- Email: [email protected]
Glutathione monoethyl ester provides neuroprotection in a rat model of stroke
Neuroscience Letters 354 (2004) 163–165 www.elsevier.com/locate/neulet
Glutathione monoethyl ester provides neuroprotection in a rat model of stroke Michelle F. Andersona,*, Michael Nilssona, Peter S. Erikssona, Neil R. Simsb b
a Institute of Clinical Neuroscience, Go¨teborg University, Go¨teborg, Sweden Centre for Neuroscience, Flinders Medical Research Institute and Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide, Australia
Received 11 July 2003; received in revised form 23 September 2003; accepted 26 September 2003
Abstract Oxidative stress plays an important role in the development of tissue damage following transient focal cerebral ischaemia. Glutathione is a central component in the antioxidant defence of cells. We have previously shown a close association between mitochondrial glutathione loss and cell death following middle cerebral artery (MCA) occlusion. Glutathione monoethyl ester increases cellular glutathione and is particularly effective in increasing the mitochondrial pool. In the present investigation, we infused glutathione monoethyl ester into the third ventricle during 2 h of MCA occlusion and 48 h of reperfusion. Infarct size was reduced from 46% of the total ischaemic hemisphere in saline-treated animals to 16% following ester treatment. Thus, glutathione monoethyl ester provides neuroprotection following transient focal cerebral ischaemia. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Focal ischaemia; Rats; Oxidative stress; Infarction; Glutathione ester; Stroke
Oxidative stress, which results from an imbalance between the generation and removal of reactive oxygen species, probably plays an important role in the development of tissue damage in stroke. This process appears to be particularly active under conditions in which the ischaemia is reversed [11], for example by thrombolysis. Glutathione is a central component in the antioxidant defence of cells, acting both to directly detoxify reactive oxygen species and as a substrate for various peroxidases [6]. We have recently shown that total (reduced plus oxidized) mitochondrial glutathione is selectively depleted during focal cerebral ischaemia and reperfusion, in a manner that is closely correlated with the pattern of cell death [4]. Mitochondrial glutathione depletion has been previously associated with cell death [8,14] and this change may be one important determinant of the likelihood of cell death following ischaemia. The exacerbation of infarct size observed when transient focal cerebral ischaemia is associated with glutathione depletion or genetic deletion of glutathione * Corresponding author. Institute of Clinical Neuroscience, Sahlgrenska University Hospital, Bla˚ Stra˚ket 7, 2nd floor, 413 45 Go¨teborg, Sweden. Tel.: þ 46-31-342-3918; fax: þ46-31-342-2467. E-mail address: [email protected] (M.F. Anderson).
peroxidase [5,10] provides further evidence that maintenance of the glutathione-mediated antioxidant defence is critical for cell survival following such an insult. Esters of glutathione can increase cellular glutathione in a variety of tissue types [1] and are particularly effective in restoring mitochondrial glutathione [2,7]. We have previously shown that a single injection of glutathione monoethyl ester into the striatum essentially restores mitochondrial glutathione content in this structure following 2 h of middle cerebral artery (MCA) occlusion in the absence of any changes in total tissue glutathione content [2]. Glutathione measurements were performed on highly enriched mitochondrial fractions [4] prepared from isolated striatal tissue at various time points following vessel occlusion and indicated that the effect of a single injection of the ester on mitochondrial glutathione content lasts for approximately 2 h. In the present investigation, we have examined the effects of glutathione monoethyl ester on ischaemic damage, using intracerebroventricular infusion of the agent as a means of producing longer lasting changes in glutathione across the affected brain tissue. Male Sprague –Dawley rats (weighing 270 – 300 g) were supplied by B&K Universal AB, Sweden. All experimental
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.09.067
164
M.F. Anderson et al. / Neuroscience Letters 354 (2004) 163–165
