| BackgroundRecent research shows that general anesthesia,especially the wide use of sevoflurane(Sev)and the others inhalation in clinical anesthesia were demonstrated to cause inflammatory response,oxidative stress and apoptosis of central neurons in infants and young children,which leading to long-term postoperative cognitive dysfunction.It is therefore important to identify the pathophysiology of sevoflurane-induced apoptosis of central neurons,and to investigate effective treatment strategies for preventing and treating sevoflurane-induced long-term cognitive dysfunction in clinical practice.However,little is known about it now.Alpha-lipoic acid(LA),which serves as effcient alipid-soluble antioxidant without obvious toxicity and side effects,is fat-soluble,can penetrate the blood-brain barrier into the brain,and has been successfully applied in mice and rats to treat diabetic neuropathy,Alzheimer's disease and other neurological degenerative diseases.Previous studies have shown that LA lead to activation of PI3K/Akt signaling pathway which was identified to play a pivotal role inmitochondria-depended apoptosis.But there is no relevant research reports on whether LA can protect the premature rats' brain neuron from injuries of Sev,relieve Sev-induced long-term postoperative cognitive dysfunction through regulating PI3K/Akt signaling pathway.Given the above,we hypothesize that mitochondria-depended neuronal apoptosis might play a role in repeated exposure to sevoflurane-induced apoptosis of hippocampus neurons in young rats.LA might block mitochondria-depended neuronal apoptosis by activating PI3K/Akt signaling pathway,and finally improve sevoflurane-induced long-term postoperative cognitive dysfunction.To verify the hypothesis,we studied the role and its possible mechanism of LA in improving sevoflurane-induced long-term postoperative cognitive dysfunction in ethological and histiocytic experiments.In the first part of our studies,we conducted the Morris water maze task to evaluate the therapeutic effect of LA in repeated exposure to sevoflurane-induced decline of learning and memory capacity in young rats.In the second part,we investigate the role of PI3K/Akt signaling pathway in mitochondria-depended neuronal apoptosis caused by sevoflurane in neonatal rats using molecular techniques as well as histopathological technology.In the third part,we determined the concrete character of PI3K/Akt signaling pathway in improving sevoflurane-induced long-term postoperative cognitive dysfunction by LA.Part 1 Effect of LA on long-term cognitive dysfunction after sevoflurane anesthesia in neonatal rats Methods:1.Experimental model constructionSprague-Dawley ratswere randomly assigned to 2 groups at the 7th day after birth(n=10):control group(C),Sevoflurane general anesthesia group(S).Rats in C group were seperated from mother rat in a box with water and 60%O2 for 2.5h.Rats in S group received general anesthesia for 2 hours with2.5%sevoflurane and 60%O2.Rats were resuscitated for 30 minutes with 60%O2 after general anesthesia.When the righting reflex recovered,let the rats come back to their mother's cages with C group immediately.At the beginning of general anesthesia(0h)and the end of general anesthesia(2h),rats under general anesthesia received cardiac puncture for the arterial blood gas analysis,the PH value,partial pressure of arterial carbon dioxide and partial pressure of arterial oxygen was measured to exclude hypoxia,hypercapnia,acidosis.2.Animalsand Drug DeliverySprague-Dawley rats were randomly assigned to4 groups at the 7th day after birth,namely Saline Control group(Saline group),saline sevoflurane general anesthesia group(Sev+saline group),LA control group(LA group)and LA pretreated sevoflurane general anesthesia group(Sev+LA group).There were 10 rats in each group.Rats in LA group and Sev+LA group were administered 20mg/kg LA by intraperitoneal injection,while the rats in Saline group and Sev+saline group received the same volume of normal saline(NS)as LA.1 hour after injection,rats in Sev+saline group and Sev+LA group received general anesthesia for 2 hours with 2.5%sevoflurane and 60%O2.Rats were resuscitated for 30 minutes with 60%O2 after general anesthesia.All treatments were administrated for 3 days at the same time for each day.3.Long-term cognitive function detection(Morris water maze test)Rats in each group received Morris water maze test at 31th day after birth to detect the long-term cognitive function.In the first 5 days,we did the place navigation test(PNT).The time from the rat entering the water to climbing onto the platform is defined as the escape latency.Those rats who cannot find the platform in 90 seconds will be guided to find the platform,put on the platform for 15 seconds and then moved away.The rats in each group were trained 4 times per day,and the interval between the two sessions was 15-20 minutes.Training sessions were carried out for 5 consecutive days,and the daily escape latency of the rats was recorded.After training,the times of crossing