title: Oxidative DNA damage precedes DNA fragmentation after experimental stroke in rat brain.
abstract: 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-hydroxy-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 mm(2)). 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 microg/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.
authors.affiliation: Department of Neurosurgery, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: J
authors.initials: J
authors.lastname: Cui
authors.forename: E H
authors.initials: EH
authors.lastname: Holmes
authors.forename: T G
authors.initials: TG
authors.lastname: Greene
authors.forename: P K
authors.initials: PK
authors.lastname: Liu
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism*; D002545:Brain Ischemia / Q000378:metabolism; D004249:DNA Damage; D053938:DNA Fragmentation; D043382:DNA-Formamidopyrimidine Glycosylase; D029968:Escherichia coli Proteins; D008297:Male; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D018384:Oxidative Stress; D051381:Rats; D020318:Rats, Long-Evans; D015427:Reperfusion Injury / Q000378:metabolism; D020521:Stroke / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli