Oxidative Stress and Pathophysiology of Ischemic ...

Send Orders of Reprints at [email protected] CNS & Neurological Disorders - Drug Targets, 2013, 12, 000-000

1

Oxidative Stress and Pathophysiology of Ischemic Stroke: Novel Therapeutic Opportunities Ramón Rodrigo*,1, Rodrigo Fernández-Gajardo1, Rodrigo Gutiérrez1, José Manuel Matamala2, Rodrigo Carrasco1, Andrés Miranda-Merchak1 and Walter Feuerhake2 1

Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Chile 2

Department of Neurological Sciences, Faculty of Medicine, University of Chile, Chile Abstract: Stroke is the second leading cause of death, after ischemic heart disease, and accounts for 9% of deaths worldwide. According to the World Health Organization [WHO], 15 million people suffer stroke worldwide each year. Of these, more than 6 million die and another 5 million are permanently disabled. Reactive oxygen species [ROS] have been implicated in brain injury after ischemic stroke. There is evidence that a rapid increase in the production of RO Simmediately after acute ischemic stroke rapidly overwhelm antioxidant defences, causing further tissue damage. These ROS can damage cellular macromolecules leading to autophagy, apoptosis, and necrosis. Moreover, the rapid restoration of blood flow increases the level of tissue oxygenation and accountsfor a second burst of ROS generation, which leads to reperfusion injury. Current measures to protect the brain against severe stroke damage are insufficient. Thus, it is critical to investigate antioxidant strategies that lead to the diminution of oxidative injury. The antioxidant vitamins C and E, the polyphenol resveratrol, the xanthine oxidase [XO] inhibitor allopurinol, and other antioxidant strategies have been reviewed in the setting of strokes. This review focuses on the mechanisms involved in ROS generation, the role of oxidative stress in the pathogenesis of ischemic stroke, and the novel therapeutic strategies to be tested to reduce the cerebral damage related to both ischemia and reperfusion.

Keywords: Antioxidants, reactive oxygen species, ischemic stroke, oxidative stress. 1. INTRODUCTION Stroke causes 9% of all deaths worldwide and is the second most common cause of death following ischemic heart disease [1]. Because life expectancy continues to increase in developed countries, the absolute number of individuals suffering from stroke will continue to rise in the near future [2]. The mortality rate in the acute phase of stroke is as high as 20% [3] and remains elevated for several years after the acute event compared to the general population [4]. Because the majority of stroke cases are nonfatal, long-term disability constitutes the major stroke-related burden [5]. Stroke can be classified into 2 categories, haemorrhagicand ischemic, the latterbeing the most prevalent form accountingfor up to 87% of all casesand is the target of most drug trials [6]. A significant proportion of ischemic stroke is caused by atherothrombosis, defined as atherosclerotic plaque disruption with superimposed thrombosis. Atherothrombosisis related to several conditions associated with oxidative stress, such as hypertension, diabetes, smoking and dyslipidaemia [7]. Current therapeutic interventions are limited to thrombolysis [8], the only intervention currently approved by *Address correspondence to this author at the Molecular and Clinical Pharmacology Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Independencia 1027, Casilla 70058, Santiago 7, Chile; Tel: 56-2-9786126; Fax: 56-2-9786126; E-mail: [email protected] 1871-5273/13 $58.00+.00

the United States Food and Drug Administration [FDA]. Thrombolysis is a treatment aimed to dissolve blood clots in order to restore blood flow before major brain damage has occurred. However, it is not effective for many patients and is only effective when applied within 4.5 hours of symptom onset [9]. In the United States, approximately 2% of stroke patients benefit from access to early thrombolysis [10]. Thus, the majority of patients may benefit from an alternative treatment. The aim of this review is to present an update of the evidence supporting the role of oxidative stress in the pathophysiology of ischemic stroke. Antioxidant system enhancement represents a potential target for novel therapies based on the prevention of oxidative stress-mediated brain injury in stroke. 2. OXIDATIVE STRESS Oxidative stress constitutes a unifying mechanism of injury of many types of disease processes. It occurs when there is an increase in the concentration of ROS and reactive nitrogen species [RNS] in the steady state. ROS and RNS are families of highly reactive species formed either enzymatically or non-enzymatically in mammalian cells. Oxidative stress arises from an imbalance between the generation of these species due to increased pro-oxidant activity over the antioxidant defence system in the body so that the latter become overwhelmed [11].

