids Res, 2013, 41(18): e170.
33	BUTT S J B, SOUSA V H, FUCCILLO M V, et al. The requirement of Nkx2-1 in the temporal specification of cortical interneuron subtypes[J]. Neuron, 2008, 59(5): 722-732.
34	GRATTON M O, TORBAN E, JASMIN S B, et al. Hes6 promotes cortical neurogenesis and inhibits Hes1 transcription repression activity by multiple mechanisms[J]. Mol Cell Biol, 2003, 23(19): 6922-6935.
35	EUN B, CHO B, MOON Y, et al. Induction of neuronal apoptosis by expression of Hes6 via p53-dependent pathway[J]. Brain Res, 2010, 1313: 1-8.
36	KOLDAMOVA R, SCHUG J, LEFTEROVA M, et al. Genome-wide approaches reveal EGR1-controlled regulatory networks associated with neurodegeneration[J]. Neurobiol Dis, 2014, 63: 107-114.
37	WANG C, QIN L N, MIN Z Q, et al. SOX7 interferes with β-catenin activity to promote neuronal apoptosis[J]. Eur J Neurosci, 2015, 41(11): 1430-1437.
38	CHENG B H, CHEN J, BAI B, et al. Neuroprotection of apelin and its signaling pathway[J]. Peptides, 2012, 37(1): 171-173.
39	ZHANG N, SU Q P, ZHANG W X, et al. Neuroprotection of dexmedetomidine against propofol-induced neuroapoptosis partly mediated by PI3K/Akt pathway in hippocampal neurons of fetal rat[J]. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 2017, 18(9): 789-796.
40	XUE H, XU Y, WANG S, et al. Sevoflurane post-conditioning alleviates neonatal rat hypoxic-ischemic cerebral injury via Ezh2-regulated autophagy[J]. Drug Des Devel Ther, 2019, 13: 1691-1706.
41	YANG Q Z, YAN W J, LI X, et al. Activation of canonical Notch signaling pathway is involved in the ischemic tolerance induced by sevoflurane preconditioning in mice[J]. Anesthesiology, 2012, 117(5): 996-1005.
42	ZHANG Y H, GAO Q S, WU Z Y, et al. Sevoflurane postconditioning ameliorates neuronal migration disorder through Reelin/Dab1 and improves long-term cognition in neonatal rats after hypoxic-ischemic injury[J]. Neurotox Res, 2021, 39(5): 1524-1542.
43	CHAI D D, CHENG Y Y, SUN Y, et al. Multiple sevoflurane exposures during pregnancy inhibit neuronal migration by upregulating prostaglandin D2 synthase[J]. Int J Dev Neurosci, 2019, 78: 77-82.
44	WANG X, SHAN Y Y, TANG Z Y, et al. Neuroprotective effects of dexmedetomidine against isoflurane-induced neuronal injury via glutamate regulation in neonatal rats[J]. Drug Des Devel Ther, 2018, 13: 153-160.
45	CHLEILAT E, SKATULLA L, RAHHAL B, et al. TGF-β signaling regulates development of midbrain dopaminergic and hindbrain serotonergic neuron subgroups[J]. Neuroscience, 2018, 381: 124-137.
46	JIANG M, TANG T X, LIANG X Y, et al. Maternal sevoflurane exposure induces temporary defects in interkinetic nuclear migration of radial glial progenitors in the fetal cerebral cortex through the Notch signalling pathway[J]. Cell Prolif, 2021, 54(6): e13042.
47	POON C L, MITCHELL K A, KONDO S, et al. The Hippo pathway regulates neuroblasts and brain size in Drosophila melanogaster[J]. Curr Biol, 2016, 26(8): 1034-1042.
48	GALDERISI U, JORI F P, GIORDANO A. Cell cycle regulation and neural differentiation[J]. Oncogene, 2003, 22(33): 5208-5219.
49	LIU S W, FANG F, SONG R X, et al. Sevoflurane affects neurogenesis through cell cycle arrest via inhibiting Wnt/β-catenin signaling pathway in mouse neural stem cells[J]. Life Sci, 2018, 209: 34-42.
50	SONG S Y, PENG K, MENG X W, et al. Single-nucleus atlas of sevoflurane-induced hippocampal cell type-and sex-specific effects during development in mice[J]. Anesthesiology, 2023, 138(5): 477-495.

[1]	Jia-qiang LUO, Liang-yu ZHAO, Chen-cheng YAO, Zi-jue ZHU, Xiao-yu XING, Peng LI, Ru-hui TIAN, Hui-xing CHEN, Jie SUN, Zheng LI. Single-cell RNA sequencing reveals the spatio-temporal expression profile of SARS-CoV-2 related receptor in human and mouse testes [J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(4): 421-426.
[2]	NI Hong-wei, HE Guang-bao, GAO Hong-mei, ZHU Yi-jun, SHI Dong-ping, HANG Yan-nan. Study on risk factors analysis and prediction model of difficult airway [J]. , 2020, 40(3): 358-.
[3]	JIANG Yun-feng,CHENG Yan-yong,SUN Yu. Role of long non-coding RNAPeg13 in apoptosis of developing primary neurons following sevoflurane injury [J]. , 2019, 39(5): 469-.
[4]	LIU Pei-pei, ZHENG Ji-jian, BAI Jie, ZHANG Ma-zhong, SUN Ying. Clinical observation on perfusion index for detection of noxious stimuli in pediatric patients under#br# general anesthesia [J]. , 2018, 38(1): 67-.
[5]	ZHOU Guo-xia, PENG Li-chao. Effects of sevoflurane anesthesia on spatial recognition and expressions of pCreb and Egr1 in hippocampus tissues of aged mice [J]. , 2015, 35(8): 1125-.
[6]	ZHANG Yan, ZHAN Qiong-hui, CHEN Jue, et al. Effects of sevoflurane pretreatment on expression of HIF-2α for renal ischemia reperfusion injury [J]. , 2015, 35(12): 1800-.
[7]	HU Xiao, WEN Da-xiang, HANG Yan-nan. Effect of sevoflurane on neuromuscular blockade of cisatracurium in elderly [J]. , 2013, 33(3): 331-.
[8]	SUN Ying, ZHU Ming, ZHANG Jan-wei, et al. Effects of sevoflurane preconditioning and postconditioning on myocardial reperfusion injury under cardiopulmonary bypass in infants [J]. , 2011, 31(9): 1316-.
[9]	SHEN Yao-feng, WU Jing-xiang, XU Mei-ying. Effects of anesthesia with propofol and sevoflurane on postoperative cognitive function of elderly patients undergoing thoracic surgery [J]. , 2011, 31(3): 322-.
[10]	XU Min, LU Zhi-jun, ZHANG Fu-jun. Effects of sevoflurane on spatial learning and memory in mice [J]. , 2011, 31(2): 182-.
[11]	SUN Ying, XU Wen-yin, HU Jie, et al. Combination effect of tramadol and low dose propofol on emergence agitation in children receiving sevoflurane for adenotonsillectomy procedure [J]. , 2010, 30(1): 73-.
[12]	SUN Ying, XU Wen-yin, HU Jie, et al. Effects of diclofenac sodium suppositories on emergence agitation after sevoflurane maintenance in children undergoing adenotonsillectomy [J]. , 2009, 29(7): 842-.