Alterations in glutathione and amino acid concentrations after hypoxia–ischemia in the immature rat brain

Developmental Brain Research 125 (2000) 51–60 www.elsevier.com / locate / bres

Research report

Alterations in glutathione and amino acid concentrations after hypoxia–ischemia in the immature rat brain Camilla Wallin a , Malgorzata Puka-Sundvall a,b , Henrik Hagberg b,c , Stephen G. Weber d , e, Mats Sandberg * a

¨ ¨ University, P.O. Box 420, SE 405 30 Goteborg , Sweden Department of Anatomy and Cell Biology, Goteborg b ¨ ¨ Department of Physiology, Goteborg University, P.O. Box 432, SE 405 30 Goteborg , Sweden c ¨ ¨ , SE 416 85, Goteborg , Sweden Department Obstetrics and Gynecology, SU /Ostra d Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA e ¨ ¨ University, P.O. Box 433, SE 405 30 Goteborg , Sweden Department of Medical Biophysics, Goteborg Accepted 19 September 2000

Abstract Hypoxic-ischemic brain injury involves an increased formation of reactive oxygen species. Key factors in the cellular protection against such agents are the GSH-associated reactions. In the present study we examined alterations in total glutathione and GSSG concentrations in mitochondria-enriched fractions and tissue homogenates from the cerebral cortex of 7-day-old rats at 0, 1, 3, 8, 14, 24 and 72 h after hypoxia–ischemia. The concentration of total glutathione was transiently decreased immediately after hypoxia–ischemia in the mitochondrial fraction, but not in the tissue, recovered, and then decreased both in mitochondrial fraction and homogenate after 14 h, reaching a minimum at 24 h after hypoxia–ischemia. The level of GSSG was |4% of total glutathione and increased selectively in the mitochondrial fraction immediately after hypoxia–ischemia. The decrease in glutathione may be important in the development of cell death via impaired free radical inactivation and / or redox related changes. The effects of hypoxia–ischemia on the concentrations of selected amino acids varied. The levels of phosphoethanolamine, an amine previously reported to be released in ischemia, mirrored the changes in glutathione. GABA concentrations initially increased (0–3 h) followed by a decrease at 72 h. Glutamine levels increased, whereas glutamate and aspartate were unchanged up to 24 h after the insult. The results on total glutathione and GSSG are discussed in relation to changes in mitochondrial respiration and microtubule associated protein-2 (MAP2) which are reported on in accompanying paper [64].  2000 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Glutathione; Cerebral hypoxia–ischemia; Mitochondria; Oxidative stress; Neonatal rat; Amino acid

1. Introduction Cerebral hypoxia–ischemia (HI) is a significant cause of perinatal brain damage with subsequent long term neurological impairment [26,81,87]. Recent data concerning the development of ischemic brain damage in animal models suggest that oxidative stress and mitochondrial dysfunction may play important roles (for reviews see [46,80]). *Corresponding author. Institute of Physiology and Pharmacology, ¨ Department of Medical Biophysics, P.O. Box 433, SE 405 30 Goteborg, Sweden. Tel.: 146-31-773-3395; fax: 146-31-412-805. E-mail address: [email protected] (M. Sandberg).

Mitochondria are an important source of reactive oxygen species, such as the superoxide radical, hydrogen peroxide and the hydroxyl radical [30]. Since these organelles lack catalase they depend on glutathione (GSH) and GSHperoxidase for inactivation of hydrogen peroxide. Glutathione is also involved in regulation of the redox status, which control the activity of a number of cellular processes, including the mitochondrial permeability transition pore [15,91]. The important role of glutathione in neurotoxicity is supported by the effects of glutathione depletion which cause: (i) increased nitric oxide synthetase activity [34], (ii) increased formation of reactive oxygen species and lipid peroxidation [18], (iii) decreased activity of

