| Objective: To detect the changes of learning and memory ability at 2h,1w,and 4w after recovery from 3.2% sevoflurane inhalation in aging model rats through testing the cognitive function with Morris water maze test and beam balance test.Detecting the relative expression of α7nAChR,ChAT and AChE mRNA and protein content in hippocampus of aging model rats through RT-qPCR and Western blot,and to explore the relationship and mechanism of cognitive function changes and central cholinergic system at 2h,1w,and 4w after recovery from sevoflurane in an aging model rats.Methods: 72 healthy adult male SD rats were given subcutaneous injection of D-galactose on the neck for 6 weeks to establish elderly models.The model rats were randomly divided into four groups: control group(abbreviated as Con group,n=18),with the carrier gas(2L/min Air+2L/min O2)was given for 6h;experimental group(abbreviated as Sev group,n=18),with the volume fraction was 3.2% sevoflurane + carrier gas for 6h;α7nAChR antagonist group(abbreviated as Sev+M group,n=18): after 24 hours of injection of antagonist,the treatment was the same as the Sev group;α7nAChR agonist group(abbreviated as Sev+P group,n=18): after 24 hours of injection of agonist,the treatment was the same as the Sev group;The Morris water maze experiment was conducted for 5d before the inhalation anesthesia experiment for each group of rats.The escape latency period of the daily navigation experiment was recorded and analyzed statistically,so as to observe whether the cognitive functions of the rats in each group were different before the inhalation of sevoflurane.After inhalation anesthesia,the four groups of rats were randomly divided into three subgroups according to the time of collection: 2h after anesthesia recovery(acute phase of anesthesia recovery),1w(medium and long-term recovery of anesthesia),4w(long-term anesthesia recovery).The Morris water maze was applied to test the spatial exploration and working memory of rats.The experiment of balance beam walking was carried out on rats by means of balance beam walking device.In this way,the learning and memory ability of rats at each time was evaluated.After the end of each period of the ethological test,6 rats in each group were selected for sampling of hippocampal tissue,and the relative mRNA expressions of 7nAChR,ChAT and AChE in the right hippocampal region of the rats at each stage were detected by real-time quantitative PCR,while the relative protein expressions in the left hippocampal region of the rats at each stage were detected by Western Blot.Results: 1.Results of the Morris Water Maze Experiment(1)Adaptive training: all rats in each group had normal swimming ability,without down syndrome.(2)Escape latency experiment: The escape latency of the rats in each group showed a shortening trend.In the test on day 2,the escape latency of the rats in each group was significantly shorter than that of the group on day 1,with statistical significance(P<0.05).On day 3,4 and 5,the escape latency of the rats in each group also showed a gradually shortened trend and entered a relatively stable period.There was no significant difference between the intra-group comparison and the previous day,indicating that the rats in each group formed a stable memory of the escaping platform after 5 days of training.There was no significant difference between the two groups(P>0.05).The baseline of learning and memory ability was consistent in all groups.(3)Working memory test after anesthesia recovery: At 2h after recovery,the latency of working memory of rats in Sev group,Sev+M group and Sev+P group was significantly longer than that in Con group,with statistically significant difference(P<0.05).The latency of working memory in the Sev+P group was significantly shorter than that in the Sev group and the Sev+M group(P<0.05).After waking up for 1w,the latency of working memory of rats in Sev group and Sev+M group was significantly prolonged compared with Con group,with statistically significant difference(P<0.05).The rats in Sev+P group were significantly shorter than those in Sev group and Sev+M group,and the difference was statistically significant(P < 0.05).At 4w after recovery,there was no significant difference in the latency of working memory between the groups(P>0.05).(4)Spatial probe test after anesthesia recovery: 2h after recovery,the number of platform crossing,effective area crossing and platform quadrant crossing of rats in Sev group decreased significantly with the comparison to Con group,with statistically significant differences(P<0.05).The number of platform crossing and effective region crossing in Sev+M group decreased significantly with comparison to Con group,and the percentage of platform quadrant crossing times increased with comparison to other groups,with statistically significant difference(P<0.05).Compared with the Sev group and Sev+M group,the number of platform crossing and effective area crossing in the Sev+P group increased significantly(P<0.05).At 1w after waking up,the number of platform quadrant crossing times of rats in Sev group was significantly reduced with comparison to Con group,with a statistically significant difference(P<0.05).There was no significant difference between other indicators and other groups(P>0.05).There was no significant difference between Sev+M group and Con group(P>0.05).The effective region crossing times and platform quadrant crossing times of the Sev+P group and the Sev+M group increased significantly(P<0.05).At 4w after recovery,there was no significant difference in the indexes among the groups(P>0.05).2.Results of the Beam Balance Test(1)Adaptive training: all the rats in each group could pass the balance bar within a specified time.