| BackgroundThe changes of mental activity,personality,social activity and cognitive ability of patients during the perioperative period are collectively referred to as perioperative neurocognitive disorders(PND).As a severe perioperative brain functional disease,PND can occur within several days,weeks or months preoperatively and postoperatively,and may last longer.Studies have shown that the incidence of perioperative neurocognitive disorders is about 15%to 45%.At present,it is believed that among the several factors related to the syndrome,anesthesia,operation and age are most closely related to the occurrence and development of PND.Experimental evidence demonstrated the anesthetics can affect the normal function of nervous system,including damage to the cognitive function of mice in the short term after anesthesia.Homocysteine(Hcy)is an important intermediate of methionine cycle metabolism.On the one hand,the metabolism of Hcy is closely related to cognitive function-related vitamins including vitamin B12 and B6;on the other hand,the accumulation of this substance will directly or indirectly cause brain cognitive impairment diseases such as AD,dementia,depression and epilepsy.Previous studies in our laboratory have found that the accumulation of Hcy will increase the susceptibility to PND,and the content of Hcy fluctuates and rises during the perioperative period.However,the pre-experimental results showed that both PND animal model and isoflurane anesthesia model could significantly decrease the content of Hcy in peripheral serum of mice in a short period of time.This time-related content difference indicates that there are complex changes in Hcy metabolism during perioperative period and after isoflurane anesthesia,and this change may be closely related to the change of cognitive function and even the occurrence of PND after anesthesia.Therefore,it is of great clinical significance to explore the temporal and spatial distribution of Hcy in mice after isoflurane anesthesia,to clarify the specific mechanism of isoflurane-induced changes in Hcy level,and to clarify the relationship between Hcy level and neurocognitive function after anesthesia.Part 1:The spatio-temporal changes of homocysteine metabolism after isoflurane anesthesiaObjective To study the temporal and spatial changes of Hcy and its metabolism-related vitamins in mice induced by isoflurane anesthesia.Methods(1)Enzyme-linked immunosorbent assay(Elisa)was used to observe the levels of Hcy and its metabolism-related vitamins(vitamin B12,folic acid)in organs and tissues related to Hcy metabolism(liver,kidney,brain,serum)immediately after isoflurane anesthesia(30min,1h,2h,4h).(2)Elisa technique was used to compare the levels of Hcy and its metabolism-related vitamins in organs and tissues(liver,kidney,brain,serum)related to Hcy metabolism in mice with different recovery time after anesthesia(0min,30min,1h,2h,4h,8h)under the condition of fixed anesthetic time(2h).Results(1)Isoflurane could decrease the levels of Hcy in liver,kidney and serum,and increase the content of Hcy in brain,but the content of vitamin B12 and folic acid related to Hcy metabolism had no fixed change in liver,kidney,brain and serum.(2)After isoflurane anesthesia for a fixed time(2 hours),with the prolongation of recovery time,the levels of Hcy in liver,kidney and serum gradually increased to that before anesthesia,while the level of Hcy in brain increased at first and then decreased,and gradually returned to the level before anesthesia,while the changes of vitamin B12 and folic acid in liver,kidney,brain and serum had no fixed rule.Part 2:Effect of isoflurane on the content and activity of Cystathionine-β-Synthase in brainObjective To study the effect of isoflurane anaesthesia on Cystathionineβ-synthase(CβS),which is the key enzyme of Hcy catabolism in mouse brain,and its distribution and function in different brain regions and nerve cells.Methods(1)The activity and content of CβS in the brain of mice were observed under different isoflurane anesthesia duration(30min,1h,2h,4h)by active enzyme continuous circulation spectroscopy and Western blotting(Western Blot).(2)The activity and content of CβS in the brain of mice under fixed anesthesia time(2 h)and different recovery time(0min,30min,1 h,2 h,4 h,8 h)were observed by active enzyme continuous circulation spectroscopy and Western blotting(Western Blot).(3)The changes of Hcy content in different brain regions of mice induced by isoflurane were observed by Elisa technique.(4)Immunofluorescence staining and primary cell culture technique were used to observe the distribution of CβS in different brain regions and different kinds of nerve cells.(5)Elisa technique and primary cell culture technique were used to observe the effect of isoflurane on the intracellular and extracellular Hcy content of neurons and astrocytes.Results(1)Isoflurane could significantly decrease the activity of CβS and increase the content of CβS in the brain of mice,and the longer the anesthesia duration,the greater the change of CβS activity and content.