| For a long time,cognitive dysfunction related to anesthesia surgery has attracted much attention.It has also been gradually recognized that sleep dysfunction is closely related to the body,especially to the damage of cognitive function.Postoperative cognitive dysfunction(POCD)is a multi-precipitating neurological complication after surgery,with the typical manifestation of decreased memory and concentration after surgery.Sleep deprivation or sleep disorder has been proved to be one of the factors affecting the postoperative cognitive function.One of the main effects of sleep deprivation on the brain is the development of memory deficits in hippocampus-dependent learning models.Sleep deprivation selectively impaired synaptic plasticity in the mouse hippocampus,which was dependent on 3’,5’-cyclic adenosine monophosphate and protein kinase A,decreased adenosine monophosphate signaling,and increased phosphodiesterase 4(an adenylate degrading enzyme)activity and protein level.Sevoflurane,a common inhaled anesthetic,is mainly used in the induction stage of pediatric anesthesia and general anesthesia,and the anesthesia induction and awakening time is short.However,studies have shown that patients with preoperative sleep deficiency will appear relatively more serious postoperative cognitive dysfunction after using sevoflurane.In the case of patients with chronic physical or psychological diseases,sleep deprivation may significantly weaken the related cognitive functions,mainly memory.Although sleep disorders have been found to affect postoperative cognitive function,the specific mechanism and how sleep affects cognitive function of patients after anesthesia are not clear.Therefore,the purpose of this study was to explore the mechanisms of different anesthesia methods on cognitive function and their interaction with sleep deprivation.PART 1:Sleep deprivation leads to cognitive dysfunction in ratsObjective:Sleep deprivation is one of the causes of long-term cognitive dysfunction,and its mechanism is still unclear.The purpose of this study was to discuss the effects of sleep deprivation on the expression levels of cognitive related proteins in the bilateral hippocampus of adult SD rats(8-12W).Method:A total of 12 male SD rats were randomly divided into Control and SD(sleep deprivation)groups,with six rats in each group.After one week’s adaptation in the standard laboratory environment,the rats in the experimental group were subjected to acute sleep deprivation for 24 hours using the modified horizontal table method.The rats in the control group were placed on a wide horizontal table to enable them to sleep under the identical environment.After 24 hours,the two groups of animals were acclimated to a dry and warm environment for 2 hours and then subjected to cognitive function tests,including the Y maze,sucrose preference test and Morris water maze test.After the behavioral experiment,the levels of Tau protein,its phosphorylation,and acetylation modification were measured in the bilateral cerebral hippocampus of two groups of rats.Results:Compared with the control group,the alternation coefficient and the number of entering the new arm in the Y maze test were decreased in the rats of the acute sleep deprivation group.2.2.The sucrose preference test showed that the sucrose intake in the sleep deprivation group was significantly reduced.3.In the Morris water maze test,the acute sleep deprivation group showed a prolonged escape latency and a decrease in the number of platform shuttles.4.Western Blot test showed that the expression level of Tau protein did not change with acute sleep deprivation.On the contrary,phosphorylated Tau(Ser404)showed a significant up-regulation after acute sleep deprivation,followed by a slight decrease in the expression level of acetylated Tau.Conclusion:Acute sleep deprivation induces long-term memory impairment,which may be related to the phosphorylation and acetylation of Tau protein.PART 2:The late effects of different anesthesia methods on cognitive impairment were not the sameObjective:Among the commonly used anesthesia methods,inhalation anesthesia represented by sevoflurane and intravenous anesthesia represented by propofol have no significant effect on cognitive function,but whether the two anesthesia methods generate new effects after acute sleep deprivation is currently unclear.The purpose of this experiment was to investigate the effects of sevoflurane and propofol anesthesia on the cognitive level of rats after acute sleep deprivation.Methods:Thirty-six male SD rats were randomly divided into six groups(n=6):Control group,acute sleep deprivation group(SD),sevoflurane anesthesia group(Sevo),propofol anesthesia group(Prop),sevoflurane