protocols were approved by the Animal Ethics Committee of Go¨teborg University. Animals were maintained under standard conditions of temperature (24 – 26 8C) and humidity (50 –60%). Rats were fasted overnight prior to all investigations. An infusion cannula was connected to an osmotic pump (brain infusion kit II and 2001 osmotic pump, Alza Scientific Products, Palo Alto, CA) containing either 0.9% sodium chloride (saline) or glutathione monoethyl ester (1 M; Sigma, St. Louis, MO) dissolved in saline and incubated at 37 8C in saline prior to placement in the animal. Anaesthesia was induced with 5% isoflurane followed by intubation and mechanical ventilation with 3% isoflurane in an O2/N2O mix (30:70). A polyethylene catheter was inserted into the left femoral artery for blood pressure and blood gas measurements. Rats were placed in a stereotaxic frame and the skull exposed. The brain catheter was placed into the dorsal third ventricle (3 mm posterior from bregma along the midline, 5 mm below the skull surface) and secured with dental cement. The pump was placed in a subcutaneous pocket in the midscapular region and the wound closed. Reversible occlusion of the right MCA was performed using a 4/0 monofilament nylon thread (Ethilon II, Johnson and Johnson AB, Sollentuna, Sweden) as previously described [3]. MCA occlusion was achieved approximately 30 min after placement of the brain catheter. Immediately following MCA occlusion, a blood sample was taken for blood gas measurements. All animals were treated post-operatively with xylocaine (10 mg/ml)/adrenaline (5 mg/ml; AstraZenica, Sweden) injected into the site of the wound. Those animals that did not exhibit anticlockwise circling at 2 h after insertion of the monofilament thread were excluded from the study. Glutathione monoethyl ester was infused (1 mmol/h) during 2 h of focal ischaemia and continued for a further 48 h to provide modulation of glutathione content during the reperfusion period when the production of harmful oxidants is particularly important. Animals were anaesthetized and decapitated after 48 h of reperfusion. Infarct volume was assessed as described previously [3] in 2.0 mm coronal slices that had been stained with 3% 2,3,5-triphenyl-2H-tetrazolium chloride (Aldrich Chemical Co., Milwaukee, WI) in saline for 20 min at 37 8C and stored in 4% paraformaldehyde. Damage was expressed relative to
Fig. 1. Infarct size in the striatum (A) and cortex (B) assessed at 48 h following 2 h of focal ischaemia. Rats were infused intracerebroventricularly with either glutathione ester (B) or vehicle (A) commencing immediately prior to occlusion of the MCA. Values are shown for individual animals in each group (**P , 0:02, *P , 0:05, Wilcoxon rank test). The mean in each group is indicated by a black bar.
the volume of the whole region (cerebral cortex or striatum) within these slices. There were no differences between the group of rats treated with glutathione ester and those treated with saline for any of the blood parameters assessed immediately following MCA occlusion (Table 1). Increases in body temperature were observed in all groups as described previously [3]. Glutathione monoethyl ester infusion produced a significant reduction in infarct size when compared to saline treatment animals. Infarct size was reduced from 46% of the total ischaemic hemisphere in saline-treated animals to 16% following glutathione ester treatment (P , 0:05, Wilcoxon rank test; data not shown). In the striatum, infarct size was reduced from 41% to 10% of the total striatal volume with ester treatment (Fig. 1). Cortical infarction was also reduced from 60% in salinetreated animals to 26% in those treated with glutathione monoethyl ester (Fig. 1). The present study demonstrates a marked neuroprotective effect of glutathione monoethyl ester on tissue infarction induced by reversed focal cerebral ischaemia. To our knowledge, this is the first report of reductions in infarct volume following treatment with a glutathione ester. An isopropyl ester of glutathione was shown previously to
Table 1 Physiological parameters for rats used to determine the effects of glutathione ethyl ester treatment on brain tissue infarction Treatment (n)
GSH ester (7) Saline (8)
Blood pressure (mmHg)
74 ^ 12 82 ^ 10
PO2 (mmHg)
106 ^ 13 110 ^ 17
PCO2 (mmHg)
41 ^ 6 44 ^ 5
pH
7.37 ^ 0.04 7.35 ^ 0.04
Temperature (8C) following MCA occlusion 1h
2h
38.4 ^ 0.6 38.9 ^ 1.0
39.4 ^ 0.7 39.8 ^ 0.7
Values are shown as the mean ^ SD. There were no statistically significant differences in any of the values for glutathione ester-treated rats compared with equivalent saline-treated rats (Student’s t-test). GSH, glutathione. All values were measured immediately following MCA occlusion.
