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Alterations in the glutathione content of mitochondria following short-term forebrain ischemia in rats
ELSEVIER
Nearoscience Letters 218 (1996) 75-78
NHROSClHC! IETTHS
Alterations in the glutathione content of mitochondria following short-term forebrain ischemia in rats E m a d Z a i d a n , N e i l R. S i m s * Department of Medical B,:ochemistry and Centre for Neuroscience, School of Medicine, Flinders University, G.P.O. Box 2100, Adelaide 5001, South Australia, Australia
Re,ceived 3 July 1996; revised version received 6 September 1996; accepted 18 September 1996
Abstract
Total glutathione was measured in mitochondria isolated following 30 rain of ischemia and recirculation periods up to 24 h. Mitochondria prepared from the dorsolateral striatum, a region containing many neurons susceptible to short ischemic periods, were compared with those front the paramedian cortex, an ischemia-resistant region. Parallel increases in glutathione content (to approximately 150% of pre-ischemic values) were seen in both regions during the first few hours of recirculation. By 24 h of recirculation, there was a decrease below pre-ischemic values in preparations from the dorsolateral striatum but not the paramedian cortex. The early increases in mitochondrial glutathione were not associated with comparable increases in total tissue glutathione. A shorter (10 min) ischemic period also produced an early increase in mitochonddal glutathione but this was reversed more rapidly to preischemic values. The observed changes indicate post-ischemic modifications of cellular oxidative defences in the two brain regions studied. Keywords: Glutathione; Mitochondria; Ischemia; Oxidative damage; Striatum; Cerebral cortex
Oxidative damage has been proposed to contribute to the delayed loss of selected neuronal subpopulations which results from a short period of global brain ischemia. Support for this proposal is provided by the ability of some antioxidants or free radical scavengers to reduce this type of neuronal loss (reviewed in [16]). A burst of production of free radicals and related metabolites which accompanies initial reperfusion [16] has attracted most attention as the likely source of the oxidative damage. However, this increased production of reactive oxygen species is shortlived and direct evidence for a role in neuronal loss is currently lacking. Delete, rious changes could also develop if ischemia or recirculation resulted in modifications of the enzymes or metabolites that normally provide defences against oxidation of cell constituents. At present, there is little information on possible changes in these defence mechanisms during ischemia and reperfusion particularly within tissue subregions containing neurons that are sensitive to short-term ischemia. * Corresponding author. Tel.: +61 8 2044242; fax: +61 8 3740139; e-mail: [email protected]
The electron transport chain of mitochondria is the main contributor to the ongoing production of small amounts of free radicals in normal cells [4,9,12]. Thus, mitochondria are a primary target of oxidative damage if the balance between production and removal of reactive oxygen species is disturbed. The function of brain mitochondria, as assessed from the capacity for respiratory activity, is similar to the pre-ischemic state during the first hour of reperfusion following 30 rain of ischemia in several brain subregions in rats [15,16]. However, in the dorsolateral striatum, a subregion containing many neurons that die within the first 24 h following such an ischemic insult, pyruvate-supported mitochondrial respiration is impaired by 3 h of reperfusion [15,16]. This loss of respiratory capacity during early recirculation, which is not seen in areas that are largely resistant to the effects of such ischemic insults, is apparently due to a selective partial loss of activity of the pyruvate dehydrogenase complex [ 19]. This enzyme complex has been found in some tissues to be particularly susceptible to inhibition under conditions of oxidative stress [3,18] probably as a result of oxidative modification of essential sulphydryl groups [3].