© 2013 Bentham Science Publishers

2

CNS & Neurological Disorders - Drug Targets, 2013, Vol. 12, No. 2

Some of the most important reactive species are superoxide, hydrogen peroxide [H2O2], hydroxyl radical, nitric oxide [NO], and peroxynitrite, among others. Superoxide is one of the most important ROS in the central nervous system [CNS], and it has the potential to harm the ROS-producing cell, as well as neighbouring cells. Superoxide is generated as a by-product of the respiratory chain or as a product of enzymes such as XO or nicotinamide adenine dinucleotide phosphate [NADPH] oxidase. Superoxide disproportionates quickly to oxygen [O2] and H2O2. In addition, various cellular oxidases, such as monoamine and amino acid oxidases, generate H2O2. In the presence of redox-active iron, highly reactive and toxic hydroxyl radicals are generated from H2O2 and superoxide in the Fenton reaction and the Haber–Weiss cycle. NO is generated by endothelial NO synthase [eNOS], neuronal NO synthase [nNOS] and inducible NO synthase [iNOS], being the latter the most important NO synthase isoform during pathologic conditions. When the level of superoxide ion is also elevated, the NO radical has dual effects: [i] to combine with superoxide anion to form highly pro-oxidant peroxynitrite and [ii] to interfere with superoxide dismutase [SOD] by reducing its antioxidant effect [12]. Antioxidant mechanisms are present in cells to remove ROS or even prevent their generation. Antioxidants such as reduced glutathione [GSH], vitamin C and vitamin E contribute to the antioxidant potential of cells. In addition, antioxidant enzymes such as SOD, catalase [CAT], GSH peroxidases [GSH-Pxs], heme oxygenase [HO], and glutathione reductase [GR] are important for the cellular defence against ROS. H2O2 generated as a product of SOD is substrate of the heme-containing CAT that converts H2O2 to O2and water. In addition, GSH-Px uses GSH to reduce H2O2 to water. Furthermore, the HO-1 system exerts three major functions in ischemia/reperfusion injury: [i] antioxidant effects; [ii] maintenance of microcirculation; and [iii] modulatory effects upon the cell cycle. HO-1 represents an important endogenous antioxidant defence mechanism against post-ischemic tissue damage [13,14]. ROS exert cell damage either directly or through behaving as intermediates in diverse signaling pathways, including deoxyribonucleic acid [DNA] damage, protein oxidation and lipid peroxidation [15]. Because of the short half-life of ROS, direct measurement is difficult in human subjects. The products formed by ROS attack to biomolecules, such as F2-isoprotanes, malondialdehyde [MDA],4-hydroxynonenal, protein carbonyl, among others, constitute a useful tool for oxidative stress assessment [16]. ROS have also an important role in physiological processes. For example, ROS play a beneficial role in the immune system by activating T lymphocytes and interleukin-2 [IL-2] production and mediating neutrophils and macrophages phagocytosis, participate in O2 homeostasis, secondary messenger system and signal transduction [17, 18]. 2.1. Oxidative Stress in the Brain Particularly in CNS, ROS and RNS are mainly generated by microglia and astrocytes and modulate synaptic

Rodrigo et al.