0165-3806 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 00 )00112-7

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mitochondrial enzymatic complexes [8], (iv) loss of mitochondrial membrane potential [92], (v) increased excitotoxic response to NMDA [10], (vi) larger infarct areas in stroke models [53] and (vii) enlargement and degeneration of mitochondria in the newborn rat [37]. Notably, a decreased glutathione concentration in brain tissue has been observed after ischemia in the adult animal [16,48,53,56,67]. We have recently demonstrated a delayed elevated efflux of glutathione after NMDA-receptor stimulation in vitro [88]. This may, at least transiently, increase the intracellular level of reactive oxygen species via a decrease in intracellular glutathione concentration. As HI brain damage is in part mediated by NMDA-receptor activation [28,50], we have evaluated here the effect of HI on the temporal profile of total glutathione and GSSG concentration in tissue and in a mitochondria enriched fraction. The effects of HI on mitochondrial respiratory control ratio (RCR) and on microtubule associated protein-2 (MAP2) were determined and are presented in an accompanying paper [64]. In order to evaluate the selectivity of HIinduced alterations we also determined the concentrations of the neurotransmitter amino acids glutamate, aspartate and GABA, as well as glutamine, a glutamate precursor synthesized in glia. Phosphoethanolamine (PEA), a soluble phospholipid precursor, was included because earlier studies have shown that this amine is specifically released with a delay after ischemia in vivo and after NMDAreceptor stimulation in vivo and in vitro [29,44,88]. All parameters were determined in both the ipsilateral (hypoxic–ischemic) and contralateral (hypoxic) hemisphere. Brain damage in this model is largely restricted to the cerebral hemisphere ipsilateral to the common carotid artery ligation [82].

2. Materials and methods

2.1. Chemicals N-ethylmaleimide, HEPES, o-phthaldialdehyde (OPA), DL-dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA; potassium salt), D-mannitol, sucrose, Tris– H 3 PO 4 , Tris–HCl and sodium azide (NaN 3 ) were obtained from Sigma (St. Louis, MO, USA).

2.2. Study design and induction of HI Inbred Wistar Furth rat pups of either sex were used (Møllegard, Denmark). A total of 109 rat pups from 12 litters were used, 52 males and 57 females. All animal procedures were approved by the local ethics committee in ¨ Goteborg, Sweden. At postnatal day 7 the pups were exposed to HI as follows: the pups were anaesthetized with halothane (3.5% for induction and 1.5% for maintenance) in a mixture of

nitrous oxide and oxygen (1:1). The left common carotid artery was dissected and cut between double ligatures of prolene sutures (6–0). The duration of anesthesia was ,10 min. The pups were left to recover with the dam for 1 h. Then the litter was placed in a chamber and exposed to 7.7060.01% oxygen in nitrogen for 65 min. Animals were decapitated 0, 1, 3, 8, 14, 24 or 72 h after HI. Untreated littermates were used as controls at postnatal days 7, 8 or 10.

2.3. Preparation of homogenate and mitochondria enriched fraction Cerebral cortex from the hemisphere ipsilateral (hypoxic–ischemic) and contralateral (hypoxic) to the ligation were dissected out and immersed in ice cold 10 mM Tris buffer (pH 7.4) containing 0.32 M sucrose and 1 mM EDTA. The tissue was rinsed and the surface cleared from meninges and pial vessels. The anterior and posterior poles were cut away and a central portion was homogenized in 1.5 ml buffer by hand (four up-and-down strokes with a total clearance of 0.12 mm and eight up-and-down strokes with a total clearance of 0.05 mm) in a 2 ml glass homogenizer (Kontes, Vineland, NJ, USA). The supernatant of a 2-min 2000 g centrifugation at 48C was centrifuged at 10 000 g for 10 min, the pellet was resuspended, recentrifuged (10 000 g, 10 min) and resuspended in a buffer (pH 7.4) that consisted of 100 mM KCl, 75 mM mannitol, 25 mM sucrose, 5 mM Tris–phosphate, 0.05 mM EDTA (potassium salt), and 10 mM Tris–HCl.

