Chapter 25 Oxidative Stress in Stroke Pathophysiology

OXIDATIVE STRESS IN STROKE PATHOPHYSIOLOGY: VALIDATION OF HYDROGEN PEROXIDE METABOLISM AS A PHARMACOLOGICAL TARGET TO AFFORD NEUROPROTECTION

Diana Amantea,* Maria Cristina Marrone,y Robert Nistico`,*,y,z Mauro Federici,y Giacinto Bagetta,*,z Giorgio Bernardi,y,} and Nicola Biagio Mercuriy,} *Department of Pharmacobiology, University of Calabria, Rende (CS), Italy y C.E.R.C.—Santa Lucia Foundation IRCCS, Rome, Italy z Section of Neuropharmacology of Normal and Pathological Neuronal Plasticity, University Centre for Adaptive Disorders and Headache (UCHAD), University of Calabria, Rende (CS), Italy } Neurological Clinic, Department of Neuroscience, ‘‘Tor Vergata’’ University, Rome, Italy

I. Introduction II. Experimental Procedures A. Animals and Drug Treatments B. Focal Cerebral Ischemia C. Neuropathology and Quantification of Ischemic Damage D. Electrophysiology E. Statistical Analysis III. Results A. Neuroprotection by MS Against Transient MCAo-Induced Brain Damage B. In Vitro Neuroprotection by MS is Mediated by Catalase IV. Discussion References

Reactive oxygen species (ROS) accumulation has been described in the brain following an ischemic insult. Superoxide anion is converted by superoxide dismutase into hydrogen peroxide (H2O2), and the latter is then transformed into the toxic hydroxyl radical, through the Haber–Weiss reaction, converted to water by glutathione peroxidase (GPx) or dismuted to water and oxygen through catalase. Accumulation of H2O2 has been suggested to exert neurotoxic eVects, although recent in vitro studies have demonstrated either physiological or protective roles of this molecule in the brain. In particular, oxidative stress is critically involved in brain damage induced by transient cerebral ischemia. Here, we demonstrate that inhibition of GPx by systemic (i.p.) administration of mercaptosuccinate (MS, 1.5–150 mg/kg) dose-dependently reduces brain infarct damage produced by transient (2 h) middle cerebral artery occlusion (MCAo) in rat. Neuroprotection was observed when the drug was administered 15 min before the ischemic insult, INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 85 DOI: 10.1016/S0074-7742(09)85025-3

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whereas no eVect was detected when the drug was injected 1 h before MCAo or upon reperfusion. Furthermore, application of MS (1 mM) to corticostriatal slices limited the irreversible functional derangement of field potentials caused by a prolonged (12 min) oxygen-glucose deprivation. This eVect was reverted by concomitant bath application of the catalase inhibitor 3-aminotriazole (20 mM), suggesting the involvement of catalase in mediating the neuroprotective eVects of MS. Thus, our findings demonstrate that MS is neuroprotective in both in vivo and in vitro ischemic conditions, through a mechanism which may involve increased endogenous levels of H2O2 and its consequent conversion to molecular oxygen by catalase.

I. Introduction

Cerebral ischemia is characterized by complex spatial and temporal events evolving over minutes or even days, leading to tissue damage in the regions supplied by the occluded vessel. Two major mechanisms involved in cellular damage following brain ischemia include amino acid excitotoxicity and oxidative stress produced by free radicals during reperfusion injury (Lo et al., 2003; Warner et al., 2004). Oxidative stress can be traced primarily to formation of superoxide and nitric oxide. Dramatic accumulation of reactive oxygen species (ROS) in the ischemic brain tissue triggers molecular pathways leading to necrosis, apoptosis, and neuroinflammation with subsequent neuronal loss and serious memory and/ or motor disturbances (Dirnagl et al., 1999). Principal sources of superoxide include electron leak during mitochondrial electron transport, perturbed mitochondrial metabolism, and inflammatory responses to injury (Warner et al., 2004). Being highly susceptible to oxidative stress, the brain possesses potent defenses against superoxide accumulation, such as free radical scavengers and, most notably, enzymatic antioxidants. Superoxide dismutase (SOD) catalyses dismutation of superoxide to hydrogen peroxide (H2O2) (Fridovich, 1995). Overexpression of SOD, as well as administration of SOD mimetics, provides significant neuroprotection in animal models of cerebral ischemia/reperfusion (see Warner et al., 2004). H2O2 can freely cross cell membranes and, although it has modest oxidative potential, it can be metabolized to produce potentially toxic free radicals, such as the hydroxyl radical (OH), through the Haber–Weiss reaction (Halliwell, 1992). Alternatively, H2O2 can be converted to water by glutathione peroxidase (GPx) or dismuted to water and oxygen through catalase (Brannan et al., 1981; De Marchesa et al., 1974). Transgenic mice overexpressing GPx are protected