transmission and non-synaptic communication between neurons and glia. In addition to these roles, ROS and RNS can induce synaptic long-term potentiation required for memory formation and evidence suggests that there is an age-dependent alteration in the role of superoxide in modulating synaptic plasticity, learning, and memory formation [19]. Unlike other organs the brain is especially vulnerable to ROS and RNS increases due to low neuron antioxidant enzymes activity and high concentrations of peroxidisable lipids, high O2consumption, and high levels of iron, all acting as pro-oxidants under pathological conditions [20]. In agreement with this view, ROS production has been reported as an important mechanism of brain injury after exposure to ischemia and reperfusion [21, 22]. 3. PATHOPHYSIOLOGY The ischemic injury leads to blood brain barrier disruption, inflammation and oxidative stress. Protein, lipids and DNA are attacked by ROS. Oxidative stress leads to apoptosis, necrosis and autophagy pathways activation, which determine the final infarct size. A schematic diagram illustrating the main contributory factors involved in the pathophysiology of the ischemic stroke is presented in Fig. (1). 3.1. Ischemia – Reperfusion Histologically, stroke is characterised by an ischemic core [infarct] surrounded by a “penumbra” [peri-infarct] region. Within ischemic core, where blood flow is most severely restricted, excitotoxic and necrotic cell death occur within minutes. In the penumbra area, where collateral blood flow can buffer the full effects of stroke, cell death occurs less rapidly through oxidative stress-mediated mechanisms such as apoptosis and inflammation [23], which are important targets for therapeutic intervention. Ischemiareperfusion refers to the restoration of blood flow to a tissue that was previously deficient in blood flow. Tissue damage occurring after reperfusion, particularly in the penumbra area, plays an important role in determining the final size of the infarct and the consequences of stroke. Ischemia initiates a complex cascade of metabolic events, several of which involve the generation of ROS and RNS that mediate most of the damage occurring in the penumbral region of infarcts [24, 25] (Fig. 1). The pathological processes involved after cerebral reperfusion are mainly oxidative stress, excitotoxicity, intracellular calcium [Ca2+] overload, inflammation and apoptosis [26]. The sequence of events begins at the mitochondrial level with a critical diminution in the electron transport chain activity with consequent adenosine-5’-triphosphate [ATP] depletion [27]. Once mitochondrial ATP synthesis is inhibited by global ischemia, ATP is consumed within minutes, causing neuronal plasma membrane depolarisation, potassium leakage into the extracellular space and sodium [Na+] entry into cells [28]. This bioenergetic impairment also prevents the plasma membrane Ca2+-ATPase from maintaining the normally low concentration of cytosolic Ca2+. Soon after the cessation of blood flow, the disrupted

Antioxidants in Ischemic Stroke Treatment


3

Fig. (1). Schematic diagram illustrating the main contributory factors involved in the pathophysiology of the ischemic stroke. The ischemic injury leads to the BBB disruption, inflammation and oxidative stress. The BBB disruption leads to oedema and leukocyte infiltration, which enhance the inflammation. Protein, lipids and DNA are attacked by ROS and RNS. Furthermore, oxygen and glucose deprivation lead to ATP depletion, resulting in glutamate release and mitochondrial dysfunction. Oxidative stress activates apoptosis, necrosis and autophagy pathways, which determine the final infarct size. BBB, blood brain barrier; ICP, intracraneal pressure; eNOS, endothelial nitric oxide synthase, nNOS, neuronal nitric oxide synthase; NADPH oxidase, reduced nicotine adenine dinucleotide phosphate oxidase.

local metabolism quickly causes a large increase in neuronal activity and enhanced glutamate release [29]. Following cerebral ischemia, neuronal glutamate release activates several pre- and post-synaptic glutamate receptors resulting in arise in intracellular Ca2+leading to excitotoxic injury (Fig. 1). Additionally, the elevated extracellular glutamate concentrations can activate nNOS as a result of Ca2+ influx [30]. High levels of intracellular Ca2+, Na+ and adenosine diphosphate [ADP] can lead to dysfunction of the mitochondria, the generation of ROS and RNS, and the activation of proteases, phospholipases, and endonucleases, leading to cell death [31, 32]. Three distinct phases of ROS generation that occur temporally within the cell have been identified in cell culture [33]. The first increase in ROS occurs during oxygen and glucose deprivation [OGD], coincides with the period of mitochondrial depolarisation, and ceases when the mitochondrial potential is lost. OGD inhibits cell respiration

