| Postoperative cognition dysfunction(POCD)is a common complication of central nervous system that occurs after surgery and/or anesthesia.Its main clinical manifestation is cognitive decline in patients after surgery and/or anesthesia.In recent years,the pathogenesis hypothesis of POCD has been proposed,including Aβ hypothesis,brain-derived nutritional factor(BDNF)hypothesis,cholinergic hypothesis,oxidative stress hypothesis,neuro-inflammatory response hypothesis,etc.Some researchers hypothesize that POCD is not a diagnostics disorder,but rather a syndrome,a group of pathological changes that exhibit common symptoms,centered on age-related memory loss and personality changes that occur after anesthesia.It has been shown that inhalation anesthetics,including isoflurane,sevoflurane and desflurane,has effects on the neuropathy of POCD and can accelerate the clinical progress of POCD.While,in recent years,researchers have discoverd,ketamine of low to medium dose can improve cognitive function.Ketamine has good analgesic effect and less influence on respiration,and is often used in clinical surgery.For this reason,isoflurane combined with ketamine may have the potential of anti-POCD application as an anesthetic in clinical surgery.However,the changes of cognitive function and specific mechanism are not clear when isoflurane is inhaled and ketamine is used simultaneously.PI3K/AKT/GSK-3β signaling pathway is an important signaling pathway and exists widely in various cells.This signal transduction pathway plays an important role in maintaining cell survival and inhibiting apoptosis,and is a classic anti-apoptosis and pro-survival pathway.This pathway change is closely related to the biological processes involved in ketamine and isoflurane.Therefore,in this study,we attempted to investigate the protective effect of ketamine on isoflurane induced cognitive impairment through the PI3K/AKT/GSK-3β pathway.Specific studies are as follows:Methods:The experimental study was conducted with 20 months-old Sprague Dawley rats.The rats were divided into six groups with 10 rats in each group and received different doses of ketamine,memantine and blank control.The details are as follows:Group Ⅰ:blank control group,only normal saline was injected;Group Ⅱ Isoflurane group:isoflurane inhalation only;Group Ⅲ:Low dose ketamine group,isoflurane inhalation,intraperitoneal injection of ketamine(2.5 mg/kg);Group Ⅳ:Middle dose ketamine group,isoflurane inhalation,intraperitoneal injection of ketamine(5 mg/kg);Group Ⅴ:High dose ketamine group,isoflurane inhalation,intraperitoneal injection of ketamine(10 mg/kg);Group VI:Memantine group,isoflurane inhalation,intraperitoneal injection with memantine(10 mg/kg).Morris water maze test and dark avoidance test were used to measure the cognitive function of the rats.Then the anesthetized rats were killed by intracardiac injection and hippocampi were taken to detect the expression of various indicators.Results:(1)In this zoological experiment,we first carried out the blank control group and isoflurane group of old rats experiment,to verify the reliability of isoflurane modeling.The experimental results showed that compared with the blank control group,the cognitive function of rats in the Isoflurane group was worse in Morris water maze test and dark avoidance test,and it could be considered that isoflurane induced cognitive dysfunction in the elderly rats.Subsequent behavioral studies with different doses of ketamine showed that ketamine significantly enhanced the cognitive function and presented a dose-dependent effect,showing a neuroprotective effect on isoflurane induced cognitive impairment.(2)Isoflurane induced an increase of Aβ and a decrease of BDNF in hippocampus of elderly rats.Ketamine treatment reduced Aβ and increased BDNF expression in a dosedependent manner.Isoflurane inhalation affected the activity of hippocampal cholinergic system,and ketamine treatment increased the activity of AchE,reflecting the increased activity of cholinergic system.Ketamine can reverse isoflurane induced oxidative stress in hippocampus of rats,decrease protein carboxyl content,decrease lipid oxidation product MDA,increase antioxidant substances SOD,GSH,CAT,etc.Ketamine can reverse isoflurane-induced nervous system inflammation,reduce isoflurane-induced pro-inflammatory cytokines IL-1β,IL-6,Tnfα,caspase-3 mRNA expression,and IL-1β,IL-6,TNF-α,caspase-3 protein expression;With the increasing of ketamine dose,the indexes were close to those of memantine treatment group.(3)The effect on histone acetylation:Histone acetylation was maladjusted in the hippocampus of rats exposed to isoflurane,showing a low concentration of AC-H3K9 and ACH4K12.After ketamine treatment,the histone deacetylation induced by isoflurane improved with the increase of ketamine dose.At the same time,histone acetylase gene and protein expression detection showed that the expression levels of Hdac1 gene mRNA and HDAC1 protein did not change with isoflurane and ketamine treatment.The expression levels of Hdac2 gene mRNA and HDAC2 protein increased in isoflurane induced group,while the expression levels of Cbp gene mRNA and CBP protein decreased.In the ketamine treatment group,the expression levels of Hdac2 mRNA and HDAC2 protein decreased,while the expression levels of Cbp mRNA and CBP protein increased.With the increasing of ketamine dose,the indexes were close to those of memantine treatment group.(4)The effect on DNA damage repair:Isoflurane induced neuronal senescence and neuronal DNA damage,which was manifested as increased expression of p53 and p21WAF1/CIP1 in rat hippocampal cells,mediating cell cycle arrest.Ketamine treatment reduced isoflurane-induced DNA damage in rat neurons and reduced the expression of p53 and p21 WAF1/CIP1.With the increasing of ketamine dose,the indexes were close to those of memantine treatment group.