2.4. Assay of GSSG Samples taken for GSSG determination were collected on ice, in sulfosalicylic acid (SSA, final concentration 2% (w / v)) to avoid spontaneous oxidation. The samples were frozen on dry ice and thawed at 48C two times, the supernatants were collected after centrifugation at 15 000 g for 20 min. The concentration of GSSG was determined after elimination of GSH with N-ethylmaleimide. The samples were mixed with N-ethylmaleimide in HEPESbuffer (final concentrations 10 and 90 mM respectively, final pH 6.8). After 2 min on ice, DTT, Na 2 EDTA, NaN 3 and HEPES-buffer were added (final concentrations: 22, 1, 5 and 50 mM respectively, final pH 8.0) in order to convert oxidized glutathione to the reduced form, GSH, and to avoid bacterial growth in the automated analysis step. After 30 min 5% acetic acid (v / v) was added to reduce the pH to 6.8. GSH was then derivatized with OPA and analyzed by HPLC using a TSK ODS-80TM column (5 mm particles, Tosoh, Tokyo, Japan). The derivatisation solution was prepared by dissolving 5 mg OPA in 50 ml methanol and diluted to 1 ml with 0.4 M borate-buffer (pH 10.0). The autoinjector was programmed to mix 30 ml of derivatisation solution with 70 ml of sample. After 2 min 20 ml of 5% acetic acid (v / v) was added to reduce the pH

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to neutrality before injection. The derivatives were eluted with a gradient from 15 to 95% methanol in sodiumacetate buffer (0.1 M, set at pH 6.85 with 50% acetic acid (v / v)) at a flow rate of 1 ml / min. Under these conditions the OPA-GSH derivative was eluted between 4.5 and 5.0 min.

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2.8. Statistical analysis Data were expressed as means6S.E.M. and were analyzed for significance using the Wilcoxon matched pairs rank sum test or the Mann–Whitney U-test for unpaired sample groups. A P value of ,0.05 was considered statistically significant.

2.5. Assay of total glutathione, amino acids and protein content 3. Results A mixture of b-mercaptoethanol, Na 2 -EDTA and NaN 3 was added to the samples, final concentrations of 20, 1 and 5 mM respectively, in order to convert all forms of oxidized glutathione to the reduced form, GSH, and to avoid bacterial growth. Thawed samples were extracted with an equal volume of 2 M HClO 4 containing 4 mM EDTA, mixed for 3 min using a vortex mixer and centrifuged at 7300 g for 10 min. 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 KHClO 4 . Total glutathione, protein amino acids, PEA, and GABA were determined using reversed phase HPLC employing automated precolumn fluorogenic labeling with OPA / b-mercaptoethanol as described earlier [72]. The column (30034.6 mm) was packed with Nucleosil 100-5 C 18 (Macherey-Nagel, Germany). The derivatives were eluted with a gradient from 0 to 90% methanol (containing 1.25% tetrahydrofuran (v / v)) in a Na-phosphate buffer (50 mM set to pH 5.40 with 1 M NaOH and containing 2.5% tetrahydrofuran (v / v)). The autoinjector (Water Wisp 717 plus) was programmed to add 25 ml OPA / b-ME to 50 ml sample. The methodology does not discriminate between thiols and disulfides, total glutathione therefore denotes the sum of the concentration of GSH, glutathione dimers (GSSG) and mixed glutathione disulfides (GSX). Tissue homogenates and mitochondria enriched fractions were dissolved in 1 M NaOH and the protein content was determined according to Lowry et al. [47].

2.6. Measurement of mitochondrial respiration The rate of mitochondrial respiration was measured as oxygen consumption in crude mitochondrial fraction according to Sims et al. [76] as described in an accompanying report [64]. RCR was defined as the ratio of respiration rates at state 3 to state 4.

2.7. Western blotting of MAP2 The MAP2 immunoreactivities in cerebral cortex homogenates were evaluated with blotting (western blot) as described by Puka-Sundvall et al. in an accompanying report [64].

