Activation of nuclear factor-kappa B in the rat brain after transient focal ischemia

Nuclear factor-kappaB (NF-kappa B) becomes activated under inflammatory con ditions and triggers induction of gene expression. Here, activation of NF-k appa B was studied after transient middle cerebral artery occlusion in the rat. Expression of p65 and p50, protein subunits of NF-kappa B, was examine d by Western blotting, and immunohistochemistry for p65 was carried out. Do uble-labelling with specific markers for astroglia and microglia was used f or cell type identification. Neurons located within and surrounding the isc hemic core were identified during the first 24 h post-ischemia by using an antibody against 72-kDa heat shock protein. NF-kappa B binding activity was evaluated at different times post-ischemia with electrophoretic mobility g el shift assays. The results showed constitutive expression of p65 and p50, and NF-kappa B binding activity. Basal p65 was seen in certain neurons and resting astrocytes. Constitutive NF-kappa B binding activity was attributa ble to one main protein complex possibly formed in neurons and astrocytes, although two minor complexes were also detected. At 1 day post-ischemia sel ective induction of p65 was seen in neurons located in a penumbra-like area . At this time, however, no disturbances of basal NF-kappa B binding activi ty were found. Western blotting showed delayed induction of p65 several day s after ischemia, whereas no changes were detected for p50. From 4 days pos t-ischemia, a substantial increase in the amount of p65 was detected due to induction in reactive astrocytes and microglia/macrophages. This was corre lated with a robust enhancement of NF-kappa B binding activity with formati on of three major specific complexes binding DNA. It is proposed that the h ighly inducible NF-kappa B complexes resulted from induction of p65 and act ivation of NF-kappa B in post-ischemic reactive glia. (C) 1999 Elsevier Sci ence B.V. All rights reserved.