M.F. Anderson et al. / Neuroscience Letters 354 (2004) 163–165
increase tissue glutathione and reduce brain oedema in response to permanent MCA occlusion [9], although possible effects on tissue infarction were not reported. Glutathione ester treatment of brain slices [12] exposed to ischaemia-like conditions also reduced mitochondrial dysfunction as well as changes in electrophysiological properties and 2-deoxyglucose uptake [13], providing further indication of the protective effects of these compounds. Previously, we showed that a single injection of glutathione monoethyl ester selectively modulated the mitochondrial pool of glutathione and temporarily blocked losses of this metabolite pool induced by ischaemia [2]. The depletion of mitochondrial glutathione produced by ischaemia [4] is likely to increase the susceptibility of these organelles to oxidative damage and promote the release of reactive oxygen species that are generated in the mitochondria as part of normal metabolism. Thus, a major component in the neuroprotective effects of the glutathione ester probably resulted from amelioration of the depletion of this metabolite in the mitochondria. The extended infusion of glutathione monoethyl ester in the present study might also have increased cytoplasmic glutathione, thus promoting a more general improvement in the ability of cells to resist ischaemia-induced oxidative stress. In the present investigation, we infused glutathione monoethyl ester for the entire ischaemia/reperfusion period. Further studies are required to define the therapeutic window and also to investigate the efficacy of systemic treatment with this compound in order to better evaluate the potential for modulating the effects of stroke. We have previously demonstrated that the ester can increase mitochondrial glutathione content in non-ischaemic tissue to approximately twice the normal values [4] suggesting that such a treatment might also have future applications for increasing the antioxidant defences in patients at risk of developing cerebral ischaemic episodes, for example during cardiac surgery.
Acknowledgements This research was supported by grants from the Swedish Medical Research Council (project number 12X-12535), the Wenner Gren Foundation, the Wenner-Grenska Foundation, the Axel Linders Foundation, the Edit Jacobsson Foundation, the Swedish Foundation for International
165
Cooperation in Research and Higher Education and the National Health and Medical Research Council of Australia.
References [1] M.E. Anderson, F. Powrie, R.N. Puri, A. Meister, Glutathione monoethyl ester: preparation, uptake by tissues, and conversion to glutathione, Arch. Biochem. Biophys. 239 (1985) 538–548. [2] M.F. Anderson, M. Nilsson, N.R. Sims, Glutathione monoethylester prevents mitochondrial glutathione depletion during focal cerebral ischemia, Neurochem. Int. 44 (2004) 153–159. [3] M.F. Anderson, N.R. Sims, Mitochondrial respiratory function and cell death in focal cerebral ischemia, J. Neurochem. 73 (1999) 1189– 1199. [4] M.F. Anderson, N.R. Sims, The effects of focal ischemia and reperfusion on the glutathione content of mitochondria from rat brain subregions, J. Neurochem. 81 (2002) 541– 549. [5] P.J. Crack, J.M. Taylor, N.J. Flentjar, J. de Haan, P. Hertzog, R.C. Iannello, I. Kola, Increased infarct size and exacerbated apoptosis in the glutathione peroxidase-1 (Gpx-1) knockout mouse brain in response to ischemia/reperfusion injury, J. Neurochem. 78 (2001) 1389– 1399. [6] R. Dringen, Metabolism and functions of glutathione in brain, Prog. Neurobiol. 62 (2000) 649–671. [7] J.C. Ferna´ndez-Checa, C. Garcı´a-Ruiz, M. Ookhtens, N. Kaplowitz, Impaired uptake of glutathione by hepatic mitochondria from chronic ethanol-fed rats. Tracer kinetic studies in vitro and in vivo and susceptibility to oxidant stress, J. Clin. Invest. 87 (1991) 397 –405. [8] J.C. Ferna´ndez-Checa, N. Kaplowitz, C. Ga´rcia-Ruiz, A. Colell, Mitochondrial glutathione: importance and transport, Semin. Liver Dis. 18 (1998) 389–401. [9] O. Gotoh, M. Yamamoto, A. Tamura, S. Sano, Effect of YM737, a new glutathione analogue, on ischemic brain edema, Acta Neurochir. Suppl. 