Holopainen I E, Järvelä J, Lopez-Picon F R, Pelliniemi L J, Kukko-Lukjanov T-K
Department of Pharmacology and Clinical Pharmacology, University of Turku, Itäinen Pitkäkatu 4, FIN-0520 Turku, Finland.
Neurochem Int. 2004 Jul;45(1):1-10. doi: 10.1016/j.neuint.2004.01.005.
Excessive activation of excitatory amino acid receptors has been implicated in neuronal death in a number of central nervous system insults. We have here investigated, the time course and mechanisms of kainate (KA)- induced neuronal death in immature organotypic hippocampal slice cultures (OHCs) using Fluoro-Jade B (FJB) staining as a marker of cell death, and immunoblotting, immunocytochemistry, and electron microscopy as methods to clarify the mechanisms. After 6 KA treatment (5 microM), no significant neuronal death was detected in any hippocampal subregion, whereas the treatment of 12, 24, and 48 h resulted in neuronal death in the CA3 regions, but not in CA1. The 48 h resting period in normal medium after KA-treatment did not rescue the cells but further increased the number of dead neurons in CA3 as compared to the corresponding acute phase. In Western blotting, the expression levels of the active, 17 kDa form of caspase-3, and the 84-85 kDa cleaved fragment of poly(ADP ribose)polymerase (PARP) were not altered from the control levels. Moreover, no active caspase-3 labelled cells were detected in immunocytochemical study 24 h after KA treatment either in the acute or resting groups. Electron microscopy showed non-apoptotic injury in the CA3a/b pyramidal neurons in KA-treated slices. Our results suggest that KA-induced neuronal death in immature OHCs is a strictly region-specific, irreversible, necrotic process.
兴奋性氨基酸受体的过度激活与多种中枢神经系统损伤中的神经元死亡有关。我们在此使用Fluoro-Jade B（FJB）染色作为细胞死亡的标志物，并采用免疫印迹、免疫细胞化学和电子显微镜等方法来阐明机制，研究了在未成熟的器官型海马脑片培养物（OHCs）中，海藻酸（KA）诱导神经元死亡的时间进程和机制。KA处理（5 microM）6小时后，在任何海马亚区均未检测到明显的神经元死亡，而处理12、24和48小时则导致CA3区神经元死亡，但CA1区未出现。KA处理后在正常培养基中48小时的静息期并未挽救细胞，反而与相应的急性期相比，进一步增加了CA3区死亡神经元的数量。在蛋白质免疫印迹分析中，活性17 kDa形式的半胱天冬酶-3以及聚（ADP核糖）聚合酶（PARP）84 - 85 kDa裂解片段的表达水平与对照水平相比没有改变。此外，在KA处理24小时后的免疫细胞化学研究中，无论是急性期还是静息期组，均未检测到活性半胱天冬酶-3标记的细胞。电子显微镜显示KA处理切片中CA3a/b锥体神经元存在非凋亡性损伤。我们的结果表明，KA诱导未成熟OHCs中的神经元死亡是一个严格区域特异性、不可逆的坏死过程。