the platform within 90 seconds were recorded for rats in each group in the probe trial.At the 36th day,we did the spacial probe test(SPT).The longer training cycle as well as the less frequency entering target quadrants within agreed time was considered to develop the depressed cognitive function.4.Data and Statistical AnalysisAll data are presented as mean±SD.The two group results were analyzed with unpaired t test,and more than two group results were analyzed with One-Way ANOVA.The analyses were performed using SPSS 13.0,and the statistical significance was determined with a criterion of P<0.05.Results1.In the blood gas analysis report,only PaO2 and OI at 2h of S group were significant different from C group(P<0.05),but in both two groups all SaO2%were equal or great than 95%.There was no hypoxemia,no hypercapia,no acidosis in rats during the anesthesia and resuscitation of each group.Other factors that cause brain neuron injury were excluded.The experemental model was built successfully.2.At the 34th,35th days from born,Compared with saline group,the Sev+saline group escape latency was longer 7.7s(P<0.05)and 15s(P<0.01).At the 36th day,platform crossing times of Sev saline group shorter 2.1 times compared with saline group(P<0.05),which means cognitive dysfunction.3.After pretreatment of LA,at the 33th,34th,35th days,compared with Sev+saline group,the escape latency of Sev+LA group were shorter 9s,14.1s,16.7s(P<0.01).And meanwhile,the times of crossing the platform increased in Sev+LA group than in Sev+saline group 2.4 times(P<0.01)at the 36th day.That means cognitive function improved.Conclusion1.Sev general anesthesia for 2 hours with 2.5%sevoflurane and 60%O2 in 7-day-old rats did not cause serious hypoxemia and Other factors that cause brain neuron injury were excluded.The experemental model was built successfully.2.Repeated exposal to Sev leads to long-term cognitive dysfunction.3.LA pretreatment improved the depressed learning and memory capacity in rats that received sevoflurane anesthesiaat an early age.Part 2 LA inhibits the sevoflurane-induced hippocampus neuron apoptosis by activiting PI3K/AktMethods:1.Detection of apoptosis in hippocampal neurons in neonatal rats after sevoflurane anesthesiaNeonatal SD rats were randomly assigned to4 groups(n=8),namely saline control group(Saline group),saline sevoflurane general anesthesia group(Sev+saline group),LA control group(LA group)and LA pretreated sevoflurane general anesthesia group(Sev+LA group).All treatments of each group were the same to the first part for 3consecutive days.After treatment,rats were sacrificed by rapid intra-cardiac infusion of tissue fixative solution under Sevoflurane anesthesia,and the brain was immediately removed and ready for DAB immunohistochemistry.Frozen sections(10?m)were cut for DAB staining to detect the distribution and proportion of Caspase-3 in hippocampal neurons.2.Determination of p-Akt activity and p-Gsk-3? expression in neonatal rat brain neurons after sevoflurane anesthesiaAfter all treatment on the last day,rats in each group were sacrificed under sevoflurane anesthesia,and the brain was immediately removed and kept in liquid nitrogen.The brain was homogenized,dissociated and centrifuged to isolate proteins.The protein concentrations of the samples were determined.Western Blot was used to detect the expression of p-Akt and p-Gsk-3? in the hippocampus.3.Data and Statistical AnalysisAll data are presented as mean±SD.The two group results were analyzed with unpaired t test,and more than two group results were analyzed with One-Way ANOVA.The analyses were performed using SPSS Statistics 13.0,and the statistical significance was determined with a criterion of P<0.05.Results1.Sev induced the hippocampus neurons apoptosis.Compared with saline group,sevoflurane anesthesia significantly increased the proportion of Caspase-3 staining in hippocampal neurons in neonatal rats.In CA1 region,Caspase-3 increased 7.9 times(P<0.01),and 8.3 times in CA3(P<0.05)region.2.LA inhibit the hippocampus neurons apoptosis caused of Sev.However,LA pretreatment reduced the CA1(P<0.05)and CA3(P<0.05)proportion of Caspase-3 staining for 38%and 57%in comparison with Sev+saline group.3.Sev blocked the PI3K signal path of rats cerebral neurons.The results of quantitative determination of protein by western blot showed that p-Akt and p-Gsk-3? reduced for 58%and 68%respectively in Sev+saline group compared with saline group(P<0.01).4.LA activated parts of the PI3K signal path of rats cerebral neurons blocked by Sev.However,LA pretreatment increased p-Akt and p-Gsk-3? expression for 59%and 85%respectively compared with SS group(P<0.01).Conclusion1.Hippocampus is important cerebral structure for study and memery.Hippocampus neurons apoptosis plays a key role of long-term cognitive dysfuction.Sevoflurane anesthesia depressed the activation of Caspase-3 in hippocampal neurons neonatal rats,and could be reversed by LA pretreatment.2.Sevoflurane anesthesia decreased the expression of p-Akt and p-Gsk-3?,two important factors