60 (1994) 318–320. [10] T. Mizui, H. Kinouchi, P.H. Chan, Depletion of brain glutathione by buthionine sulfoximine enhances cerebral ischemic injury in rats, Am. J. Physiol. 262 (1992) H313 –H317. [11] C.A. Piantadosi, J. Zhang, Mitochondrial generation of reactive oxygen species after brain ischemia in the rat, Stroke 27 (1996) 327–331. [12] T. Sasaki, M. Senda, T. Ohno, S. Kojima, A. Kubodera, Effect of in vitro ischemic or hypoxic treatment on mitochondrial electron transfer activity in rat brain slices assessed by gas-tissue autoradiography using [15O] molecular oxygen, Brain Res. 890 (2001) 100 –109. [13] S. Shibata, K. Tominaga, S. Watanabe, Glutathione protects against hypoxic/hypoglycemic decreases in 2-deoxyglucose uptake and presynaptic spikes in hippocampal slices, Eur. J. Pharmacol. 273 (1995) 191–195. [14] U. Wu¨llner, J. Seyfried, P. Groscurth, S. Beinroth, S. Winter, M. Gleichmann, M. Heneka, P.A. Lo¨schmann, J.B. Schulz, M. Weller, T. Klockgether, Glutathione depletion and neuronal cell death: the role of reactive oxygen intermediates and mitochondrial function, Brain Res. 826 (1999) 53–62.
Glutathione monoethyl ester provides neuroprotection in a rat model of stroke Michelle F. Andersona,*, Michael Nilssona, Peter S. Erikssona, Neil R. Simsb b
a Institute of Clinical Neuroscience, Go¨teborg University, Go¨teborg, Sweden Centre for Neuroscience, Flinders Medical Research Institute and Department of Medical Biochemistry, School of Medicine, Flinders University, Adelaide, Australia
Received 11 July 2003; received in revised form 23 September 2003; accepted 26 September 2003
Abstract Oxidative stress plays an important role in the development of tissue damage following transient focal cerebral ischaemia. Glutathione is a central component in the antioxidant defence of cells. We have previously shown a close association between mitochondrial glutathione loss and cell death following middle cerebral artery (MCA) occlusion. Glutathione monoethyl ester increases cellular glutathione and is particularly effective in increasing the mitochondrial pool. In the present investigation, we infused glutathione monoethyl ester into the third ventricle during 2 h of MCA occlusion and 48 h of reperfusion. Infarct size was reduced from 46% of the total ischaemic hemisphere in saline-treated animals to 16% following ester treatment. Thus, glutathione monoethyl ester provides neuroprotection following transient focal cerebral ischaemia. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Focal ischaemia; Rats; Oxidative stress; Infarction; Glutathione ester; Stroke
Oxidative stress, which results from an imbalance between the generation and removal of reactive oxygen species, probably plays an important role in the development of tissue damage in stroke. This process appears to be particularly active under conditions in which the ischaemia is reversed [11], for example by thrombolysis. Glutathione is a central component in the antioxidant defence of cells, acting both to directly detoxify reactive oxygen species and as a substrate for various peroxidases [6]. We have recently shown that total (reduced plus oxidized) mitochondrial glutathione is selectively depleted during focal cerebral ischaemia and reperfusion, in a manner that is closely correlated with the pattern of cell death [4]. Mitochondrial glutathione depletion has been previously associated with cell death [8,14] and this change may be one important determinant of the likelihood of cell death following ischaemia. The exacerbation of infarct size observed when transient focal cerebral ischaemia is associated with glutathione depletion or genetic deletion of glutathione * Corresponding author. Institute of Clinical Neuroscience, Sahlgrenska University Hospital, Bla˚ Stra˚ket 7, 2nd floor, 413 45 Go¨teborg, Sweden. Tel.: þ 46-31-342-3918; fax: þ46-31-342-2467. E-mail address: [email protected] (M.F. Anderson).