3.1. Total glutathione and GSSG 3.1.1. Hypoxic-ischemic hemisphere The concentration of total glutathione was transiently decreased from 6.9160.4 nmol / mg protein (non-HI controls) to 5.07 nmol / mg protein (P,0.05) immediately after HI in the mitochondrial fraction (72.867.7% of the contralateral concentration, Fig. 1A). No such effect was observed in the tissue samples (Fig. 1B). Between 1 and 8 h after HI total glutathione levels were not significantly different when compared to concentrations of the contralateral hemisphere or from non-operated animals. At 14–72 h after HI the concentrations of total glutathione were decreased both in the mitochondrial fraction and in the tissue, minima were observed at 24 h after HI (55.167.2 and 60.268.9%, respectively). The concentration of GSSG increased selectively in the mitochondrial fraction immediately after HI from 0.2560.02 nmol / mg protein (non-HI controls) to 0.3560.02 nmol / mg protein (P,0.05, 140.968.0% of the contralateral concentration, Fig. 1C). 3.1.2. Hypoxic hemisphere The level of GSSG was |4% of total glutathione both in the mitochondrial fraction and in the tissue (Table 1). No significant changes were observed in total glutathione or GSSG concentrations in the hypoxic hemisphere, compared to values from corresponding control animals (Table 2). 3.2. RCR and MAP2 The results on RCR and microtubule associated protein2 (MAP2) are presented by Puka-Sundvall et al. in accompanying paper [64]. Briefly, the mitochondrial RCR was decreased immediately after HI in the ipsilateral hemisphere, recovered at 3 h with a secondary drop at 8–72 h (Fig. 2). The decreased RCR was due to reduced state 3 respiration (i.e. in the presence of ADP). A drop in MAP2 immunoreactivity was notable, although not significant, from 0 to 8 h of reperfusion. A significant decrease was observed after 14 h, and at 24 h post insult it was almost depleted (Fig. 2). RCR and MAP2 immunoreactivity were unaltered in the contralateral cerebral cortex.

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Fig. 1. Total glutathione, GSSG and PEA levels in mitochondria enriched fraction and tissue from HI cortex, expressed as percent of contralateral cortex, means6S.E.M. (n56–12). The concentrations were determined in controls (postnatal day 7, 8 and 10) and at indicated times after HI. * P,0.05 compared to contralateral hemisphere, § P,0.05 compared to non-HI controls.

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Table 1 Contents of total glutathione, GSSG and amino acids in control 7-day-old rats (n56–12)a

Total glutathione GSSG PEA a

Tissue

Mitochondrial fraction

14.4960.51 0.6560.04 102.2065.22

6.9160.4 0.2560.02 44.4762.33

Tissue Aspartate Glutamate GABA Glutamine

29.1161.03 89.5262.81 12.3760.89 32.2361.39

Values are given as nmol / mg protein (means6S.E.M.)

3.3. Amino acids 3.3.1. HI hemisphere (expressed as percent of contralateral hemisphere) The concentrations of PEA (Fig. 1E and F) changed similarly to total glutathione both in the mitochondrial fraction and in the tissue. The level was 73.665.2% at 0 h after HI in the mitochondrial fraction, and minima of 54.964.3% and 47.165.8% were reached after 14 h in the mitochondrial fraction and after 24 h in the tissue, respectively. The concentrations of glutamate and aspartate (Fig. 3) remained unchanged in the tissue from 0 to 24 h after HI and were decreased at 72 h after HI (66.266.1%, and

67.8612.6% respectively). The tissue concentrations of glutamine (Fig. 3) were increased from 3 to 72 h after HI (maximum at 8 h, 162.3618.6%). The GABA concentrations (Fig. 3) were increased in the tissue at 0, 1 and 3 h after HI (maximum at 3 h, 175.669.7%), and decreased 72 h after HI (55.866.8%).

