Neuronal Formation of Free Radicals Plays a Minor Role in Hypoxic Cell Death in Human NT2-N Neurons

Abstract

Free radicals are suggested to play an important role in hypoxic-ischemic neuronal death. However, the importance in human disease is not known. Furthermore, whether posthypoxic free radical formation mainly occurs in endothelium and neutrophils, or whether neuronal production is important, is not finally determined. To study this we differentiated human Ntera2 teratocarcinoma cells into postmitotic NT2-N neurons and exposed them to free radicals, hypoxia, or oxygen and glucose deprivation. These cells are devoid of nitric oxide synthase, and we hypothesized that free radicals are important mediators downstream of N-methyl-D-aspartate stimulation. Production of free radicals, evaluated with the fluorescent dyes dihydrorhodamine and 2',7'-dichlorodihydrofluorescein, was significantly higher in neurons deprived of oxygen and glucose after 40 min of reoxygenation than in normoxic cells. The antioxidant trolox, the flavonoid quercetin, thiopental, and the N-methyl-D-aspartateglutamate receptor antagonist MK-801 reduced the formation of free radicals. Treatment with the flavonoid rutin (86 Ϯ 16% of hypoxic cells without drug, p Ͻ 0.01), trolox (86 Ϯ 20%, p Ͻ 0.01), and MK-801 (57 Ϯ 12%, p Ͻ 0.01) reduced lactate dehydrogenase release after 6 h of hypoxia. Trolox, salicylate, and quercetin also significantly reduced lactate dehydrogenase release after 3 h of oxygen and glucose deprivation. The protection offered by these antioxidants was, however, limited compared with the effect of MK-801. We conclude that oxygen and glucose deprivation causes a moderate increase in the formation of free radicals in NT2-N neurons that can be Several experiments have indicated a role for free radicals in the pathogenesis of hypoxic-ischemic cell death. The xanthine oxidase inhibitor and hydroxyl radical scavenger allopurinol reduces hypoxic-ischemic brain damage in rats (1), and van Bel et al. (2) demonstrated beneficial effects of allopurinol in asphyxiated infants. Infarct size and brain edema after focal cerebral ischemia are attenuated in adult transgenic mice overexpressing CuZn SOD (3). However, brain injury after hypoxia-ischemia was exacerbated in a similar newborn mice model (4).During reperfusion after cerebral ischemia in pigs, superoxide anion is generated (5). Production of free radicals after ischemia has been reported from rat brain microvessels (6) and from fetal bovine endothelial cells (7). A substantial part of posthypoxic free radical formation in endothelium has been attributed to xanthine oxidase (7). On the other hand, ScmidElsaesser et al. (8)

Acidosis has opposite effects on neuronal survival during hypoxia and reoxygenation

“…In the present study, acidosis (during both ischemia and reoxygenation) increased the formation of isoprostanes by 50%. We have previously found that neuronal free radical formation during and after OGD (with neutral medium) plays a minor role in this model (Almaas et al 2002); the current study confirms these findings as there were no significant difference in the levels of isoprostanes between oxygen-and glucose-deprived neurones with neutral medium compared with normoxic cells with neutral medium. The effects of antioxidants were not tested in the current experiments, so whether the acidosis-induced 50% enhancement of free radical formation is an important cause of the deleterious effect of acidosis during reoxygenation has not been settled.…”

“…In the present study, acidosis (during both ischemia and reoxygenation) increased the formation of isoprostanes by 50%. We have previously found that neuronal free radical formation during and after OGD (with neutral medium) plays a minor role in this model (Almaas et al . 2002); the current study confirms these findings as there were no significant difference in the levels of isoprostanes between oxygen‐ and glucose‐deprived neurones with neutral medium compared with normoxic cells with neutral medium.…”

Acidosis during Reoxygenation Has an Early Detrimental Effect on Neuronal Metabolic Activity

“…Acidosis causes a 50% increase in free radical formation in the current model (1). However, as we have previously found a rather limited role for free radicals in hypoxic cell death of NT2-N neurons, this may not sufficiently explain the deleterious effect of acidosis during reoxygenation (3).…”

“…A possible aggravating effect of acidosis during reoxygenation could be through an increase in free radical formation (1,23), but we have not found oxygen radicals to be important mediators of cell death in our model (3). Another possible detrimental effect of acidosis during reoxygenation could be through a negative effect on mitochondrial function.…”

“…Several mechanisms with opposing effects may be involved. In our hypoxia model, cell death is mediated through NMDA and non-NMDA receptors, and pretreatment with MK-801 offers almost complete protection (1)(2)(3)(4). Whereas the protective effect of acidosis during hypoxia is probably to a large extent mediated through an inhibitory effect on glutaminergic NMDA receptors (1,5), the mechanisms behind the detrimental effect of acidosis during reoxygenation are less clear.…”