Matsushita K, Meng W, Wang X, Asahi M, Asahi K, Moskowitz M A, Lo E H
Department of Neurology, Harvard Medical School, Massachusetts General Hospital East, Charlestown 02129, USA.
J Cereb Blood Flow Metab. 2000 Feb;20(2):396-404. doi: 10.1097/00004647-200002000-00022.
The overall hypothesis that cell death after intracerebral hemorrhage is mediated in part by apoptotic mechanisms was tested. Intracerebral hemorrhage was induced in rats using stereotactic infusions of 0.5 U of collagenase (1-microL volume) into the striatum. After 24 hours, large numbers of TUNEL-positive stained cells with morphologies suggestive of apoptosis were present in the center and periphery of the hemorrhage. Double staining with Nissl and immunocytochemical labeling with antibodies against neuronal nuclei and glial fibrillary acidic protein suggested that these TUNEL-positive cells were mostly neurons and astrocytes. Electrophoresis of hemorrhagic brain extracts showed evidence of DNA laddering into approximately 200-bp fragments. Western blots showed cleavage of the cytosolic caspase substrate gelsolin. The density of TUNEL-positive cells at 24 and 48 hours after hemorrhage was significantly reduced by treatment with the broad-spectrum caspase inhibitor zVADfmk. It was unlikely that apoptotic changes were due to neurotoxicity of injected collagenase because TUNEL-positive cells and DNA laddering were also obtained in an alternative model of hemorrhage where autologous blood was infused into the striatum. Furthermore, equivalent doses of collagenase did not induce cell death in primary neuronal cultures. These results provide initial evidence that apoptotic mechanisms may mediate some of the injury in brain after intracerebral hemorrhage.
脑出血后细胞死亡部分由凋亡机制介导这一总体假说得到了验证。采用立体定向向大鼠纹状体内注入0.5 U胶原酶（1微升体积）诱导脑出血。24小时后，在出血中心和周边出现大量形态提示凋亡的TUNEL阳性染色细胞。用尼氏染色和针对神经元核及胶质纤维酸性蛋白的抗体进行免疫细胞化学标记双重染色表明，这些TUNEL阳性细胞大多是神经元和星形胶质细胞。出血性脑提取物的电泳显示有DNA梯状条带形成约200 bp的片段。蛋白质免疫印迹显示胞质半胱天冬酶底物凝溶胶蛋白被裂解。用广谱半胱天冬酶抑制剂zVADfmk处理后，出血后24小时和48小时TUNEL阳性细胞的密度显著降低。凋亡变化不太可能是由于注射的胶原酶具有神经毒性，因为在将自体血注入纹状体的另一种脑出血模型中也获得了TUNEL阳性细胞和DNA梯状条带。此外，同等剂量的胶原酶在原代神经元培养物中并未诱导细胞死亡。这些结果提供了初步证据，表明凋亡机制可能介导脑出血后大脑的部分损伤。
