Isoflurane Exposure During Pregnancy On The Offspring Of Rats Brain Cell Apoptosis And The Effects Of Learning And Memory Ability

Objective:During pregnancy,the mother may need to accept drug therapy for various reasons,each year,about 2%of mothers during pregnancy had accepted general anesthetics,so the safety of the use of anesthetic drugs during pregnancy is one of the urgent problems,because of the ethical constraints and clinical experiment operation is very difficult,the little information at present such research.Previous animal experiments from behavior and histology confirmed the anesthetic of immature neurons have toxic effects,the result let human matrix of obstetric surgery during pregnancy and infant surgery,concern for the application of anesthetics.On the other hand,because they cannot obtain histological evidence directly,neurotoxic effect of drug is not yet confirmed in humans,but there are some retrospective study on the cognitive dysfunction or learning disabilities such as circumstantial evidence,that anesthetics have toxic effects to human infants and young children the central nervous system,usually think the anesthetic of the immature animal central nervous system toxicity effect and dose,duration,effects and whether there is a combination of a variety of factors.The rapid development of the human brain begins early in the embryo and lasts about three years after birth.Animal and human in the number of nerve,structure,there exist huge differences developmental stage,such as on the basis of synapse formation,rodents from late pregnancy to 2 weeks after birth,the equivalent of human pregnancy after 3 months to 3 years after birth.This study through the late pregnancy rats inhaled anesthetics exposure,comparison of isoflurane the clinical commonly used inhalation anesthesia drugs affect children's brain development,and to explore its influence on learning and memory ability,find a smaller effect on brain development and learning memory ability of drugs,has clinical significance.Methods:1.choose healthy pregnant 20 days Sprague-Dawley?SD?rats,weight of 380g-420 g,intraperitoneal injection of 2%sodium pentobarbital 50 mg/kg for anesthesia,the internal carotid artery catheter into smooth and soft,intraductal use a small amount of heparin anticoagulation,then from the catheter to rat subcutaneous tissue near the back of the neck of a subcutaneous tunnel,subcutaneous tissue to pass through the subcutaneous tunnel through the indwelling catheter to the back of the neck and fixed,in order to prevent the rats to take off the catheter.The next day,the 21-day SD rats were randomly divided into four groups:O₂group,Iso1 group,Iso2 group and Iso3group.According to experimental design,the anesthesia cases in accordance with the requirements for each experiment prefilled gas:the O₂group?50%oxygen+50%nitrogen?,the Iso1 group?isoflurane 1.3%,50%oxygen and 50%nitrogen as carrier gas?,the Iso2 group?isoflurane 2.0%,50%oxygen and 50%nitrogen as carrier gas?,the Iso3group?isoflurane 2.6%,50%oxygen and 50%nitrogen as carrier gas?.Subsequently,the anaesthesia box was placed in the water bath box,and each group of rats were placed into their respective anaesthesia boxes,all of which retained their own breathing.Iso1group,Iso2 group and Iso3 group rats after anesthesia,anal placement temperature measuring electrode,according to the anus temperature adjust the water temperature of water bath,are keeping rat anal temperature of 36?³7?,the O₂group rats into anus temperature measuring electrode,the water bath temperature setting use Iso1group,Iso2 group and Iso3 group rats the average temperature of the water bath.The catheter placed in the internal carotid artery of the rat was extended through the operation hole above the anaesthesia box for the use of arterial blood.Monitoring indicators during anaesthesia:the concentration of anaesthesia,pulse oximetry?SpO₂?,direct arterial pressure?MAP?,heart rate?HR?.After 1 hour,all four groups of pregnant rats were selected to test arterial blood gas?p H,PaCO₂,PaO₂?.In addition,Iso1 group,the Iso2 group and Iso3 group pregnant rat out anesthesia cases respectively,apply the mask to continue breathing in each corresponding concentrations of drug,at the same time for cutting leather,laparotomy exploration stimulation such as surgery,placing the catheter through the arteries connected directly pressure suite,observe its direct arterial pressure,heart rate changes,and presence of body movement.Three hours later,the four groups of pregnant rats were again selected to test arterial blood gas?pH,PaCO₂,PaO₂?,and then stopped inhaling anesthetics and killing rats.2.Choose healthy pregnant 21 days SD rats,weight 380 g-420 g,randomly divided into three groups:O₂group,Iso1 group,Iso2 group.According to experimental design,the anesthesia cases in accordance with the requirements for each experiment prefilled gas:the O₂group?50%oxygen+50%nitrogen?,the Iso1 group?isoflurane 1.3%,50%oxygen and 50%nitrogen as carrier gas?,the Iso2 group?isoflurane 2.0%,50%oxygen and 50%nitrogen as carrier gas?.Subsequently,the anaesthesia box was placed in the water bath box,and each group of rats were placed into their respective anaesthesia boxes,all of which retained their own breathing.Iso1 and Iso2 group rats after anesthesia,the anus in temperature measuring electrode,according to the anus temperature adjust the water temperature of water bath,are keeping rat anal temperature of 36?