Möckel V, Fischer G
Pharma Division, Preclinical Research, F. Hoffmann-La Roche, Basel, Switzerland.
Brain Res. 1994 Jun 13;648(1):109-20. doi: 10.1016/0006-8993(94)91911-9.
Rat embryonic hippocampal neurons cultured on astrocyte feeder-layers were sensitive to different excitotoxic stimuli after 10-12 DIV. Almost all neurons (approximately 95%) died within 20 h after a transient exposure for 10 min to 50 microM glutamate, a continuous exposure to either 25 microM NMDA or 250 microM kainate or after a 15-min deprivation of glucose and oxygen. Dizocilpine at 10 microM protected neurons against the glutamate- and NMDA-mediated toxicity as well as against 30 min glucose and oxygen deprivation. However, it failed to protect against kainate toxicity and prolonged glucose/oxygen deprivation (60 min). An additional treatment with CNQX (100 microM) protected neurons even under the latter two conditions. This indicates that the vast majority of neurons was sensitive to different excitotoxic stimuli acting through different types of glutamate receptors leading to calcium overload of the cells which might be the common denominator of triggering cell death under these conditions. Expression of calcium-binding proteins, such as calbindin D28K or calretinin, might increase the intracellular calcium buffer capacity of neurons, thus, rendering them more resistant to calcium overload. Therefore, we analysed whether neurons expressing these calcium-binding proteins would survive these toxic stimuli. Indeed, a small population of the neurons (3-5%) survived, including a subpopulation of calretinin-positive but not calbindin D28K-positive neurons. This implies that the expression of calcium-binding proteins per se does not render neurons more resistant towards these excitotoxic stimuli. Moreover, most of the surviving calretinin-positive neurons showed morphological damage as indicated by loss of neurites. When cytotoxicity due to calcium overload was induced by an exposure of the cells to the calcium ionophore 4-bromo-A23187 rather than by activation of glutamate receptors, calretinin-positive cells were found not to be significantly more resistant than the vast majority of neurons. This may indicate that the lower sensitivity of a subpopulation of calretinin-positive neurons to excitotoxic stimuli may be due to a lower expression of glutamate receptors.
在星形胶质细胞饲养层上培养的大鼠胚胎海马神经元在培养10 - 12天后对不同的兴奋性毒性刺激敏感。短暂暴露于50微摩尔谷氨酸10分钟、持续暴露于25微摩尔NMDA或250微摩尔海人酸10分钟或在葡萄糖和氧气剥夺15分钟后，几乎所有神经元（约95%）在20小时内死亡。10微摩尔的地佐环平可保护神经元免受谷氨酸和NMDA介导的毒性以及30分钟的葡萄糖和氧气剥夺。然而，它不能保护神经元免受海人酸毒性以及延长的葡萄糖/氧气剥夺（60分钟）。即使在后面两种情况下，额外用CNQX（100微摩尔）处理也能保护神经元。这表明绝大多数神经元对通过不同类型谷氨酸受体起作用的不同兴奋性毒性刺激敏感，导致细胞钙超载，这可能是在这些条件下触发细胞死亡的共同因素。钙结合蛋白如钙结合蛋白D28K或钙视网膜蛋白的表达可能会增加神经元的细胞内钙缓冲能力，从而使它们对钙超载更具抗性。因此，我们分析了表达这些钙结合蛋白的神经元是否能在这些毒性刺激下存活。确实，一小部分神经元（3 - 5%）存活下来，包括一群钙视网膜蛋白阳性但钙结合蛋白D28K阴性的神经元。这意味着钙结合蛋白本身的表达并不能使神经元对这些兴奋性毒性刺激更具抗性。此外，大多数存活的钙视网膜蛋白阳性神经元表现出形态损伤，如神经突丧失。当通过将细胞暴露于钙离子载体4 - 溴 - A23187而非激活谷氨酸受体来诱导因钙超载引起的细胞毒性时，发现钙视网膜蛋白阳性细胞并不比绝大多数神经元更具抗性。这可能表明钙视网膜蛋白阳性神经元亚群对兴奋性毒性刺激的较低敏感性可能是由于谷氨酸受体表达较低。
