Stretch injury causes calpain and caspase-3 activation and necrotic and apoptotic cell death in septo-hippocampal cell cultures
Stretch injury causes calpain and caspase-3 activation and necrotic and apoptotic cell death in septo-hippocampal cell cultures
复制标题
DOI：
10.1089/neu.2000.17.283
复制
发表时间：
2000-04-01
影响因子：
4.2
通讯作者：
Hayes, RL
中科院分区：
文献类型：
--
作者：
Pike, BR;Zhao, XR;Hayes, RL
创伤性脑损伤(TBI)导致许多中枢和全身反应，使对原发机械性创伤的影响的解释复杂化。为此，最近发展了几种机械性细胞损伤的体外模型，可以比体内更精确地控制细胞内和细胞外环境。尽管我们最近报道了大鼠颅脑损伤后钙蛋白酶和caspase-3酶的激活，但在任何体外的机械性细胞损伤模型中，还没有检测到钙蛋白酶和/或caspase-3的作用。在这项研究中，我们使用了不同程度的快速机械拉伸来检测隔海马细胞培养中细胞骨架蛋白α-血影蛋白(280 KDa)被Calain处理成145 kDa的标志性片段以及caspase-3将其处理成与凋亡相关的120 kDa的片段。此外，还检测了牵张损伤对细胞存活率和形态的影响。损伤后1h，胞浆乳酸脱氢酶的最大释放量和核内碘化丙啶摄取量的最大值与各个损伤水平下钙蛋白特异的145 kDa片段对α-血影蛋白的峰值积聚有关。钙激活的急性期(1~6h)与核形态改变的亚群有关，表现为坏死性(深染)或凋亡性(核浓缩、缩小)。相反，caspase-3对凋亡相关的120 kDa片段的处理仅在中度损伤后24小时才检测到，但未检测到轻度或严重损伤。Caspase-3激活期主要与细胞核萎缩、碎裂和以细胞凋亡为特征的凋亡体形成有关。这项研究的结果表明，隔-海马区细胞培养的快速机械牵张损伤复制了体内脑损伤中常见的几种重要的生化和形态变化，尽管也注意到了重要的差异。
Traumatic brain injury (TBI) results in numerous central and systemic responses that complicate interpretation of the effects of the primary mechanical trauma. For this reason, several in vitro models of mechanical cell injury have recently been developed that allow more precise control over intra- and extracellular environments than is possible in vivo. Although we recently reported that calpain and caspase-3 proteases are activated after TBI in rats, the role of calpain and/or caspase-3 has not been examined in any in vitro model of mechanical cell injury. In this investigation, varying magnitudes of rapid mechanical cell stretch were used to examine processing of the cytoskeletal protein alpha-spectrin (280 kDa) to a signature 145-kDa fragment by calpain and to the apoptotic-linked 120-kDa fragment by caspase-3 in septo-hippocampal cell cultures. Additionally, effects of stretch injury on cell viability and morphology were assayed. One hour after injury, maximal release of cytosolic lactate dehydrogenase and nuclear propidium iodide uptake were associated with peak accumulations of the calpain-specific 145-kDa fragment to alpha-spectrin at each injury level. The acute period of calpain activation (1-6 h) was associated with subpopulations of nuclear morphological alterations that appeared necrotic (hyperchromatism) or apoptotic (condensed, shrunken nuclei). In contrast, caspase-3 processing of alpha-spectrin to the apoptotic-linked 120-kDa fragment was only detected 24 h after moderate, but not mild or severe injury. The period of caspase-3 activation was predominantly associated with nuclear shrinkage, fragmentation, and apoptotic body formation characteristic of apoptosis. Results of this study indicate that rapid mechanical stretch injury to septo-hippocampal cell cultures replicates several important biochemical and morphological alterations commonly observed in vivo brain injury, although important differences were also noted.