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

Abstract

In response to oxidative stress, the ischemic brain induces immediate early genes when its nuclear genes contain gene damage. Antioxidant that reduces gene damage also reduces cell death. To study the mechanism of neuronal sensitivity, we investigated the transcription of the c-fos gene after brain injury of the ischemia-reperfusion type using focal cerebral ischemia-reperfusion in Long-Evans hooded rats. We observed a significant (p < 0.01) increase in c-fos mRNA in the ischemic cortex immediately after brain injury. However, the c-fos transcript was sensitive to RNase A protection assay (RPA) upon reperfusion. The transcript became significantly resistant to RPA (42%, p < 0.03) when 3-bromo-7-nitroindazole (25 mg/kg, i.p.), known to abolish nitric oxide, gene damage and neuronal sensitivity, was injected. Our data suggest that neuronal nitric oxide synthase and aberrant mRNA from genes with oxidative damage could be associated with neuronal sensitivity.

Fig. 1

Transcription of c-fos mRNA after 60 min of vessel occlusion in an FCIR model of brain injury using RT-PCR (18 cycles). The primers for c-fos cDNA amplification were: 5′-GCATGGGCTCCCCTGTCAAC-3′ (upstream) and 5′-GCCCAGGTCATTGGGGATC-3′ (downstream). One of the c-fos primers was ³²P labeled as previously described [5, 23]. The left (L or contralateral) and right (R or ipsilateral) cortical RNA was amplified from animals (n = 4 in each lane) with a reperfusion time of 30 min (lane 1), 60 min (lane 2), 90 min (lane 3), 120 min (lane 4), 3 days (lane 5), 2 weeks (lane 6) and no ischemia (lane 7). M = Marker c-fos from a cloned cDNA that was used for in situ RPA.

Fig. 2

Transcription of c-fos mRNA (arrows) after FCIR (a: 60 min of ischemia followed by 30 min of reperfusion, or 60/30, singlegene amplification; b: 60/15, coamplification) using RT-PCR (30 cycles). N = Normal (no FCIR); I = FCIR treated; M = KB DNA size marker.

Fig. 3

Topographical presentation of NGF mRNA expression using in situ RPA. a Expression of FCIR-induced NGF mRNA. b Expression of NGF mRNA in the non-FCIR brain. The expression of intact NGF genes in the ischemic brains was determined in 8 animals. The tissue sections were hybridized to the ³²P-cRNA probe [5], followed by treatments with RNase A. The autoradiogram was developed in the same cassette at -70°C for 2 days. The intensity of mRNA from each sample (resistant to RNase A treatment) was determined and statistically analyzed as described in the text.

Fig. 4

Increased intact c-fos mRNA after treatment with 3Br7NI. The expression of intact c-fos mRNA after FCIR (60/15), using in situ RPA, increased significantly (p < 0.03) in animals treated with 3Br7Nl (n = 6). Oil alone (a) or the drug in oil (b) was injected 5 min after vessel occlusion. Four brain sections (20 μm thick, each separated by 100 μm) per animal were tested. An autoradiogram was developed at the same time at -70 ° C for 7 h. The image on the autoradiogram from each animal was quantitatively measured using Alphalmager 3.2, and the data were analyzed using GraphPad Prism. No signal was detected using the sense mRNA probe. A small effect was observed in the contralateral cortex. The intensity of ischemia-induced intact (RNase A-resistant) c-fos mRNA in the brain using in situ RPA (in pixel values) was 599 ± 49 in animals treated with oil (n = 4) and 843 ± 67 in animals treated with 3Br7NI (c). * p < 0.03 (t test).