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Recent evidence implicates tumor necrosis factor (TNF), a cytokine with both cytotoxic and cytoprotective activities, in the pathogenesis of cerebral ischemia. The development of TNF cytotoxicity is dependent upon the balance between the activities of intracellular signaling pathways that mediate either apoptotic or anti-apoptotic effects. One critical protective signaling mechanism is the activation of nuclear factor (NF)-kappaB, a ubiquitous transcription factor that regulates expression of anti-apoptotic gene products. Here we show the distribution and kinetics of NF-kappaB activation and the correlation between loss of NF-kappaB activity, TNF up-regulation, and apoptosis in a standardized rat model of focal cerebral ischemia. We observed a rapid and progressive ischemia-induced loss of p65 immunoreactivity within the ischemic core and nearby penumbra. These findings were confirmed by Western blot analysis of nuclear extracts and by electrophoretic mobility shift assay. The anatomical area of suppressed NF-kappaB activity overlapped significantly with the zones of TNF overexpression and apoptosis. Loss of NF-kappaB activity and increased TNF expression preceded the onset of cell death. Direct evidence that loss of NF-kappaB activity can sensitize brain cells to TNF cytotoxicity was obtained in vitro by co-administration of MG-132, an inhibitor of NF-kappaB activation, and TNF to neuronal-like and glial-like cell cultures. Inhibition of NF-kappaB significantly increased the sensitivity of these cultures to TNF cytotoxicity, indicating that the observed loss of neuronal NF-kappaB activity during cerebral ischemia can participate in the development of TNF-induced cytotoxicity.