The Mechanism Of Downregulating The Expression Of Myelin Basic Protein In Zebrafish Embryos Exposed To Propofol

In our country,with development of anesthetic technology and the promulgation of the"two child"policy,thousands of newborns,infants and even pregnant women need to undergo the necessary diagnostic tests,operation and analgesia.It was considered that general anesthesia is a reversible change in consciousness in the past.In recent decades,animal experiments and retrospective human studies have demonstrated that general anesthetic may have potential risks to the neurodevelopment of the fetus and children,which is a hot issue for more and more clinical anesthesiology workers,parents and society.Propofol,most commonly used intravenous anesthetics now,which has a rapid onset,fast recovery,no accumulation and fewer adverse reactions after continuous infusion,is widely used in various types of surgery.Animal experiments of propofol on neurotoxicity showed that it could induce increased apoptosis of neuronal,affected synaptogenesis and plasticity,and even cause short-term or long-term behavioral abnormalities.Neurons plays a major role in nervous system cells,whereas glial cells accounted for a total of 50%nerve cells,and it will take a higher proportion of nerve cells in higher species.Oligodendrocytes accounted for 70%of glial cells,with its main function in central nervous system,including formation of insulating myelin sheath around the axon and maintenance of normal function of neurons.Myelin sheath is an important structure to ensure fast,accurate and unidirectional transmission of neural signals.Myelin basic protein(MBP)is a marker protein of mature oligodendrocytes to play an important role in myelination as a major component of myelin sheath.It is regulated by many signals during oligodendrocyte maturation,such as transcription factors olig2,is mainly involved in development and differentiation of oligodendrocytes in early stage,while Oligl plays a role in the final stage,regulating myelin formation and myelin regeneration.Furthermore,both of them have a positive regulation of MBP promoter sequence.Recent animal experiments releaved that neonatal or fetal Macaca mulatta exposed to propofol for 5h could induce increased apoptosis of fetal brain neurons,and induce oligodendrocyte apoptosis in the initial stage of forming myelin sheath,in addition,the damage in fetal brain cells was more serious than it was in newborn.It is the initial period of all kinds of nerve cells and the key point for oligodendrocyte proliferation,differentiation and maturation in fetal phase.At this time,whether exposure to propofol will have adverse effects on the maturation and myelination of oligodendrocytes in fetal brain,is continued to be explored.Previously,we established the model of embryonic zebrafish exposed to propofol,found that propofol could lead to increased apoptosis of nerve cells in 3dpf and downregulate the expression of MBP in the early stage of formation.In this study,we will further observe the behavioural changes at different developmental stages and investigate the mechanism of downregulation of MBP induced by propofol exposure to embryonic zebrafish.Objective1.To observe the effects of zebrafish embryos exposed to propofol on MBP expression in larvae at different developmental stages.2.To observe behavioral changes of larvae at different developmental stages by exposure to propofol in zebrafish embryos.3.To explore the mechanism of downregulation of MBP expression caused by propofol exposure in zebrafish embryos.Materials and Methods1.Experimental animals and groups:The adult wild zebrafish strain,Tg(mbp:GFP)and Tg(olig2:DsRed2)transgenic lines,half male and half female,has a fish age of 6 month to 1 year.At 18:00 in the afternoon,adult male and female fish will be matched according to the proportion by 1:1 or 1:2.The next morning,collecting and placing embryo in an incubator at 28.5 after female fish lay their eggs.6h later,dead embryo wrer removed and the rest were randomly divided into six well plates,30 eggs per hole.The fish water containing 20?g/m and 30?g/ml propofol was added for 5 ml per hole,set up control group,DMSO group(0.014%)at the same time.At 24hpf,removing dead eggs and replacing each group by half solution.All groups were replaced with fresh fish water at 48hpf.0.003%PTU should be added to inhibit the pigments and keeplarvaetransparent for photograph.2.Detection of mRNA and protein expression of MBP and Olig2:Larvae were collected and extracted at different developmental stages.mRNA and protein expression of MBP and Olig2 were detected by real-time fluorescence quantitative PCR and Western blotting.3.Behavior test of larvae:The Noldus behavior tracking system and analysis software were used to derive the motion parameters of larvae to analyze the change of behavior4.TUNEL detection of oligodendrocyte apoptosis:Tg(mbp:GFP)transgenic larvae at different developmental stages were taken and made into frozen sections for TUNEL-double immunofluorescence staining to detect the apoptosis of oligodendrocyte.5.Detection of oligodendrocyte precursor cell apoptosis:Acridine orange vital staining on 48hpf Tg(olig2:DsRed2)transgenic larvae were carried out to detect apoptosis of oligodendrocyte precursor cells.6.EDU detection of oligodendrocyte precursor cells proliferation:EDU were added to label S-phase proliferating cells before collection.Frozen sections of Tg(olig2:DsRed2)transgenic larvae were made followed by immunofluorescence staining to detect proliferation of the oligodendrocyte precursor cells.7.Statistical analysis:Statistical analysis was carried out using SPSS 21.0 statistical software,and the measurement data were expressed in mean number of standard deviations(x±s)One-way ANOVA with LSD or Tamhane was performed for heterogeneity of variance;P<0.05 has statistical significance for differences.Graph Pad Prism 6 were used for plotting and image J for gray value analysis.Results1.MBP mRNA decreased in 3dpf?7dpf(P<0.05).Expression of MBP protein were significantly decreased in 3dpf?7dpf?10dpf,compared with control group(P<0.05).For Olig2 mRNA and protein expression,both of them downregulated in 48hpf(P<0.05).DMSO group had no statistical significance at all times compared with the control group(P>0.05).2.The activity and ability adapted to the environment of larvae in 5dpf were obviously weaken(P<0.05).The adaptation ability and response speed in 7dpf were decreased(P<0.05).There was no statistical difference in the results of 10dpf larvae(P>0.05).3.There were significantly higher apoptosis of oligodendrocytes in 3dpf for all experimental group and in 7dpf for higher concentration group(P<0.05).Apoptosis of oligodendrocyte precursor cells in 48hpf increased in a concentration dependent after exposure to propofol at embryonic stage(P<0.05).4.There were no significant changes in proliferation of oligodendrocyte precursor cells after exposure to propofol(P>0.05).Conclusion1.Zebrafish exposed to propofol at embryonic stage could downregulate the expression of MBP,which is sustainable to 10dpf.2.Zebrafish exposed to propofol at embryonic stage could lead to behavioral changes in 5dpf and 7dpf.3.The decrease of MBP expression in larvae after exposed to propofol at embryonic stage may be related to downregulation of transcription factor Olig2 and the increase of apoptosis in oligodendrocytes and their precursor cells.4.Zebrafish exposed to propofol at the embryonic stage does not inhibit the proliferation of oligodendrocyte precursor cells.