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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 18, 2003 as doi:10.1096/fj.02-1086fje. |
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Departamento de Farmacología, Facultad de Medicina, Universidad Complutense de Madrid (UCM), Madrid. Spain
2Correspondence: Departamento de Farmacología, Facultad de Medicina, UCM, 28040 Madrid. Spain. E-mail: neurona{at}med.ucm.es
SPECIFIC AIMS
Reversal of neuronal glutamate transporters caused by ATP fall and subsequent imbalance of membrane ionic gradients accounts for most glutamate release and subsequent excitotoxicity after occlusion of cerebral arteries. We have studied whether antioxidants are neuroprotective in experimental stroke by delaying ischemia-induced ATP fall and therefore decreasing glutamate release.
PRINCIPAL FINDINGS
1. The antioxidants glutathione (GSH), the superoxide dismutase analog MnTBAP, and tocopherol improve stroke outcome
Infarct volume was measured in brains of adult male Fischer rats 48 h after being exposed to permanent middle cerebral artery occlusion (MCAO). Intraperitoneal administration of GSH, MnTBAP, or tocopherol 2 h before MCAO caused a reduction in infarct volume, an effect that was parallel to a lesser neurological damage (Fig. 1
A).
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2. The neuroprotective effect of GSH, MnTBAP, and tocopherol correlates with a decrease in brain glutamate concentrations and an increase in brain ATP after MCAO
Permanent MCAO caused an increase in brain glutamate concentrations immediately after occlusion. This increase was inhibited by previous intraperitoneal administration of GSH, MnTBAP, or tocopherol (Fig. 1B
). In addition, MCAO caused a reduction in brain ATP levels that was partially prevented by prior treatment with GSH, MnTBAP, or tocopherol (Fig. 2
A). These compounds also caused an increase in brain ATP levels of sham-operated (Fig. 2A
) and control animals (137±19, 186±14, and 189±23% in GSH-, MnTBAP-, and tocopherol-treated animals, respectively, P<0.05, n=4).
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3. Antioxidants enhance brain ATP by increasing mitochondrial respiratory chain (MRC) complex I-III respiration
To ascertain the source of the increased brain ATP levels, ATP production by isolated rat mitochondria was investigated. GSH, SOD/CAT, and tocopherol all increased glutamate/malate/ADP (MRC complex I-, III-, and IV) -dependent ATP synthesis (Fig. 2B
). We therefore studied the effects of GSH, SOD/CAT, and tocopherol on the activity and respiration of MRC complexes using rat brain submitochondrial particles (SMP), a preparation in which MRC is uncoupled from ATP synthase. All the antioxidants, but not oxidized glutathione, increased MRC complex I-III activity and respiration without affecting either complex II-III or complex IV.
4. Antioxidants scavenge reactive oxygen species (ROS) but do not reverse established mitochondrial oxidative damage
To characterize the antioxidant mechanism of the compounds tested, the effect of prior or subsequent addition of antioxidants on complex I-III was studied after exposure of SMP to H2O2 (40 µM). H2O2 caused a decrease in complex I-III respiration that was not affected by subsequent addition of antioxidants. However, SOD/CAT and tocopherol prevented H2O2-induced inhibition of complex I-III respiration when added before H2O2.
CONCLUSIONS
Previous evidence has shown that strategies able to prevent ischemia-induced ATP fall are neuroprotective in both in vitro and in vivo models of brain ischemia. The MRC, which is coupled to the synthesis of ATP for the oxidative phosphorylation, is a major source of reactive oxygen species and has been shown to be vulnerable to oxidative stress. Therefore, antioxidants may be neuroprotectants by increasing ATP levels and thus preventing ATP fall induced by ischemia.
We first tested the effect of several antioxidants on infarct outcome. In in vivo experiments, SOD was substituted by its molecular weight analog MnTBAP. Our results show that administration of GSH, MnTBAP, or tocopherol prior to MCAO was effective in improving stroke outcome, as demonstrated by a reduction in infarct volume and an improvement of neurological scores.
As is well known, glutamate plays a predominant role in the pathogenesis of ischemic brain injury; this excitatory amino acid is released in high concentrations in the core of the cerebral infarction and in the penumbral tissue, leading to a massive influx of calcium that activates a variety of catabolic processes that subsequently produce cell death. Therefore, we tested the correlation between the neuroprotective effect of antioxidants and brain glutamate levels; indeed, these compounds reduced MCAO-induced increase in brain glutamate concentrations.
Glutamate release induced by severe ischemia is largely due to reversed operation of neuronal glutamate transporters. This reversal results from the depletion in ATP levels caused by ischemia. To search for the mechanism of this action of antioxidants, we tested their effects on brain ATP levels. These compounds caused an increase in brain ATP levels in control, sham-operated, and MCAO-exposed animals and enhanced ATP production in isolated brain mitochondria stimulated by complex I-III substrates, indicating that mitochondrial oxidative phosphorylation is the source of the increased brain ATP levels after administration of antioxidants.
To investigate the action of antioxidants on mitochondrial ATP synthesis, we tested their effects on MRC complexes respiration and activity using rat brain SMP. In these experiments, SOD was used in combination with catalase (CAT) in order to remove H2O2 formed from superoxide anion dismutation. We found that GSH, SOD/CAT, and tocopherol increased complex I-III activity and respiration without affecting complex II-III or IV activity. These results indicate that antioxidants target mitochondrial respiratory chain complex I-III, resulting in an increased respiration and ATP production that delays ischemia-induced ATP fall and subsequent reversal of glutamate transporters (Fig. 3
). These data indicate that this action of antioxidants takes place only in those cells with sufficient oxygen pressure for oxidative phosphorylation, implying that a neuroprotective effect of antioxidants should be expected on neurones with sufficient oxygen pressure, such as those in the ischemic penumbra or after recanalization.
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We have also characterized the anti-oxidative mechanism of these compounds by studying their effects on MRC complex I-III inhibition of respiration caused by a pro-oxidative agent, H2O2. Our results show that antioxidants are able to prevent H2O2-induced inhibition of complex I-III respiration when added before but not after the addition of H2O2, indicating that they are able to scavenge the formation of reactive oxygen species (ROS) but fail to reverse established oxidative damage, at least at this level.
It has been demonstrated that 
2–5% of the electron flow in healthy isolated brain mitochondria produces superoxide anion and H2O2. It is plausible that this basal ROS production constitutes a physiological 
brake
of ATP synthesis from oxidative phosphorylation; its removal by antioxidants may be beneficial in situations in which cellular energetic levels are compromised, such as brain ischemia.
Our results show that antioxidants exhibit a neuroprotective effect that occurs concomitant to an inhibition of both ischemia-induced increase in glutamate release and a decrease in brain ATP levels.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1086fje; doi: 10.1096/fj.02-1086fje 
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  J. Mallolas, O. Hurtado, M. Castellanos, M. Blanco, T. Sobrino, J. Serena, J. Vivancos, J. Castillo, I. Lizasoain, M. A. Moro, et al. A polymorphism in the EAAT2 promoter is associated with higher glutamate concentrations and higher frequency of progressing stroke J. Exp. Med., March 20, 2006; 203(3): 711 - 717. [Abstract] [Full Text] [PDF]
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-tocopherol on ATP production by glutamate/malate-dependent respiration (control value: 11.8±1.0 µmol/g of mitochondrial protein) in isolated mitochondria. Data are mean ± SE, n= 4, *P< 0.05 vs. control.
, activation; 
, inhibition).