³7?,the O₂group rats into anus temperature measuring electrode,the water bath temperature setting to use Iso1and Iso2 group rats the average temperature of the water bath.After 3 hours,each group of pregnant mice stopped inhaling anesthetics and took out the anaesthesia box so that it would wake up naturally at room temperature.For 6 hours after cesarean section,fetal rats,1/2 of the fetal rat brain,beheaded executed immediately after the ice quickly isolate the hippocampus,the hippocampus with protein liquid cracking cracking after weighing,and then use kit BCA protein quantitative determination of total protein concentration,with pyrolysis liquid level concentration-80?,detect activation of Cleaved cysteinyl aspartate specific proteinase 3?Cleaved-Caspase 3?protein levels with western blotting.The other half to death after the broken ends of fetal rats brain,and in salt containing 4?phosphate buffer solution?PBS?in hippocampus,and the preparation of single cell suspension,using flow cytometry Annexin V/propidine iodide?AV/PI?double staining to detect hippocampal neuron apoptosis.3.Choose healthy pregnant 21 days SD rats,weight 380 g-420 g,randomly divided into three groups:O₂group,Iso1 group,Iso2 group.According to experimental design,the anesthesia cases in accordance with the requirements for each experiment prefilled gas:the O₂group?50%oxygen+50%nitrogen?,the Iso1 group?isoflurane 1.3%,50%oxygen and 50%nitrogen as carrier gas?,the Iso2 group?isoflurane 2.0%,50%oxygen and 50%nitrogen as carrier gas?.Subsequently,the anaesthesia box was placed in the water bath box,and each group of rats were placed into their respective anaesthesia boxes,all of which retained their own breathing.Iso1 and Iso2 group rats after anesthesia,the anus in temperature measuring electrode,according to the anus temperature adjust the water temperature of water bath,are keeping rat anal temperature of 36?³7?,the O₂group rats into anus temperature measuring electrode,the water bath temperature setting to use Iso1and Iso2 group rats the average temperature of the water bath.After 3 hours,each group of pregnant mice stopped inhaling anesthetics and took out the anaesthesia box so that it naturally woke up at room temperature and continued to be raised until delivery.After the natural delivery of the pregnant rats,the number of births per pregnant mouse was recorded and the healthy male mice were selected for the number.Random half of male offspring breeding to 28 days after birth,each team will then line Mirros water maze experiment,the space of three groups of animals are in Mirros water maze exploration experiment 2 hours after the brain,including death,half the offspring of break immediately take brain tissue,rapid separation of hippocampus on the ice,with protein liquid cracking cracking after weighing,and then use kit BCA protein quantitative determination of total protein concentration,use after cracking,such as concentration of leveling-80?preservation,detect the cAMP-response element binding protein?CREB?and Phosphorylated cAMP-response element binding protein?pCREB?protein levels with western blotting.The other half of their male offsprings after cardiac perfusion with 4%paraformaldehyde,stripping out the brain tissue,and after fixed in4%paraformaldehyde,using immunohistochemical method to detect the late offspring after CREB,pCREB expression in the hippocampus.After another 1/2 of the male offspring from birth has been raised to 90 days,each team will then line Mirros water maze experiment,the space of three groups of animals are in Mirros water maze exploration experiment 2 hours after the brain,including death,half the offspring of break immediately take brain tissue,rapid separation of hippocampus on the ice,with protein liquid cracking cracking after weighing,and then use kit BCA protein quantitative determination of total protein concentration,use after cracking,such as concentration of leveling-80?preservation,detected CREB and pCREB protein levels with western blotting;The other half of their male offspring after cardiac perfusion with 4%paraformaldehyde,stripping out the brain