Dexmedetomidine-mediated Wnt Pathway Inhibits Sevoflurane-induced Cognitive Impairment in Neonatal Rats

Abstract

Objective To explore the effect of dexmedetomidine(Dex)on sevoflurane-induced cognitive impairment in neonatal rats through Wnt signaling pathway. Methods Sixty 7-day-old SD rats were assigned into five groups:control group(without any intervention),Dex group(intraperitoneal injection of 25 μg/kg Dex),sevoflurane group(3% sevoflurane treatment for 4 hours),sevoflurane+Dex group(inhalation of 3% sevoflurane after injection of 25 μg/kg Dex for 4 hours),and sevoflurane+Dex+Wnt inhibitor group(Wnt inhibitor XAV393 and 25 μg/kg Dex were injected and 3% sevoflurane was inhaled for 4 hours).Three weeks later,Morris water maze was used to detect the cognitive function;TdT-mediated dUTP nick end labeling(TUNEL)staining was performed to detect the apoptosis of hippocampal neurons;neuronal nuclei (NeuN) staining was conducted to detect the survival of hippocampal neurons;Western blot was carried out to detect the expression of apoptosis-related proteins.The expression of the factors involved in Wnt/GSK-3β/β-catenin signaling pathway was detected by fluorescence quantitative polymerase chain reaction,and Western blot. Results Compared with the control group,there was no significant difference in the escape latency of Dex group(t=0.304,P=0.768);the escape latency in sevoflurane group(t=5.823,P=0.002),sevoflurane+Dex group(t=3.188,P=0.010),and sevoflurane+Dex+Wnt inhibitor group(t=5.784,P=0.002)was significantly prolonged.Compared with that in the sevoflurane group,the escape latency in sevoflurane+Dex group(t=3.646,P=0.005)was significantly shortened.Compared with that in sevoflurane+Dex group,the escape latency in sevoflurane+Dex+Wnt inhibitor group(t=3.296,P=0.008)was prolonged.Compared with that in the control group,the times of crossing platform in sevoflurane group(t=5.179, P=0.004),sevoflurane+Dex group(t=2.309,P=0.043),and sevoflurane+Dex+Wnt inhibitor group(t=3.871, P=0.003)decreased.Compared with that in sevoflurane group,the times of crossing platform in sevoflurane+Dex group(t=3.296,P=0.008)significantly increased.Compared with that in sevoflurane+Dex group,the times of crossing platform in sevoflurane+Dex+Wnt inhibitor group(t=2.361, P=0.041)reduced.Compared with the control group,there was no significant difference in the number of apoptotic cells in Dex group(t=1.920,P=0.127),and the number of apoptotic cells in sevoflurane group,sevoflurane+Dex group,and sevoflurane+Dex+Wnt inhibitor group increased by 16%(t=13.436,P=0.002),5%(t=7.752, P=0.001),and 11.5%(t=12.612,P=0.002),respectively.Compared with that in the sevoflurane group,the number of apoptotic cells in sevoflurane+Dex group and sevoflurane+Dex+Wnt inhibitor group decreased by 11%(t=8.521,P=0.002)and 5.5%(t=3.123,P=0.036),respectively.Compared with that in the sevoflurane+Dex group,the number of apoptotic cells in sevoflurane+Dex+Wnt inhibitor group increased by 6.5%(t=6.250,P=0.003).Compared with that in the control group,the number of positive cells in 0.15 mm2 area did not show significant difference in Dex group(t=0.898,P=0.136)and sevoflurane+Dex group(t=0.203,P=1.519),and that in sevoflurane group and sevoflurane+Dex+Wnt inhibitor group decreased by 31(t=4.702,P=0.009)and 26(t=3.948,P=0.014),respectively.Compared with that in sevoflurane group,the number of positive cells in sevoflurane+Dex group and sevoflurane+Dex+Wnt inhibitor group increased by 17(t=3.415,P=0.018)and 5(P=0.001),respectively.Compared with that in sevoflurane+Dex group,the number of positive cells in sevoflurane+Dex+Wnt inhibitor group decreased by 12(t=3.010,P=0.039).Western blot was used to detect