3.3.2. Hypoxic hemisphere (expressed as percent of nonHI controls, Table 2) No changes were observed in the concentrations of PEA or glutamate. The aspartate level was decreased in the tissue at 0 h and increased at 72 h after HI. The glutamine

Table 2 Total glutathione, GSSG and amino acid levels in contralateral, hypoxic cortex expressed as percent of untreated controls, means6S.E.M. (n56–12)a

0 1 3 8 14 24 72

h h h h h h h

Time after HI 0 1 3 8 14 24 72

h h h h h h h

Time after HI 0 1 3 8 14 24 72 a

h h h h h h h

Total glutathione Mitochondria

GSSG Mitochondria

PEA Mitochondria

104.5613.1 84.8610.1 96.4612.2 88.4610.6 84.165.2 97.664.7 96.765.6

100.2610.3 93.269.4 91.3612.0 97.066.5 102.669.8 97.7610.3 103.4611.6

125.2621.0 119.0616.1 104.0618.9 107.4616.3 89.867.9 104.964.6 88.967.1

Total glutathione Tissue 92.564.7 107.067.3 92.166.2 102.868.7 90.663.8 97.667.2 107.661.4

GSSG Tissue 104.164.3 96.865.5 96.265.3 89.769.8 93.467.5 105.365.2 97.264.3

PEA Tissue 89.567.5 112.0612.3 91.269.0 104.7615.0 83.5613.9 92.568.9 113.061.4

Aspartate Tissue 38.462.9 * 88.463.8 81.862.8 87.3610.8 85.965.1 95.269.6 119.862.5 *

Glutamate Tissue 94.865.2 103.865.6 96.867.1 93.769.1 91.464.4 101.069.5 105.664.5

GABA Tissue 145.3618.3 * 178.1628.8 * 95.8611.9 86.1615.8 71.362.7 * 82.267.7 120.762.14 *

Concentrations were determined in the mitochondria enriched fraction and in the tissue at indicated times after the insult.

Glutamine Tissue 86.668.9 116.269.8 110.0611.8 113.5611.2 112.967.0 121.368.62 126.463.4 *

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4. Discussion

4.1. Total glutathione and GSSG

Fig. 2. GSH (total glutathione) and RCR (respiratory control ratio) was measured in the mitochondrial fraction. RCR is defined as the oxygen consumption in the presence (state 3) / absence (state 4) of ADP. MAP2 (microtubule associated protein-2) was calculated as total immunoreactivity of MAP2 (280 kDa 170 kDa fragments) in cortex tissue. GSH and RCR are expressed as percent of non-HI controls, MAP2 is expressed as percent of contralateral cortex. The values of RCR and MAP2 originate from Puka-Sundvall et al. [64].

concentration was increased at 72 h after HI in the tissue. The concentration of GABA was increased at 0, 1 and 72 h, but decreased 14 h after HI.

Fig. 3. Tissue levels of aspartate, glutamate, glutamine and GABA in HI cortex, expressed as percent of contralateral cortex, means6S.E.M. (n5 6–12). * P,0.05.

