Strong neuroprotection by inhibition of NF-kappaB after neonatal hypoxia-ischemia involves apoptotic mechanisms but is independent of cytokines.

Abstract

Interactions between excitotoxic, inflammatory, and apoptotic pathways determine outcome in hypoxic-ischemic brain damage. The transcription factor NF-kappaB has been suggested to enhance brain damage via stimulation of cytokine production. There is also evidence that NF-kappaB activity is required for neuronal survival. We used the NF-kappaB inhibitor NBD, coupled to TAT to facilitate cerebral uptake, to determine the neuroprotective capacity of NF-kappaB inhibition in neonatal hypoxia-ischemia (HI) and to identify its contribution to cerebral inflammation and damage. Brain damage was induced in neonatal rats by unilateral carotid artery occlusion and hypoxia and analyzed immunohistochemically; NF-kappaB activity was analyzed by EMSA. We analyzed cytokine mRNA levels and activation of apoptotic pathways by Western blotting. In vitro effects of TAT-NBD were determined in a neuronal cell line. Inhibition of cerebral NF-kappaB activity by TAT-NBD had a significant neuroprotective effect; brain damage was reduced by more than 80% with a therapeutic window of at least 6 hours. In contrast to earlier suggestions, the protective effect of TAT-NBD did not involve suppression of early cytokine upregulation after HI. Moreover, NF-kappaB inhibition prevented HI-induced upregulation and nuclear as well as mitochondrial accumulation of p53, prevented mitochondrial cytochrome-c release and activation of caspase-3. Finally, TAT-NBD could directly increase neuronal survival because TAT-NBD was sufficient to inhibit death in a neuronal cell line. A nonactive mutant peptide did not have any effect. Inhibition of NF-kappaB has strong neuroprotective effects that involve downregulation of apoptotic molecules but are independent of inhibition of cytokine production.