The Effect Of Midazolam On The Proliferation Of Neural Stem Cells And The Preliminary Exploration Of Underlying Mechanism

Part1The Effect of Midazolam on the Proliferation of NeuralStem Cells and the Preliminary Exploration of UnderlyingMechanism (in-vitro experiments)Objective To investigate the effect of midazolam on the proliferation of neuralstem cells (NSCs) isolated from the hippocampus of rats and the underlyingmechanism. Methods We firstly isolated neural stem cells from Sprague-Dawleyrats within12hours of birth and then cultured them in Dulbecco's Modified EagleMedium: Nutrient Mixture F-12(DMEM/F-12) media supplemented withmitogen and B27supplement. Immunocytochemistry was used to identify theexpression of nestin, a marker of neural stem cells. Two classic experiments—cellcounting kit8(CCK8) and5-bromo-2'-deoxyuridine(BrdU)incorporation testwere used to test the effect of midazolam on the proliferation of NSCs. Cell cycledistribution analysis was also used to analyse the percentage of cells in the Sphase which had a positive correlation with proliferation activity. We also appliedGABA_Areceptor antagonist bicuculline and Na-K-2Cl cotransport blockerfurosemide in our experimrnts to investigate the mechanism of the effect inducedby midazolam. Calcium sensitive fluorescent dye Fluo3-AM was used to tracethe calcium influx induced by midazolam. Western blot was also used to identifythe expression pattern of phosphorylated cAMP response element binding protein(pCREB), a crucial transcription factor for neurogenesis in response to midazolam.Results The majority (>70-80%)of cells raised from this method expressed nestin. In the CCK8and BrdU incorporation test, the proliferation activity was reduced inresponse to90μM midazolam (ultimate concentration) and these effects werepartly or entirely rescued by the pretreatment with GABA_Areceptor antagonistbicuculline and Na-K-2Cl cotransport blocker furosemide. The intracellularcalcium concentration was elevated in response to the administration ofmidazolam (30-90μM) and this effect was completely blocked by the pretreatmentwith GABA_Areceptor antagonist bicuculline. The expression of pCREB of NSCscultured in pro-differentiation culture media was enhanced by continuousinfluence by30μM midazolam. Conclusion Midazolam decreased theproliferation of NSCs isolated from hippocampus of rats and this effect was atleast partly induced by GABA_Areceptor. The enhanced expression of pCREB inNSCs might be triggered by the calcium influx induced by midazolam. Part2The Effects of Midazolam on the Proliferation of NSCsLocated in the Hippocampus of Mice and the HippocampalDependent Spatial Memory (in-vivo experiments)Objective To investigate the effects of midazolam on the proliferation of NSCslocated in the hippocampus of mice and the hippocampal-dependent spatialmemory in vivo. Methods3-4weeks old mice were randomly devided into twogroups—midazolam group (mid group) and normal saline group (veh group). Weused BrdU to label the cells in S phase in vivo and immumohistochemistry(floating frozen-section) was also used to display these cells. Morris water mazewas used to test the effect of midazolam on the the hippocampal dependent spatialmemory of mice. Results3times administration of midazolam (20mg/kg, with2hinterval) decreased the cells incorporatin BrdU in mice dentate gyrus. However,we didn't detect any statistical significance between the two groups in the performance of Morris water maze with respect to escape latency, residence timeand crossing frequency. Conclusion Midazolam limitedly influence theneurogenesis of mice hippocampus or has different effects on the different phaseof NSCs. Further researches are required before we get a systemic evaluation onthe effect of midazolam on the neurogenesis of hippocampus. Part3The Effect of Phosphorylated CREB on the Expressionof Neurogenesis Related MicroRNAsObjective To investigate the relationship of expression of phosphorylated CREBand neurogenesis related microRNA and further explore the mechanism throughwhich pCREB regulate the expression of miR-124. Methods Firstly, western blotwas used to identify the different expression of pCREB in the NSCs cultured intwo different media. Special real-time quantitative PCR to microRNA was used toquanify the expression of3neurogenesis related microRNAs (miR-124, miR-9,let-7b) in the NSCs cultured in two different media. Then, bioinformatics wasused to predict the potential binding sites in the upstream loci of primarymicroRNA-124. Then, chromatin immunoprecipitation (ChIP) was used toidentify these potential binding sites in the promoters. Results When cultured inpro-differentiation media, the NSCs significantly expressed higher pCREB andneurogenesis related microRNAs (miR-124, miR-9, let-7b). Bioinformaticsindicated that there were potential binding sites in upstream loci of primarymicroRNA-124(pri-mir-124-2and pri-mir-124-3). Chromatin immuno-precipitation identified the same results with those predicted.Conclusion In theearly phase of NSCs differentiation, the enhanced expression of pCREB promotesthe differentiation of NSCs and may be the inducement of many neurogenesisrelated microRNAs. CREB binds constitutively to the upstream loci ofpri-mir-124-2and pri-mir-124-3and the phosphorylation of CREB may promotethe transcription of these two primary microRNAs. Conclusion190μM Midazolam decreased the proliferation of NSCs isolated fromhippocampus of rats and this effect was at least partly induced by GABAAreceptor.2The enhanced expression of pCREB in NSCs might be triggered by thecalcium influx induced by midazolam.320mg/kg (repeatedly administered for3times with2hours interval)midazolam limitedly influence the neurogenesis of mice hippocampus or hasdifferent effects on the different phase of NSCs. Further researches are requiredbefore we get a systemic evaluation on the effect of midazolam on theneurogenesis of hippocampus.4CREB constitutively binds to the upstream loci of pri-mir-124-2andpri-mir-124-3and the phosphorylation of CREB may promote the transcription ofthese two primary microRNAs.