Strong Neuroprotection by Inhibition of NF-B After Neonatal Hypoxia-Ischemia Involves Apoptotic Mechanisms but Is Independent of Cytokines Cora H.A. Nijboer, MSc; Cobi J. Heijnen, PhD; Floris Groenendaal, MD, PhD; Michael J. May, PhD; Frank van Bel, MD, PhD; Annemieke Kavelaars, PhD Background and Purpose—Interactions between excitotoxic, inflammatory, and apoptotic pathways determine outcome in hypoxic-ischemic brain damage. The transcription factor NF-B has been suggested to enhance brain damage via stimulation of cytokine production. There is also evidence that NF-B activity is required for neuronal survival. We used the NF-B inhibitor NBD, coupled to TAT to facilitate cerebral uptake, to determine the neuroprotective capacity of NF-B inhibition in neonatal hypoxia-ischemia (HI) and to identify its contribution to cerebral inflammation and damage. Methods—Brain damage was induced in neonatal rats by unilateral carotid artery occlusion and hypoxia and analyzed immunohistochemically; NF-B 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. Results—Inhibition of cerebral NF-B 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-B 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. Conclusions—Inhibition of NF-B has strong neuroprotective effects that involve downregulation of apoptotic molecules but are independent of inhibition of cytokine production. (Stroke. 2008;39:2129-2137.) Key Words: nuclear factor– kappa-B  inflammation  neonatal  neuroprotection P erinatal cerebral hypoxia-ischemia (HI) is a major cause of neonatal morbidity and mortality. 1 The mechanisms underlying HI brain damage are only partially understood and involve excitotoxicity, apoptosis, and inflammation. To date, effective therapeutic strategies to combat HI brain injury are lacking. 2 Nuclear factor kappa B (NF-B) is a ubiquitously ex- pressed transcription factor that regulates expression of genes involved in inflammation, cell survival, and apoptosis. 3,4 In resting cells, NF-B is sequestered in the cytoplasm by binding to inhibitory IB proteins typified by IB. Signal- induced phosphorylation of IB by a high molecular-weight complex of proteins named the IB-kinase (IKK)-complex is a key step in NF-B activation. The IKK complex consists of 2 kinases, IKK and IKK, and the regulatory protein NEMO (NF-B essential modulator). Phosphorylated IB becomes ubiquitinated and is proteasome-degraded after which free NF-B enters the nucleus to regulate transcription. 4,5 NF-B activation has been described in various in vivo and in vitro models of brain injury (reviewed in 6 ), but its role in cerebral damage is complex as it functions in both protective and damaging pathways. In neurons, NF-B supports sur- vival by increasing the expression of antioxidants, growth factors, and antiapoptotic molecules. However, NF-B also upregulates expression of proapoptotic factors such as p53. 7–9 Reciprocal interaction between proapoptotic activity of p53 and antiapoptotic signals provided by NF-B further compli- cate the prediction of the effect of NF-B activity on brain damage. Finally, glial NF-B activation induces production of proinflammatory cytokines, a pathway proposed to pro- mote neuronal death in vivo. 10 Previous studies investigating the role of NF-B in cere- bral damage have used either nonselective pharmacological NF-B inhibitors or mice harboring targeted deletions of elements of the IKK/NF-B pathway (eg, 11–17 ). The results of the latter studies are conflicting, and the precise effects of Received September 11, 2007; final revision received October 17, 2007; accepted November 22, 2007. From the Laboratory of Psychoneuroimmunology (C.H.A.N., C.J.H., A.K.) and the Department of Neonatology (C.H.A.N., F.G., F.v.B.), University Medical Center Utrecht, The Netherlands; and the Department of Animal Biology (M.J.M.), University of Pennsylvania, School of Veterinary Medicine, Philadelphia. Correspondence to Cobi J. Heijnen, University Medical Center Utrecht, Laboratory of Psychoneuroimmunology, Lundlaan 6, KC 03.068.0, 3584 EA Utrecht, The Netherlands. E-mail C.Heijnen@umcutrecht.nl © 2008 American Heart Association, Inc. Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.107.504175 2129 at Universiteitsbibliotheek Utrecht on October 16, 2015 http://stroke.ahajournals.org/ Downloaded from