peroxidase [5,10] provides further evidence that maintenance of the glutathione-mediated antioxidant defence is critical for cell survival following such an insult. Esters of glutathione can increase cellular glutathione in a variety of tissue types [1] and are particularly effective in restoring mitochondrial glutathione [2,7]. We have previously shown that a single injection of glutathione monoethyl ester into the striatum essentially restores mitochondrial glutathione content in this structure following 2 h of middle cerebral artery (MCA) occlusion in the absence of any changes in total tissue glutathione content [2]. Glutathione measurements were performed on highly enriched mitochondrial fractions [4] prepared from isolated striatal tissue at various time points following vessel occlusion and indicated that the effect of a single injection of the ester on mitochondrial glutathione content lasts for approximately 2 h. In the present investigation, we have examined the effects of glutathione monoethyl ester on ischaemic damage, using intracerebroventricular infusion of the agent as a means of producing longer lasting changes in glutathione across the affected brain tissue. Male Sprague –Dawley rats (weighing 270 – 300 g) were supplied by B&K Universal AB, Sweden. All experimental
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.09.067
164
M.F. Anderson et al. / Neuroscience Letters 354 (2004) 163–165
protocols were approved by the Animal Ethics Committee of Go¨teborg University. Animals were maintained under standard conditions of temperature (24 – 26 8C) and humidity (50 –60%). Rats were fasted overnight prior to all investigations. An infusion cannula was connected to an osmotic pump (brain infusion kit II and 2001 osmotic pump, Alza Scientific Products, Palo Alto, CA) containing either 0.9% sodium chloride (saline) or glutathione monoethyl ester (1 M; Sigma, St. Louis, MO) dissolved in saline and incubated at 37 8C in saline prior to placement in the animal. Anaesthesia was induced with 5% isoflurane followed by intubation and mechanical ventilation with 3% isoflurane in an O2/N2O mix (30:70). A polyethylene catheter was inserted into the left femoral artery for blood pressure and blood gas measurements. Rats were placed in a stereotaxic frame and the skull exposed. The brain catheter was placed into the dorsal third ventricle (3 mm posterior from bregma along the midline, 5 mm below the skull surface) and secured with dental cement. The pump was placed in a subcutaneous pocket in the midscapular region and the wound closed. Reversible occlusion of the right MCA was performed using a 4/0 monofilament nylon thread (Ethilon II, Johnson and Johnson AB, Sollentuna, Sweden) as previously described [3]. MCA occlusion was achieved approximately 30 min after placement of the brain catheter. Immediately following MCA occlusion, a blood sample was taken for blood gas measurements. All animals were treated post-operatively with xylocaine (10 mg/ml)/adrenaline (5 mg/ml; AstraZenica, Sweden) injected into the site of the wound. Those animals that did not exhibit anticlockwise circling at 2 h after insertion of the monofilament thread were excluded from the study. Glutathione monoethyl ester was infused (1 mmol/h) during 2 h of focal ischaemia and continued for a further 48 h to provide modulation of glutathione content during the reperfusion period when the production of harmful oxidants is particularly important. Animals were anaesthetized and decapitated after 48 h of reperfusion. Infarct volume was assessed as described previously [3] in 2.0 mm coronal slices that had been stained with 3% 2,3,5-triphenyl-2H-tetrazolium chloride (Aldrich Chemical Co., Milwaukee, WI) in saline for 20 min at 37 8C and stored in 4% paraformaldehyde. Damage was expressed relative to
Fig. 1. Infarct size in the striatum (A) and cortex (B) assessed at 48 h following 2 h of focal ischaemia. Rats were infused intracerebroventricularly with either glutathione ester (B) or vehicle (A) commencing immediately prior to occlusion of the MCA. Values are shown for individual animals in each group (**P , 0:02, *P , 0:05, Wilcoxon rank test). The mean in each group is indicated by a black bar.
the volume of the whole region (cerebral cortex or striatum) within these slices. There were no differences between the group of rats treated with glutathione ester and those treated with saline for any of the blood parameters assessed immediately following MCA occlusion (Table 1). Increases in body temperature were observed in all groups as described previously [3]. Glutathione monoethyl ester infusion produced a significant reduction in infarct size when compared to saline treatment animals. Infarct size was reduced from 46% of the total ischaemic hemisphere in saline-treated animals to 16% following glutathione ester treatment (P , 0:05, Wilcoxon rank test; data not shown). In the striatum, infarct size was reduced from 41% to 10% of the total striatal volume with ester treatment (Fig. 1). Cortical infarction was also reduced from 60% in salinetreated animals to 26% in those treated with glutathione monoethyl ester (Fig. 1). The present study demonstrates a marked neuroprotective effect of glutathione monoethyl ester on tissue infarction induced by reversed focal cerebral ischaemia. To our knowledge, this is the first report of reductions in infarct volume following treatment with a glutathione ester. An isopropyl ester of glutathione was shown previously to
Table 1 Physiological parameters for rats used to determine the effects of glutathione ethyl ester treatment on brain tissue infarction Treatment (n)
GSH ester (7) Saline (8)
Blood pressure (mmHg)
74 ^ 12 82 ^ 10
PO2 (mmHg)
106 ^ 13 110 ^ 17
PCO2 (mmHg)
41 ^ 6 44 ^ 5
pH
7.37 ^ 0.04 7.35 ^ 0.04
Temperature (8C) following MCA occlusion 1h
2h
38.4 ^ 0.6 38.9 ^ 1.0
39.4 ^ 0.7 39.8 ^ 0.7
Values are shown as the mean ^ SD. There were no statistically significant differences in any of the values for glutathione ester-treated rats compared with equivalent saline-treated rats (Student’s t-test). GSH, glutathione. All values were measured immediately following MCA occlusion.
M.F. Anderson et al. / Neuroscience Letters 354 (2004) 163–165
increase tissue glutathione and reduce brain oedema in response to permanent MCA occlusion [9], although possible effects on tissue infarction were not reported. Glutathione ester treatment of brain slices [12] exposed to ischaemia-like conditions also reduced mitochondrial dysfunction as well as changes in electrophysiological properties and 2-deoxyglucose uptake [13], providing further indication of the protective effects of these compounds. Previously, we showed that a single injection of glutathione monoethyl ester selectively modulated the mitochondrial pool of glutathione and temporarily blocked losses of this metabolite pool induced by ischaemia [2]. The depletion of mitochondrial glutathione produced by ischaemia [4] is likely to increase the susceptibility of these organelles to oxidative damage and promote the release of reactive oxygen species that are generated in the mitochondria as part of normal metabolism. Thus, a major component in the neuroprotective effects of the glutathione ester probably resulted from amelioration of the depletion of this metabolite in the mitochondria. The extended infusion of glutathione monoethyl ester in the present study might also have increased cytoplasmic glutathione, thus promoting a more general improvement in the ability of cells to resist ischaemia-induced oxidative stress. In the present investigation, we infused glutathione monoethyl ester for the entire ischaemia/reperfusion period. Further studies are required to define the therapeutic window and also to investigate the efficacy of systemic treatment with this compound in order to better evaluate the potential for modulating the effects of stroke. We have previously demonstrated that the ester can increase mitochondrial glutathione content in non-ischaemic tissue to approximately twice the normal values [4] suggesting that such a treatment might also have future applications for increasing the antioxidant defences in patients at risk of developing cerebral ischaemic episodes, for example during cardiac surgery.
