Tumor necrosis factor alpha (TNFα) plays a key role in pathogenesis of brain injury. However, TNFα exhibits no cytotoxicity in primary cultures of brain cells. This discrepancy suggests that other pathogenic stimuli that exist in the setting of brain injury precipitate TNFα cytotoxicity. The hypothesis was tested that reactive oxygen species (ROS), that are released early after brain injury, act synergistically with TNFα in causing cell death.

Cultured human and rat brain capillary endothelial cells (RBEC), and cortical astrocytes were treated with TNFα alone or together with different doses of H₂O₂, and apoptotic cell death and DNA fragmentation were measured by means of 3′-OH-terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Hoechst fluorescence assay, respectively. The effect of H₂O₂ on TNFα-induced activation of nuclear factor kappa B (NF-κB) was measured by Western blots of cytoplasmic and nuclear extracts of RBEC using anti-inhibitor of NF-κB (IκB) and anti-p65 subunit of NF-κB antibodies. Nuclear translocation of NF-κB was investigated by immunofluorescent staining of RBEC with anti-p65 antibodies.

TNFα alone had no cytotoxic effect in brain endothelial cells and astrocytes at concentrations up to100 ng/ml. Co-treatment with 5–10 µM of H₂O₂ caused a two-fold increase in the number of apoptotic cells 24 hr later. Similar doses (1–3 µM) of H₂O₂ initiated early DNA fragmentation. H₂O₂ inhibited TNFα-induced accumulation of p65 in the nucleus, although it had no effect on degradation of the IκB in cytoplasm. Immunostaining confirmed that H₂O₂ inhibited p65 transport to the nucleus.

Reactive oxygen species could act synergistically with TNFα in causing cytotoxicity via inhibition of a cytoprotective branch of TNFα signaling pathways, which starts with NF-κB activation.