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PI1 S0304-3940(96) 13128-1
76
E. Zaidan, N.R. Sims / Neuroscience Letters 218 (1996) 75-78
Thus, the post-ischemic mitochondrial changes in brain may indicate a delayed development of selective oxidative damage in the dorsolateral striatum in the post-ischemic period. Glutathione plays a central role in the antioxidant defences of cells. Depletion of glutathione in various tissues including brain results in oxidative tissue damage in which mitochondria are particularly affected [6,9]. One major role of glutathione is in the reactions catalyzed by glutathione peroxidase that are involved in removal of hydrogen peroxide and lipid peroxides [12]. This enzyme is particularly important in mitochondria as these organelles lack catalase, an enzyme that contributes to the removal of hydrogen peroxide at other subcellular sites. Hydrogen peroxide is produced in a number of cellular reactions including the removal of the superoxide radical [4]. Accumulation of hydrogen peroxide is undesirable as, under some conditions, it is a precursor of the hydroxyl radical and other highly reactive species capable of producing widespread oxidative damage of many cell components [4]. Hydrogen peroxide may also directly modify some cell components resulting in more subtle and selective changes within cells. Cellular glutathione can be depleted by release of the oxidized form, a process which is accelerated under conditions of oxidative stress [12]. In brain mitochondria, glutathione content has also been reported to decrease under some conditions through the irreversible formation of protein-glutathione mixed disulphides [11]. Small reductions (13-27%) of tissue glutathione have been detected in samples of whole forebrain or cortex removed during the first few hours of recirculation following 30 rain of forebrain ischemia [2,13]. A more recent study examined glutathione changes in three brain regions and reported both larger post-ischemic reductions in tissue glutathione and some modest differences in the magnitude of the change between the regions investigated [17]. None of these studies specifically compared subregions containing the neurons which are particularly susceptible to shortterm ischemia with subregions containing ischemia-resistant neurons nor did they address the possibility that the mitochondrial glutathione content may be differentially affected. The aim of the present study was to determine whether forebrain ischemia or recirculation produces alterations in the size of the glutathione pool in mitochondria that could contribute to an increased susceptibility to oxidative damage in the dorsolateral striatum. Mitochondria isolated from this ischemia-sensitive subregion were compared with those from the paramedian cortex, a subregion in which essentially all neurons survive following a short period of cerebral ischemia. The procedure used for subfractionation of the small tissue samples yields mitochondria exhibiting highly active, well-coupled respiratory function [14,16] and other characteristics which indicate good preservation of properties from those in the intact
issue [14,16]. The subfractionation conditions incorporated features (EDTA in the buffers and restricted washing of the preparation) which were reported previously to optimize recovery of mitochondrial glutathione [8]. Thus, the characteristics of the isolation procedure, taken together with observations that efflux of glutathione from mitochondria occurs only slowly even under some abnormal conditions [12] suggest that the measurements of total glutathione in these mitochondria is likely to closely reflect that in the brain subregions prior to fractionation. Male Porton rats (250-350 g) were surgically prepared and near-complete forebrain ischemia induced for either 10 or 30 min using the 'four-vessel occlusion' method as described previously [ 10,19]. Body temperature was monitored with a rectal probe and maintained above 36.5°C with a heating lamp during ischemia and the first hour of recirculation. Control animals were surgically prepared and manipulated as for the ischemic group but either were not made ischemic or had the occlusion reversed within the first 2 rain. Rats were killed by decapitation at recirculation times between 0 and 24 h, the brains transferred to ice-cold buffer and the subregions dissected in a cold room. Mitochondria were prepared from the dorsolateral striatum and paramedian cortex using a rapid isolation procedure based on Percoll-density gradient centrifugation as described previously [14,19] except that the fraction obtained following gradient centrifugation was washed only once. The loose pellet obtained from this wash was frozen immediately (-80°C) and glutathione content assayed within 3 days. Thawed samples were extracted with an equal volume of 2 M HC104 containing 4 mM EDTA, mixed for 3 rain using a vortex mixer and centrifuged at 7300 g for 10 rain (Eppendorf 5415C microfuge). The supernatant was neutralized with a solution containing 2 M KOH and 0.3 M 3(N-morpholino)propanesulphonic acid and the sample recentrifuged (10 min, 7300 g) to remove precipitated KHC104. Total glutathione (reduced plus oxidized glutathione) in the supernatant was determined by measuring the rate of formation of 5-thio-2-nitrobenzoate from 5,5'dithio-bis 2-[nitrobenzoate] in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione reductase [1]. The reaction was performed at 30°C and followed spectrophotometrically at 412 nm (using a Shimadzu UV-3000 spectrophotometer). Glutathione in each sample was calculated from a linear standard curve (0-0.6 nmol glutathione) measured in parallel with the mitochondrial extracts on each day of assay. The pelleted material following perchloric acid extraction was solubilized using 400/~1 2 M NaOH and the protein content determined [7]. The glutathione content was similar in mitochondria from the two subregions in non-ischemic rats (Fig. 1) and fell within the range of values reported previously for normal brain mitochondria [5,11,20]. The glutathione
E. Zaidan, NJ~. Sims / Neuroscience Letters 218 (1996) 75-78
41 e-
6
~E
o,-- 2 :i 1
Control 30rnln ~20mln Ischemla
1h
3h
8h
24hi
Reclrculation
Fig. 1. Effect of 30 rain of ischemia and recirculation periods up to 24 h on the glutathione content of mitochondria from the dorsolateral striatum and paramedian cortex. Values are shown as the mean + SD (n = 8 for control group; n = 4 - 5 for ischemic and postischemic groups). **P < 0.01 compared with corresponding control value (one-way analysis of variance with Student-Newman-Keuls test for multiple comparisons).