the gray values of caspase-3,Bax,and Bcl-2 in the hippocampus,which showed no significant difference between Dex group and control group(t=0.612,P=0.573; t=1.225,P=0.288;t=0.961,P=0.391).Compared with that in the control group,the expression of Bax(t=13.440,P=0.002;t=8.520,P=0.001; t=13.320,P=0.002)and caspase-3(t=9.860,P=0.001;t=6.120,P=0.004;t=11.620,P=0.003)were up-regulated and that of Bcl-2(t=7.671,P=0.002;t=2.880,P=0.045;t=6.280,P=0.003)was down-regulated in sevoflurane group,sevoflurane+DEX group,and sevoflurane+Dex+Wnt inhibitor group.Compared with that in sevoflurane group,the expression of Bax(t=8.130,P=0.001)and caspase-3(t=7.120,P=0.002)was down-regulated and that of Bcl-2(t=6.201,P=0.003)was up-regulated in sevoflurane+Dex group.Compared with that in sevoflurane+Dex group,the expression of Bax(t=7.310,P=0.002)and caspase-3(t=7.750,P=0.002)were up-regulated and that of Bcl-2(t=4.206,P=0.013)was down-regulated in sevoflurane+Dex+Wnt inhibitor group.The mRNA expression levels of Wnt3a,GSK-3β,and β-catenin in hippocampus were detected by fluorescence quantitative polymerase chain reaction.The mRNA expression levels of Wnt3a,GSK-3β,and β-catenin in the control group did not differ significantly from those in Dex group(t=1.230,P=0.290;t=0.901,P=0.418;t=1.837,P=0.140);the mRNA expression levels of Wnt3a and β-catenin in the control group did not differ significantly from those in sevoflurane+Dex group(t=1.102,P=0.332;t=1.030,P=0.361).Compared with that in the control group,the protein expression of Wnt3a and β-catenin in sevoflurane group(t=5.790,P=0.004;t=7.130,P=0.002)and sevoflurane+Dex+Wnt inhibitor group(t=7.130,P=0.002; t=5.500,P=0.005)was down-regulated,and the GSK-3β expression was up-regulated in sevoflurane group(t=4.800,P=0.009),sevoflurane+Dex group(t=2.940,P=0.045),and sevoflurane+Dex+Wnt inhibitor group(t=3.100,P=0.042).Compared with that in sevoflurane group,the mRNA expression of Wnt3a(t=4.460,P=0.011)and β-catenin(t=6.390,P=0.003)was up-regulated while that of GSK-3β(t=4.160,P=0.004)was down-regulated in sevoflurane+Dex group.Compared with that in sevoflurane+Dex group,the mRNA expression of Wnt3a(t=5.730,P=0.005)and β-catenin(t=4.640,P=0.010)was down-regulated while that of GSK-3β(t=3.240,P=0.117)was up-regulated in sevoflurane+Dex+Wnt inhibitor group.Compared with that in the control group,the protein expression of Wnt3a and β-catenin in Dex group(t=0.735,P=0.503;t=0.245,P=0.819)and sevoflurane+Dex group(t=1.623,P=0.180;t=1.159,P=0.311)did not differ significantly,while that in sevoflurane group(t=7.280,P=0.002; t=5.640,P=0.005)and sevoflurane+Dex+Wnt inhibitor group(t=7.240,P=0.002;t=4.970,P=0.008)was down-regulated. Compared with that in sevoflurane group,the protein levels of Wnt3a(t=6.410,P=0.003)and β-catenin(t=4.640,P=0.015)were up-regulated in sevoflurane+Dex group.Compared with that in sevoflurane+Dex group,the protein expression of Wnt3a(t=6.360,P=0.003)and β-catenin(t=4.640,P=0.016)was down-regulated in sevoflurane+Dex+Wnt inhibitor group.Compared with that in the control group,the expression(gray value)of P(ser9)-GSK-3β/GSK-3β in sevoflurane group(t=11.280,P=0.002),sevoflurane+Dex group(t=7.080,P=0.002),and sevoflurane+Dex+Wnt inhibitor group(t=9.970,P=0.001)were down-regulated.Compared with that in the sevoflurane group,the expression of P(ser9)-GSK-3β/GSK-3β were up-regulated in sevoflurane+Dex group(t=8.310,P <0.001).Compared with that in sevoflurane+Dex group,the expression of P(ser9)-GSK-3β/GSK-3β in sevoflurane+Dex+Wnt inhibitor group was down-regulated(t=5.510,P=0.005). Conclusion Dex can mediate Wnt/GSK-3β/β-catenin signaling pathway to inhibit sevoflurane-induced cognitive impairment in neonatal rats.