GLOBAL CEREBRAL-ISCHEMIA ACTIVATES NUCLEAR FACTOR-KAPPA-B PRIOR TO EVIDENCE OF DNA FRAGMENTATION

The oxidative stress responsive transcription factor nuclear factor-ka ppa B (NF-kappa B) consists of a p50 (50 kDa) and p65/RelA (65 kDa) co mponent and can be activated in vitro by TNF alpha, IL1 beta, hydrogen peroxide and oxygen radicals. All of the above factors are also known to be elevated at certain times after transient global ischemia. The present study was performed to determine if NF-kappa B was activated i n vivo by transient global forebrain ischemia. Adult male rats were su bjected to 30 min of 4-vessel occlusion (4-VO) and sacrificed at selec ted post-ischemic time points. Levels of NF-kappa B p50 and p65 subuni ts were determined by immunocytochemistry, Western blot and electropho retic mobility-shift analysis. The enhancer complex was also confirmed by immuno-gel-shift analysis. Specific labeling of DNA strand breaks and DNA fragmentation was examined in situ by means of the terminal de oxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) met hod. Western blot analysis of hippocampus showed induction of p50 and p65. A time course of NF-kappa B induction in hippocampus showed a p50 -specific band at 6 h that increased in intensity over 12, 48 h and th en decreased by 96 h post-ischemia. Immunocytochemistry revealed at 24 h post-ischemia that p65 and p50 immunoreactivity was present in neur onal nuclei of hippocampal CA1 neurons as well as all other hippocampa l regions and several other forebrain regions which were not vulnerabl e to transient forebrain ischemia. At 72 h post-ischemia, nuclear NF-k appa B immunoreactivity had disappeared in all brain areas except in h ippocampal CA1 neurons which were degenerating. No evidence for DNA fr agmentation as revealed by TUNEL staining could be observed at 24 h. H owever, at 72 h, hippocampal CA1 neurons were heavily labeled. The res ults of this study demonstrate that global forebrain ischemia causes a transient activation of NF-kappa B in many forebrain regions. NF-kapp a B remains persistently activated in the vulnerable hippocampal CA1 s ector. Because of the persistent activation of NF-kappa B in these neu rons, the possibility exists that NF-kappa B has a role in programmed cell death in hippocampal CA1 neurons.