(5)The effect on PI3K/AKT/GSK-3β pathway protein expression:In isoflurane induced group,the p-AKT/AKT level was decreased.After ketamine treatment,,the level of p-AKT/AKT increased,which depended on ketamine concentration.The expression of GSK-3β was inhibited in the isoflurane induced group,and up regulated after ketamine treatment,which depended on ketamine concentration.Isoflurane inhibited PI3K/AKT/GSK-3β pathway and damaged cognitive function.Ketamine activated PI3K/AKT/GSK-3β pathway induced by isoflurane.With the increasing of ketamine dose,the indexes were close to those of memantine treatment group.Discussion:Morris water maze experiment and dark avoidance experiment are the most classic learning and memory experiments,which are widely used in animal experiments.The results of this behavioral experiment showed that isoflurane induced cognitive impairment in rats,and ketamine therapy improved the cognitive function of rats.The result is in line with our expectations.In pathological situations,Aβ will fold and accumulate in error and become neurotoxic.BDNF may be a key factor in cognitive performance,especially in learning and memory.The expression and signal changes of BDNF may lead to neurodegenerative changes.In this experiment,isoflurane inhalation induced the increase of Aβ and BDNF in hippocampus of rats.Ketamine therapy could reduce Aβ and increase the expression of BDNF.The continuous production of various endogenous and exogenous reactive oxygen species leads to the continuous and accumulated oxidative damage of cells,and changes many cell functions.Protein carbonylation is one of the most harmful irreversible oxidative protein modification,and it is the main sign of oxidative stress related diseases.Ketamine can reverse isoflurane induced oxidative stress in brain,decrease protein carboxyl content,decrease lipid oxidation product MDA,and increase antioxidant substances including SOD,GSH and CAT.In the pathogenesis of POCD,inflammation plays an important role.Cytokines are closely related to learning and memory,especially IL-1β,IL-6 and TNF-α.Ketamine can reverse the neuroinflammation caused by isoflurane.Lysine acetylation and deacetylation of histone are the key regulatory factors of gene expression,which regulate the growth and proliferation of neurons and play an important role in cognitive function.Lysine acetylation is catalyzed by lysine acetyltransferase(KATs),which usually promotes gene transcription and cognitive ability.On the contrary,histone lysine deacetylase(HDAC)catalyzes,antagonizes gene transcription and negatively regulates cognition in many brain regions.HDAC2 is the main medium of HDAC inhibition on cognitive function,and HDAC1 has little influence on cognitive function.In this study,isoflurane could induce the histone deacetylation in hippocampus of rats,which showed that the concentration of AC-H3K9 and AC-H4K12 decreased,HDAC2 level increased and CBP level decreased.Ketamine treatment can improve this change,reduce histone deacetylation,and improve cognitive function.Neuronal cell senescence refers to cell cycle arrest,mainly occurring in the G0/G1 phase of the cell cycle,which can lead to neurodegenerative diseases under pathological conditions.One of the most important inducement is the DNA damage repair response.In the process of injury,p53 gene is activated,and the expression of p53 can induce the transcription expression of P21(CIP1/WAF1),induce cell G1 phase senescence,prevent damaged DNA from entering S phase,and ensure the correct transmission of genetic information during cell division.The PI3K/AKT/GSK-3β signaling pathway plays multiple roles in aging and many neurodegenerative diseases,including promoting the hyperphosphorylation of Tau protein,regulating the transcription of BDNF and other neuropeptides,participating in the progression of neuroinflammation,affecting mitochondrial function and synaptic plasticity,regulating the CREB-CBP pathway and participating in histone acetylation,and regulating the growth,proliferation,differentiation and apoptosis of neurons,etc.Inhibition of the PI3K/AKT/GSK3β signaling pathway in the cortex and hippocampus leads to neurodegeneration and spatial learning disabilities in animal studies.We found that isoflurane induced inhibition of the PI3K/AKT/GSK-3β pathway and impaired cognitive function.Ketamine activated isoflureinhibited PI3K/AKT/GSK-3β pathway and improved cognitive function.Morris Water maze test is a classic learning and memory experiment,as well as dark avoidance test.Both of them are widely used in rat/mouse animal experiments to detect their cognitive functions.The results of this behavioral experiment showed that isoflurane induced cognitive impairment in rats,and ketamine treatment improved the cognitive function of rats.It was in line with our expectations.In summary,we concluded that ketamine played an anti-apoptotic,anti-inflammatory and antioxidant role,improved histone deacetylation and DNA damage,and thus prevented cognitive impairment caused by isoflurane anesthesia.The effect of ketamine may be realized through PI3K/AKT/GSK-3β pathway.The limitations of this study are the relatively small sample size and the lack of specific studies on the interior of the PI3K-AKT-GSK-3β pathway,such as whether isoflurane and ketamine inhibit/activate this pathway through a common site.These all need further study.In addition,ketamine has several side effects,including mental symptoms and ulcerative cystitis.Ketamine metabolite(R)-ketamine has fewer side effects.Further research is needed to explore whether(R)-ketamine can play the same cognitive protective role. |
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