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We evaluated the expression of DNA repair proteins, redox factor-1 (Ref-1) and X-ray repair cross-complementing protein 1 (XRCC1), relevant to neurodegeneration following kainic acid-induced seizures in rats. Neurons with oxidative DNA damage exhibited increased expression and colocalization of Ref-1 and XRCC1. Upregulation of DNA repair proteins was also associated with p53 induction and TUNEL. Coexpression of DNA repair proteins and cell death markers following seizures suggests that the DNA repair response may not be sufficient to prevent excitotoxin-induced neurodegeneration.

Activation of the p53-stress response pathway has been implicated in excitotoxic neuronal cell death. Recent studies have demonstrated an age-dependent induction of both p53 mRNA and protein in the rat brain following lithium-pilocarpine-mediated status epilepticus (LPSE). We investigated whether other proteins that have been shown to participate in the p53 cascade are induced by LPSE. We used immunohistochemistry to examine the expression of Mdm2, Bax, CD95/Fas/APO-1, ATM, Ref-1 and ubiquitin. A significant increase in nuclear Mdm2 immunoreactivity, which colocalized with p53, was observed in cells within hippocampal pyramidal cell layers, dentate gyrus, piriform cortex, amygdala and thalamus. Dual immunofluorescence microscopy revealed a reduction in free ubiquitin expression in cells with p53 and Mdm2 accumulation. Increased immunoreactivity for CD95/Fas/APO-1 and Bax was also detected in the same p53-positive cells. Moreover, expression of Ref-1 and ATM, which are involved in the response to oxidative stress-induced DNA damage and regulation of p53 function, were increased. Colocalization of Ref-1 and p53 suggests that Ref-1 might activate p53 function in LPSE-induced neurodegeneration. In contrast, ATM immunoreactivity was predominantly cytoplasmic suggesting that ATM may not directly modulate p53 activity in injured neurons. These results extend our previous observations with regard to activation and stabilization of p53 in injured central nervous system neurons. The data indicate that p53 induction following LPSE may activate downstream pro-apoptotic genes leading to neurodegeneration.

Activation of the tumor suppressor gene, p53, has been strongly implicated in selective neuronal cell death. This study investigated p53 expression in the immature and adult rat brain following status epilepticus induced by the administration of lithium-pilocarpine (LPSE). Both p53 mRNA and protein were examined in relation to neuronal degeneration using in situ hybridization and immunohistochemistry, respectively. Injured cells with eosinophilic cytoplasm with increased p53 mRNA were observed in hippocampal subfields, piriform cortex, amygdala and thalamus. p53 mRNA levels reached a peak by 8 h and returned to baseline by 24 h after the onset of LPSE. The magnitude of p53 mRNA induction was greatest in 21-day-old rats. In contrast to the cellular expression pattern of p53 mRNA, immunohistochemistry demonstrated that p53 protein was increased in all of the eosinophilic cells. Further, double-labeling studies revealed that p53 protein was elevated in neurons that were degenerating. This was supported by colocalization of activated caspase 3 in some cells with damaged DNA. These results provide additional evidence for a critical role for the p53 pathway in excitotoxic neuronal cell death due to status epilepticus.

The mechanisms underlying kainate (KA) neurotoxicity are still not well understood. We previously reported that KA-mediated neuronal damage in organotypic cultures of hippocampal slices was associated with p53 induction. Recently, both bax and caspase-3 have been demonstrated to be key components of the p53-dependent neuronal death pathway. Caspase activation has also been causally related to the release of mitochondrial cytochrome c (Cyto C) in the cytoplasm as a result of the collapse of the mitochondrial membrane potential (Deltapsi(M)) and the opening of mitochondrial permeability transition pores (mPTP). In the present study, we observed a rapid induction of bax in hippocampal slice cultures after KA treatment. In addition, the levels of Cyto C and caspase-3 were increased in the cytosol while the level of the caspase-9 precursor was decreased. There was also a complete reduction of Rhodamine 123 fluorescence after KA treatment, an indication of Deltapsi(M) dissipation. Furthermore, inhibition of mPTP opening by cyclosporin A partially prevented Cyto C release, caspase activation and neuronal death. These data suggest the involvement of bax, several caspases, as well as Cyto C release in KA-elicited neuronal death. Finally, inhibition of caspase-3 activity by z-VAD-fmk only partially protected neurons from KA toxicity, implying that multiple mechanisms may be involved in KA excitotoxicity.

One-sided lateral funiculotomy at the C-2 segment induced axon reaction in the contralateral red nucleus of adult cats. Two to 60 days postoperatively the animals were sacrificed and the mesencephalon was fixed in ethanol-acetic acid, 3:1. Ten micrometer paraffin sections including both red nuclei were stained for RNA with azure B after incubation in DNAse. Cytophotometric measurements of RNA content of neurons from the caudal 600-1000 micrometer of each red nucleus were made with a Zeiss Cytoscan system using an automatic scanning stage. In contrast to the heightened RNA synthesis that has been reported for axotomized peripheral (extrinsic) neurons, the axotomized central (intrinsic) neurons of the red nucleus showed no evidence of accumulation of cytoplasmic or nucleolar RNA. Rather depletion of cellular RNA occurred. Further indication of the regressive nature of rubral axon reaction derived from morphometric measurements that showed cytoplasmic, nuclear and nucleolar atrophy of the neurons of the red nucleus contralateral to operation with the exception of a possible transient cytoplasmic enlargement 9 days postoperatively. From the cytophotometric and morphometric data here reported we are led to suggest that the frequently observed failure of axonal repair in mammalian CNS results from the innately regressive nature of the axon reaction of many mammalian central neurons.