Acknowledgements This research was supported by grants from the Swedish Medical Research Council (project number 12X-12535), the Wenner Gren Foundation, the Wenner-Grenska Foundation, the Axel Linders Foundation, the Edit Jacobsson Foundation, the Swedish Foundation for International
165
Cooperation in Research and Higher Education and the National Health and Medical Research Council of Australia.
References [1] M.E. Anderson, F. Powrie, R.N. Puri, A. Meister, Glutathione monoethyl ester: preparation, uptake by tissues, and conversion to glutathione, Arch. Biochem. Biophys. 239 (1985) 538–548. [2] M.F. Anderson, M. Nilsson, N.R. Sims, Glutathione monoethylester prevents mitochondrial glutathione depletion during focal cerebral ischemia, Neurochem. Int. 44 (2004) 153–159. [3] M.F. Anderson, N.R. Sims, Mitochondrial respiratory function and cell death in focal cerebral ischemia, J. Neurochem. 73 (1999) 1189– 1199. [4] M.F. Anderson, N.R. Sims, The effects of focal ischemia and reperfusion on the glutathione content of mitochondria from rat brain subregions, J. Neurochem. 81 (2002) 541– 549. [5] P.J. Crack, J.M. Taylor, N.J. Flentjar, J. de Haan, P. Hertzog, R.C. Iannello, I. Kola, Increased infarct size and exacerbated apoptosis in the glutathione peroxidase-1 (Gpx-1) knockout mouse brain in response to ischemia/reperfusion injury, J. Neurochem. 78 (2001) 1389– 1399. [6] R. Dringen, Metabolism and functions of glutathione in brain, Prog. Neurobiol. 62 (2000) 649–671. [7] J.C. Ferna´ndez-Checa, C. Garcı´a-Ruiz, M. Ookhtens, N. Kaplowitz, Impaired uptake of glutathione by hepatic mitochondria from chronic ethanol-fed rats. Tracer kinetic studies in vitro and in vivo and susceptibility to oxidant stress, J. Clin. Invest. 87 (1991) 397 –405. [8] J.C. Ferna´ndez-Checa, N. Kaplowitz, C. Ga´rcia-Ruiz, A. Colell, Mitochondrial glutathione: importance and transport, Semin. Liver Dis. 18 (1998) 389–401. [9] O. Gotoh, M. Yamamoto, A. Tamura, S. Sano, Effect of YM737, a new glutathione analogue, on ischemic brain edema, Acta Neurochir. Suppl. 60 (1994) 318–320. [10] T. Mizui, H. Kinouchi, P.H. Chan, Depletion of brain glutathione by buthionine sulfoximine enhances cerebral ischemic injury in rats, Am. J. Physiol. 262 (1992) H313 –H317. [11] C.A. Piantadosi, J. Zhang, Mitochondrial generation of reactive oxygen species after brain ischemia in the rat, Stroke 27 (1996) 327–331. [12] T. Sasaki, M. Senda, T. Ohno, S. Kojima, A. Kubodera, Effect of in vitro ischemic or hypoxic treatment on mitochondrial electron transfer activity in rat brain slices assessed by gas-tissue autoradiography using [15O] molecular oxygen, Brain Res. 890 (2001) 100 –109. [13] S. Shibata, K. Tominaga, S. Watanabe, Glutathione protects against hypoxic/hypoglycemic decreases in 2-deoxyglucose uptake and presynaptic spikes in hippocampal slices, Eur. J. Pharmacol. 273 (1995) 191–195. [14] U. Wu¨llner, J. Seyfried, P. Groscurth, S. Beinroth, S. Winter, M. Gleichmann, M. Heneka, P.A. Lo¨schmann, J.B. Schulz, M. Weller, T. Klockgether, Glutathione depletion and neuronal cell death: the role of reactive oxygen intermediates and mitochondrial function, Brain Res. 826 (1999) 53–62.