| Objective To investigate the effects of different concentrations of sevoflurane on the survival, proliferation and differentiation of rat fetal neural stem cells and the preliminary mechanisms, thus provide some guidance for the clinical selection and management of sevoflurane.Methods Rat fetal neural stem cells(FNSC) isolated from the cortexes of the fetal(embryonic day 14) Sprague-Dawley rats were bought from Invitrogen and cultured in the neural stem cells complete serum free medium(SFM). To keep the condition consistent, all experiments were carried out on cells between passages of 2 and 4.Approximately 90% of the cells were stained positive by the undifferentiated FNSC markers, Nestin and Sox2, by immunofluorescence at passage 4.1. Rat fetal neural stem cells were maintained as an monolayer and adherent culture and then randomly divided into 4 groups: control group(group C) which inhaled 5% carbon dioxied with humidified air; low concentration of sevoflurane group( group SL which inhaled 1.2% sevoflurane or 0.5 MAC sevoflurane); middle concentration of sevoflurane group( group SM which inhaled 2.4% sevoflurane or 1MAC sevoflurane);a higher concentration of sevoflurane group( group SH which inhaled 4.8% sevoflurane or 2MAC sevoflurane). After exposured to sevoflurane for 6 hours, 12 hours or 24 hours,respectively,the number of viable cells were evaluated by 3-(4, 5-dimethyithiazol-2-yl)-2, 5-diphenyl-tetra- zolium bromide(MTT) reduction assay.2. Firstly use Annexinn V / PI flow cytometry to detect the effects of different durations of group SH on rat fetal neural stem cells’ apoptosis,and then western blotting to measure the apoptosis execution protein cleaved caspase-3 and apoptosis-related protein bcl-2 and bax levels, to further clarify the mechanism of sevoflurane induced apoptosis.3. Neural stem cells were exposed to different concentrations of sevoflurane for 6 hours.Cell proliferation was assessed immunocytochemically using 5-ethynyl-2-deoxyuridine(Ed U) incorporation immediately end the anesthesia and differentiation by Sox2 staining 24 hours later.4. Western blotting assay was used to detect p-JNK and JNK levels after different durations of group SH,to find out the possible mechanism of sevoflurane on the neural stem cells’ survival,proliferation and differentiation.Results 1.There were no statistics differences in cell viability between group C and group SL at any durations(P>0.05),however,group SM had a significant differences compared with group C at 24 hours(P<0.0001).Group SH could time dependently reduce the number of viable cells(P<0.05).2. Compared with group C, group SH treated for 12 hours and 24 hours led to a time-dependent increase of apoptotic cells, increased levels of cleaved caspase-3 at 12 hours and decreased bcl-2 / bax level at 12 hours and 24 hours with statistically significance(P<0.05).3. Compared with group C, group SH had decreased cell proliferation indicated by Ed U incorporation assay, and increased cell differentiation by Sox2 staining(P<0.0001),whereas group SL and group SM had no difference(P>0.05).4. Compared with group C, group SH could time dependently induce increased expression of p-JNK and p-JNK / JNK ratio, but had no significant effect on the level of total protein JNK(P<0.05).Conclusion High concentrations of sevoflurane can induce FNSC apoptosis with time dependently and also can inhibit cells proliferation and induce its spontaneous differentiation, however low concentrations don’t have this effect. On the other hand,sevoflurane can only decreased cell viability with concentration dependently. The effects of sevoflurane on FNSC viability, proliferation and differentiation might related to activate the JNK pathway. |
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