Activation of nuclear factor-kappaB in the rat brain after transient focal ischemia.
Nuclear factor-kappaB (NF-kappaB) becomes activated under inflammatory conditions and triggers induction of gene expression. Here, activation of NF-kappaB was studied after transient middle cerebral artery occlusion in the rat. Expression of p65 and p50, protein subunits of NF-kappaB, was examined by Western blotting, and immunohistochemistry for p65 was carried out. Double-labelling with specific markers for astroglia and microglia was used for cell type identification. Neurons located within and surrounding the ischemic core were identified during the first 24 h post-ischemia by using an antibody against 72-kDa heat shock protein. NF-kappaB binding activity was evaluated at different times post-ischemia with electrophoretic mobility gel shift assays. The results showed constitutive expression of p65 and p50, and NF-kappaB binding activity. Basal p65 was seen in certain neurons and resting astrocytes. Constitutive NF-kappaB binding activity was attributable to one main protein complex possibly formed in neurons and astrocytes, although two minor complexes were also detected. At 1 day post-ischemia selective induction of p65 was seen in neurons located in a penumbra-like area. At this time, however, no disturbances of basal NF-kappaB binding activity were found. Western blotting showed delayed induction of p65 several days after ischemia, whereas no changes were detected for p50. From 4 days post-ischemia, a substantial increase in the amount of p65 was detected due to induction in reactive astrocytes and microglia/macrophages. This was correlated with a robust enhancement of NF-kappaB binding activity with formation of three major specific complexes binding DNA. It is proposed that the highly inducible NF-kappaB complexes resulted from induction of p65 and activation of NF-kappaB in post-ischemic reactive glia.
Gabriel
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C
Departament of Farmacologia i Toxicologia, IIBB, CSIC-IDIBAPS, Jordi Girona 18-26, 08034, Barcelona, Spain.
Justicia
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C
Camins
A
A
Planas
A M
AM
D000818:Animals; D001157:Arterial Occlusive Diseases / Q000503:physiopathology; D015153:Blotting, Western; D001923:Brain Chemistry / Q000502:physiology*; D002545:Brain Ischemia / Q000503:physiopathology*; D002561:Cerebrovascular Disorders / Q000503:physiopathology; D017931:DNA Primers; D005786:Gene Expression Regulation / Q000502:physiology; D007150:Immunohistochemistry; D008297:Male; D016328:NF-kappa B / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D009457:Neuroglia / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley
D017931:DNA Primers; D016328:NF-kappa B