(2)After isoflurane anesthesia for a fixed time(2h),with the prolongation of recovery time,the activity of CβS in the brain gradually increased to the pre-anesthesia level,while its content increased at first and then decreased,and gradually returned to the pre-anesthesia level.(3)The content of Hcy in different brain regions of mice was different under isoflurane anesthesia.The content of Hcy in cortex and hippocampus increased significantly after anesthesia(P<0.05).(4)CβS was highly expressed in neurons in cortex and hippocampus of mice,but rarely expressed in astrocytes in mouse brain.(5)Isotlurane could increase the content of Hcy in primary cultured neurons,but had no significant effect on the level of extracellular and extracellular Hcy in astrocytes.Part 3:Effects of regulating cystathionine-β-synthase in neurons on neurocognitive functionObjective With the help of S-AdenosylMethionine(SAM)and CβS-flox transgenic animals,pharmacological methods and gene-regulation techniques were used to specifically interfere with the activity and content of CβS in the brain of mice.Multiple cognitive-related behavioral paradigms were used to study the internal relationship between CβS and cognitive impairment after isoflurane anesthesia.Methods(1)Before the primary cultured neurons were treated with isoflurane(iso 1.4%,2h),they were pre-incubated with SAM(200mM)for 1h,and the changes of CβS enzyme activity after isoflurane treatment were observed.(2)SAM(2 μl,200mM)was injected into the lateral ventricle of mice 1 hour before isoflurane anesthesia.Open field test,elevated maze test and contextual fear conditioning test were used to observe the differences of basic locomotor ability,anxiety and contextual fear memory in different experimental groups at the time of 1 hour after anesthesia,to explore whether SAM can improve the cognitive function impairment caused by isoflurane.(3)Combined with CβS-flox transgenic animals and virus stereotaxic injection technique,CβS of neurons in bilateral dorsal hippocampal regions of mice were specifically knocked out.Open field test and elevated plus maze were used to detect the changes of basic motor ability and anxiety after knockout,contextual fear conditioning test were used to detect the changes of contextual fear memory.(4)The effects of isoflurane(1.4%,2 h)anesthesia on the cognitive function of bilateral dorsal hippocampal CβS-knockout mice were observed by the combined application of intracerebroventricular injection and virus stereotaxic injection,and SAM(2 μl,200mM)was injected into the lateral ventricle 1 hour before isoflurane anesthesia.The effect of SAM on cognitive impairment in CβS-knockout mice was studied by related cognitive behavioral changes.Results(1)Pre-administration of SAM could improve the decrease of CβS enzyme activity in primary cultured neurons induced by isoflurane.(2)Intracerebroventricular injection of SAM before anesthesia could improve the decrease of contextual fear memory in mice induced by isoflurane.(3)Bilateral dorsal-hippocampal CβS-knockout increased the anxiety of mice,but had no significant effect on their locomotor ability and contextual fear memory.(4)Isoflurane could impair the ability of contextual fear memory of bilateral dorsal-hippocampal CβS-knockout mice,and this effect was more significant than that of non-knockout mice,but had no significant effect on their basic motor ability and anxiety level.While intracerebroventricular injecting of SAM had no significant effect on the locomotor ability,anxiety level and contextual fear memory ability of isoflurane-anesthetized CβS-knockout mice.Part 4:The mechanism of isoflurane inhibiting cystathionine-β-synthase activityObjective To study the mechanism of isoflurane-induced changes of CβS activity.Methods(1)The changes of CβS activity purified by E.coli system under the action of different concentrations of isoflurane were observed by active enzyme continuous cycle spectroscopy.(2)The interaction mode between isoflurane and CβS at molecular level was explored by NMR--WaterLOGSY technique.Results(1)Isoflurane could immediately cause a significant decrease in the activity of CβS protein purified in E.coli system.(2)NMR-WaterLOGSY showed that isoflurane molecule could directly bind to CβS protein and inhibit the activity of CβS protein.Through the study of the above four parts,we could draw the following conclusions:Conclusion(1)After isoflurane anesthesia,the learning and memory function of mice declined and the level of Hcy in blood,liver and kidney decreased,while the level of Hcy in brain increased.(2)Isoflurane could significantly decrease the activity and increase the content of CβS,the key enzyme of Hcy catabolism in mouse brain,and CβS was highly enriched in mouse cortex and hippocampal neurons.(3)The neurocognitive function of hippocampal neurons in CβS-knockout mice was not impaired,and the alteration of neurocognitive function induced by isoflurane anaesthesia depended on the inhibitory effect of CβS activity rather than its content expression,while the cognitive impairment induced by isoflurane could be improved by CβS agonist SAM.(4)Isoflurane could directly bind to CβS protein and inhibited its activity immediately. |
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