Linnik M D, Zobrist R H, Hatfield M D
Department of Pharmacology, Marion Merrell Dow Research Institute, Kansas City, Mo.
Stroke. 1993 Dec;24(12):2002-8; discussion 2008-9. doi: 10.1161/01.str.24.12.2002.
Cells die by one of two mechanisms, necrosis or programmed cell death. Necrosis has been implicated in stroke and occurs when the cytoplasmic membrane is compromised. Programmed cell death requires protein synthesis and often involves endonucleolytic cleavage of the cellular DNA. We assessed the potential contribution of programmed cell death to ischemia-induced neuronal death.
Cycloheximide (protein synthesis inhibitor; 1 mg/kg per 24 hours) or vehicle (1 mL/kg per 24 hours) was continuously infused into the right cerebral ventricle of spontaneously hypertensive rats. Neocortical focal ischemia was produced by tandem occlusion of the right common carotid artery and the ipsilateral middle cerebral artery. After 24 hours the brain was stained with 2% 2,3,5-triphenyltetrazolium and the ischemic zone quantitated. Protein synthesis was determined by [3H]methionine incorporation into acid-precipitated protein. DNA integrity was determined in isolated DNA by gel electrophoresis and in whole cells by flow cytometry.
Continuous cycloheximide infusion caused approximately 70% reduction in cortical protein synthesis. Cycloheximide also reduced the size of the infarction produced by focal cerebral ischemia when compared with controls (ischemic brain volume, 147.5 +/- 25.9 and 188.7 +/- 16.8 mm3 for cycloheximide and saline, respectively; P < .01), suggesting that protein synthesis may contribute to cell death. Purified DNA from the ischemic zone showed evidence of endonucleolytic degradation when fractionated by gel electrophoresis. Flow cytometric analysis demonstrated increased propidium iodide fluorescence in intact cells isolated from ischemic cortex, indicating an increased accessibility of degraded DNA to the intercalating dye.
New protein synthesis appears to contribute to ischemic cell death in which endonucleolytic DNA degradation is apparent. These observations implicate programmed cell death in ischemic injury and may open unique therapeutic approaches for the preservation of neurons in stroke.
细胞通过坏死或程序性细胞死亡这两种机制之一死亡。坏死与中风有关，当细胞质膜受损时就会发生。程序性细胞死亡需要蛋白质合成，并且通常涉及细胞DNA的内切核酸酶切割。我们评估了程序性细胞死亡对缺血诱导的神经元死亡的潜在作用。
将环己酰亚胺（蛋白质合成抑制剂；每24小时1毫克/千克）或溶剂（每24小时1毫升/千克）持续注入自发性高血压大鼠的右侧脑室。通过结扎右侧颈总动脉和同侧大脑中动脉造成新皮质局灶性缺血。24小时后，用2%的2,3,5-三苯基四氮唑对大脑进行染色，并对缺血区域进行定量。通过[3H]甲硫氨酸掺入酸沉淀蛋白来测定蛋白质合成。通过凝胶电泳在分离的DNA中以及通过流式细胞术在全细胞中测定DNA完整性。
持续注入环己酰亚胺导致皮质蛋白质合成减少约70%。与对照组相比，环己酰亚胺还减小了局灶性脑缺血产生的梗死灶大小（环己酰亚胺组和生理盐水组的缺血脑体积分别为147.5±25.9和188.7±16.8立方毫米；P<0.01），这表明蛋白质合成可能导致细胞死亡。从缺血区域纯化的DNA经凝胶电泳分离后显示出内切核酸酶降解的证据。流式细胞术分析表明，从缺血皮质分离的完整细胞中碘化丙啶荧光增加，表明降解的DNA对嵌入染料的可及性增加。
新的蛋白质合成似乎导致了缺血性细胞死亡，其中内切核酸酶介导的DNA降解很明显。这些观察结果表明程序性细胞死亡参与了缺血性损伤，并可能为中风时神经元的保护开辟独特的治疗途径。