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against transient focal brain ischemia damage (Weisbrot-Lefkowitz et al., 1998); whereas increased infarct size and exacerbated apoptosis is observed in GPx knockout mice (Crack et al., 2001), possibly due to accumulation of H2O2 in the ischemic/reperfused brain tissue. Interestingly, in addition to possible damaging eVects, it has been suggested that H2O2 generates suYcient molecular oxygen within the rodent spinal cord and in rat hippocampal slices to support synaptic transmission during hypoxia (Fowler, 1997; Walton and Fulton, 1983). Moreover, we have recently demonstrated that the neuroprotective eVect of H2O2 against oxygen glucose deprivation (OGD) in rat substantia nigra or hippocampal slices is due to production of molecular oxygen through catalase (Geracitano et al., 2005; Nistico` et al., 2008). Thus, in conditions of reduced oxygen supply, H2O2 may exert a protective role through its metabolic degradation to O2. However, to date, there is no information on whether H2O2 may contribute to neuroprotection against brain ischemia in vivo. Here, we demonstrate that systemic administration of mercaptosuccinate (MS), a GPx inhibitor, significantly reduces brain infarct damage produced by transient middle cerebral artery occlusion (MCAo) in rat. Neuroprotection is also observed in corticostriatal slices subjected to OGD, where it is inhibited by the catalase inhibitor 3-aminotriazole (3-AT). Thus, our findings suggest that increased endogenous levels of H2O2 during an ischemic insult may provide protection via production of molecular oxygen through catalase.

II. Experimental Procedures

A. ANIMALS AND DRUG TREATMENTS Adult male Wistar rats (Charles River, Calco, Como, Italy) were housed under controlled environmental conditions with ambient temperature of 22  C, relative humidity of 65%, and 12 h light:12 h dark cycle, with free access to food and water. Mercaptosuccinic acid (1.5–150 mg/kg, Sigma-Aldrich, Milan, Italy) or vehicle (0.01 M phosphate buVered saline (PBS), 1 ml/kg) were administered i.p. 15 min or 1 h before MCAo, or at the onset of reperfusion. All the experimental procedures were carried out in accordance with the European Community Council Directive on 24 November, 1986 (86/609/ EEC), included in the D.M. 116/1992 of the Italian Ministry of Health. All eVorts were made to minimize the number of animals used and their suVering.

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B. FOCAL CEREBRAL ISCHEMIA Brain ischemia was induced by occlusion of the middle cerebral artery in rats weighing 280–320 g by intraluminal filament, using the relatively noninvasive technique previously described by Longa et al. (1989). Briefly, rats were anaesthetized with 5% isoflurane in air, and were maintained with the lowest acceptable concentration of the anaesthetic (1.5–2%). Body temperature was measured with a rectal probe and was kept at 37  C during the surgical procedure with a heating pad. Under an operating microscope, the external and internal right carotid arteries were exposed through a neck incision. The external carotid artery was cut approximately 3 mm above the common carotid artery bifurcation and a silk suture was tied loosely around the external carotid stump. A silicone-coated nylon filament (diameter: 0.37 mm, Doccol Corporation, Redlands, CA, USA) was then inserted into the external carotid artery and gently advanced into the internal carotid artery, approximately 18 mm from the carotid bifurcation, until mild resistance was felt, thereby indicating occlusion of the origin of the middle cerebral artery in the Willis circle. The silk suture was tightened around the intraluminal filament to prevent bleeding. The wound was then sutured and anaesthesia discontinued. To allow reperfusion, rats were briefly reanaesthetized with isoflurane, and the nylon filament was withdrawn 2 h after MCAo. After the discontinuation of isoflurane and wound closure, the animals were allowed to awake and were kept in their cages with free access to food and water. Cerebral blood flow (CBF) was monitored in the cerebral cortex of the ischemic hemisphere corresponding to the supply territory of the middle cerebral artery by laser-doppler flowmetry (DRT4, Moor Instruments, Devon, UK). To this aim, a rectangular bent laser-doppler probe was glued onto the parietal bone (2 mm posterior and 5 mm lateral from bregma) and local CBF was continuously measured from 20 min before the onset of ischemia until 10 min after reperfusion, keeping the animal under isoflurane anaesthesia. Flow values were collected every 5 min before MCAo and after reperfusion; whereas data were collected at 10 min intervals during occlusion.

