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.

Figure 1

The ability of E. coli DNA polymerase-I (Kornberg pol-I) to extend synthesis of the 3′-end generated by E. coli Fpg protein. A synthetic DNA sequence of c-fos gene (5′-CATCATGGTCZTGGTTTGGGCA-3′, where Z is the oh8dG) was labeled on the 5′ end using [γ]-³²P-ATP (21), then was hybridized to the complementary strand, which C was opposite Z. The double-stranded DNA (7.5 × 10⁶ cpm/pmol, 100 fmol) was treated with buffer (lanes 1, 2) or with Fpg protein (0.15 μg, lanes 3–10) in 37°C for 10 min, followed by heating at 80°C for 10 min. All of the reaction products were then incubated with dNTP (40 μM) (lanes 1–10) and additional endonuclease-free DNA polymerase-I (2.5 U, two different preparations of Kornberg enzyme [lanes 5, 6, 9, 10] or endonuclease-free Klenow enzyme [lanes 7, 8]) at 37°C for 5 min. The reaction was stopped by heating and then resolved in 10% sequencing PAGE gel to analyze single-strand DNA.

Figure 2

FPGSS in the cortex after FCIR. FPGSS in the left (A) and the right (B) cortices from a Long-Evans rat treated with FCIR (90/15). FPGSS appear as the white fluorescent signal. Bar = 20 μm.

Figure 3

The incorporation of dig-dUTP by Kornberg DNA polymerase-I is dependent on SSB bearing 3′-OH ends. The incorporation of dig-dUTP to 3′-OH termini (white fluores-cent signal) in the sham-operated control brains (one of four is presented here) was tested using only Fpg protein (A), Kornberg DNA polymerase-I (B), or DNase I and then Kornberg DNA polymerase-I (C). The fluorescent signal that can be observed in panels A and B comes mostly from the background of the cytoplasm. Similar results were noted in another set of experiments when Kornberg enzyme was replaced with Klenow or terminal transferase (not shown). Bar = 35 μm.

Figure 4

FPGSS in rat brain after FCI and reperfusion. Four representative ipsilateral cortices from Table 2 show FPGSS in sham-operated animals and animals treated with FCI (90 min) and reperfusion of various time intervals. The tissue in the left panels (A—D) was treated with buffer and the Kornberg polymerase-I only; the tissue in panels E—H was treated with Fpg protein then the Kornberg polymerase-I. The green fluorescent signal indicates the FPGSS and is the strongest at 15 min of reperfusion (90/15) and at 90/30 min of reperfusion. Perinuclear signals (asterisks) are shown in the 90/60 groups. At 30 and 60 min of reperfusion, some signal appears in tissue without Fpg protein (non-FPGSS or SSB/5′DSB, arrows). Bar = 40 μm.

Figure 5

8-OH-G antigen in the cortex after FCI-reperfusion. The 8G-14 IgM antibody was used to demonstrate the presence of oxidative DNA lesions after transient ischemia. The figure shows the right hemisphere surrounding the lateral ventricle at low magnification. The green fluorescence indicates the presence of the FITC-antibody-antigen complex; the background was stained yellow by the fluorescent antifade-mounting medium (Sigma, two particulates are present in the image). The cortical samples are from one of three animals treated with 90 min of FCI and 15 min of reperfusion. A higher magnification is shown in Fig. 6. Bar = 100 μm.

Figure 6

Immunoreactivity of oh8G/oh8dG is stronger in cytosol than in nuclei after FCIR. A higher magnification of typical ipsilateral brain tissue from each group of animals with 90/15 FCIR (A—C, E, F) or without FCIR (D) are shown: A, D—F) frontal cortex; B) parietal cortex; C) corpus callosum. The smaller patch of green signal could be from overlapping astrocytes. The IgM antibody in panels E, F was either preadsorbed with the 8-OH-g-BSA conjugates (50 ng, E) or deleted in the assay (F ), and they serve as the negative controls. The FITC signal was strong enough that preadsorption did not completely eliminate the green FITC signal in panel E. Arrows show signals in the cytoplasm and perhaps nuclei such that the intensity of yellow antifade was reduced. Bar = 10 μm.

Figure 7

Specificity of 8G-14-antibody binding for the oh8G-BSA conjugate using radioimmunoassay (see Materials and Methods). Four determinations in two separate experiments were performed. The mean (means) and sd (bars) at each dilution are shown.

Figure 8

The immunoreactivity of oh8dG (IgM-8G-14) remains visible after RNase treatment. A typical image of the right cerebral cortices from one of three animals underwent FCIR (60/0) and RNase A (25 μg/ml, 30 min at room temperature, bottom panel) treatment before the addition of the 8G-14 IgM. The green fluorescent signal was visible in area surrounding the nuclei.

Figure 9

Immunoreactivity of oh8dG using commercially available IgG and pretreatment of RNase A. The FCIR cortex after 60 min of FCI (n=3) is shown. Tissue was pretreated with RNase A before the IgG antibody against oh8G/oh8dG. Arrow shows strong oh8dG-immunoreactivity in the location where GFAP-positive cells occupied (see Fig. 11). Stars show the rims around the nuclei, a possible indication of mitochondrial DNA damage (see Fig. 8). Bar = 65 μm.

Figure 10

GFAP-positive and GFAP-negative cells contain oh8dG-immunoreactivity. This figure shows the double-stain for GFAP- (top panels) and oh8dG- (bottom panels) immunoreactivity. Arrows indicate the location of GFAP-positive cells; asterisks indicate location of GFAP-negative cells. A) sham-operated control; B) 90/01 of FCIR. Bar = 10 μm.

Figure 11

TUNEL-positive stain after FCIR. Coronal sections with HE stain (A) or with double staining for TUNEL and GFAP immunoreactivity (B—D) in the right cerebral cortex (the ischemic core and border area surrounding the core) from one of four typical animals with 4 day reperfusion after 30 min FCIR. Green fluorescent: TUNEL staining; red fluorescent: astrocytes. Asterisks: neurons at different magnifications; arrows: GFPA-postive cells in area where oh8dG- (FPGSS-)positive cells were observed (Fig. 9, 4G). Bar = 50 μm (A, B) or 10 μm (C, D).