Chapter 25 Oxidative Stress in Stroke Pathophysiology: Validation of Hydrogen Peroxide Metabolism as a Pharmacological Target to Afford Neuroprotection

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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 effects, 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, whereas no effect 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 effect was reverted by concomitant bath application of the catalase inhibitor 3‐aminotriazole (20 mM), suggesting the involvement of catalase in mediating the neuroprotective effects 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.

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 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 effects, it has been suggested that H2O2 generates sufficient 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 effect 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, Nisticò 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.

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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 buffered 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

Neuroprotection by MS Against Transient MCAo‐Induced Brain Damage

Systemic (i.p.) administration of the GPx inhibitor MS (1.5–150 mg/kg) dose‐dependently 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 shown in Fig. 1C. Ischemic damage in vehicle treated animals involved brain regions supplied by the middle cerebral

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

References (30)

  • M.V. Avshalumov et al.

    Activation of ATP‐sensitive K+ (KATP) channels by H2O2 underlies glutamate‐dependent inhibition of striatal dopamine release

    Proc. Natl. Acad. Sci. USA

    (2003)
  • T.S. Brannan et al.

    Regional distribution of catalase in the adult rat brain

    J. Neurochem.

    (1981)
  • P. Calabresi et al.

    Long‐term synaptic depression in the striatum: Physiological and pharmacological characterization

    J. Neurosci.

    (1992)
  • B.T. Chen et al.

    H2O2 is a novel, endogenous modulator of synaptic dopamine release

    J. Neurophysiol.

    (2001)
  • P.J. Crack et al.

    Increased infarct size and exacerbated apoptosis in the glutathione peroxidase‐1 (Gpx‐1) knockout mouse brain in response to ischemia/reperfusion injury

    J. Neurochem.

    (2001)
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