In situ detection of AP sites and DNA strand breaks bearing 3'-phosphate termini in ischemic mouse brain

Abstract

Our aims were to examine whether oxidative DNA damage was elevated in brain cells of male C57BL/6 mice after oxidative stress, and to determine whether neuronal nitric oxide synthase (nNOS) was involved in such damage. Oxidative stress was induced by occluding both common carotid arteries for 90 min, followed by reperfusion. Escherichia coli exonuclease III (Exo III) removes apyrimidinic or apurinic (AP) sites and 3'-phosphate termini in single-strand breaks, and converts these lesions to 3'OH termini. These ExoIII-sensitive sites (EXOSS) can then be postlabeled using digoxigenin-11-dUTP and Klenow DNA polymerase-I, and detected using fluorescein isothiocyanate-IgG against digoxigenin. Compared with the non-ischemia controls, the density of EXOSS-positive cells was elevated at least 20-fold (P < 0.01) at 15 min of reperfusion, and remained elevated for another 30 min. EXOSS mainly occurred in the cell nuclei of the astrocytes and neurons. Signs of cell death were detected at 24 h of reperfusion and occurred mostly in the neurons. Both DNA damage and cell death in the cerebral cortical neurons were abolished by treatment with 3-bromo-7-nitroindazole (30 mg/kg, intraperitoneal), which specifically inhibited nNOS. Our results suggest that nNOS, its activator (calcium), and peroxynitrite exacerbate oxidative DNA damage after brain ischemia.-Huang, D., Shenoy, A., Cui, J., Huang, W., Liu, P. In situ detection of AP sites and DNA strand breaks bearing 3'-phosphate termini in ischemic mouse brain.

Figure 1

In situ EXOSS detection using E. coli ExoIII and Klenow DNA polymerase-I. Cerebral cortices from animals with no FbIR (A), or FbIR of 90/0 (B), 90/15 (C, D). All panels were from samples treated with ExoIII, except panel D, before incorporation of dig-dUTP using Klenow DNA polymerase-I. The green fluorescence represents signs of EXOSS, and the orange signal indicates nuclei as counter-stained using PI. Bar = 50 μm.

Figure 2

Extension synthesis by Klenow DNA polymerase-I on single-stranded DNA breaks. The AP site in ³²P-DS oligomer D was treated with (lanes 2–5) or without (lane 1) E. coli ExoIII (A). The reaction mixtures were then incubated either with buffer (lanes 1–3) or dNTP (40 μM) plus endonuclease-free Klenow DNA polymerase-I (lanes 4 and 5 for two different lots of enzymes) at 37°C for 5 min. The oh8dG removal in ³²P-DS oligomer Z was treated with (lanes 2–6) or without (lane 1) E. coli Fpg protein at 37°C for 10 min (B). The PO₄ terminus in ³²P-DS oligomer Z was incubated with (lanes 3, 5, 6) or without (lanes 1, 2, 4) E. coli Exo III at 37°C for 10 min before primer extension using Klenow DNA polymerase-I. The reaction was stopped by heating and further analyzed in sequencing PAGE (10%). +, Enzyme added, -, no enzyme (buffer only). Note that DNA polymerase-I could not perform primer extension synthesis (lane 4) unless the Fpg-protein products (3′-PO₄ termini) were pretreated with ExoIII (lanes 5 and 6, for two different lots of DNA pol-I).

Figure 3

In situ EXOSS detection using E. coli ExoIII and Klenow DNA polymerase-I. Cerebral cortices from animals as in Fig. 1, but with FbIR 90/30 (A), 90/45 (B), and 90/60 (C). All panels were from samples treated with ExoIII before incorporation of dig-dUTP using Klenow DNA polymerase-I. The animals (90/15 FbIR) in panel D were treated with 3BR7NI after vessel occlusion. The green fluorescent represents EXOSS, and the orange signal indicates nuclei as counter-stained using PI. Bar = 50 μm.

Figure 4

EXOSS in mouse brain after FbIR. Quantitative analysis of cerebral cortical cells (EXOSS-positive per mm²) at 0, 15, 30, 45, and 60 min after 90 min of forebrain ischemia. Intraperitoneal soybean oil control (open bars, n=44 total, see text) or 3BR7NI in oil (closed bars, n=4 for each of 15, 30, and 45 min). ***P < 0.01.

Figure 5

The effect of 3BR7NI (30 mg/kg) on brain NOS activity in cytoplasm from the mouse brain. The mean ± SE of NOS activity (three determinations in each animal) from three or four animals (n) are shown. The time after drug injection is indicated. *p < 0.001.

Figure 6

EXOSS in neurons and astrocytes after FbIR. Coronal sections with double staining for EXOSS (top, green signals) or GFAP (bottom, red signals) immunoreactivity from animals that underwent 90/15 FbIR. Boxes, GFAP-negative (neurons); arrows, GFAP-positive (astrocytes); ARN, arcuate nuclei of the hypothalamus. Bars = 25 μm.

Figure 7

EXOSS in the arcuate nuclei of the hypothalamus after FbIR. The surrounding area of the third cerebral ventricle shows clusters of EXOSS-positive signal (the white signal) in the arcuate nuclei of the hypothalamus (FbIR, 90/15), whereas the adjacent hypothalamus showed less fluorescent signal. Bar = 100 μm.

Figure 8

NADPHd-staining in the arcuate nuclei of the hypothalamus after FbIR. Composite low-magnification image (bar = 200 μm) of NADPHd staining (the dark signals) in the right hemisphere of a mouse brain. A, B) Higher magnification of two types of NADPHd-positive staining (bar = 20 μm). C) Negative staining in the projection of arcuate nuclei of the hypothalamus (bar = 120 μm).

Figure 9

TUNEL-positive staining after FbIR with and without 3BR7NI. Typical TUNEL-positive brain tissue (the white fluorescent signal) in the cerebral cortex 1 day after 90 min of ischemia (FbIR, two magnifications were shown in panels A and B), compared with the same tissue from a no-FbIR control (C) and an FbIR animal with 3BR7NI (D). Because we could not obtain images at the low magnification for the animals with no FbIR and with FbIR + 3BR7NI, we obtained images with a higher magnification for panels B—D. Bar = 100 μm in panel A and 25 μm in panels B—D.

Figure 10

Coronal sections with double staining for TUNEL and GFAP immunoreactivity in the cerebral cortex and the hippocampal formation from animals with 1 reperfusion day after 90 min FbIR (similar to Fig. 8, A & B). Arrows, neurons; arrowheads, astrocytes. Most TUNEL-positive staining was found in non-astrocytes. Bar = 20 μm.