Selective Inhibition of Nuclear Factor-κB Activation After Hypoxia/Ischemia in Neonatal Rats Is Not Neuroprotective

Abstract

Activated nuclear factor-B (NFB) has been shown to increase transcription of several genes that could potentially contribute to neuronal damage, such as proinflammatory cytokines, chemokines, and inducible nitric oxide synthase. The aim of our study was to investigate whether inhibition of NFB activation could prevent hypoxia/ischemia (HI)-induced cerebral damage in neonatal rats. We used a cell permeable peptide (NEMO binding domain [NBD] peptide) that is known to prevent the association of the regulatory protein NEMO with IKK, the kinase that activates NFB. Via this mechanism, the NBD peptide can specifically block the activation of NFB, without inhibiting basal NFB activity. Cerebral HI was induced in neonatal rats by occlusion of the right carotid artery followed by 90 min of hypoxia (FiO 2 ϭ 0.08). Immediately upon reoxygenation, as well as 6 and 12 h later, rats were treated with vehicle or NBD peptide (20 mg/kg i.p.). Histologic analysis of brain damage was performed at 6 wk after HI. To assess NFB activation, electromobility shift assays (EMSAs) were performed on brain nuclear extracts obtained 6 h after reoxygenation. Increased NFB activity could be shown at 6 h after HI in both hemispheres. Peripheral administration of NBD peptide prevented this HI-induced increase in NFB activity in both hemispheres. Histologic analysis of long-term cerebral damage revealed that inhibition of NFB activation by administration of NBD peptide at 0, 6, and 12 h after HI resulted in an increment of neuronal damage. In conclusion, our data suggest that inhibition of NFB activation using NBD peptide early after HI increases brain damage in neonatal rats. (Pediatr Res 59: 232-236, 2006) P erinatal HI is a common cause of neonatal mortality and morbidity. Long-term effects can be cerebral palsy and/or impaired neurodevelopmental outcome (1,2). During HI and in particular upon reperfusion and reoxygenation, a cascade of potentially destructive pathways is activated eventually leading to neuronal cell death (3,4).Activation of the transcription factor NFB regulates the production of pro-and anti-inflammatory cytokines, chemokines, and inducible nitric oxide synthase and of adhesion molecules, which together will promote apoptosis and neuronal cell death (5,6). In resting cells, the heterodimeric NFB, consisting of a p50 and a p65 protein, is retained in the cytoplasm through interaction with the inhibitory protein IB. Activation of NFB requires phosphorylation of IB by the I-kinase complex, consisting of IKK␣, IKK␤, and the regulatory protein NEMO. Phosphorylated IB will be ubiquitinated and degraded via the proteasome pathway. The free or activated NFB can then translocate to the nucleus of the cell and will bind to the promoter sequences of many target genes (7).Evidence of a role for NFB activation in HI-induced cerebral damage comes from animal models. In NFB-p50 knockout mice, neuronal damage was significantly reduced in an adult model of stroke by occlusion of the middle cerebral artery (8). Similarly, inhibitio...

Strong Neuroprotection by Inhibition of NF-κB After Neonatal Hypoxia-Ischemia Involves Apoptotic Mechanisms but Is Independent of Cytokines

“…In our previous study using NBD 19 and not TAT-NBD, we did not observe neuroprotection by treatment with NBD at 0, 6, and 12 hours after HI. The most important difference with the present study is that we now administered the peptide at 0 and 3 hours after HI, suggesting that 0-, 6-, and 12-hour treatment may lead to adverse (aspecific) effects.…”

“…at 20 mg/kg. 19 Rats were terminated by 300 mg/kg pentobarbital and perfused with 4% paraformaldehyde in PBS or brains were snap frozen.…”

“…This treatment schedule did not reduce HI-induced brain damage. 19 The aim of the present study was to further explore the possible neuroprotective effect of IKK/NF-B inhibition. In addition, we analyzed the contribution of IKK/NF-B activity to cytokine production and regulation of apoptotic cell death to delineate the role of NF-B in the pathophysiology of HI brain damage.…”

A Dual Role of the NF-κB Pathway in Neonatal Hypoxic-Ischemic Brain Damage

“…at 20 mg/kg. [11][12][13] Rats were terminated by 300 mg/kg pentobarbital and perfused with 4% paraformaldehyde in PBS or were decapitated.…”

“…injection at 0/6/12 hours after HI) did not protect, but rather aggravated, neonatal HI brain damage. 13 The marked difference between the strong protective effect of 0/3-hour TAT-NBD treatment and the aggravating effect of 0/6/12-hour NBD treatment prompted us to further investigate the possible contribution of coupling of the TAT sequence to NBD. In addition, we further investigated the kinetics of NF-B activation and the mechanism of action of TAT-NBD in neonatal HI brain damage.…”