content was unaffected by both 30 rain of ischemia and the first 20 rain of recirculation. However, by 1 h of recirculation, mitochondria from the two subregions showed a similar increase in glutathione (to approximately 150% of preischemic values) which persisted at 3 h and was reversed by 6 h (Fig. 1). This increase during early recirculation was not simply a result of a general increase in the content of glutathione in the cells as glutathione in unfractionated tissue measured at 3 h of recirculation was not significantly different from non-ischemic controls in either the dorsolateral striatum (control, 17.7 + 1.0 nmol/mg protein; 3 h recirculation, 16.9 + 1.4 nmol/mg protein) or the paramedian cortex (control, 15.9 + 1.0 nmol/mg protein; 3 h recirculation, 16.4 + 1.7 nmol/mg protein). Measurements of mitochondrial glutathione at selected recirculation times following a 10 rain period of ischemia revealed an increase at l h of recirculation (Fig. 2) which was of a similar magnitude to that seen following 30 min of ischemia. However, the increase following 10 min of ischemia was more short-lived, being essentially fully reversed by 3 h. In contrast to the parallel changes in mitochondrial glutathione in the two subregions during the initial hours of recirculation, different responses were observed at 24 h of recirculation following the 30 min ischemic period (Fig. 1). At this time, the glutathione content was unaltered from the 6 h values in mitochondria from the paramedian cortex whereas preparations from the dorsolateral striatum showed a significant reduction (to 52% of pre-ischemic values). The preservation of mitochondrial glutathione during the first 6 h of recirculation, and indeed the increases observed at some time points, does not provide support for the proposal that susceptibility to oxidative damage
77
may be enhanced by reduced availability of this metabolite post-ischemically. These findings during the initial recirculation period are unlikely to have been compromised by alterations in the purity of the mitochondria isolated following ischemia. The isolation procedure has been shown previously to produce mitochondrial preparations from small tissue samples that contain little contamination and are at least comparable in purity to fractions obtained from much larger amounts of tissue with other commonlyemployed fractionation procedures [14]. Furthermore, the purity of the fraction obtained from the two subregions is apparently unaffected by 30 rain of ischemia and recirculation periods up to 6 h, as shown by preservation of both the amount of protein recovered and the activity of a number of mitochondrial markers [ 16,19]. Only at 24 h of recirculation were significant reductions detected in the glutathione content of mitochondria from the dorsolateral striatum. Based on previous histological and biochemical studies, the majority of neurons in the dorsolateral striatum progress to advanced stages of degeneration between 6 and 24 h of recirculation and marked changes in several other mitochondrial properties have been found to develop during this period [15,16,19]. Thus, the delayed decrease in glutathione content which differentiated the dorsolateral striatum from the ischemiaresistant paramedian cortex was probably a consequence of the cellular changes accompanying neuronal death rather than a contributor to this process. There was apparently no direct relationship between the increases in glutathione content during early recirculation and the selectivity of neuronal loss as the observed changes were similar in the two regions studied despite marked differences in sensitivity of the constituent neurons to ischemia. Furthermore, a similar increase in glutathione content was also seen, albeit of shorter duration, ll Dorsolateral strixturn I
~ _mE
[] Paramedlan cortex
'f 4
c o
Control
]
lh
3h
]
Recirculatlon Fig. 2. Effects of 10 min of ischemia and recirculation for 1 h or 3 h on the glutathione content of mitochondria from the dorsolateral striatum and paramedian cortex. Values are shown as the mean + SD (control, n = 8; 1 h, n = 6; 3 h, n = 5). These measurements were obtained in parallel with those in Fig. 1 and a common control group has been used. **P < 0.01 compared with corresponding control (one-way analysis of variance with Student-Newman-Keuls test for multiple comparisons).
