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 i
psilateral cortex were demonstrated using Escherichia coli formamidopyrimid
ine DNA N-glycosylase (Fpg protein)-sensitive sites (FPGSS), as labeled in
situ using digoxigenin-dUTP and detected using antibodies against digoxigen
in. Because Fpg protein removes 8-hydroxy-2'-deoxyguanine (oh8dG) and other
lesions in DNA, FPGSS measure oxidative DNA damage. The number of FPGSS-po
sitive 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 in
creased 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 mu g/ml) to the tissue reduced, but did not abolish, the oh8d
G signal, The number of animals with positive FPGSS or oh8dG was significan
tly (P<0.01) higher in the FCIR group than in the sham-operated control gro
up. We detected few FPGSS of oh8dG-positive cells in the animals treated wi
th FCIR of 90/60, No terminal UTP nicked-end labeling (TUNEL)-positive cell
s, as a detection of cell death, were detected at this early reperfusion ti
me. Our data suggest that early oxidative DNA lesions elicited by experimen
tal stroke could be repaired. Therefore, the oxidative DNA lesions observed
in the nuclear and mitochondrial DNA of the brain are different from the D
NA fragmentation detected using TUNEL.