Effects And Mechanisms Of Sevoflurane On Learning And Memory In Transgenic Mice Model Of Alzheimer's Disease

BackgroundAlzheimer's disease(AD)is a common neurodegenerative disease,however,the etiology of AD is not very clear.The incidence of AD increases gradually in recent years,and population aging might be an important causative factor.The present study shows that morbidity of AD is 60% to 70%,seriously affecting the health and life qualilty of the elderly,which has become one of the most intractable problems in the current gerontology.With the continuous improvement of medical techniques,general anesthesia surgery is widely performed in the elderly patients.It highlighted the effects of general anesthesia effects on alzheimer's patients and became a hot research direction in the field of anesthesiology.Researches on animals have shown inhalation anesthesia can increase the neurons filament winding and induce apoptosis of neural cells.Epidemiological investigations showed that the application of general anesthesia was associated with the increased prevalence rate of AD.General anesthesia is a potential risk for the cause of postoperative cognitive dysfunction and AD.However,a recent meta-analysis by Dallas et al.showed that according to the present studies,general anesthesia may not increase the morbidity of AD,although high quality and long-term follow-up studies are not enough.Sevoflurane is the most widely used inhalation anesthetic.In Vitro studies have shown that sevoflurane anesthesia could induce neurotoxicity in the normal brain tissues of adult and pregnant mice,and exacerbate AD neuro pathogenesis by inducing apoptosis and accumulation of ?-amyloid protein.However,Callaway et al.suggested that sevoflurane anesthesia did not impair learning ability or memory function in rat pups and aged rats.The other studies also showed that sevoflurane may improve the cognitive function postoperately.To clarify these contradictions,it is important to further understand the mechanism of the effects of sevoflurane on AD patients.Therefore,the present project planed to establish inhalation anesthesia AD animal models by using Sweden family beta amyloid precursor protein(APPswe)transgenic mice.The methods of molecular biology and behavior were used to study the effects and mechanisms of sevoflurane on learning and memory function of the AD mice,which may provide theoretical basis for the clinical application of anesthesia in patients with AD.Part One: Identification of APPswe transgenic mice and establishment of inhalation anesthesia animal modelObjectiveTo identify APPswe transgenic mice and establish inhalation anesthesia animal models.MethodsAt 10-15 days after birth,PCR amplification was used to detect the expression of APPswe gene by using the end tail tissues of APPswe transgenic mice.90 APPswe transgenic mice were randomly divided into three groups including the control group without any intervention,sham group(mice were palced in 100% O2 anesthesia box for 2h)and sevoflurane group(mice were placed in box containing 2.5% sevoflurane and 100% O2 for 2h).Animal spontaneous breathing,anesthetics and oxygen concentration remain unchanged.Temperature(37±0.2 ?)was kept by using a heating pad and monitored by a rectal thermometer every 20 mins.Blood pressure was measured non-invasively(BP-2000;VisitechSystems,Inc.,Apex,NC,USA).Heart rate and oxygen saturation were tested by pulse oximeter.Blood gas analysis was performed by getting arterial blood.ResultsPCR results showed that the expression of APPswe gene was positive and the product contained 344 kb bases.During the procedure of anesthesia,all mice were alive and all the vital sighs were normal.ConclusionThrough this step,we identified APPswe transgenic mice and successfully established inhalation anesthesia animal models,which was a solid foundation for subsequent tests.Part Two: The effects of sevoflurane anesthesia on the learning memory function in APPswe transgenic miceObjectiveTo monitor the ability of learning and memory in mice models by using Morris water maze and Y-maze experiments.MethodsIn this study,we used Morris water maze to evaluate the learning and memory in mice models.Morris water maze test apparatus is deep circular pool with diameter of 160 cm,height of 50 cm and water height of 30 cm.The water temperature is maintained at 23±1?.The pool is divided into four quadrants and the black wall was marked with four equidistant points namely east,south,west,north.A southwest quadrant of the platform with diameter of 10 cm was fixed,and the water level is 1 cm higher than the platform.Before the experiments,mice were placed in a closed,quiet,dark environment to adapt to half an hour.24 hours after the end of anesthesia,the computer system will automatically record the whereabouts of video analysis software to process the results.Swimming speed,the