Establishment Of Primary Cultured Human Embryo Neuron Anoxia Model & Preliminary Research Of Naloxone On Ischemic Neuron Injury In Vitro

Objects: To establish an in vitro ischemic model of primary cultured human embryo cerebral neuron and using it to study the cellular and molecular mechanism of naloxone on neuron anoxia injury. Materials & methods: The mixed cell culture riching in neurons were deprived both oxygen and glucose using common incubator and self-made tightly closed container. The pH, PCO2, PO2 of supernatant culture medium were measured at different time pre and post anoxia. Using MTT assay, NSE ELISA, HPLC and RT-PCR methods to study the changes of lactate dehydrogenase(LDH), neuro-specific enolase(NSE) and glutamate concentrations and the expression of NSE, SCF/c-kitR of anoxia neurons and neurons pretreated with different concentrations of naloxone. Results : (D PO2 decreased to 29.4-34. lmmHg(P<0.01) while pH has no significant changes. PCO2 increased significantly(P<0.05), but still within normal range(38.6~43.2mmHg). (2) One hour of oxygen-glucose deprivation was associated with a large increase in extracellular LDH, NSE and glutamate concentrations and a significantly decrease of cell vitality(P<0.01). Although the 3 or 5 hr groups had also increased significantly(P<0.01), but there's no differences among these three time groups(P>0.05). 〢s for the one-hour oxygen-glucose deprivation group, the extracellular concentration of LDH, NSE and glutamatedecreased significantly with the increase of pretreated concentration of naloxone, while the intracellular NSE and cell vitality presented adverse changes梚ncreased with the concentration of pretreated naloxone. Until the maximum concentration of naloxone(10u g/ml), all of these indexes went back to control level. ㊣mmediately after slight anoxia for 30min, the expression of NSE and SCF/c-kit of neurons increased significantly(P<0.01). Among those, the expression of SCF presented a double-peak value within 24hr. After pretreated with different concentrations of naloxone, the peak value of each group were delayed to appear and went down with the increase of naloxone concentration. All of these had significantly differences with control group(P<0.01). Conclusion: The results indicates that with these methods of cell culture and oxygen-glucose deprivation for one hour, an in vitro neuron injury model is available. It can served as a simplified model which may be very useful in elucidating cellular and molecular mechanisms involved in cerebral ischemia. These observations suggest that naloxone plays a protective role on neuron ischemia which may be related to the inhibition of excitotoxicity of excitatory amino acids and regulation of multi-protective factors expression.