The Effect And Mechanism Of Sevoflurane In Adult Neurogenesis

Backgroud and Study ObjectiveOne of the central dogmas of neuroscience,that the adult mammalian brain was unable to facilitate neurogenesis,and that nerve injury or damage was irreversible.In 1960s and 1970s,some researchers have pointed out that mammals may the ability of neurogenesis,however,these results were not accepted by the neuroscience community because there was insufficient evidence that the labeled cells were neurons.In recent years,studies have shown that the adult neuro system have the ability of neurogenesis.Neurogenesis is composed by neural stem cll proliferation, differentiation,maturation and integration.GABA signal pathway has a close relationship with neurogenesis,and it play a very important role in neural stem cell proliferation,survival,differentiation,etc.Sevoflurane,which is induced rapidly, comfortable smelling and easy to control the depth of anesthesia,is widely used in clinical.So it is widely used clinically.Now,the study on the role of it in neurogenesis was only observed in vivo.But,in vivo micro-environment,there may have some complex implications.The purpose of this study is through preparation of four-vessel occlusion rat model of global cerebral ischemia to observe the role of sevoflurane on neurogenesis;and through culturing the the rat adult neural stem cells(ANSCs) in vitro to observe whether sevoflurane has effect on it.We have chosen CREB as a candidate signaling molecule,which participate in the GABA and the Wnt signaling pathway simultaneously,to explore the potential molecular mechanisms of sevoflurane. MATERIALS AND METHODS1,Experimental groups50 SPF-grade SD male rats,weight 250±20g,were randomly divided into 5 groups,each 10.The first group is Four-vessel occlusion rat cerebral ischemia model group with sevoflurane(3.5ml/kg).The second group is Sham-operated group with sevoflurane(3.5ml/kg).The third group is Four-vessel occlusion rat cerebral ischemia model with sevoflurane(7ml/kg).The fourth group is Sham-operated group with sevoflurane(7ml/kg).And the last group is Four-vessel occlusion rat cerebral ischemia model with physiologic saline.Animals in each group were continuously administered before surgery for 7 days,intraperitoneal injection.And make the model at the first hour after the 7^th day administration.2.Experimental methods2.1 Preparation of four-vessel occlusion rat model of global cerebral ischemiaRats were fasted for 24h the day before the production model to ensure homogeneity of the blood glucose levels.Before surgery,10%chloral hydrate to rats by intraperitoneal injection(40mg/100g weight).Select 3 groups randomly to prepare models.The first day,to separate and expose artery,vertebral artery,and block vertebral artery.The next day,the carotid was occluded for 15 minutes to cause brain ischemia injury in rats.And then restore cerebral blood flow in rats,and form ischemia-reperfusion injury.Another 2 groups,which has not been dealt with,as a sham-operated group.2.2BrdU label and prepare hippocampus frozen sectionGive each group of rats by intraperitoneal injection of BrdU(100mg/kg),for 3 consecutive days after the surgical treatment.28 days later,rats were injected intraperitoneally 10%chloral hydrate(40mg/100g weight).Pruning the brain tissue and then 4%paraformaldehyde fixed for 4-6 hours,rinse 1-2 hours.Next,put the brain block into 30%sucrose PBS solution 2-3 days,until it sink to the bottom.Cut into thin slices,8mm.Affixed to the glass slide,dried at room temperature for 2 hours, bake 5 minutes.2.3 Immunohistochemical staining a.Slices in each group first 2N HC1 at 37℃treatment 30min,then 0.1M borate buffer(pH8.5) 10min;b.0.1M PBS(pH7.4) Rinse 3×10min;c.incubate at room temperature for 2h(0.3%Triton X-100,0.1%BSA and 2% normal goat serum dissolved in PBS);d.BrdU:anti-BrdU 4℃incubate 24h;NeuN:anti-NeuN 4℃incubate 24h;BrdU / NeuN double-labeled:At the same time,anti-BrdU and anti-Neun 4℃incubated 24h.e.0.1M PBS(pH7.4) rinse 3×10minf.BrdU:anti-anti-BrdU dark incubation at room temperature 2h;NeuN:anti-anti -NeuN dark incubated at room temperature 2h;BrdU / NeuN double-labeled:At the same time,anti- anti-BrdU and anti- anti-Neun dark incubation at room temperature 2h;g.0.1M PBS(pH7.4) rinse 3×10min;h.Scrub the slide film,with 24h air dryingi.Make it transparent with xylene,then mount it with neutral resin,dry it,and prepare for examinationeach group have set up not add antigen and double-antigen groups,as control.3 Adult neural stem cells cultivation and identificationAfter SCR022 recovered,make it suspended in culture medium containing 20ng/mL FGF-2,cultured in the culture plate containing L-ornithine,and change the fluid every day.Immunocytochemical identification methods are as follows:remove the Culture medium,cultured cells fixed with pre-cooling 