Cerebral HI evoked an immediate decrease of total glutathione in the mitochondria-enriched fraction in cortex of the immature rat. Possible mechanisms mediating the decrease include increased oxidation, release from the mitochondria and / or decreased import from the cytosol. The increased GSSG concentration indicates oxidative stress during the HI period, but only accounts for a minor part of the early loss of mitochondrial glutathione. Although increased free radical formation occurs during the reoxygenation period [12,61], it is not known whether this is the case also during the HI period. The increase in GSSG could also be due to insufficient GSSG reductase activity or lack of NADPH, the reducing substrate in this reaction [38,51]. Mitochondria do not synthesize glutathione, but it is imported from the cytosol [25,55]. There was no decrease in the concentration of total glutathione in the tissue immediately after HI, ruling out decreased cytosolic glutathione levels as a cause of the initial glutathione loss in the mitochondria-enriched fraction. However, glutathione uptake in the mitochondria may be reduced as such uptake is stimulated by ATP [13,19,55] and ATP is decreased during and immediately after the HI period [58,90,94]. An event known to cause increased efflux of mitochondrial glutathione in vitro is opening of the mitochondrial permeability transition pore [66,73]. Although pore opening is likely to occur after HI it is not known if it occurs during the HI period in the immature animal. Glutathione is an anion at neutral pH, another possibility is thus efflux via anion selective channels which are present in the inner membrane [20]. The function of these channels are unknown but have been suggested to be involved in regulation of mitochondrial volume [20]. Opening of anion specific channels in the mitochondria could also explain the decrease of the anion PEA in the mitochondrial fraction (see also below). The early alterations in the mitochondrial fraction of total glutathione and GSSG were coincident with a decreased RCR but any cause–effect relationship has not been proven. The crude mitochondrial fraction used in this study contains mitochondria, synaptosomes and myelin. The contribution of the non-mitochondrial elements is unknown but is likely to be relatively small due to the immature state of the tissue. The rationales for using the crude fraction are the low amount of tissue available in each animal, and that isolation of mitochondria may involve a selection of mitochondrial subpopulations. The early reduction in RCR after HI in this study was similar to that observed in tissue homogenates of cerebral cortex in the immature rat [24]. These findings indicate that the preparation used in this study does not further damage the mitochondria. The later drop in the concentration of total glutathione,

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observed in both the mitochondrial fraction and in the tissue at 14–72 h, was not paralleled by increased GSSG. This is in agreement with studies on GSH and GSSG tissue concentrations after ischemia in the mature brain [16,67,75]. The decreased tissue glutathione concentration is possibly due to efflux of glutathione followed by catabolism. Efflux of glutathione is increased after ischemia [2,41,57], paralleled by increased extracellular levels of cysteine and g-glutamyl-glutamate[45,57], products of g-glutamyl transpeptidase activity. Likewise, neuronal energy impairment, induced by 1-methyl-4phenylpyridinium (MPP 1 ), causes massive efflux of glutathione followed by extracellular hydrolysis of glutathione by g-glutamyl transpeptidase [33]. The molecular mechanisms of glutathione transport have not yet been clearly identified for any cell type in the brain. A carrier has been suggested to be involved in the efflux of glutathione in astrocytes [70], and bidirectional transport of intact glutathione was recently observed in synaptosomes [36]. It is not settled if the relatively well characterized glutathione transport in liver cells (for reviews see [4,39]) also operate in brain cells. The decreased concentration of total glutathione may also be related to decreased ATP dependent synthesis as the time course is similar to the secondary decrease in RCR and the high energy phosphate level [6,24,64]. In agreement, glutamate cause a parallel decrease in glutathione, ATP and impairment of mitochondrial function in cultured neurons [1]. A correlation between the tissue concentration of glutathione and ATP concentration is also observed after ischemia in the adult animal [67]. The decrease in tissue and mitochondrial glutathione concentrations was also accompanied by a marked loss of the dendrosomatic neuronal cytoskeleton protein MAP2, which is considered to be a marker for neuronal damage after HI [23,49]. The secondary decrease in mitochondrial glutathione may be due to decreased glutathione uptake from the diminished cytosolic concentration and / or due to poor function of mitochondria. In accordance, ultrastructural studies do show that swelling of mitochondria and accumulation of calcium occur prior to the secondary decrease in glutathione [62]. Glutathione could also be released from mitochondria via pore opening or activation of anion channels in the inner membrane, as discussed above. The decreased glutathione concentration may also be related to production of cytokines, which increase early after HI [27,78]. Application of tumor necrosis factor (TNF-a) or interleukin-1-b to astrocytes lead to depletion of glutathione, an event suggested to cause apoptosis [77]. To our knowledge it is not known if cytokines also initiate depletion of glutathione in neuronal cells. The degeneration of neurones after HI in the immature animal probably involves both apoptosis and necrosis [62,95]. In the HI hemisphere caspase 3 activation, a hallmark of apoptosis, peaks around 24 h post-insult [64], i.e. at the time-point of maximal glutathione depletion. Apoptosis induced in

