Effects Of Ropivacaine-induced Convulsion On Bax, Bcl-2Expression Of Hippocampus And Learning And Memory In Developing Rats

Ropivacaine is becoming more and more popular in clinical anesthesia and ache therapy. Because of lower cardiotoxicity, better separation of sensation and motion, and better blockade of nerve impulses than bupivacaine, it gains widespread use in clinical practices. It is widely used in spinal anesthesia and regional anesthesia for children. However, systemic and localized toxic reactions can occur, usually as a result of accidental intravascular or intrathecal injection or administration of an excessive dose.The systemic developing phases of infants and children, especially Central Nervous System (CNS), are different from adults. It is discovered that adverse effects of nerve apoptosis are associated with the use of anesthetic drugs in developing key point of CNS. Certain hippocampus nerves would be damaged by anesthesia-induced convulsion. Memory and message accumulation are association with hippocampus nerves, which play a significant role on memory transition.So far, it has not reported whether infants with different ages have different toxicity effects of local anesthetics and of learning and memory. It is of great importance to do some experimental researches to work it out. Based on previous studies, we hypothesized that different developing phases of CNS have different effects of ropivacaine-induced convulsion. The aim of the present study was to determine convulsion ED50of ropivacaine in developing rats with sequential experimental method to investigate the relationship between ages and convulsion ED50of ropivacaine; determine the expressions of bax and bcl-2at different time by immunohistochemistry method; observe the effects of ropivacaine-induced convulsion on spatial memory and learning ability of developing rats when they growed up.Part I:Determination of convulsion ED50of ropivacaine in developing rats with sequential experimental method1Methods44rats were divided into two groups:15-day-old group (Group D15,n=22) and60-day-old group (Group D60, n=22). Drugs were intraperitoneally injected to SD rats and observed the behaviors in30minutes. Determined the dosage with sequential change. Convulsion was defined as that it achieved classification III or more and happened within30minutes. The ED50and the95%confidence interval were calculated.2ResultsThe ED50of Group D15is54.95mg/kg and the95%confidence interval is50.52mg/kg-59.77mg/kg, while the Group D60is34.36mg/kg and the95%confidence interval is31.81mg/kg～37.1lmg/kg. T-test of two groups lgED50was accounted and the result is7.84, P<0.01, the differences of them have statistical significance.3Conclusions3.1The sequential experimental method for determination of ED50is simple, efficient and reliable. 3.2The convulsion ED50induced by ropivacaine in15-day-old rats is54.95mg/kg while in60-day-old rats is34.36mg/kg. It shows a negative correlation between developing ages and convulsion ED50induced by ropivacaine.Part II Determination the effects of ropivacaine-induced convulsion on hippocampus expressions of bax and bcl-2at different time by immunohistochemistry method1Methods194rats were divided into two groups:15-day-old group (GroupD15, n=97) and60-day-old group(Group D60, n=97).Then divided the groups into two parts. Rats in one part, called Group NS, were i.p injection of N.S. The other were i.p injection of convulsion ED50of ropivacaine. Then the rats were put in a box with30%oxygen. SpO2and HR were monitored. Observed the behaviors in30minutes, according to the behaviors, the rats which injected ropivacaine were divided into two groups: convulsion stating group(Group SC) and non-convulsion group. If the rats were in convulsion stating more than twenty minutes, i.p. inject10%chloralhydrate (350mg/kg). At2h、6h、24h、3d、7d after convulsion, chose five rats of Group-SC randomly, anesthetized with intraperitoneal (i.p.) injection10%chloralhydrate (350mg/kg). Then perfused heats, fetched the brains, saved the tissues with hippocampus about3-4mm. Embedded them with paraffin, siced them up for immunohistochemistry experiments. Did the same disposal to the Group NS of Group-D15and Group-D60. Calculated the positive expressions of Bax and Bcl-2in hippocampus with microscope after immunohistochemistry experiments.2ResultsConvulsion latency and persistence time of Group-D15were longer than that of Group-D60. The SpO2of all convulsion rats maintained more than90%during convulsion process.The expressions of Bax and Bcl-2of Group-D15and Group-D60with N.S ip.were on an even keel, while of Group-SC increased at the second hour after convulsion, at the twenty-fourth hour reached the top and then declined.The ratio of Bax and Bcl-2Group-D15and Group-D60with N.S ip.were between1.00and1.05. while of Group-SC increased at the second hour after convulsion, at the twenty-fourth hour reached the top and then declined. The Group-SC of15-day-old rats declined to the normal level at the third day after convulsion, while that of60-day-old rats were still higher than normal level at the seventh day after convulsion.3ConclusionIt suggested that it had different effects of rat’s ages on the expressions of Bax and Bcl-2and their ratio of hippocampus apoptosis induced by ropivacaine convulsion. It had less hippocampus apoptosis on the yonger rats.PartⅢ Observe the effects of ropivacaine-induced convulsion on spatial memory and learning ability of developing rats on the eighth and forty-fifth postoperative day1MethodsChose ten rats in every Group NS and Group SC randomly, observe the effects of ropivacaine-induced convulsion on spatial memory and learning ability of developing rats on the eighth and forty-fifth postoperative day. The rats were trained in Morris Water Maze in the first three days. Dropped in water from every quadrant, let it stay on the platform for10seconds after it landed. Help was needed if it can not find the platform within120seconds, marked120seconds. Tested them spatial memory and learning ability from the fourth day to the sixth day. Calculated and marked their means of escape latency. Calculated the times of crossing platform within60seconds after rats were dropped in water from the farthest quadrant.2ResultsWhile the training increased, the time of escape latency (EL) was decreased and crossing times (CT) were increased.On the eighth postoperative day, the results of EL and CT showed that there were no statistical significance in the comparison Group SC with Group NS of15-day-old rats(P>0.05); but compared by Group NS of60-day-old rats, Group SC of60-day-old rats had less scores in the results of EL and CT(P<0.05).On the forty-fifth postoperative day, the results of EL and CT showed that there were no statistical significance in the comparison Group SC with Group NS of15-day-old rats and60-day-old rats(P>0.05).3ConclusionsIt suggested that it had no different effects of ropivacaine-induced convulsion on spatial memory and learning ability in rats of different developing phases on the forty-fifth postoperative day; but some differences existed on the eighth postoperative day.Paper Conclusions1The convulsion ED50induced by ropivacaine in15-day-old rats is54.95mg/kg while in60-day-old rats is34.36mg/kg. It shows a negative correlation between developing ages and convulsion ED50induced by ropivacaine.2It suggested that it had different effects of rat’s ages on the expressions of Bax and Bcl-2and their ratio of hippocampus apoptosis induced by ropivacaine convulsion. It had less hippocampus apoptosis on the yonger rats.3It suggested that it had no different effects of ropivacaine-induced convulsion on spatial memory and learning ability in rats of different developing phases on the forty-fifth postoperative day; but some differences existed on the eighth postoperative day.