Effects Of Different Concentrations Of Isoflurane Anesthesia On Expression Of P-GSK-3β/β-catenin And Proliferation Of Neural Stem Cells In Hippocampus Of Developmental Rats

Background:Volatile anesthetics are widely used in the pediatric population because of their analgesic effects,good controllability,and maintain the steadiness of haemodynamics.Isoflurane is a volatile anesthetic usually used in clinical anesthesia.There is a great deal of retrospective clinical studies indicate that children who had been exposed to isoflurane in the first three years of life are more prone to learning disabilities when they mature.The hippocampal dentate gyrus plays an important role in spatial learning and memory processing,appropriate level of neurogenesis in the early postnatal days is the base of its functions.Recent rodent studies have demonstrated that isoflurane exposure may have neurotoxic effects on the developing brains,which could inhibit the proliferation of neuron stem cells,lead to neurodegeneration and impair the ability of spatial learning and memory processing related to hippocampus,the underlying mechanisms have not been clarified clearly.The canonical Wnt signaling pathway is an important pathway in the development of mammalian nervous system,which plays critical roles in maintaining normal neuron function,regulating the proliferation and transference of neural stem cells,as well as neuronal polarization and so on.The phosphorylation of glycogen synthase kinase-3 beta(P-GSK-3β)and P-catenin are the two most important elements in the canonical Wnt signaling pathway,any changes in expression levels or activity of P-GSK-3β/β-catenin could affect the expression of Wnt target genes,thus affecting the NSCs’ normal functions.Previous research has shown that the intravenous anesthetic ketamine induced neurotoxic effects on developing brains is associated with a temporal decrease in P-GSK-3β,if P-GSK-3β/β-catenin also play a role in the neurotoxic effects of isoflurane on developing brains has not been clearly studied yet.Objective:1.To investigate the effects of different concentrations of isoflurane on the expression of P-GSK-3β and β-catenin of neonatal rats.2,To investigate the effects of different concentrations of isoflurane on the proliferation of neural stem cell in hippocampal dentate gyrus of neonatal rats.Methods:Forty-eight post-natal day(P)7 Sprague-Dawley(SD)rats,weighing 15～20g,were randomly divided into five groups(n=12):control group(group C),0.5%isoflurane(group 10.5),1%isoflurane(group I1),1.5%isoflurane(group 11.5).Rats in group C inhaled 30%oxygen for 4h and four isoflurane groups were anesthetized with 0.5%,%,1.5%isoflurane in 30%oxygen for 4h respectively(group 10.5,11,11.5).Meanwhile,six rats were selected randomly from every gro 定容至 receive intraperitoneal injection of 5-bromodeoxyuridine(BrdU)200mg/kg just before isoflurane anesthesia to assess the progenitor proliferation of dentate gyrus.The rats were sacrificed at 6h after anesthesia and hippocampal tissues were collected.Immunohistochemical staining was used to detect the number of BrdU positive cells in dentate gyrus,Western blot analysis was used to detect the expression of β-catenin and the phosphorylation of GSK-3β(P-GSK-3β).Results:1.The expression of β-catenin,P-GSK-3β were measured by Western blotting.Compared with group C,the expression of β-catenin,P-GSK-3β were decreased in I1 and I1.5 groups(P<0.05).However there was no statistical difference(P>0.05)in 10.5 group(P<0.05).2.Proliferation of NSCs in hippocampal dentate gyrus of neonatal rats.The NSCs in hippocampus of developmental rats are mainly concentrated in the hilus and SGZ of the dentate gyrus,the number of BrdU positive cells were counted in these two areas.Compared with group C,the number of BrdU positive cells were decreased in I1 and 11.5 groups(P<0.05).However there was no statistical difference(P>0.05)in 10.5 group.Conclusion:In the present study,we compared the effects of different concentrations of isoflurane on the proliferation of NSCs in hippocampal dentate gyrus of neonatal rats and the expression ofβ-catenin and P-GSK-3β,we found that high concentrations of isoflurane(1%and 1.5%)can suppress the proliferation of NSCs in the dentate gyrus of neonatal rats,but the effect of low concentration(0.5%)is inapparent.The mechanism may be related to the inhibition of P-catenin expression and phosphorylation of GSK-3β.