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human monocytic cells and in jurkat cells leads to a dramatic reduction of intracellular glutathione via extrusion [21,22,85]. Whether efflux is involved in the apoptotic process is unclear [22,86] but several studies indicate a relationship between glutathione depletion and apoptosis in neuronal cells in vitro [7,17,52,65]. A possible link between diminished glutathione concentration and cell death, either by apoptosis or necrosis, is by altered redox state. The activities of caspases are redox regulated, low concentrations of H 2 O 2 activates caspases, whereas high concentrations of H 2 O 2 suppress caspase activity and leads to necrosis [32,84]. Glutathione and glutathione-related enzymes are present in both glia and neurons [40,42,59,68,71]. The neuron is the primary site of HI injury [87]. Our results on a more or less complete loss of the neuronal marker MAP2 [64] thus indicate that the decrease in glutathione is at large neuronal. Although the cause and effect relationship remains to be found many reports do show that depletion of glutathione can be detrimental for mitochondrial function and aggravate ischemic nerve cell damage (see introduction).

4.2. Amino acids The more or less perfect match between the decreased concentrations of PEA and glutathione following HI is very intriguing. After NMDA-, and kainate-receptor stimulation in hippocampal slices there is a parallelism between glutathione and PEA efflux [88]. Increased efflux of PEA is a regular finding after insults such as ischemia, epilepsy and hypoglycemia [29,43,72]. Efflux of PEA after ischemia is partly sensitive to anion channel blockers [60]. One plausible explanation for the decrease in PEA (and glutathione) is thus elevated efflux via anion channels or by a mechanisms that are anion channel dependent. The decrease in PEA is likely to be neuronal as the PEA concentration correlates well with neuronal but not with glial protein markers [31]. The biological significance of a lowered PEA concentration is not known. The increased glutamine concentration beginning at 3 h after HI is puzzling. One putative explanation is that HI causes a disturbance in the neuronal-glial glutamateglutamine homeostasis in favor of increased synthesis of glutamine in glia. General proliferation of glial cells is a later phenomenon starting 24 h after HI [9,11]. Glutamine synthesis is ATP-dependent and was not reduced at any time point after HI which further strengthen the view that the impairments in energy production is at large neuronal. Immediately after HI the tissue concentrations of aspartate were similarly decreased in the HI (ipsilateral) and hypoxic (contralateral) hemisphere compared to non-hypoxic controls, which is in agreement with a previous report [3]. This is likely due to accumulation of pyruvate during hypoxia [93] which favors synthesis of a-ketoglutarate via aminotransferase activity. The increase in a-ketoglutarate

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may in turn lower tissue aspartate through the activity of aspartate aminotransferase as proposed earlier [93]. The initial increase of GABA at 0 and 1 h after HI may be due to the enhanced activity of glutamate decarboxylase by low pH and low ATP [74,79,83]. Another possible mechanism is by inhibition of GABA transaminase, which requires NAD 1 and has a high pH optimum [5]. The later decrease which was significant at 72 h may be due to loss of GABA-ergic neurons [69]. In conclusion, HI leads to a transient decrease of total glutathione and an increase of GSSG in the mitochondrial fraction immediately after the insult. Total glutathione levels were also significantly decreased from 14 to 72 h post-insult in both the mitochondrial fraction and in the tissue. Both the early and the late decreases in total glutathione preceded declining levels of the neurotransmitters GABA and glutamate and were parallel to a decrease in RCR, PEA and MAP2 levels. The reduced glutathione levels after HI may be of pathophysiological relevance by rendering the cells more vulnerable to oxidative stress and / or by affecting redox-regulated factors, such as mitochondrial permeability transition [14], caspases [32,84] and transcription factors [35,54].

Acknowledgements This work was supported by the National Institute of Health Grant GM 44842, the Swedish Natural Science ˚ ´ Research Council (No. 1905-310) and Ahlen-Stiftelsen.

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