Rapid Nuclear Factor κB Activation and Cytokine and Heme Oxygenase-1 Gene Expression in Edematous White Matter After Porcine Intracerebral Hemorrhage

Abstract

63 Introduction: Lobar intracerebral hemorrhage (ICH) induces secondary events in perihematomal white matter including oxidative stress, edema and demyelination. Pro-inflammatory cytokines may play a role, since tumor necrosis factor-α (TNF-α)and interleukin-1β (IL-1β) induce vasogenic edema and TNF-α induces apoptosis in oligodendrocytes. Also, inhibition of heme oxygenase-1 (HO-1) reduces ICH-induced edema (Wagner et al. 2000). Since the oxidatively sensitive transcription factor, nuclear factor κB(NF-κB), regulates these genes, we hypothesize that NF-κB is activated and these mRNAs are expressed in perihematomal white matter early after ICH. Methods: We infused 2.5 ml of autologous blood into the frontal white matter of pentobarbital anesthetized pigs (N=12)and monitored and controlled physiologic variables. We froze brains in situ between 0.5 and 24 hrs post-ICH. We sampled tissue from perihematomal edematous and similarly located control white matter. Nuclear proteins were extracted for Western blotting (NF-κBp65 subunit) and for electrophoretic mobility shift assays (EMSA) and total RNA was extracted for RT-PCR. We used porcine-specific TNF-α, IL-1β, HO-1 and β-actin primers. Relative changes in densitometric data were normalized to β-actin. Results: NF-κB was activated at 30 min in edematous white matter and remained elevated during the 24 hrs following ICH. NF-κBp65 subunit levels were increased to 160±9% of control (mean±SD, N=3)in nuclear extracts between 0.5 and 2 hrs. A further significant (p<0.01) increase to 233±26% (N=3) occurred between 4 and 24 hrs. Including an inhibitor of NF-κB translocation in the infused blood prevented NF-κBp65 appearance in nuclear extracts. Increased NF-κB DNA binding activity (EMSA)was present at 2 hrs. Expression of TNF-α, IL-1β and HO-1 mRNAs were significantly (p<0.05) upregulated at 2 hrs (N=6) to 640±257%, 649±248% and 236±39% of control, respectively. Conclusion: Early NF-κB activation and cytokine and HO-1 gene expression may underlie delayed edema, DNA fragmentation and demyelination in perihematomal white matter after ICH.