78
E. Zaidan, N.R. Sims / Neuroscience Letters 218 (1996) 75-78
following 10 m i n of ischemia. In contrast to 30 m i n of ischemia, this lesser ischemic period has been found in previous studies to be insufficient to initiate damage in most n e u r o n s of the dorsolateral striatum. Although there is n o direct association with the selectivity of neuronal loss, the increases in mitochondrial glutathione in the absence of changes in total cell glutathione, do indicate generalized modifications to the antioxidant defences in the post-ischemic period. Mitochondria are u n a b l e to synthesize glutathione [8]. Rather, the cellular glutathione contained within these organelles is obtained by specific uptake from the cytoplasmic pool [9]. Thus, a modification of the m o v e m e n t of glutathione b e t w e e n cytoplasm and mitochondria provides the most straightforward explanation for the increased glutathione content in mitochondria. This m a y have arisen as a delayed cellular response to the burst of free radical production during initial reperfusion or be indicative of o n g o i n g oxidative stress during the first few hours of recirculation. The control of glutathione m o v e m e n t s in brain m i t o c h o n d r i a have not been extensively studied. Thus, further investigation is needed to identify factors capable of m o d i f y i n g the mitochondrial glutathione pool in the post-ischemic period. This work was supported by grants from the National Health and Medical Research Council (Australia), Flinders Research F o u n d a t i o n and Flinders University. [1] Akerboom, T.P.M. and Sies, H., Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples, Methods Enzymol., 77 (1981) 373-382. [2] Cooper, A.J.L., Pulsinelli, W.A. and Duffy, T.E., Glutathione and ascurbate during ischemia and postischemic reperfusion in rat brain, J. Neurocbem., 35 (1980) 1242-1245. [3l Crane, D, Haussinger, D., Graf, P. and Sies, H., Decreased flux through pymvate dehydrogenase by thiol oxidation during t-butyl hydroperoxide metabolism in perfused rat liver, Hoppe-Seyler Z. Physiol. Chem., 364 (1983) 977-987. [4] Halliwell, B., Reactive oxygen species and the central nervous system, J. Neurochem., 59 (1992) 1609-1623. [5] Huang, J. and Philben, M.A., Distribution of glutathione and glutathione-related enzyme systems in mitochondria and cytosol of cultured cerebellar astrocytes and granule cells, Brain Res., 680 (1995) 16-22.
[6] Jain, A., Martensson, J., Stole, E., Auld, P.A.M. and Meister, A., Glutathione deficiency leads to mitochondrial damage in brain, Proc. Natl. Acad. Sci. USA, 88 (1991) 1913-1917. [7] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. [8] M~"tensson, J., Goodwin, C.W. and Blake, R., Mitochondrial glutathione in hyperrnetabolic rats following bum injury and thyroid hormone administration: evidence of a selective effect on brain glutathione by burn injury, Metabolism, 41 (1992) 273-277. [9] Meister, A., Mitochondrial changes associated with glutathione deficiency, Biochim. Biophys. Acta, 1271 (1995) 35-42. [10] Pulsinelli, W.A. and Duffy, T.E, Regional energy balance in rat brain after transient forebrain ischemia, J. Neurochem., 40 (1983) 1500-1503. [11] Ravindranath, V. and Reed, D.J., Glutathione depletion and formation of glutathione-protein mixed disulfide following exposure of brain mitochondria to oxidative stress, Biochem. Biophys. Res. Comm., 169 (1990) 1075-1079. [12] Reed, D.J., Glutathione: toxicological implications, Annu, Rev. Pharmacol. Toxicol., 30 (1990) 603-631. [•3] Rehncrona, S., Folbergrova, J., Smith, D.S. and SiesjO, B.K., Influence of complete and pronounced incomplete cerebral ischemia and subsequent recirculation on cortical concentrations of oxidized and reduced glutathione in the rat, J. Neurochem., 34 (1980) 477486. [14] Sims, N.R., Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation, J. Neurochem., 55 (1990) 698-707. [15] Sims, N.R. and Pulsinelli, W.A., Altered mitochondrial respiration in selectively vulnerable brain subregions following transient forebrain ischemia in the rat, J. Neurochem., 49 (1987) 1367-1374. [16] Sims, N.R. and Zaidan, E., Biochemical changes associated with selective neuronal death following short-term cerebral ischaemia, Int. J. Biochem. Cell Biol., 27 (1995) 531-550. [17] Shivakumar, B.R., Kolluri, S.V.R. and Ravindranath, V., Glutathione and protein thiol homeostasis in brain during reperfusion after cerebral ischemia, J. Pharmacol. Exp. Ther., 274 (1995) 1167-1173. [18] Vlessis, A.A., Muller, P., Bartos, D. and Tmnkey, D., Mechanisms of peroxide-induced cellular injury in cultured adult cardiac myocytes, FASEB J., 5 (1991) 2600-2605. [19] Zaidan, E. and Sims, N.R., Selective reductions in the activity of the pyruvate dehydrogenase complex in mitochondria isolated from brain subregions following forebrain ischemia in rats, J. Cerebral Blood Flow Metab., 13 (1993) 98-104. [20] Zoccarato, F., Cavallini, L., Deana, R. and Alexandre, A., Pathways of hydrogen peroxide generation in guinea pig cerebral cortex mitochondria, Biochem. Biophys. Res. Comm., 154 (1988) 727734.
Nearoscience Letters 218 (1996) 75-78
NHROSClHC! IETTHS
Alterations in the glutathione content of mitochondria following short-term forebrain ischemia in rats E m a d Z a i d a n , N e i l R. S i m s * Department of Medical B,:ochemistry and Centre for Neuroscience, School of Medicine, Flinders University, G.P.O. Box 2100, Adelaide 5001, South Australia, Australia
Re,ceived 3 July 1996; revised version received 6 September 1996; accepted 18 September 1996
Abstract
Total glutathione was measured in mitochondria isolated following 30 rain of ischemia and recirculation periods up to 24 h. Mitochondria prepared from the dorsolateral striatum, a region containing many neurons susceptible to short ischemic periods, were compared with those front the paramedian cortex, an ischemia-resistant region. Parallel increases in glutathione content (to approximately 150% of pre-ischemic values) were seen in both regions during the first few hours of recirculation. By 24 h of recirculation, there was a decrease below pre-ischemic values in preparations from the dorsolateral striatum but not the paramedian cortex. The early increases in mitochondrial glutathione were not associated with comparable increases in total tissue glutathione. A shorter (10 min) ischemic period also produced an early increase in mitochonddal glutathione but this was reversed more rapidly to preischemic values. The observed changes indicate post-ischemic modifications of cellular oxidative defences in the two brain regions studied. Keywords: Glutathione; Mitochondria; Ischemia; Oxidative damage; Striatum; Cerebral cortex
Oxidative damage has been proposed to contribute to the delayed loss of selected neuronal subpopulations which results from a short period of global brain ischemia. Support for this proposal is provided by the ability of some antioxidants or free radical scavengers to reduce this type of neuronal loss (reviewed in [16]). A burst of production of free radicals and related metabolites which accompanies initial reperfusion [16] has attracted most attention as the likely source of the oxidative damage. However, this increased production of reactive oxygen species is shortlived and direct evidence for a role in neuronal loss is currently lacking. Delete, rious changes could also develop if ischemia or recirculation resulted in modifications of the enzymes or metabolites that normally provide defences against oxidation of cell constituents. At present, there is little information on possible changes in these defence mechanisms during ischemia and reperfusion particularly within tissue subregions containing neurons that are sensitive to short-term ischemia. * Corresponding author. Tel.: +61 8 2044242; fax: +61 8 3740139; e-mail: [email protected]
The electron transport chain of mitochondria is the main contributor to the ongoing production of small amounts of free radicals in normal cells [4,9,12]. Thus, mitochondria are a primary target of oxidative damage if the balance between production and removal of reactive oxygen species is disturbed. The function of brain mitochondria, as assessed from the capacity for respiratory activity, is similar to the pre-ischemic state during the first hour of reperfusion following 30 rain of ischemia in several brain subregions in rats [15,16]. However, in the dorsolateral striatum, a subregion containing many neurons that die within the first 24 h following such an ischemic insult, pyruvate-supported mitochondrial respiration is impaired by 3 h of reperfusion [15,16]. This loss of respiratory capacity during early recirculation, which is not seen in areas that are largely resistant to the effects of such ischemic insults, is apparently due to a selective partial loss of activity of the pyruvate dehydrogenase complex [ 19]. This enzyme complex has been found in some tissues to be particularly susceptible to inhibition under conditions of oxidative stress [3,18] probably as a result of oxidative modification of essential sulphydryl groups [3].
