해마 조직 절편 배양을 이용한 무산소 손상에 대한 MK-801, CNQX, Cycloheximide 및 BAPTA-AM의 효과

초록

objective：Glutamate induced excitotoxicity is one of the leading causes of cell death under pathologic condition. However, there is controversy whether excitotoxicity may also participate in the neuronal death under low intensity insult such as simple hypoxia or hypoglycemia. To investigate the role of NMDA receptor in low intensity insult, we chose anoxia as the method of injury and used organotypically cultured hippocampal slice as the material of experiment. Materials & Methods：The hippocampal slices cultured for 2-3 weeks were exposed to 60 minutes of complete oxygen deprivation(anoxia). Neuronal death was assessed with Sytox stain. Corrected optical density of fluorescence in gray scale, used as cellular death indicator, was obtained from pictures taken at 24 and 48 hours following the insult. The well-known in vivo phenomenon of regional difference in susceptibility of hippocampal sub-fields to ischemic insult was reproduced in HOSC(hippocampal organotypic slice culture) by complete oxygen deprivation injury. Results：CA1 was the most vulnerable to complete oxygen deprivation in hippocampus while CA3 was resistant. Oxygen deprivation for 10 and 20 minutes with glucose(6.5g/l) present was insufficient to induce neuronal death in the cultured hippocampal slice. However, after 30 minutes exposure under anoxic condition, neuronal death was able to be detected in the center of CA1 area. The intensity and area of fluorescence indicating cell death correlated with the duration of oxygen deprivation. NMDA receptor and non-NMDA receptor blocking with MK-801(30 & 60μM) and CNQX(100μM) did not provide cellular protection to HOSC against damage induced by oxygen deprivation, but increased intracellular calcium buffering capacity with BAPTA-AM(10μM) was effective in preventing neuronal death (p＝0.01, Student’s t-test). Cycloheximide(1μg/ml, 10μg/ml) provided no protection to HOSC against insult of complete oxygen deprivation for 60 minutes and combined therapy of MK-801(30 & 60μM) and cycloheximide(1 & 10μg/ml) was also ineffective in preventing neuronal death. Conclusion：The results of this study show that the another mechanism not associated with glutamate receptor(NMDA & non NMDA) may play major role in cell death mechanisms induced by complete oxygen deprivation and increased intracellular calcium during anoxia may participate in the neuronal death mechanism of oxygen deprivation. Further investigation of the calcium entry channel activated during oxygen deprivation is necessary to understand the neuronal death of anoxia.

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