| IntroductionCentral nervous system (CNS) has a complicated feedback mechanism between excitatory and inhibitory to maintain the stability of neuron membrane.γ-Aminobutyric acid(GABA) and glutamate(GLU) are thought to be the most important inhibitory and excitatory amino acid neurotransmitters in the mammal.Glutamic acid decarboxylase decarboxylase(GAD) is the key enzyme for the conversion of glutamic acid into gamma aminobutyric acid(GABA),the main inhibitory neurotransmitter in the central nervous system(CNS).GABA content exceed 40%,the seizure occurred.Spontaneously epileptic rat (SER), is a homozygous double mutant obtained by mating heterozygous Tremor rats (tm) (tm/+) and homozygous Zitter rats (zi) (zi/zi). SER exhibits spontaneous tonic convulsions and absence-like seizures after the age of 8 weeks. The profiles of conventional antiepileptic drugs in SER are quite similar to the efficacy profile in human epilepsy. They are an. ideal model for researching human inheritant epilepsy.Therefore, as parental generation of SER,the Tremor rat also had high research value.This research compared the content change of two kinds of amino acid between Tremor rats and Wistar rats.also,this study investigated the mRNA and protein expressions of GAD65 and GAD67 hippocampus of Tremor rats in order to reveal the probable pathogenesis revolved in epilepsy.Materials and Methods 1,Animals and materialsTremor rats and normal Wistar rats at the age of 13-15 weeks were housed in individual cages under a controlled environment (12:12 hlight/dark cycle,50-70% humidity,24℃). Food and water were available ad libitum. Each group includes 5 rats. the GAD65 and GAD67 antibodies were purchased from Santa.OPA(Amresco,USA),GLU,GAB A standard (sigma)2,Determination of glutamate and GABA inTremor rat by HPLCTremor rat was anesthetize with ethylether,,decollated,exfoliated the hippocampus, low-temperature homoenate, took supernatant fluid after centrifugated,then determinate the content of GLU and GABA in Tremor rat by HPLC after reacted with OPA mixture.3,RT-PCR analyzes the expression ofGAD65,GAD67 mRNATotal RNAs of rat's hippocampus were prepared with the method of TRIzol, RT-PCR was taken according to instruction. After 1.5% agarose gel electrophoresis, the results were analyzed by FluorChemTM maging System.4,Western blotting analyzes the expression of GAD65,GAD67 proteinTotal proteins of rat's hippocampus were prepared. Quantitate the protein with Bradford method. After 10% SDS-PAGE gel electrophoresis, antibodies incubated, immunoreactive bands were visualized using ECL.5,Statistical analysisMean and standard deviation were calculated for all measurements. Student's t-test was performed to determine statistical significance, which was evaluated at P<0.01.Results1,The HPLC result shows:GLU content in hippocampus of Tremor rat is 2242.0±100.1μg·g-1, GAB A content in hippocampus of Tremor is 530.8±28.4μg·g-1;GLU content in hippocampus of Wistar rat is 1801.5±37.1μg·g-1, GABA content in hippocampus of Wistar rat is 420.9±17.9μg·g-1,compare with Wistar rat the GLU and GABA content in Tremor rat are apparently higher. There were significant difference of content expression of GLU and GABA in hippocampus between Tremor rats and Wistar rats (P<0.01).2,RT-PCR result shows:There were no significant difference of GAD65 and GAD67 mRNA expression in hippocampus between Tremor rats and Wistar rats (P>0.05).3,Western blotting result shows:There were no significant difference of GAD65 and GAD67 protein expression in hippocampus between Tremor rats and Wistar rats(P>0.05).Conclusion1,Compare with Wistar rat the GLU content in Tremor rat is apparently higher, the increase of GLU may be one reason duing the epilepsy course.2,The increased GABA in hippocampus of Tremor rat may be a compensatory neuroprotective mechanism in our body when seizure happends.3,There were no significant difference of GAD65 and GAD67 protein expression in hippocampus between Tremor rats and Wistar rats (P>0.05).
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