C. NEUROPATHOLOGY AND QUANTIFICATION OF ISCHEMIC DAMAGE Cerebral infarct volume was evaluated 22 h after reperfusion in rats subjected to 2 h MCAo. Animals were sacrificed by decapitation and the brains were rapidly removed. Eight serial sections from each brain were cut at 2-mm intervals from the frontal pole using a rat brain matrix (Harvard Apparatus, MA, USA). To measure ischemic damage, brain slices were stained in a solution containing 2% 2,3,5-triphenyltetrazolium chloride (TTC) in saline, at 37  C. After 10 min incubation, the slices were transferred to 10% neutral buVered formaldehyde and

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stored at 4  C prior to analysis. Images of TTC-stained sections were captured using a digital scanner and analyzed using an image analysis software (ImageJ, version 1.30). Infarct volume (mm3) was calculated by summing the infarcted area (unstained) of the eight sections and multiplying by the interval thickness between sections (Li et al., 2000). D. ELECTROPHYSIOLOGY Male Wistar rats (280–320 g of weight) were deeply anaesthetized with halothane and subsequently decapitated. Brain slices were prepared according to a previously described procedure (Geracitano et al., 2003). Briefly, the brain was rapidly removed and corticostriatal coronal slices (300 mm) were cut from a tissue block with the use of a vibratome (at 20–25  C) in artificial cerebrospinal fluid solution (ACSF). Slices containing the neostriatum and the neocortex were incubated in a reservoir (1 h, 33  C) and separately transferred into a recording chamber, completely submerged in a continuously flowing ACSF (32.5–33  C, 2.5–3 ml/ min) and gassed with a 95% O2–5% CO2 mixture. The composition of this ACSF was (in mM): NaCl, 126; NaH2PO4, 1.2; MgCl2, 1.2; CaCl2, 2.4; KCl, 2.5; NaHCO3, 18; glucose, 10. To study the eVects of in vitro ischemia, slices were deprived of both oxygen and glucose by omitting glucose from standard ACSF and saturating it with a gas mixture of 95% N2 and 5% CO2. Field potentials were recorded using extracellular glass microelectrodes filled with ACSF (5–10 M ) and placed within the dorsal striatum. Signals were fed to an Iso-DAM8 amplifier (WPI), filtered at 1 kHz, acquired and analyzed with the ‘‘LTP program’’ (Anderson and Collingridge, 2001). A bipolar concentric NiCr-insulated stimulating electrode was placed in the white matter between the cortex and the striatum to activate corticostriatal fibers. Test stimuli were delivered every 60 s, at half-maximal intensity. The field potential amplitude was defined as the mean amplitude of the peak negativity, measured from the peak of the early and the late positivity (Calabresi et al., 1992). MS and 3-amino-1,2,4-triazole (3-AT), obtained from Sigma-Aldrich (Milan, Italy), were dissolved to their final concentrations in ACSF. E. STATISTICAL ANALYSIS Data are reported as means  S.E.M. and statistical analysis was performed by the Student’s t-test or by ANOVA followed by Dunnett’s post hoc test, as appropriate. Experimental data were elaborated by means of Prism 3 program (GraphPAD Software for Science, San Diego, CA, USA), and diVerences were considered statistically significant for P < 0.05.

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III. Results

A. NEUROPROTECTION BY MS AGAINST TRANSIENT MCAO-INDUCED BRAIN DAMAGE Systemic (i.p.) administration of the GPx inhibitor MS (1.5–150 mg/kg) dosedependently reduced brain infarct area and volume produced by 2 h MCAo, as assessed by TTC staining 22 h after reperfusion (Fig. 1A–C). A representative image of the infarcted (pale) areas throughout the brain of rats treated with MS (150 mg/kg) or vehicle (PBS, 1 ml/kg), administered i.p. 15 min before MCAo, is

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FIG. 1. Systemic administration of MS dose-dependently reduces brain infarct damage produced by transient MCAo in rat. Brain infarct areas (A) and volumes (B) were measured in TTC-stained brain coronal sections from rats treated with mercaptosuccinate (MS), i.p., 15 min before transient (2 h) MCAo. Brain damage was evaluated after 22 h of reperfusion. Values are expressed as mean  S.E.M.; * indicates P < 0.05 versus vehicle (one-way ANOVA followed by Dunnett’s posttest; n ¼ 4–6 rats per experimental group). (C) Representative brain coronal sections (2 mm thick), stained with TTC, showing the infarct area (unstained) in rats treated with MS (150 mg/kg) or vehicle (PBS, 1 ml/kg) i.p., 15 min before transient (2 h) MCAo followed by 22 h reperfusion. Compared to vehicle-treated animals, MS

