INDUCTION OF DNA FRAGMENTATION AFTER 10 TO 120 MINUTES OF FOCAL CEREBRAL-ISCHEMIA IN RATS
INDUCTION OF DNA FRAGMENTATION AFTER 10 TO 120 MINUTES OF FOCAL CEREBRAL-ISCHEMIA IN RATS
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DOI：
10.1161/01.str.26.7.1252
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发表时间：
1995-07-01
期刊：
影响因子：
8.3
通讯作者：
ZALOGA, C
中科院分区：
文献类型：
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作者：
LI, Y;CHOPP, M;ZALOGA, C
背景与目的研究了大鼠大脑中动脉短暂性闭塞不同时间对神经元坏死的诱导作用。本研究的目的是测量显示凋亡小体的细胞数量和解剖分布，作为DNA断裂和凋亡细胞死亡的指示，作为大鼠短暂性MCA闭塞持续时间的函数。方法雄性Wistar大鼠（n=24），分别用单纤管封堵10、20、30、60、90、120 min（每组4例），再灌注48 h。使用末端脱氧核苷酸转移酶（TdT）介导的dutp -生物素缺口末端标记（TUNEL）方法在石蜡切片中测量DNA片段。邻近切片采用苏木精和伊红染色分析缺血细胞损伤，免疫组织化学双染色法进行细胞鉴定。以假手术大鼠（n=4）和未接受任何手术的正常大鼠（n=4）为对照进行细胞凋亡检测。结果在5 μ m厚的冠状切片内，正常、假手术大鼠及缺血大鼠对侧脑半球各有0 ~ 3个凋亡细胞出现DNA片段。MCA闭塞10 ~ 20分钟后，视前区和纹状体选择性神经元坏死区出现DNA断裂（10 ~ 20）的凋亡细胞增多。缺血30 ~ 60分钟后，分散的凋亡细胞（30 ~ 60分钟）出现DNA断裂，并扩展到皮质选择性神经元坏死区域。阻断90 ~ 120分钟后，凋亡细胞组（70 ~ 200，约95%神经元）主要定位于梗死内边界区。结论一系列轻度至重度缺血再灌注刺激可诱导核小体间DNA切割。短暂性脑闭塞后出现DNA断裂的凋亡细胞的存在和解剖位置表明细胞凋亡伴随着神经元坏死。
Background and Purpose The induction of neuronal necrosis has been studied after various durations of transient middle cerebral artery (MCA) occlusion in the rat. The objective of the present study was to measure the numbers and anatomic distribution of cells exhibiting apoptotic bodies as an indication of DNA fragmentation and apoptotic cell death as a function of duration of transient MCA occlusion in the rat.Methods The MCA of male Wistar rats (n=24) was occluded for 10, 20, 30, 60, 90, and 120 minutes (n=4 per group) with the use of an intraluminal monofilament, and reperfusion was instituted for 48 hours. DNA fragmentation was measured in paraffin sections with the use of a terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) method. Adjacent sections were stained with hematoxylin and eosin for analysis of ischemic cell damage, and immunohistochemical double staining methods were used for cell identification. Sham-operated rats (n=4) and normal rats not subjected to any surgical procedure (n=4) were used as controls for apoptosis detection.Results Within 5-mu m-thick coronal sections, DNA fragmentation was present in 0 to 3 apoptotic cells in each hemisphere of normal, sham-operated rats as well as in the contralateral hemisphere of ischemic rats. After 10 to 20 minutes of MCA occlusion, apoptotic cells exhibiting DNA fragmentation (10 to 20) increased in the regions of selective neuronal necrosis in the preoptic area and in the striatum. After 30 to 60 minutes of ischemia, scattered apoptotic cells (30 to 60) exhibited DNA fragmentation and expanded into areas of selective neuronal necrosis in the cortex. After 90 to 120 minutes of occlusion, groups of apoptotic cells (70 to 200, >95% neurons) were primarily localized to the inner boundary zone of the infarct.Conclusions A range of mild to severe ischemia-reperfusion stimuli induce internucleosomal DNA cleavage. The presence and anatomic location of apoptotic cells exhibiting DNA fragmentation after transient cerebral occlusion indicate that apoptosis accompanies neuronal necrosis.