Preconditioning-induced neuroprotection is mediated by reactive oxygen species and activation of the transcription factor nuclear factor-kappaB.

Preconditioning by a sublethal stimulus induces tolerance to a subsequent, otherwise lethal insult and it has been suggested that reactive oxygen species (ROS) are involved in this phenomenon. In the present study, we determined whether preconditioning activates the transcription factor nuclear factor-kappaB (NF-kappaB) and how this activation contributes to preconditioning-induced inhibition of neuronal apoptosis. Preconditioning was performed by incubating mixed cultures of neurons and astrocytes from neonatal rat hippocampus with xanthine/xanthine oxidase or FeSO4 for 15 min followed by 24 h of recovery which protected the neurons against subsequent staurosporine-induced (200 nM, 24 h) apoptosis. The cellular ROS content increased during preconditioning, but returned to basal levels after removal of xanthine/xanthine oxidase or FeSO4. We detected a transient activation of NF-kappaB 4 h after preconditioning as shown by immunocytochemistry, by a decrease in the protein level of IkappaBalpha as well as by electrophoretic mobility shift assay. Preconditioning-mediated neuroprotection was abolished by antioxidants, inhibitors of NF-kappaB activation and cycloheximide suggesting the involvement of ROS, an activation of NF-kappaB and de novo protein synthesis in preconditioning-mediated rescue pathways. Furthermore, preconditioning increased the protein level of Mn-superoxide dismutase which could be blocked by antioxidants, cycloheximide and kappaB decoy DNA. Our data suggest that inhibition of staurosporine-induced neuronal apoptosis by preconditioning with xanthine/xanthine oxidase or FeSO4 involves an activation of NF-kappaB and an increase in the protein level of Mn-superoxide dismutase.

Preconditioning-induced neuroprotection is mediated by reactive oxygen species.

The current study was performed to determine the role of reactive oxygen species (ROS) in preconditioning against different forms of neuronal damage. Primary cultures of chick embryonic neurons were treated with either FeSO(4) (100 microM; 15 min) to generate hydroxyl radicals or xanthine/xanthinoxidase (10 microM/0.5 mU ml(-1); 15 min; =X/XO (pre)) to produce superoxide radicals. Both stimuli moderately enhanced ROS formation as measured by fluorescence microscopy. This preconditioning significantly protected the neurons against subsequent glutamate (1 mM)-induced excitotoxic damage, staurosporine (200 nM)-induced neuronal apoptosis and oxidative damage caused by exposure to xanthine/xanthinoxidase (500 microM/5 mU ml(-1); 1 h; =X/XO (dam)). The antioxidants vitamin E (10 microM) and 2-OH-estradiol (1 microM), present during the 15-min preconditioning period, completely abolished the protective effect of X/XO (pre). Furthermore, glutamate, staurosporine or X/XO (dam) markedly enhanced oxygen radical formation. Preceding preconditioning by mild ROS stimulation with X/XO (pre) or Fe(2+) reduced this oxygen radical burst. Again, the effect of X/XO (pre) could be blocked by coadministration of vitamin E or 2-OH-estradiol. However, the FeSO(4)-mediated preconditioning was not abolished by the radical scavengers. To address this phenomenon, the effect of vitamin E and 2-OH-estradiol on Fe(2+)- and X/XO (pre)-induced ROS formation kinetics within the 15 min of preconditioning was monitored. The moderate rise of intracellular ROS content during preconditioning was only reduced permanently by the antioxidants, when the neurons were treated with X/XO (pre), but not when Fe(2+) was used. Thus, an immediate and constant radical scavenging seems to be indispensable to abolish the ROS-induced neuronal preconditioning. The current results indicate that preconditioning by moderate ROS-stimulation protects cultured neurons against different damaging agents and prevents against the subsequent massive oxygen radical formation.

Neuroprotection by estrogens in a mouse model of focal cerebral ischemia and in cultured neurons: evidence for a receptor-independent antioxidative mechanism.

Estrogens have been suggested for the treatment of neurodegenerative disorders, including stroke, because of their neuroprotective activities against various neurotoxic stimuli such as glutamate, glucose deprivation, iron, or beta-amyloid. Here, the authors report that 17beta-estradiol (0.3 to 30 mg/kg) and 2-OH-estradiol (0.003 to 30 mg/kg) reduced brain tissue damage after permanent occlusion of the middle cerebral artery in male NMRI mice. In vitro, 17beta-estradiol (1 to 10 micromol/L) and 2-OH-estradiol (0.01 to 1 micromol/L) reduced the percentage of damaged chick embryonic neurons treated with FeSO4. In these primary neurons exposed to FeSO4, the authors also found reactive oxygen species to be diminished after treatment with 17beta-estradiol (1 to 10 micromol/L) or 2-OH-estradiol (0.01 to 10 micromol/L), suggesting a strong antioxidant activity of the estrogens that were used. Neither the neuroprotective effect nor the free radical scavenging properties of the estrogens were influenced by the estrogen receptor antagonist tamoxifen. The authors conclude that estrogens protect neurons against damage by radical scavenging rather than through estrogen receptor activation.

Enalapril and moexipril protect from free radical-induced neuronal damage in vitro and reduce ischemic brain injury in mice and rats.

Angiotensin-converting enzyme inhibitors have been demonstrated to protect spontaneously hypertensive rats from cerebral ischemia. The present study investigated the protective effect of enalapril and moexipril in models of permanent focal cerebral ischemia in normotensive mice and rats. To elucidate the mechanism of neuroprotection the influence of these angiotensin-converting enzyme inhibitors on glutamate-, staurosporine- or Fe2+/3+-induced generation of reactive oxygen species and neuronal cell death in primary cultures from chick embryo telencephalons was studied. Treatment with moexipril or enalapril dose-dependently reduced the percentage of damaged neurons, as well as mitochondrial reactive oxygen species generation induced by glutamate, staurosporine or Fe2+/3+. Furthermore, moexipril and enalapril attenuated staurosporine-induced neuronal apoptosis as determined by nuclear staining with Hoechst 33258. In mice, 1 h pretreatment with enalapril (0.03 mg/kg) or moexipril (0.3 mg/kg) significantly reduced brain damage after focal ischemia as compared to control animals. Additionally, moexipril (0.01 mg/kg) was able to reduce the infarct volume in the rat model after focal cerebral ischemia. The results of the present study indicate that the angiotensin-converting enzyme inhibitors enalapril and moexipril promote neuronal survival due to radical scavenging properties.