anesthesia group after sleep deprivation(SD+Sevo)and propofol anesthesia group after sleep deprivation(SD+Prop).The experiment was started after one week adaptation to the standard laboratory environment.The rats in the sevoflurane anesthesia group were anesthetized by inhalation under the condition of 3%sevoflurane+60%O2 for two hours.In the propofol anesthesia group,propofol was injected at a uniform rate of 2ml/h through the tail vein for 1 h.In the compound group,acute sleep deprivation was performed before anesthesia.The rats in the experimental group were subjected to acute sleep deprivation for 24 hours using the modified horizontal table method,while those in the non-deprivation group were placed on a wide horizontal table to enable them to sleep under the same environment.After 24 hours,all the rabbits were removed from each group and acclimated to a dry and warm environment for 2 hours before cognitive function tests were performed,including the Y maze,sucrose preference test and Morris water maze test.After the behavioral experiment,bilateral cerebral hippocampus tissues of two groups of rats were selected and the levels of inflammatory factors TNF-α and IL-1 were detected using Elisa kit.Results:Compared with the control group,the cognitive level of rats in the sevoflurane anesthesia group was slightly damaged,while that of rats in the propofol anesthesia group was not significantly affected.2.Compared with the acute sleep deprivation group,sevoflurane anesthesia caused cognitive impairment similar to acute sleep deprivation.3.Compared with the acute sleep deprivation group,the sevoflurane anesthesia group after sleep deprivation showed significantly reduced alternation times and neoarm time in the Y maze,and the Morris water maze showed increased escape latency and decreased alternation times,as well as a significant decrease in sucrose intake.Elisa showed increased expression of the neuroinflammatory factors TNF-α and IL-1 in the hippocampus.4.Compared with the acute sleep deprivation group,the sleep deprivation and propofol anesthesia groups showed a surprising reversal of cognitive impairment.The Y maze,Morris water maze and sucrose preference experiments all showed that the cognitive level of rats in the SD+Prop group(including memory ability and long-term learning ability)was significantly improved compared with that in the simple sleep deprivation group.Elisa showed that the expression levels of neuroinflammatory factors in bilateral hippocampus were decreased.Conclusion:The cognitive function is slightly damaged by sevoflurane inhalation anesthesia.However,in the inhalation anesthesia after acute sleep deprivation,sevoflurane aggravates the cognitive dysfunction caused by sleep deprivation,and the mechanism is not clear.Also for acute sleep deprivation,propofol as a mainstream intravenous anesthetic showed a different reversal of cognitive impairment than sevoflurane.PART 3:Differentially expressed proteins were found in both acute sleep deprivation and anesthesiaObjective:Acute sleep deprivation and inhalation anesthesia can cause differential expression of a variety of related proteins in a series of pathways including cognitive function and hyperalgesia.When sleep deprivation and anesthesia successively occur,the differential expression of proteins is still unknown.We designed this part of the experiment to explore the differential expression of the same proteins involved in sleep deprivation and inhalation anesthesia.Methods:The data of datasets GSE34424 and GSE64617 were used to analyze differentially expressed genes using the method of generating trust.The analysis of differentially expressed genes was based on the limma[PMID:25605792]function package of R language(version3.5.2,the same below).In GSE34424,differentially expressed genes were screened based on the criteria of absolute value of log-transformed differential expression multiple(Log2FC)>1 and FDR≤0.05.Differentially expressed genes were screened in GSE64617 based on the absolute value of log-transformed differential expression multiple(Log2FC)>0.5 and FDR≤0.05.Results:Using the qualified data sets to analyze the differential genes,we obtained a total of 74 differentially expressed genes in GSE34424 between the sleep deprivation sample and the normal sample,including 40 up-regulated genes and 34 down-regulated genes,where the 74 differentially expressed genes were four coincident with all genes in the Mapk pathway;There were six coincident genes with all genes in the pathway where Mapt located.Genes that do not intersect with all genes in the pathway in which other genes reside.Intersecting genes included the SIK3,phosphorylated and acetylated forms of Tau