Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain
Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain
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DOI：
10.1096/fasebj.14.7.955
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发表时间：
2000-05
期刊：
影响因子：
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通讯作者：
J. Cui;E. Holmes;Thomas G. Greene;Philip K. Liu
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文献类型：
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作者：
J. Cui;E. Holmes;Thomas G. Greene;Philip K. Liu
在雄性Long-Evans大鼠中，通过双侧闭塞颈总动脉和右侧大脑中动脉30-90分钟，随后进行不同时间的再灌注，使用局灶性脑缺血和再灌注（FCIR）模型诱导实验性卒中。使用大肠杆菌甲酰胺嘧啶DNA N-糖基化酶（Fpg蛋白）敏感位点（FPGSS）证明同侧皮质中的氧化DNA损伤，使用地高辛-dUTP原位标记并使用地高辛抗体检测。由于Fpg蛋白去除了DNA中的8-羟基-2 ′-脱氧鸟嘌呤（oh 8dG）和其他损伤，因此FPGSS可以测量氧化性DNA损伤。假手术对照组皮质中的FPGSS阳性细胞数量为3 ± 3（平均值± sd/mm 2）。在接受90分钟闭塞和15分钟再灌注（FCIR 90/15）的动物中，FPGSS阳性细胞显著增加了200倍。通过使用针对8-羟基-鸟苷（oh 8 G）和oh 8dG的单克隆抗体证实了氧化性DNA损伤。RNA酶A（100 μg/ml）预处理组织减少，但没有消除，oh 8dG信号。FCIR组中FPGSS或oh 8dG阳性的动物数量显著（P<0.01）高于假手术对照组。我们在用90/60的FCIR处理的动物中检测到很少的oh 8dG阳性细胞的FPGSS。在此早期再灌注时间未检测到作为细胞死亡检测的末端UTP缺口末端标记（TUNEL）阳性细胞。我们的数据表明，实验性中风引起的早期氧化DNA损伤可以修复。因此，在脑的核和线粒体DNA中观察到的氧化DNA损伤与使用TUNEL检测到的DNA片段化不同。崔，J.，Holmes，E. H、格林，T. G.，Liu，P. K.大鼠实验性脑卒中后DNA氧化损伤先于DNA断裂
Experimental stroke using a focal cerebral ischemia and reperfusion (FCIR) model was induced in male Long‐Evans rats by a bilateral occlusion of both common carotid arteries and the right middle cerebral artery for 30–90 min, followed by various periods of reperfusion. Oxidative DNA lesions in the ipsilateral cortex were demonstrated using Escherichia coli formamidopyrimidine DNA N‐glycosylase (Fpg protein)‐sensitive sites (FPGSS), as labeled in situ using digoxigenin‐dUTP and detected using antibodies against digoxigenin. Because Fpg protein removes 8‐hy‐droxy‐2′‐deoxyguanine (oh8dG) and other lesions in DNA, FPGSS measure oxidative DNA damage. The number of FPGSS‐positive cells in the cortex from the sham‐operated control group was 3 ± 3 (mean ± sd per mm2). In animals that received 90 min occlusion and 15 min of reperfusion (FCIR 90/15), FPGSS‐positive cells were significantly increased by 200‐fold. Oxidative DNA damage was confirmed by using monoclonal antibodies against 8‐hydroxy‐guanosine (oh8G) and oh8dG. A pretreatment of RNase A (100 μg/ml) to the tissue reduced, but did not abolish, the oh8dG signal. The number of animals with positive FPGSS or oh8dG was significantly (P<0.01) higher in the FCIR group than in the sham‐operated control group. We detected few FPGSS of oh8dG‐positive cells in the animals treated with FCIR of 90/60. No terminal UTP nicked‐end labeling (TUNEL)‐positive cells, as a detection of cell death, were detected at this early reperfusion time. Our data suggest that early oxidative DNA lesions elicited by experimental stroke could be repaired. Therefore, the oxidative DNA lesions observed in the nuclear and mitochondrial DNA of the brain are different from the DNA fragmentation detected using TUNEL.—Cui, J., Holmes, E. H., Greene, T. G., Liu, P. K. Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain.