Jones Paul Alexander, May Gillian Ruth, McLuckie Joyee Ann, Iwashita Akinori, Sharkey John
Fujisawa Institute of Neuroscience, Division of Neuroscience, University of Edinburgh, 1 George Square, Edinburgh, UK.
Cell Res. 2004 Jun;14(3):241-50. doi: 10.1038/sj.cr.7290225.
Characterising the mechanisms of cell death following focal cerebral ischaemia has been hampered by a lack of an in vitro assay emulating both the apoptotic and necrotic features observed in vivo. The present study systematically characterised oxygen-glucose-deprivation (OGD) in primary rat cortical neurones to establish a reproducible model with components of both cell-death endpoints. OGD induced a time-dependent reduction in cell viability, with 80% cell death occurring 24 h after 3 h exposure to 0% O2 and 0.5 mM glucose. Indicative of a necrotic component to OGD-induced cell death, N-methyl-D-aspartate (NMDA) receptor inhibition with MK-801 attenuated neuronal loss by 60%. The lack of protection by the caspase inhibitors DEVD-CHO and z-VAD-fmk suggested that under these conditions neurones did not die by an apoptotic mechanism. Moderating the severity of the insult by decreasing OGD exposure to 60 min did not reduce the amount of necrosis, but did induce a small degree of apoptosis (a slight reduction in cell death was observed in the presence of 10 uM DEVD-CHO). In separate experiments purported to enhance the apoptotic component, cells were gradually deprived of O2, exposed to 4% O2 (as opposed to 0%) during the OGD period, or maintained in serum-containing media throughout. While NMDA receptor antagonism significantly reduced cortical cell death under all conditions, a caspase-inhibitor sensitive component of cell death was not uncovered. These studies suggest that OGD of cultured cortical cells models the excitotoxic, but not the apoptotic component of cell death observed in vivo.
由于缺乏一种能够模拟体内观察到的凋亡和坏死特征的体外检测方法，局灶性脑缺血后细胞死亡机制的研究受到了阻碍。本研究系统地对原代大鼠皮质神经元中的氧-葡萄糖剥夺（OGD）进行了表征，以建立一个具有两种细胞死亡终点成分的可重复模型。OGD诱导细胞活力随时间下降，在暴露于0%氧气和0.5 mM葡萄糖3小时后24小时，80%的细胞死亡。用MK-801抑制N-甲基-D-天冬氨酸（NMDA）受体可使神经元损失减少60%，这表明OGD诱导的细胞死亡有坏死成分。半胱天冬酶抑制剂DEVD-CHO和z-VAD-fmk缺乏保护作用，表明在这些条件下神经元不是通过凋亡机制死亡。将OGD暴露时间减少至60分钟以减轻损伤的严重程度，并没有减少坏死的数量，但确实诱导了轻微程度的凋亡（在存在10 μM DEVD-CHO的情况下观察到细胞死亡略有减少）。在旨在增强凋亡成分的单独实验中，细胞逐渐被剥夺氧气，在OGD期间暴露于4%氧气（而不是0%），或在整个过程中维持在含血清培养基中。虽然NMDA受体拮抗在所有条件下都显著减少了皮质细胞死亡，但未发现细胞死亡的半胱天冬酶抑制剂敏感成分。这些研究表明，培养的皮质细胞的OGD模拟了体内观察到的细胞死亡的兴奋毒性成分，但不是凋亡成分。