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shown in Fig. 1C. Ischemic damage in vehicle treated animals involved brain regions supplied by the middle cerebral artery, namely the striatum and the frontoparietal cortex; penumbral regions corresponding to the medial striatum and the motor cortex were rescued as a result of MS treatment (Fig. 1C). Moreover, at the highest neuroprotective dose tested (150 mg/kg), the drug did not significantly aVect CBF assessed by laser-doppler flowmetry in the frontoparietal cortex (Fig. 1D). Neuroprotection was only observed when MS was administered 15 min before MCAo. In fact, administration of the drug 1 h before the induction of ischemia or at the beginning of reperfusion resulted in an infarct volume which was not significantly diVerent from vehicle treated animals (Fig. 2). This suggests that a limited time-window exists to provide reduction of brain infarct damage by GPx inhibition.

B. IN VITRO NEUROPROTECTION BY MS IS MEDIATED BY CATALASE Electrophysiological experiments showed that when corticostriatal slices were bathed in an ischemic medium, there was a decrease in the field excitatory post synaptic potential (f EPSP) response that was dependent on the duration of the oxygen and glucose deprivation. The eVects of diVerent periods of OGD are shown in Fig. 3A: after 7 min of OGD the f EPSP depression was transient and fully reversible (n ¼ 4) upon reperfusion with normal oxygenated ACSF; conversely, synaptic transmission failed to recover when 12 min OGD was applied (n ¼ 11). A significant recovery of the f EPSP was observed (61.22  1%, paired Student’s t-test P < 0.05, Fig. 3B, n ¼ 11) following superfusion with MS (1 mM), applied 15 min before and during OGD. Longer administration times or higher doses of MS were not able to ameliorate the percentage of f EPSP recovery (data not shown). In order to confirm and extend previous observations (Geracitano et al., 2005; Nistico` et al., 2008), suggesting a potential involvement of the catalase pathway in mediating the neuroprotective eVect of MS, we applied the catalase inhibitor 3-AT. The simultaneous administration of 3-AT (20 mM) with MS (1 mM) completely reverted the recovery promoted by the administration of MS alone (n ¼ 5, Fig. 3C). To exclude the possibility that 3-AT could per se contribute to the suppression of synaptic transmission during OGD, we show full recovery (Fig. 3D,

administration produced a significant reduction of brain infarct damage in penumbral areas including the medial striatum and the motor cortex. (D) Regional CBF was measured by laser-doppler flowmetry over the ischemic parietal cortex. For each rat, CBF decreased to approximately 20% of the baseline value during the 2-h period of ischemia (MCAo) and recovered to baseline during reperfusion (R). There were no significant diVerences in regional CBF between vehicle-treated controls (n ¼ 3) and MS (150 mg/kg)-treated animals (n ¼ 3). Values are expressed as mean  S.E.M.

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FIG. 2. Time-window for the neuroprotection aVorded by MS against MCAo-induced brain damage. Brain infarct volume produced by transient (2 h) MCAo followed by 22 h of reperfusion in rats treated with MS (150 mg/kg) or vehicle (PBS, 1 ml/kg) i.p., 1 h or 15 min before the induction of ischemia, or upon reperfusion. Infarct volume was detected by staining consecutive 2-mm-thick coronal brain slices with TTC as described in the methods section. Data from vehicle-treated animals were pooled together, being the infarct volume values not significantly aVected by the treatment schedule. Values are expressed as mean  S.E.M.; * indicates P < 0.05 versus vehicle (one-way ANOVA followed by Dunnett’s posttest; n ¼ 4–6 rats per experimental group).

n ¼ 7) of f EPSPs when 3-AT was superfused during 7 min OGD. This eVect was similar to that obtained when 7 min OGD was applied alone (paired Student’s t-test, P ¼ 0.22).

IV. Discussion

The main finding of the present manuscript is that pharmacological inhibition of the GPx activity, reduces the extent of ischemic damage produced by transient MCAo in the rat brain and limits the irreversible functional derangement of field potentials in corticostriatal slices caused by a prolonged (12 min) oxygen-glucose deprivation. Although there is a general agreement that an ischemic insult facilitates an excessive generation of hydroxyl radicals and, therefore, GPx plays an important role in the defense against H2O2-induced damage (Crack et al., 2001; Hoehn et al.,

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FIG. 3. (A) Exposure to 7 min OGD (n ¼ 4, white squares) causes a transient fEPSP depression that is always reversible upon reoxygenation, whereas exposure to 12 min OGD (black squares) causes an irreversible loss of the fEPSP (n ¼ 1). (B) Treatment with MS (1 mM), 15 min before and during OGD, protects synaptic responses from the fatal insult (n ¼ 11). (C) Administration of 3-AT (20 mM, n ¼ 5) reverts the neuroprotection by MS; while (D) it was ineVective on synaptic responses per se (n ¼ 7). Bars indicate the time duration of the drug treatment and of the OGD.