(2)Beam balance test: 2h after waking up,rats in Sev group and Sev+M group had significantly longer starting time of the balance bar than those in Con group and Sev+P group(P<0.05).After waking up for 1w,the rats in the Sev group had a shorter starting time than those in the Con group,with a statistically significant difference(P<0.05).The rats in the Sev+M group had a longer starting time than those in the other groups,and the difference was statistically significant(P<0.05).There was no significant difference between Sev+P group and Con group(P>0.05).At 4w after waking up,there was no significant difference between rats in Sev group and Con group and Sev+P group(P>0.05).The rats in Sev+M group started to move after crossing the bar and the other groups were all prolonged,and the difference was statistically significant(P<0.05).3.Results of Inter-Group Comparison of α7nAChR,ChAT and AChEmRNA Expression(1)2h after waking up,the expression levels of α7nAChR,ChAT,and AChE mRNA in hippocampus of Sev group rats were all down-regulated with comparison to those in Con group,and the difference was statistically significant(P<0.05);The expression level was down-regulated with comparison to Sev group rats,and AChE mRNA expression was up-regulated with comparison to Sev group,and the difference was statistically significant(P<0.05).The expression levels of α7nAchR and ChAT mRNA in hippocampus of Sev+P group rats were up-regulated with comparison to those in Sev group and Sev+M group,and the expression levels of AChE mRNA were down-regulated with comparison to Sev M group rats.(2)1w after waking up,the expressions of α7nAChR and ChAT mRNA in hippocampus of Sev group rats were down-regulated with comparison to those in Con group,and the expressions of AChE mRNA were up-regulated in rats of Con group,and the difference was statistically significant(P<0.05);α7nAChR,ChAT,AChE mRNA expressions in hippocampus of Sev+M group rats.Compared with Sev group,rats were up-regulated;α7nAChR mRNA expression in hippocampus of Sev+P group was up-regulated with comparison to Sev group and Sev+M group,while ChAT and AChE mRNA expression in hippocampus were down-regulated with comparison to Sev group,and the difference was statistical significance(P<0.05).(3)4w after waking up,the expression levels of α7nAChR,ChAT and AChE mRNA in hippocampus of Sev group rats were all up-regulated with comparison to Con group,and the difference was statistically significant(P<0.05).The expression of AChE mRNA in hippocampus of Sev+M group was lower than that of Sev group,but the expressions of α7nAChR and ChAT mRNA were up-regulated with comparison to Sev+P group.The difference was statistically significant(P<0.05).The expression of α7nAChR,ChAT,and AChE mRNA in hippocampus of Sev+P group rats was down-regulated with comparison to Sev group rats,and the difference was statistically significant(P<0.05).4.Results of Inter-Group Comparison of α7nAChR,ChAT and AChE Protein Relative Expression(1)2 hours after waking up,The contents of α7nAchR,ChAT and AChE in hippocampus of Sev group rats were down-regulated with comparison to those in Con group,and the difference was statistically significant(P<0.05).The α7nAChR protein content in hippocampus of Sev+P group rats was up-regulated with comparison to Sev group and Sev+M group rats,and the difference was statistically significant(P<0.05).(2)1w after waking up,the content of α7nAChR,ChAT and AChE in hippocampus of Sev group rats had no significant change with comparison to Con group,and the difference was not statistically significant(P>0.05).The α7nAChR protein content in hippocampus of Sev+M group rats was down-regulated with comparison to Sev group and Sev+P group rats,and the difference was statistically significant(P <0.05).(3)4w after waking up,There was no significant up-regulation or down-regulation of α7nAChR,ChAT,and AChE protein in the hippocampus of each group,and the difference was not statistically significant(P>0.05).Conclusion:1.Aging model rats were exposed to 3.2% sevoflurane,and cognitive dysfunction occurred in the acute stage of recovery after resuscitation and extended to the medium and long term.2.After inhalation of 3.2% sevoflurane in aged rats,the hippocampus ChAT,AChE,7nAChR mRNA and protein metabolism were disturbed in the acute stage of recovery.3.PNU,a 7NachR-specific agonist,can up-regulate mRNA and protein expression of 7nAChR in the hippocampus of rats and improve short-term cognitive dysfunction. |
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