of the PI3K antiapoptotic pathway,in the hippocampus in neonatal rats,and could be up-regulated by LA pretreatment.Protein p-Akt and p-Gsk-3? are key factor of antiapoptosis pathway PI3K.LA up-regulated p-Akt and p-Gsk-3?shows that LA activated parts of the PI3K signal path of rats cerebral neurons blocked by Sev.Part 3 The PI3K/Akt signaling pathway mediates LA improvement long-term cognitive dysfunction caused by sevoflurane.Methods:1.Animals and Drug Delivery7-day-old rats were randomly assigned to6 groups(n=8).And detailed grouping and processing are as follows:1)Saline Control group(Saline' group):Rats were injected intracerebroventricle(ICV)the same volume of saline as Wortmannin treated in other groupsby.2)Saline sevoflurane general anesthesia group(Sev+saline' group):Rats were administered the same volume of saline as Wortmannin treated in other groups by ICV.1 hour after injection,rats received sevoflurane general anesthesia for 2 hours with 2.5%sevoflurane and 60%O2.Rats were resuscitated for 30 minutes with 60%O2 after general anesthesia.3)Wortmannin control group(WM group):Rats were injected lmg/kg(2mg/ml WM),a sepcific inhibitor to PI3K/Akt,by intobilateral intracerebroventricle(ICV)separately guided by brain stereotactic apparatus.4)LA pretreated sevoflurane general anesthesia group(Sev+LA+saline' group):Rats were administered 20mg/kg LA(10mg/ml)by intraperitoneal injection and administered the same volume of saline as Wortmannin treated in other groups by ICV.One hour later,the rats received general anesthesia for 2 hours with 2.5%sevoflurane and 60%O2.Rats were resuscitated for 30 minutes with 60%O2 after general anesthesia.5)LA IP+Wortmannins ICV pretreated+sevoflurane general anesthesia group(Sev+LA+WM group):Rats were administered 20mg/kg LA(10mg/ml)by intraperitoneal injection and lmg/kg(2mg/ml WM)ICV injection.And 1 hour later,rats received Sev general anesthesia for 2 hours with2.5%sevoflurane and 60%O2.Rats were resuscitated for 30 minutes with 60%O2 after general anesthesia.6)Sevoflurane general anesthesia plus ICV Wortmannin group(Sev+WM group):Rats were administered lmg/kg(2mg/ml WM)ICV injection.And1 hour later,rats received general anesthesia for 2 hours with 2.5%sevoflurane and 60%O2.Rats were resuscitated for 30minutes with 60%O2 after general anesthesia.All treatments were administrated for 3 days at the same time for each day.2.The Morris water maze test was used to detect the cognitive function of rats in each group.The specific method was described in the first part.3.After all treatment,rats were sacrificed by rapid intra-cardiac infusion of tissue fixative solution under sevoflurane anesthesia,and the brain was immediately removed and ready for fluorescence immunohistochemistry.Frozen sections(10?m)were cut for DAB staining to detect the distribution and proportion of Caspase-3 in hippocampal neurons.4.After all treatment(the method as before)on the last day,the other 6 groups rats were sacrificed under sevoflurane anesthesia,and the brain was immediately removed and kept in liquid nitrogen.The brain was homogenized,dissociated and centrifuged to isolate proteins.The protein concentrations of the samples were determined.western blot was used to detect the expression of p-Akt and p-Gsk-3? in the hippocampus.5.Data and Statistical AnalysisAll data are presented as mean±SD.The two group results were analyzed with unpaired t test,and more than two group results were analyzed with One-Way ANOVA.The analyses were performed using SPSS Statistics 13.0,and the statistical significance was determined with a criterion of P<0.05.Results1.On the 33-35 day of birth,the escape latency of rats in Sev+LA+WM group increased(P<0.01)than that of Sev+LA group.And mean while,the times of crossing the platform in 90 seconds decreased(P<0.05).WM+saline' and Sev+WM were not different from control groups.2.Caspase3 staining apoptotic cells increased for 57%and 90%in CA1 and CA3 of Sev+LA+WM group than Sev+LA group(P<0.01).3.The results of protein quantitative test by Western Blotmethod revealed a decreased expression of p-Akt(P<0.01).and p-Gsk-3?(P<0.05).for 38%and 41%in Sev+LA+WM group than in Sev+LA group.Conclusion1.Wortmannin can effectively reduce the expression of p-Akt in the hippocampus of rats which received sevoflurane anesthesia after LA pretreatment.2.Wortmannin ICV of the rapeutic dose didn't cause the hippocampus neurons apoptosis and long-term cognitive disorder.3.Wortmannin can reverse the effect of LA pretreatment on the decline of long-term learning and cognitive function induced by sevoflurane anesthesia in rats.4.Wortmannin significantly reduced the inhibition of LA on the apoptosis of hippocampal neuronsinduced by sevoflurane in rats,and thusinhibited the neuroprotective effect of LA on sevoflurane general anesthesia.Summary:LA inhibition of Sev induced apoptosis by activiating antiapoptosis pathway PI3K/Akt,plays a key role of neonatal nueronprotection from long-term cognitive dysfuction. |
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