average percentage of time absconded initial quadrant.The experiments were recorded for five consecutive days(four times / day).Y-maze test: 24 hours after the end of anesthesia,the mice were place in Ymaze.The three-arm lamp was turned on and the mice were allowed to adapt 3 minutes.and then open the mouse is not an arm lamp(random light),5 seconds dwell(lights as a safe area),power remaining arms(voltage 45-55V),when the mice used in the security zone time(effective within 30 seconds,otherwise fail).Light stays on for 30 seconds,then turn off,one end of the experiment,the interval between the two experiments 1 minute,followed by repeating 20 times a day at a fixed training,continuous training three days the same time period.In this experiment Y-maze judging standards for electric shock in mice that go directly to the safe area known as the "correct response",escaped any arm unlit are considered "wrong response." Record the number of correct responses(20 times a day training the correct number of responses),and the number of active avoidance,(not light,escape response times to complete within 5s).ResultsMorris water maze test showed that there was no significant differences on swimming time and the escape latency among the three groups before the application of sevoflurane.After sevoflurane anesthesia,no differences on the escape latency and time spent first quadrant were seen between the sham and control groups.However,compared to control and sham groups,the escape latency was longer(p<0.01),and time spent first quadrant ws shorter(p<0.05)in sevoflurane group.In addition,Y-maze experiment showed compared with the control group and the sham group,reduced numbers of correct responses and active avoidance were observed the transgenic mice of sevoflurane group with statitically significant difference(p <0.01).ConclusionsThrough the water maze and Y maze experiments,the results showed that sevoflurane inhalation anesthesia can affect learning and memory ability of AD mice.Part Three: The effects of sevoflurane anesthesia on hippocampal neuron apoptosis and related signaling pathway in in APPswe transgenic miceObjectiveIn this part,we observed the apoptotic changes on hippocampal CA1,CA3 and DG regions by using TUNEL in situ apoptosis detection kit.Western blotting was used to detect the protein expression of Bcl-xL and Caspase-3 genes.MethodsTUNEL experiments was performed according to the manufacturer's recommendation(US Roche apoptosis detection kit).Mice were anesthetized by pentobarbital sodium,and the brain tissue sections were sampled from CA1,CA3 and DG regions.The samples were dewaxed,dehydrated,incubated with proteinase K at 37 ? for 30 mins,immersed in a 5% peroxide hydrogen blocking buffer and blocked for 15 mins at room temperature.The reaction mixture was prepared and incubated at room temperature for color development for 10 mins.Five viewing fields were evaluated and counted under fluorescence microscope.Western blotting was used to detect the expression of Bcl-xL and Caspase-3 genes.Tissue protein was extracted,electrophoresed on 10% SDS gel,transferred to a PVDF membranes.The membrane was blocked,incubated with anti-caspase antibody,anti-Bcl-xL antibody(1:1000)at 4? overnight,incubated with horseradish peroxidase(HRP)labeled goat anti-rabbit or anti-mouse IgG secondary(dilution of 1: 2000)at room temperature for lh.The films were developed.ResultsIn order to observe the effects of sevofluraneon on hippocampal neurons,we carried out TUNEL assay.Apoptotic cell number was counted in the CA1,CA3 and DG regions.It was found that the apoptotic cell numbers of the CA1 and CA3 region in the sevoflurane group were significantly higher than those in the sham group and control group(P< 0.01).However,there was no significant difference in the DG region among the three groups.Our results suggested that sevoflurane further aggravated the learning and memory ability by inducing hippocampus neuron apoptosis.To further explore the related mechanisms,we verified the protein expression levels of Bcl-xL and caspase-3 in the mice hippocampus in each group by Western blotting.The results showed that the protein expression levels of caspase-3 were significantly higher,and the Bcl-xL levels were significantly lower in the sevoflurane group than in the control group and sham group(P< 0.01)ConclusionsIn AD mice models with sevoflurane inhalation anesthesia,the results from TUNEL assays showed increased neural cell apoptosis in hippocampus CA1,CA3 regions.By Western blotting,increased expression of caspase-3 protein and decreased expression of Bcl-xL protein were observed.It suggested that sevoflurane may damage the ability of learning and memory in AD mice models,and activation of caspase-3 and downregulation of Bcl-xL may be involved in the neural cell apoptosis induced by application of sevoflurane inhalation anesthesia.