Oxidative Damage to the c‐fos Gene and Reduction of Its Transcription After Focal Cerebral Ischemia

Abstract

Abstract:We investigated oxidative damage to the c-fos gene and to its transcription in the brain of Long-Evans rats using a transient focal cerebral ischemia and reperfusion (FCIR) model. We observed a significant ( p Ͻ 0.001) increase in the immunoreactivity to 8-hydroxy-2Ј-guanine (oh8G) and its deoxy form (oh8dG) in the ischemic cortex at 0 -30 min of reperfusion in all 27 animals treated with 15-90 min of ischemia. Treatment with a neuronal nitric oxide synthase (nNOS) inhibitor, 3-bromo-7-nitroindazole (60 mg/ kg, i.p.), abolished the majority but not all of the oh8G/ oh8dG immunoreactivity. Treatment with RNase A reduced the oh8G immunoreactivity, suggesting that RNA may be targeted. This observation was further supported by decreased levels of mRNA transcripts of the c-fos and actin genes in the ischemic core within 30 min of reperfusion using in situ hybridization. The reduction in mRNA transcription occurred at a time when nuclear gene damage, detected as sensitive sites to Escherichia coli Fpg protein in the transcribed strand of the c-fos gene, was increased 13-fold ( p Ͻ 0.01). Our results suggest that inhibiting nNOS partially attenuates FCIR-induced oxidative damage and that nNOS or other mechanisms induce nuclear gene damage that interferes with gene transcription in the brain. Key Words: Stroke-DNA repair-Oxidative stress-Gene expression-Neuroregeneration-Apoptosis. J. Neurochem. 73, 1164Neurochem. 73, -1174Neurochem. 73, (1999.An elevated sensitivity to oxidative stress in certain populations of brain cells may be a key mechanism underlying several neurological disorders (Liu et al., 1989;Jenner et al., 1992;Neve, 1996;Parshad et al., 1996;Kisby et al., 1997). Numerous studies have correlated an increase in calcium influx, glutamate, and reactive oxygen species (ROS) with neuronal death. ROS levels are known to be elevated after brain injury of the ischemia-reperfusion type (Hall and Braughler, 1989;Rosenthal et al., 1992;Poiries, 1994;Yoshida et al., 1994;Zhang et al., 1994;O'Neill et al., 1996;Smith et al., 1996) and following administration of excitotoxic drugs (Schulz et al., 1995;Ayata et al., 1997;Back et al., 1998). ROS are known to damage mitochondrial DNA (Driggers et al., 1993;Mecocci et al., 1993;Yakes and Van Houten, 1997;Murakami et al., 1998), nuclear genes (Liu et al., 1996;Taffe et al., 1996;Chen et al., 1997), and RNA (Liu et al., 1996;Nunomura et al., 1999). Damage to nucleic acids may lead to abnormal gene expression and premature neuronal death (Wolozin et al., 1996;Citron et al., 1997;Lamb, 1997;Iadecola et al., 1999).Activation of gene transcription in the ischemic brain following oxidative stress appears to signify a need for gene products to repair the injury. For example, transcription of one of the immediate early genes, the c-fos gene, is activated after head injury Yang et al., 1994). The product of the c-fos transcript then forms activator protein-1, which in turn activates various cellular functions, including the production of growth factors and DNA repair en...

Characterization and Comparison of the NR3A Subunit of the NMDA Receptor in Recombinant Systems and Primary Cortical Neurons

“…In situ hybridizations were performed on mouse brains as previously described (Cui et al 1999) using cRNA encoding the antisense sequence of the unique NR3A-2 exon. Control experiments using the sense sequence probe were performed in parallel and produced no signal.…”

Neuronal NOS inhibitor that reduces oxidative DNA lesions and neuronal sensitivity increases the expression of intact c-fos transcripts after brain injury

“…Antioxidant that specifically inhibits neuronal nitric oxide synthase, 3Br7NI, was injected intraperitoneally in oil at a dose of 25 mg/kg 5 min after vessel occlusion [4,14]. Body temperature was maintained at 37 ± 0.5 ° C during surgery and the postoperative period until the animals recovered fully from anesthesia.…”

“…For in situ hybridization analyses, the animals were perfused with 200 ml each of saline followed by two fixatives in sequence (fixative A: 0.8 g of NaOH, 8 g of paraformaldehyde and 1.64 g of sodium acetate in 200 ml of distilled H 2 O, adjusted with 50% glacial acetic acid to pH 6.5; fixative B: 1.4 g of NaOH, 14 g of paraformaldehyde and 13.35 g of borax, adjusted to pH 9.5 with 50% HC1) and cryoprotected in 20% sucrose in fixative B overnight at 4°C as previously described [3][4][5]. Brain slices were prepared within 24 h and stored at -20°C for no longer than 4 weeks before analysis.…”

Noninvasive delivery of gene targeting probes to live brains for transcription MRI

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