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved PI1 S0304-3940(96) 13128-1
76
E. Zaidan, N.R. Sims / Neuroscience Letters 218 (1996) 75-78
Thus, the post-ischemic mitochondrial changes in brain may indicate a delayed development of selective oxidative damage in the dorsolateral striatum in the post-ischemic period. Glutathione plays a central role in the antioxidant defences of cells. Depletion of glutathione in various tissues including brain results in oxidative tissue damage in which mitochondria are particularly affected [6,9]. One major role of glutathione is in the reactions catalyzed by glutathione peroxidase that are involved in removal of hydrogen peroxide and lipid peroxides [12]. This enzyme is particularly important in mitochondria as these organelles lack catalase, an enzyme that contributes to the removal of hydrogen peroxide at other subcellular sites. Hydrogen peroxide is produced in a number of cellular reactions including the removal of the superoxide radical [4]. Accumulation of hydrogen peroxide is undesirable as, under some conditions, it is a precursor of the hydroxyl radical and other highly reactive species capable of producing widespread oxidative damage of many cell components [4]. Hydrogen peroxide may also directly modify some cell components resulting in more subtle and selective changes within cells. Cellular glutathione can be depleted by release of the oxidized form, a process which is accelerated under conditions of oxidative stress [12]. In brain mitochondria, glutathione content has also been reported to decrease under some conditions through the irreversible formation of protein-glutathione mixed disulphides [11]. Small reductions (13-27%) of tissue glutathione have been detected in samples of whole forebrain or cortex removed during the first few hours of recirculation following 30 rain of forebrain ischemia [2,13]. A more recent study examined glutathione changes in three brain regions and reported both larger post-ischemic reductions in tissue glutathione and some modest differences in the magnitude of the change between the regions investigated [17]. None of these studies specifically compared subregions containing the neurons which are particularly susceptible to shortterm ischemia with subregions containing ischemia-resistant neurons nor did they address the possibility that the mitochondrial glutathione content may be differentially affected. The aim of the present study was to determine whether forebrain ischemia or recirculation produces alterations in the size of the glutathione pool in mitochondria that could contribute to an increased susceptibility to oxidative damage in the dorsolateral striatum. Mitochondria isolated from this ischemia-sensitive subregion were compared with those from the paramedian cortex, a subregion in which essentially all neurons survive following a short period of cerebral ischemia. The procedure used for subfractionation of the small tissue samples yields mitochondria exhibiting highly active, well-coupled respiratory function [14,16] and other characteristics which indicate good preservation of properties from those in the intact
issue [14,16]. The subfractionation conditions incorporated features (EDTA in the buffers and restricted washing of the preparation) which were reported previously to optimize recovery of mitochondrial glutathione [8]. Thus, the characteristics of the isolation procedure, taken together with observations that efflux of glutathione from mitochondria occurs only slowly even under some abnormal conditions [12] suggest that the measurements of total glutathione in these mitochondria is likely to closely reflect that in the brain subregions prior to fractionation. Male Porton rats (250-350 g) were surgically prepared and near-complete forebrain ischemia induced for either 10 or 30 min using the 'four-vessel occlusion' method as described previously [ 10,19]. Body temperature was monitored with a rectal probe and maintained above 36.5°C with a heating lamp during ischemia and the first hour of recirculation. Control animals were surgically prepared and manipulated as for the ischemic group but either were not made ischemic or had the occlusion reversed within the first 2 rain. Rats were killed by decapitation at recirculation times between 0 and 24 h, the brains transferred to ice-cold buffer and the subregions dissected in a cold room. Mitochondria were prepared from the dorsolateral striatum and paramedian cortex using a rapid isolation procedure based