| BackgroundHypoxia, i.e., decrease in oxygen availability, affects a number of cellular processes, including the release of various kinds of transmitters and messenger molecules from neuronal cells. It is well known that PAF, a membrane lipid derivative molecule, is increased duing ischemia and hypoxia, which stimulates the further release of glutamate at presynaptic endings. Ginkgolide B, one of the component of Ginkgo Biloba Leaves, is a potent PAF receptor antagonist. Its neuroprotective effect may be due, at least in part, to interact with presynaptic binding sites and inhibit glutamate and other transmitters release. Numerous in vivo studies have shown that Ginkgolide B protect neuronal damage following hypoxia, but there is few evidence showing that PAF receptor antagonists have direct neuroprotective actions. On the other hand, central to the idea of the neurotoxic effects of transmitters is excessively high extracellular concentrations of neurotransmitters, including amino acids, cause tissue damage by excessive excitation. Total tissue content, information coming from majority of the current in vivo studies, is therefore a poor index of extracellular concentration.Objectivesl.to examine how hypoxia affect hippocampal transmitter release;2.to attempt to obtain information on the extracellular concentrations of transmitters releasing from hypoxic hippocampus;3.to assess the importance of extracellular calcium ion in endogenous release from hippocampus when exposed to hypoxia;4.to explore whether Ginkgolide B affect the release of transmitters from hypoxic hippocampal slices, providing an direct evidence of its neuroprotective effects.Methodsin vitro hypoxia model was used to evaluate the effect of hypoxic insult on neuronal function in the present study. rp-HPLC coupled with an UV detector and pie-column derivation of OPA techniques were used to assay amino acid neurotransmitters. Altogether 8 amino acids were separated by baseline within 20 min of retention time. They are asp, glu, asn, gin, gly, tau, GABA, and ala. The separation and determination of dopamine and norepinephrine were examined by rp-HPLC with an electrochemical detector.Group division: Experiments were performed in 3 different conditions, i.e., normal4medium, high K* stimulated medium, and Ca2+-free medium. Hippocampal slices from a same rat were then divided into 3 treatment groups: control group梥lices were oxygenated with 95%O2/5%CC>2 and incubated at 35~36癈 for 30min; hypoxia group梥lices were oxygenated and incubated on the same conditions as the control group, then the mixture gases change into 95%O2/5%CO2- Hypoxic condition were remained for lOmin; Ginkgolide B preincubation group梥lices will be preincubated at the normal oxygenated medium containing lOOmM Ginkgolide B, 10 min later, repeated hypoxic treatment. More KC1 was added into medium to raise the K* concentration from 3.5 mM to 50 mM, and group divisions and treatments are as same as the unstimulated incubations. Ca2+-free medium were the same as the other conditions. Altogether 9 different conditioning experiments were performed.Data analysis: SPSS 10.0 and SAS6.12 were used. The total significant differences among groups were compared by two way ANOVA, factoring treatment group and incubation medium level. Post hoc testing were used to evaluate the significance of subgroup differences by LSD and SNK methods, significant correlation between every two transmitters was analyzed by Pearson Correlation. Alpha equals to 0.05, two tailed.Resultsl.Our results demonstrated that high K+ stimulated incubation increased most transmitter release from hippocampal slices except taurine, of which glutamate and GAB A reached the highest level (J><0.01).2.Gln, tau, and gly effluxes from rat hippocampal slices are calcium-dependent under normal oxygen supply condition, while the other amino acids release are calcium-independent3.10 min of hypoxia treatment increased amino acids release in normal aCSF medium except gin and ala. Glu and GABA...
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