tissue,and after fixed in 4%paraformaldehyde,using immunohistochemical method to detect the late offspring after CREB,pCREB expression in the hippocampus.Results:1.The results of blood gas analysis of 1 hour and 3 hours after anesthesia showed that there was no significant difference in PaO₂in each group.There was no significant difference in p H between the O₂group,Iso1 group and Iso2 group,and significantly higher than the Iso3 group?P<0.05?.There was no significant difference in PaCO₂between the O₂group,Iso1 group and Iso2 group,and was significantly lower than the Iso3 group?P<0.05?.There was no significant difference between MAP and HR before anaesthesia.1 hour after anesthesia,there was no significant difference in MAP and HR between the O₂group,Iso1 group and Iso2 group,and significantly higher than the Iso3 group?P<0.05?.In the surgical exploration,there was no significant difference in MAP and HR between Iso2 group and Iso3 group rats,and significantly lower than the Iso1 group?P<0.05?.In the Iso1 group,MAP and HR were significantly higher than the initial anesthesia and 1 hour after anesthesia?P<0.05?.2.The results of flow cytometry showed that the proportion of early apoptotic cells in the O₂group,Iso1 group and Iso2group was 32.45%±10.27%,35.94%±11.10%,46.53%±10.99%.There was no significant difference in the apoptosis rate between the O₂group and the Iso1 group,and was lower than the Iso2 group?P<0.05?.The results showed that there was no significant difference between O₂group and Iso1 group Cleaved-Caspase 3 protein expression,while the expression level of Cleaved-Caspase 3 in the Iso2 group was significantly higher than that in the O₂group and the Iso1 group?P<0.05?.3,Rats of P28 Mirros water maze results showed that O₂,Iso1 group,Iso2 group platform of the offspring rats quadrant??quadrant?distance percentage of 43.41%±15.66%,44.94%±14.65%,40.23%±9.46%,three groups was no significant difference of statistics.The platform crossing times of the O₂group,Iso1 group and Iso2 group were 4.0±1.9,4.1±2.0,2.2±1.5.Respectively,the O₂group and Iso1 group were higher than the Iso2group,with statistical significance?P<0.05?.The immunohistochemical results showed that the expression levels of pCREB were similar in the O₂group and Iso1 group,and were higher than that in the Iso2 group?P<0.05?.The results showed that the expression of pCREB protein was similar in the O₂group and Iso1 group,and higher than that in the Iso2 group?P<0.05?.There was no significant difference in the expression of CREB protein in the three groups.Rat of P90 Mirros water maze results showed that the O₂group,Iso1 group,Iso2 group platform of the offspring rats quadrant??quadrant?distance percentage of 41.48%±8.69%,39.50%±11.24%,38.40%±10.61%,three groups was no significant difference of statistics.The platform crossing times of the O₂group,Iso1 group and Iso2 group were 5.0±2.0,4.8±2.1,3.3±1.7,and the O₂group and Iso1group were higher than the Iso2 group,with statistical significance?P<0.05?.The immunohistochemical results showed that the expression levels of p CREB were similar in the O₂group and Iso1 group,and were higher than that in the Iso2 group?P<0.05?.The results showed that the expression of p CREB protein was similar in the O₂group and Iso1 group,and higher than that in the Iso2 group?P<0.05?.There was no significant difference in the expression of CREB protein in the three groups.Conclusion:1.Pregnant rats inhaled 1.3%isoflurane?equivalent to 1 MAC?had little effect on respiration and circulatory system,but it was difficult to tolerate surgery;Inhalation of 2.0%isoflurane?equivalent to 1.5 MAC?can withstand surgery,while it has little effect on respiratory and circulatory systems.Inhalation of 2.6%isoflurane?equivalent to 2 MAC?was able to withstand surgery,but its respiration and circulatory function were significantly inhibited.2,pregnant rats inhaled 1.3%isoflurane 3 hours for all the children the growth of hippocampal neurons has no obvious apoptosis,pregnant rats inhaled 2.0%isoflurane 3 hours,the offspring hippocampal neuron apoptosis increased significantly.The effects of isoflurane exposure during pregnancy may be positively correlated with isoflurane inhalation concentration.3.Pregnant rats inhaled1.3%isoflurane for 3 hours,and their offspring were not affected in the early stage and adult stage to distinguish the general spatial position and spatial memory accuracy.Pregnant rats inhaled 2.0%isoflurane for 3 hours,and their children were not affected by the ability to distinguish the general spatial position,but their spatial memory accuracy was impaired.The mechanism of the damage to the memory accuracy of isoflurane during pregnancy during pregnancy may be related to the inhibition of phosphorylation of the hippocampal dentate back CREB.