| Objective: To establish and improve the method for determination of the amino acid neurotransmitters in brain hippocampus of rat by precolumn derivatization reversed-phase high performance liquid chromatography(RP-HPLC)and study the changes of Glutamic acid and Gamma Aminobutyric acid in brain hippocampus of PTZ-induced epileptic rats.Methods: A model of temporal lobe epilepsy was induced via PTZ kindling in SD male rats by hypodermic injection.The rats were devided into normal control group and epilepsy group.When amino acids in samples were pretreated with methanol and acetonitrile,the chromatographic separation was performed on a Red Classical AQ-18 column(250*4.6mm,5um)using 9-fluorenylmethyl chloroformate(FMOC-CL)as pre-column derivatization and using A[50mmol/L sodiumacetate(PH4.8)410m L+water85m L+diethyleneoxide5m L]and B[acetonitr ile500 m L] as mobile phase at a current velocity being 1.0 m L/min.This method used gradient elution and the chromatogram was detected at a wave-length of265 nm with the column temperature being from 30℃-32℃。Result: Glutamic acid and Gamma Aminobutyric acid were completely separated with 30 minutes.The linear relation was well in the range from20μg/m L to 200μg/m L,their concentrations had a high linear relationship with its peak area(r>0.9995).The sample adding standard recoveries of this two amino acid were all more than 100%,and the relative standard deviation of sample adding standard recoveries were both less than 2%.Conclution: The method has a series of advantages,such as good stability,simpleness,high sensitivity,promptness,good specificity and repeatabiltity.After improving,the precolumn derivatization reverse phase high performance liquid chromatography with a reaction with FMOC-CL can effectively separate and measure glutamic acid and gamma aminobutyric acid in brain hippocampus of rat.Objective: To observe the changes of contents in glutamic amino acid(Glu)andγ-aminobutyric amino acid(GABA)in the hippocampus of kindled rats in 30 minutes after epileptic seizure happens,in order to explore the terminational mechanism of epilepsy.Methods: A model of acute epileptic seizure was induced via PTZ kindling in SD male rats,in order to establish the acute epileptic seizure model of rats.Killed the rats immediately while the rats appeared and reached the level of IV-V,cutted the heads of the rats and took out the hippocampuses.The other groups of the rats were dealed with respectively by the same way after the rats appeared and reached the level of IV-V symptoms of epileptic seizure for five minutes、ten minutes、fifteen minutes、twenty minutes、twenty-five minutes、thirty minutes.The contents of Glu and GABA in the hippocampus were detected with high performance liquid chromatography(HPLC).The normal control group of the rats were established via normal saline hypodermic injection.Result: Compared to the normal control group,the levels of Glu in the hippocampal tissues in the 0 min group、5min group、10min group、15min group、20min group and 25 min group were higher(P<0.05)when the epileptic seizure reached the level of IV-V,and the content of Glu in 25 min group rised obviously,it reached the peak(P<0.05).The content of Glu was not significantly different between the 30 min group and the normal control group(P>0.05).The level of GABA rised significantly in 5minutes(P<0.05)and began to decline after 5minutes,it came to the minimum value at 10 minutes,but it began to lift at 15 minutes,until 25 minutes,the content of GABA arrived the peak(P<0.05),and it declined after 25 minutes.Conclusion: The research reveals that there is a rule in the contents of Glu and GABA which changes with the time after epileptic seizure happens,and the balance between Glu and GABA is one of the reasons which result in the termination of epilepsy,the changes of the contents of Glu and GABA are significant in terminating the happening of epilepsy. |
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