on Percoll-density gradient centrifugation as described previously [14,19] except that the fraction obtained following gradient centrifugation was washed only once. The loose pellet obtained from this wash was frozen immediately (-80°C) and glutathione content assayed within 3 days. Thawed samples were extracted with an equal volume of 2 M HC104 containing 4 mM EDTA, mixed for 3 rain using a vortex mixer and centrifuged at 7300 g for 10 rain (Eppendorf 5415C microfuge). The supernatant was neutralized with a solution containing 2 M KOH and 0.3 M 3(N-morpholino)propanesulphonic acid and the sample recentrifuged (10 min, 7300 g) to remove precipitated KHC104. Total glutathione (reduced plus oxidized glutathione) in the supernatant was determined by measuring the rate of formation of 5-thio-2-nitrobenzoate from 5,5'dithio-bis 2-[nitrobenzoate] in the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione reductase [1]. The reaction was performed at 30°C and followed spectrophotometrically at 412 nm (using a Shimadzu UV-3000 spectrophotometer). Glutathione in each sample was calculated from a linear standard curve (0-0.6 nmol glutathione) measured in parallel with the mitochondrial extracts on each day of assay. The pelleted material following perchloric acid extraction was solubilized using 400/~1 2 M NaOH and the protein content determined [7]. The glutathione content was similar in mitochondria from the two subregions in non-ischemic rats (Fig. 1) and fell within the range of values reported previously for normal brain mitochondria [5,11,20]. The glutathione
E. Zaidan, NJ~. Sims / Neuroscience Letters 218 (1996) 75-78
41 e-
6
~E
o,-- 2 :i 1
Control 30rnln ~20mln Ischemla
1h
3h
8h
24hi
Reclrculation
Fig. 1. Effect of 30 rain of ischemia and recirculation periods up to 24 h on the glutathione content of mitochondria from the dorsolateral striatum and paramedian cortex. Values are shown as the mean + SD (n = 8 for control group; n = 4 - 5 for ischemic and postischemic groups). **P < 0.01 compared with corresponding control value (one-way analysis of variance with Student-Newman-Keuls test for multiple comparisons).
content was unaffected by both 30 rain of ischemia and the first 20 rain of recirculation. However, by 1 h of recirculation, mitochondria from the two subregions showed a similar increase in glutathione (to approximately 150% of preischemic values) which persisted at 3 h and was reversed by 6 h (Fig. 1). This increase during early recirculation was not simply a result of a general increase in the content of glutathione in the cells as glutathione in unfractionated tissue measured at 3 h of recirculation was not significantly different from non-ischemic controls in either the dorsolateral striatum (control, 17.7 + 1.0 nmol/mg protein; 3 h recirculation, 16.9 + 1.4 nmol/mg protein) or the paramedian cortex (control, 15.9 + 1.0 nmol/mg protein; 3 h recirculation, 16.4 + 1.7 nmol/mg protein). Measurements of mitochondrial glutathione at selected recirculation times following a 10 rain period of ischemia revealed an increase at l h of recirculation (Fig. 2) which was of a similar magnitude to that seen following 30 min of ischemia. However, the increase following 10 min of ischemia was more short-lived, being essentially fully reversed by 3 h. In contrast to the parallel changes in mitochondrial glutathione in the two subregions during the initial hours of recirculation, different responses were observed at 24 h of recirculation following the 30 min ischemic period (Fig. 1). At this time, the glutathione content was unaltered from the 6 h values in mitochondria from the paramedian cortex whereas preparations from the dorsolateral striatum showed a significant reduction (to 52% of pre-ischemic values). The preservation of mitochondrial glutathione during the first 6 h of recirculation, and indeed the increases observed at some time points, does not provide support for the proposal that susceptibility to oxidative damage
77
