| Objective: To observe the effect of dexmedetomidine pretreatment on propofol-induced neurotoxicity in the developing brain in rat,and to explore the role of PI3K/Akt/GSK-3? signaling pathway in the neuroprotection of dexmedetomidine.Methods: Two hundred and fifty seven-day-old(P7)neonatal SpragueDawley rats were randomly divided into 10 groups(n=10,each): group N,group F,group D1,group D2,group P,group PD25,group PD50,group PD75,group LYPD and group TDPD.Rats were intraperitoneally injected(i.p.)with saline,intralipid,or 10% dimethyl sulfoxide(DMSO)in 100 ?l in group N,group F,or group D2,respectively.And 5 ?l 10% DMSO was injected intracerebroventricularly(i.c.v.)in group D1.In group P,rats were intraperitoneally injected with 100mg/kg propofol in the absence of dexmedetomidine pretreatment.In group PD25,group PD50,or group PD75,25,50 or 75 ?g/kg dexmedetomidine were administered by intraperitoneal injection respectively 30 min before the exposure to 100mg/kg propofol.In group LYPD or group TDPD,rats were treated with the PI3 K inhibitor LY294002(25 ?g/?l,i.c.v.)or the GSK-3? inhibitor TDZD-8(1 mg/kg i.p.)respectively 30 min before pretreatment with 75 mg/kg dexmedetomidine and then propofol exposure.At 1 h after recovery from anesthesia,some rats were used for blood sampling from the left cardiac ventricle for blood gas(n=5).Some rats were sacrificed by decapitation and their hippocampi were used for Semi-quantitative reverse transcription-PCR and Western blot studies(n=10)at 1h after recovery from anesthesia.For immunofluorescence studies,some rats were perfused transcardially with 4% paraformaldehyde(n=5),and the hippocampi of the rest rats were isolated for transmission electron microscope(TEM)(n=5).The expression of PI3 K mRNA,Akt mRNA and GSK-3? mRNA in hippocampus was detected by Semi-quantitative reverse transcription-PCR.The protein levels of Akt,pAkt(ser473),GSK-3? and pGSK-3?(ser9)in hippocampus were detected by Western blot.The expression and cellular localization of pAkt(ser473)protein and pGSK-3?(ser9)protein in hippocampus was observed by immunofluorescence staining.And the ultrastructure of hippocampal neurons was examined by Transmission electron microscope.Results: Compared with group N,the level of GSK-3? mRNA was significantly increased in group P(P<0.05),while the level of GSK-3? mRNA in group TDPD,and the levels of PI3 K mRNA and Akt mRNA in group LYPD were decreased(P<0.05).Compared with group P,the levels of PI3 K mRNA and Akt mRNA were reduced in group LYPD,the level of GSK-3? mRNA was reduced in group PD75 and group TDPD.The expression of pAkt(ser473)and pGSK-3?(ser9)was down-regulated in group P,group PD25,group PD50,group PD75 and group LYPD as compared with group N(P<0.05).The expression of pAkt(ser473)in group PD75 and group TDPD,and the expression of pGSK-3?(ser9)in group PD25,group PD50,group PD75 and group TDPD was up-regulated as compared with group P(P<0.05).Compared with group PD75,the expression of pAkt(ser473)and pGSK-3?(ser9)in group LYPD was decreased,while the expression of pGSK-3?(ser9)in group TDPD was increased(P<0.05).The ratio of pAkt(ser473)/Akt in group P,group PD25,group PD50,group PD75,group LYPD and group TDPD,and the ratio of pGSK-3?(ser9)/GSK-3? in group P,group PD25,group PD50 and group LYPD was decreased as compared with group N(P<0.05).Compared with group P,the ratio of pAkt(ser473)/Akt in group PD75 and group TDPD,and the ratio of pGSK-3?(ser9)/ GSK-3? in group PD25,group PD50,group PD75,group LYPD and group TDPD was significantly increased(P<0.05).Besides,the ratio of pAkt(ser473)/Akt and the ratio of pGSK-3?(ser9)/ GSK-3? in group LYPD were lower,while the ratio of pAkt(ser473)/Akt and the ratio of pGSK-3?(ser9)/ GSK-3? in group TDPD were higher than those in group PD75(P<0.05).The results of immunofluorescence showed that pAkt(ser473)and pGSK-3?(ser9)were localized mainly in the cytoplasm.Compared with group N,the expression of pAkt(ser473)in group P,group PD25,group PD50 and group LYPD was downregulated(P<0.05).Compared with group P,the expression of pAkt(ser473)and pGSK-3?(ser9)in group TDPD,and the expression of pAkt(ser473)in group PD75 was up-regulated(P<0.05),while the expression of pGSK-3?(ser9)in group LYPD was down-regulated(P<0.05).The expression of pAkt(ser473)and pGSK-3?(ser9)in group LYPD was decreased(P<0.05),while the expression of pGSK-3?(ser9)in group TDPD was increased(P<0.05)as compared with group PD75.The ultrastructure of hippocampal neurons was normal in group N,group F,group D1 and group D2.While chromatin margination,pyknosis,mitochondrial vacuolar degeneration and synaptic damage were observed in group P.Ultrastructural damage in hippocampal neurons and synapsis in group PD25,group PD50,group PD75 and group TDPD was attenuated,while in group LYPD it was aggravated.Conclusion: Propofol could induce hippocampal neurons injury.Dexmedetomidine could attenuate propofol-induced neurotoxicity in the developing brain in rats,which is mediated by activation of PI3K/Akt/GSK-3? signaling pathway. |
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