protein,and PSD95.Conclusion:Coincidence protein appears in the genes with different expression levels after acute sleep deprivation and sevoflurane inhalation,which can be used as the research object of the next experimental part.PART 4:Sevoflurane inhalation anesthesia and acute sleep deprivation affect dendritic spine density by upregulating SIK3Objective:Many studies have shown that Tau protein is phosphorylated at multiple sites under the influence of a variety of protein kinases,of which the serine position,Ser404,is easy to mediate neurofibrillary tangles,further causing long-term cognitive impairment.Sevoflurane inhalation anesthesia exacerbated the cognitive dysfunction caused by acute sleep deprivation,and the analysis of letters revealed the cross-over of differential protein expression between sleep deprivation and sevoflurane inhalation anesthesia.Therefore,this experiment aims to explore the changes of expression levels of differentially expressed proteins SIK3,p-Tau(Ser-404),ace-Tau and postsynaptic dense substance PSD-95 in the sleep deprivation combined with sevoflurane inhalation anesthesia group as indicated by the signaling analysis.Methods:Golgi staining was use to detect that changes of synaptic plasticity in the brain-filled hippocampus of rats.Western Blot was also used to detect the changes of expression levels of SIK3,Tau,phosphorylated Tau,acetylated Tau and PSD95 in the hippocampus of rats with cognitive dysfunction after inhalation anesthesia,intravenous anesthesia and sleep deprivation-inhalation/intravenous anesthesia.The changes of cognitive behavior were detected by Y maze and Morris water maze after intraperitoneal injection of SIK3 inhibitor YKL-05-099.The density and morphological changes of dendritic spines in rat hippocampal neurons after administration of YKL-05-099 were detected by Golgi staining.The changes in the expression of Tau,phosphorylated/acetylated Tau,and PSD95 in the hippocampus of rats after inhibition by the inhibitor SIK3 were determined by Western Blot.Results:The density and structural change of hippocampal dendritic spines in rat with sleep deprivation-sevoflurane cognitive impairment were detected.that number and density of dendritic spines in the sleep deprivation group were significantly increase,and the number of dendritic spines in the SD+Sevo group was larger than that in the SD group,and the results were statistically significant.2.2.WB was used to detect the expression of related proteins.The results showed that,compared with con group,the expression levels of SIK3,Tau,phosphorylated/acetylated Tau and PSD95 in SD group were increased.The expression levels of each protein in Sevo group and Prop group were also increased slightly,to the same extent as that in SD group.The expression levels of each protein in SD+Sevo group were increased significantly,which was statistically significant compared with the above groups.It was noted that each protein in SD+Prop group remained at the baseline level.3.After the application of SIK3 inhibitors,compared with baseline,the behavioral results in the sleep deprivation group showed no statistical difference,and the cognitive impairment in the SD+Sevo group was alleviated.Golgi staining results showed that the number of dendritic spines in the SIK3 inhibitor group was decreased,indicating that the specific inhibition of SIK3 expression could effectively down-regulate the number of dendritic spines.WB results showed that the expression level of Tau protein in the inhibitor group was decreased.Conclusion:SD+sevoflurane group exhibited obvious cognitive impairment and significant changes in the expression levels of related proteins.Although sevoflurane inhalation did not significantly affect cognitive function,sleep deprivation aggravated the density and morphological changes of hippocampal dendritic spines in the sevoflurane inhalation group,resulting in aggravated postoperative cognitive dysfunction.After inhibition of SIK3,behavioral experiments revealed that the density and morphology of dendritic spines stained by Golgi as well as the expression levels of related proteins were significantly changed,with the density of dendritic spines relatively reduced.Behavioral experiments indicated that the cognitive impairment was alleviated compared with that before the inhibitor,and this inhibitor did not affect the time to restore baseline after sleep deprivation and the Baseline itself.PART 5:Propofol intravenous anesthesia reverses cognitive impairment caused by acute sleep deprivationObjective:The above experiments have confirmed that sevoflurane anesthesia aggravated the cognitive dysfunction caused by sleep deprivation by affecting the expression of