2003; Sheldon et al., 2008), there are also experimental data suggesting that an increased production of H2O2 may, instead, represent a critical component of the neuroprotective processes that occur during or after an ischemic episode (Fowler, 1997; Geracitano et al., 2005). Here, we have tested the hypothesis that the pharmacological blockade of GPx activity, by reducing the degradation of H2O2, might provide more substrate for the action of catalase in converting H2O2 into H2O and O2. Thus, by using MS, a potent and specific inhibitor of selenium-dependent GPx (Chaudiere et al., 1984), we have observed that this compound protects the brain from the ischemic damage caused under either in vivo or in vitro experimental conditions.

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Systemic administration of MS resulted in a significant reduction of brain infarct volume produced by transient MCAo. The eVect of the drug was dosedependent, although limited to a specific time-window before (15 min) the ischemic event. Since GPx represents a crucial enzyme involved in H2O2 elimination in the brain, the net eVect of its inhibition by MS is accumulation of H2O2 (Warner et al., 2004). Previous studies have suggested that H2O2 may reduce release of neurotransmitters, including glutamate, dopamine, and -aminobutyric acid through activation of ATP-sensitive Kþ channels (Avshalumov and Rice, 2003; Chen et al., 2001). Although this mechanism has been suggested to underlie the beneficial eVects of H2O2 during metabolic stress, under our experimental conditions neuroprotection by MS appears to be due to an increased formation of H2O2 and, most notably, to its subsequent transformation into O2 and H2O by catalase. This hypothesis is supported by the loss of the MS-induced protective eVects on the ischemic derangement of the field potentials following a pretreatment with the catalase inhibitor 3-AT. Accordingly, we have already shown in a model of in vitro ischemia (OGD) that a positive modulation of the catalase activity plays an essential role in the H2O2dependent neuroprotection, supplying for the lack of O2 that occurs in the tissue after an ischemic event (Geracitano et al., 2005; Nistico` et al., 2008). Our results are in line with those obtained by Vanella et al. (1993), showing that a pretreatment with buthionine sulfoximine (BSO), a drug that inhibits GPx activity by reducing glutathione synthesis, prolongs the survival time of rats subjected to 20-min cerebral ischemia. At this point, we have to mention that our results suggest that an acute inhibition of GPx by MS is neuroprotective. In spite of this, data in the literature show that the deleterious eVects on cellular metabolism and neuronal survival after a stroke episode, appear when this enzyme is chronically inactivated or genetically ablated (Crack et al., 2001, 2006). Indeed, we have to consider the possibility that various changes in endogenous antioxidant enzymes might occur during cerebral ischemia and reperfusion (Kumari Naga et al., 2007; Ter Horst et al., 1994), giving rise to a complex scenario. It is worth noting that neurodegenerative processes likely occur after chronic inactivation of GPx associated with ischemia; while under conditions of a transient GPx inhibition, as that produced in our experimental settings, the early toxic events leading to tissue damage after ischemia (Lipton, 1999), are probably inhibited. The lack of toxicity due to transient GPx inhibition is also suggested by the analysis of the field potential that was not modified by the application of MS, demonstrating that basal synaptic transmission and neuronal activity remains viable in spite of the pharmacological inhibition of this enzyme. Therefore, our results suggest a pattern of an inverse relationship between the susceptibility of neurons to ischemia and the transient (beneficial) and chronic (detrimental) inhibition of GPx activity. Accordingly, the transient and rapid

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reduction of the activity of this antioxidant enzyme enhances H2O2 formation in the ischemia-reoxygenation phase (Lievre et al., 2000; Vanden Hoek et al., 1997), consequently the newly formed H2O2 acts as a substrate for catalase in producing the rescuing molecule O2. On the other hand, a sustained (chronic) inactivation of GPx reduces cellular viability by reducing neuronal resistance to harmful events. With regard to the protective eVects observed under in vivo ischemia, the laserdoppler investigation showing no flow modification after treatment with MS rules out that rheological modifications are involved in the neuroprotective action of the drug. This is also confirmed in in vitro experiments where an in situ rescuing eVect of this compound mediated by catalase was demonstrated. In conclusion, our findings suggest a neuroprotective role for the acute GPx inhibition against transient brain ischemia and are consistent with an involvement of H2O2 formation, and its consequent conversion into O2 by catalase, in mediating the beneficial eVect of MS.

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