4%formaldehyde PBS.To seal by 5%BSA containing 0.3%Triton,for 60 minutes at 25℃.Incubated overnight with anti- Nestin,then anti-anti TRITC,DAPI-stained nuclei.To observe under fluorescence microscope,the red fluorescence-positive cell is Nestin-positive adult neural stem cells,blue for DAPI staining of nuclei.4.Experimental methods4.1 Treat neural stem cells with Sevoflurane Cells were kept in purpose-built airtight,temperature-controlled,cell-culture chambers.Preconditioning conditions were achieved with the desired concentration of sevoflurane 1.3%,1.9%,2.7%,3.3%(containers also containing 5%CO2,20%O2, and at an atmospheric pressure using nitrogen balance).1.3%for the low concentration group,1.9%and 2.7%for concentration,3.3%for the high concentration group,sevoflurane does not contain as a blank control group.Incubate cells for 2h.4.2 Neural stem cell staining and cell countingAfter Cells fixed,dye with DAPI for 15 minutes.For cell counting,each group counts 4 hole,each hole were randomly selected four horizons,to sum as the the total number of pore cells.4 hole cell count sum,the average is the Group's total number of cells.And the cells of blank group were standardized to 100.4.3 Western blotExpression levels of CREB and pCREB were measured by Western blot.The cell is divided into three groups:blank control group(without any treatment),low concentration of sevoflurane group(1.3%sevoflurane treatment) and in the concentration of sevoflurane group(1.9%).Precooling PBS,containing 1mM Na3VO4 wash the cells 2 times,and then spallate cells.Collect the cell lysate,after SDS-PAGE electrophoresis separation,transfer on PVDF membrane,closed by 5% skimmed milk.Incubate PVDF membrane in 3%BSA containing anti-CREB or anti-pCREB at 4℃overnight.And then add two anti-and chemiluminescent reagents Separately,to obtain images.Observe the gray ratio of pCREB and CREB,judge the level of CREB phosphorylation.Blank control group,the ratio is standardized to 100, so the other group.5.Statistical methodsData in each group use SPSS 13.0 statistical software to analysis,all measurement are expressed as mean±standard deviation.After homogeneity of variance test(P＞0.05), using one-way ANOVA statistical method,P＜0.05 was considered significant difference.Multiple comparisons of each group mean are using LSD method (Least-significant difference test). RESULTS1.The impact of sevoflurane on dentate gyrus neurons proliferationThe variance between group was analyzed by LSD.There are significant differences between Ischemic group with sevoflurane(3.5ml/kg) and sham-operated group,Ischemic group and saline control group.So are Ischemic group with sevoflurane(7ml/kg).Significant difference exist between Sevoflurane 3.5ml/kg and 7ml/kg ischemic groups.There is no statistical difference between Two sham-operated group and saline control group.2.Culture in vitro and identification of adult rat hippocampal neural stem cellsIn vitro culture of adult neural stem cells expressed Nestin protein,the positive rate was 99%.3.Sevoflurane to promote proliferation of adult neural stem cellsLow concentration sevoflurane group(1.3%) do not promote proliferation ANSCs, the concentration group(1.9%and 2.7%) compared with control group can promote ANSCs proliferation obviously(P＜0.05).High concentration group(3.3%) compared with the concentration of group ANSCs can also promote the proliferation significantly(P＜0.05).This shows that Sevoflurane promoted the proliferation of ANSCs in a dose-dependent manner.4.Sevoflurane increase the level of CREB phosphorylationIn order to identify the role of sevoflurane in promoting ANSCs proliferation, Westernblot was exploited to detect the phosphorylation level of CREB and pCREB. Medium concentrations of sevoflurane significantly increase the ratio of p-CREB/CREB Medium ANSCs.CONCLUSION1.Given sevoflurane(3.5ml/kg and 7ml/kg) to four-vessel occlusion rat model of global cerebral ischemia by intraperitoneal injection,the number of rat dentate gyrus newborn neurons could be affeced.This showed that sevoflurane can facilitate nerve regeneration,moreover,the promotion may have a dose-dependent manner.In this study,sevoflurane had no significant effect on nerve regeneration of sham-operated group adult rats.This is probably because sevoflurane need certain stimulation to promote adult nerve regeneration2.In vitro experiments of this study,we found that sevoflurane at concentrations (1.9%and 2.7%) and high concentration(3.3%) can promote the proliferation ANSCs; and at blank control group and low dose(1.3%) had no significant effect.This proves that sevoflurane can promote the proliferation of ANSCs.3.CREB phosphorylation and ANSCs proliferation has always been closely related. The results of this study show that sevoflurane probably promote ANSCs proliferation by changing the activity of CREB.