Pulera M R, Adams L M, Liu H, Santos D G, Nishimura R N, Yang F, Cole G M, Wasterlain C G
Department of Neurology, Veteran's Affairs Medical Center, Sepulveda, Calif., USA.
Stroke. 1998 Dec;29(12):2622-30. doi: 10.1161/01.str.29.12.2622.
The mechanisms of excitotoxic cell death in cerebral ischemia are poorly understood. In addition to necrosis, apoptotic cell death may occur. The purpose of this study was to determine whether an established model of cerebral hypoxia-ischemia in the neonatal rat demonstrates any features of apoptosis.
Seven-day-old neonatal rats underwent bilateral, permanent carotid ligation followed by 1 hour of hypoxia, and their brains were examined 1, 3, and 4 days after hypoxia-ischemia. The severity of ischemic damage was assessed in the dentate gyrus and frontotemporal cortex by light microscopy. Immunocytochemistry was performed to detect the cleavage of actin by caspases, a family of enzymes activated in apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reactivity was examined in the cortical infarction bed and dentate gyrus. Neonatal rat brain DNA was run on agarose gel electrophoresis to detect DNA fragmentation. Ethidium bromide-staining and electron microscopy were used to determine whether apoptotic bodies, 1 of the hallmarks of apoptosis, were present.
The frontotemporal cortex displayed evidence of infarction, and in most rats the dentate gyrus showed selective, delayed neuronal death. Immunocytochemistry demonstrated caspase-related cleavage of actin. TUNEL and DNA electrophoresis provided evidence of DNA fragmentation. Ethidium bromide-staining and electron microscopy confirmed the presence of chromatin condensation and apoptotic bodies.
Features of apoptosis are present in the described model of cerebral hypoxia-ischemia. Apoptosis may represent a mode of ischemic cell death that could be the target of novel treatments that could potentially expand the therapeutic window for stroke.
脑缺血中兴奋性毒性细胞死亡的机制尚不清楚。除坏死外，可能会发生凋亡性细胞死亡。本研究的目的是确定新生大鼠已建立的脑缺氧缺血模型是否表现出凋亡的任何特征。
7日龄新生大鼠双侧永久性结扎颈动脉，随后进行1小时缺氧，在缺氧缺血后1天、3天和4天检查其大脑。通过光学显微镜评估齿状回和额颞叶皮质缺血损伤的严重程度。进行免疫细胞化学检测半胱天冬酶对肌动蛋白的切割，半胱天冬酶是在凋亡中被激活的一类酶。在皮质梗死灶和齿状回检查末端脱氧核苷酸转移酶介导的dUTP生物素缺口末端标记（TUNEL）反应性。将新生大鼠脑DNA进行琼脂糖凝胶电泳以检测DNA片段化。使用溴化乙锭染色和电子显微镜确定是否存在凋亡小体，凋亡小体是凋亡的标志之一。
额颞叶皮质显示梗死证据，在大多数大鼠中齿状回显示出选择性、延迟性神经元死亡。免疫细胞化学显示肌动蛋白的半胱天冬酶相关切割。TUNEL和DNA电泳提供了DNA片段化的证据。溴化乙锭染色和电子显微镜证实存在染色质浓缩和凋亡小体。
在所述的脑缺氧缺血模型中存在凋亡特征。凋亡可能代表一种缺血性细胞死亡模式，可能成为新型治疗的靶点，从而有可能扩大中风的治疗窗口。