may be enhanced by reduced availability of this metabolite post-ischemically. These findings during the initial recirculation period are unlikely to have been compromised by alterations in the purity of the mitochondria isolated following ischemia. The isolation procedure has been shown previously to produce mitochondrial preparations from small tissue samples that contain little contamination and are at least comparable in purity to fractions obtained from much larger amounts of tissue with other commonlyemployed fractionation procedures [14]. Furthermore, the purity of the fraction obtained from the two subregions is apparently unaffected by 30 rain of ischemia and recirculation periods up to 6 h, as shown by preservation of both the amount of protein recovered and the activity of a number of mitochondrial markers [ 16,19]. Only at 24 h of recirculation were significant reductions detected in the glutathione content of mitochondria from the dorsolateral striatum. Based on previous histological and biochemical studies, the majority of neurons in the dorsolateral striatum progress to advanced stages of degeneration between 6 and 24 h of recirculation and marked changes in several other mitochondrial properties have been found to develop during this period [15,16,19]. Thus, the delayed decrease in glutathione content which differentiated the dorsolateral striatum from the ischemiaresistant paramedian cortex was probably a consequence of the cellular changes accompanying neuronal death rather than a contributor to this process. There was apparently no direct relationship between the increases in glutathione content during early recirculation and the selectivity of neuronal loss as the observed changes were similar in the two regions studied despite marked differences in sensitivity of the constituent neurons to ischemia. Furthermore, a similar increase in glutathione content was also seen, albeit of shorter duration, ll Dorsolateral strixturn I
~ _mE
[] Paramedlan cortex
'f 4
c o
Control
]
lh
3h
]
Recirculatlon Fig. 2. Effects of 10 min of ischemia and recirculation for 1 h or 3 h on the glutathione content of mitochondria from the dorsolateral striatum and paramedian cortex. Values are shown as the mean + SD (control, n = 8; 1 h, n = 6; 3 h, n = 5). These measurements were obtained in parallel with those in Fig. 1 and a common control group has been used. **P < 0.01 compared with corresponding control (one-way analysis of variance with Student-Newman-Keuls test for multiple comparisons).
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E. Zaidan, N.R. Sims / Neuroscience Letters 218 (1996) 75-78
following 10 m i n of ischemia. In contrast to 30 m i n of ischemia, this lesser ischemic period has been found in previous studies to be insufficient to initiate damage in most n e u r o n s of the dorsolateral striatum. Although there is n o direct association with the selectivity of neuronal loss, the increases in mitochondrial glutathione in the absence of changes in total cell glutathione, do indicate generalized modifications to the antioxidant defences in the post-ischemic period. Mitochondria are u n a b l e to synthesize glutathione [8]. Rather, the cellular glutathione contained within these organelles is obtained by specific uptake from the cytoplasmic pool [9]. Thus, a modification of the m o v e m e n t of glutathione b e t w e e n cytoplasm and mitochondria provides the most straightforward explanation for the increased glutathione content in mitochondria. This m a y have arisen as a delayed cellular response to the burst of free radical production during initial reperfusion or be indicative of o n g o i n g oxidative stress during the first few hours of recirculation. The control of glutathione m o v e m e n t s in brain m i t o c h o n d r i a have not been extensively studied. Thus, further investigation is needed to identify factors capable of m o d i f y i n g the mitochondrial glutathione pool in the post-ischemic period. This work was supported by grants from the National Health and Medical Research Council (Australia), Flinders Research F o u n d a t i o n and Flinders University. [1] Akerboom, T.P.M. and Sies, H., Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples, Methods Enzymol., 77 (1981) 373-382. [2] Cooper, A.J.L., Pulsinelli, W.A. and Duffy, T.E., Glutathione and ascurbate during ischemia and postischemic reperfusion in rat brain, J. Neurocbem., 35 (1980) 1242-1245. [3l Crane, D, Haussinger, D., Graf, P. and Sies, H., Decreased flux through pymvate dehydrogenase by thiol oxidation during t-butyl hydroperoxide metabolism in perfused rat liver, Hoppe-Seyler Z. Physiol. Chem., 364 (1983) 977-987. [4] Halliwell, B., Reactive oxygen species and the central nervous system, J. Neurochem., 59 (1992) 1609-1623. [5] Huang, J. and Philben, M.A., Distribution of glutathione and glutathione-related enzyme systems in mitochondria and cytosol of cultured cerebellar astrocytes and granule cells, Brain Res., 680 (1995) 16-22.
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