SIK3.Therefore,the purpose of this experiment was to explore the potential effect of propofol intravenous anesthesia on cognitive dysfunction after acute sleep deprivation.Methods:The changes of synaptic plasticity in rat brain-filled hippocampus were detected by Golgi staining.Western Blot and Elisa were used to detect the changes of expression levels of SIK3,Tau,phosphorylated Tau,acetylated Tau and PSD95 in the hippocampus of rats with cognitive dysfunction after inhalation anesthesia,intravenous anesthesia and sleep deprivation—inhalation/intravenous anesthesia.AGEs was used to increase Tau modification,followed by Y-maze and Morris water maze to detect the cognitive level and Western Blot to identify the expression changes of phosphorylated/acetylated Tau and PSD95 in rat hippocampus.Results:After the phosphorylation of Tau protein was increased by AGEs,the Y maze and Morris water maze were used to detect the degree of cognitive dysfunction and Western Blot was used to detect the expression levels of SIK3,phosphorylated Tau(Ser-404),acetylated Tau and PSD95 in the hippocampus of rats.Conclusion:1.Compared with the simple sleep deprivation group,the propofol anesthesia group after sleep deprivation showed increased alternating percentage and new arm entry times in the Y maze,and the Morris water maze showed that the escape latency of the sleep deprivation combined with intravenous anesthesia group was shortened,suggesting that the long-term learning and memory ability was repaired.In the sucrose preference test,the sucrose intake of rats in the deprivation combined with intravenous anesthesia group was significantly increased.2.The expression levels of related proteins were measured in the hippocampus of bilateral cerebral regions of each rat.Compared with the sleep deprivation group,the expression level of SIK3 was not different.The total Tau and Tau protein phosphorylation were down-regulated,while acetylated Tau was slightly increased.The expression of postsynaptic dense substance PSD-95 was up-regulated to a certain extent.3.There is no significant difference in the expression level of SIK3 after the application of AGEs.The previously increased Tau,p-Tau,is restored to high expression after the application of the inhibitor,accompanied by the down-regulation of the expression of acetylated Tau and the increase of the expression level of PSD-95.As a common intravenous anesthetic,propofol does not cause obvious damage to the cognitive function when used alone,and the cognitive function of rats in the SD+propofol group is even better than that in the sleep deprivation group,suggesting that propofol may have certain brain protective effects on patients with sleep disorders.Summary1.SD will cause mild cognitive impairment,sevoflurane and propofol simple application has little effect on cognitive function2.The SD+sevoflurane group exhibited significant cognitive impairment and significant changes in the expression levels of related proteins.Although sevoflurane inhalation did not significantly affect cognitive function,sleep deprivation aggravated the density and morphological changes of hippocampal dendritic spines in the sevoflurane inhalation group,resulting in aggravated postoperative cognitive dysfunction.3.As a common intravenous anesthetic,propofol does not cause obvious damage to the cognitive function when used alone,and the cognitive function of rats in the SD+propofol group is even better than that in the sleep deprivation group,suggesting that propofol may have certain brain protection effects on patients with sleep disorders.4.After inhibition of SIK3,behavioral experiments showed that the density and morphology of dendritic spines stained by Golgi as well as the expression levels of related proteins were significantly changed,among which the density of dendritic spines was relatively reduced.Behavioral experiments indicated that the cognitive impairment was alleviated compared with that before the inhibitor,and this inhibitor did not affect the time to restore baseline after sleep deprivation and the baseline itself,suggesting that inhibition of SIK3 might be an effective measure to prevent the cognitive impairment in general anesthesia patients with sleep disorders.5.After the serine phosphorylation modification of Tau was promoted,the effects of sleep deprivation and cognitive function repair induced by propofol intravenous anesthesia disappeared,and the cognitive level recovered to the same level as that of pure acute sleep deprivation,suggesting that Tau protein modification might play a role as a key site for propofol to reverse the cognitive impairment induced by sleep deprivation. |
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