Long-term Sevoflurane Exposure Reduces The Differentiation Potential And Hypoxia Tolerance Potential Of Neural Stem Cells

Objectives To study the effects of exposure to the same concentration of sevoflurane for different durations on neural stem cell activity,differentiation potential and hypoxia tolerance potential,and provide certain guidance for controlling the time of clinical anesthesia for infants and young children.Methods Primary cell extraction and culture technology was used to extract and culture primary NSC from C57/BL mice,and the neural stem cell marker Nestin was detected using Western Blot（WB）to determine whether it was NSC.The obtained cells were randomly divided into three groups:control group（group C）,4h group,and 8h group.The culture environment of group C was 21%O₂,5%CO₂ and balanced N₂ without inhalation of sevoflurane;the culture environment of group C was 21%O₂,5%CO₂,balanced N₂ and4.1%sevoflurane for 4 hours;the culture environment of 8h group It is 21%O₂,5%CO₂,balanced N₂and 4.1%sevoflurane for 8 hours.The cell spheroidization experiment and immunofluorescence（IF）are used to detect the differentiation potential of neural stem cells.The cell viability is detected by the CCK-8 experiment.The expression of related proteins is achieved by Western blotting.The cell cycle detection is by Realized by flow cytometry,the detection of apoptosis is carried out by TdT-mediated d UTP gap end labeling（TUNEL）.Enzyme-linked immunosorbent assay（ELISA）is used to detect the expression of lactate dehydrogenase（LDH）.The WB images obtained in the experiment are all quantified with image J,and data obtained from the experiment and treated with SPSS24.0 software Graph Pad Prism8.3 software.All experimental data are the results of four parallel experiments,expressed as mean±SD.Data were compared by t-test for independent samples.P<0.05 represents statistically significant difference;Comparative analysis of variance using one-way（ANOVA）among the three groups,and P<0.05represents statistically significant difference.Results 1 Compared with the control group,the neural stem cell spheroidization experiment results of the sevoflurane exposure group for 8 hours were significantly different,and it was statistically significant（P<0.05）.However,although the results of the neural stem cell spheroidization experiment of the sevoflurane exposure group for 4 hours were slightly different from those of the control group,they were not statistically significant（P<0.05）.And the results of western blotting also confirmed this result.CCK-8detects the activity of each group,there is no difference between group C and group 4h,but group C and group 8h are significantly different,with statistical significance（P<0.05）.2The results of cell cycle detection by flow cytometry showed that the number of cells in the G2 phase of the 8h group was increased compared with that of the control group,and the Western blot results showed that the expression of GFAP and NSE increased and the expression of Nestin decreased in the 8h group,which was statistically significant.（P<0.05）.3 IF results showed that Nestin expression in the 8h group decreased,and the expression of NSE and GFAP increased.4 Western blot results showed that the expression of hypoxia-related proteins in neural stem cells in the group exposed to sevoflurane for 8hours was reduced,and it was statistically significant（P<0.05）.The number of apoptosis in the 8h group increased,which was statistically significant（P<0.05）.The expression of LDH increased in the 8h group,which was statistically significant（P<0.05）.Conclusion Long-term exposure to sevoflurane（8 hours）will promote the premature differentiation of NSC,and reduce its pluripotency,reserve and hypoxia tolerance.From the perspective of central nervous system reserve cells,it reveals the children caused by long-term exposure to sevoflurane the root cause of damage to the nervous system.Figure 8;Table 8;Reference 142...