at complex IV, causing an accumulation of the reduced intermediates of the respiratory chain that enables electron leakage to generate ROS. The second rise in ROS occurs approximately 25–35 min after OGD or chemical ischemia as a result of XO activation and is evidenced by the strong decrease in ROS levels following exposure to oxypurinol and allopurinol. Intracellular ATP depletion is necessary to occur as a previous step since it leads to the conversion of adenine nucleotides into hypoxanthine and xanthine, substrates for XO. Rapid restoration of blood flow increases the level of tissue oxygenation, leading to the third phase of ROS generation, which is the major contributor to reperfusion injury [33]. This phase can be blocked by NADPH oxidase inhibitors and is absent in NADPH oxidase subunit–knockout mice [33]. Additionally, activation of NADPH oxidase through a Ca2+-dependent pathway is partly attributable to glutamate release and excitotoxicity.

4


It must be emphasised that additional mechanisms contribute to the generation of ROS in vivo such as infiltrating neutrophils, microglia⁄macrophages and endothelial cells. Accordingly, ROS generation during reperfusion has also been demonstrated in the endothelial and smooth muscle cells of small blood vessels [34] and cancontribute to blood brain barrier [BBB] disruption and inflammatory processes in in vivo models. Indeed, elimination of NADPH oxidase 4 [NOX4] in stroke patients was shown to bebeneficial in the absence of arterial recanalization [35], which suggests that NADPH oxidase also participates in ROS generation prior to reperfusion. XO was identified early onto be a source of ROS during neuronal reperfusion in human patients. However, additional intracellular ROS sources, particularly NADPH oxidase, have been identified that contribute to oxidative stress during reperfusion [36]. NOX4 is the most abundant vascular isoform of NADPH oxidases; its expression is higher in cerebral than in peripheral blood vessels [37] and is further elevated at both the messenger ribonucleic acid [mRNA] and protein level following stroke [35, 38]. NOX4 -/- but not NOX1 y/- or NOX2 y/- mice are protected from both transient and permanent ischemic stroke [35]. Therefore, it has been hypothesised that NOX4 is the most relevant source of ROS in stroke. 3.2. Inflammation Interruption of blood flow to the brain leads to energy loss and necrotic cell death in the ischemic core. This initiates an immune response characterised by a rapid activation of resident microglial cells and astrocytesand brain infiltration by neutrophils and macrophages, as demonstrated in both animal models and stroke patients [3942].

Rodrigo et al.

brain injury. Furthermore, NO is responsible for cytotoxicity through the inhibition of ATP-producing enzymesand stimulation of pro-inflammatory enzymes such as cyclooxygenase-2 [COX-2] [12, 50]. Robust precedent exists in the literature that emphasises a key role for COX-2 in cerebral ischemic injury in animal models [51, 52]. Disruption of the COX-2 gene provided protection against ischemic brain injury in rodents [53, 54] whereas COX-2 overexpressing mice had larger infarcts after experimental strokes [55]. Inflammatory cells generate ROS and RNS, which in turn are capable of activating these same inflammatory cells, resulting in a vicious cycle (Fig. 1). ROS and RNS can activate these inflammatory cells through several mechanisms including ROS activation of nuclear factor kappa B [NF-B], which is a highly redox-sensitive transcription factor mediating the activation of microglia, neutrophils and macrophages. Furthermore, local ROSinduced expression of cytokines from astrocytes also mediates the activation of microglia [56]. 3.3. Blood-Brain Barrier Disruption It has been largely confirmed in cell culture models of the BBB that ROS and RNS play a key role in increasing permeability of the cerebral endothelium. H2O2 induces increased paracellular permeability of BBB that is accompanied with redistribution of occludin and ZO-1 and increased protein expression of occludin and actin, which accounts for the BBB disruption [57, 58].

Infiltrating neutrophils and microglia/macrophages may generate toxic levels of NO mainly via the iNOS isoform, which is strongly induced following ischemic injury both in animal models [45, 46] and in stroke patients [47]. However, the contribution of nNOS [48] and NADPH oxidase expressed at the plasma membrane of microglia can also contribute to bursts of ROS generation [49].

There are two phases in BBB disruption:[i] early disruption of the BBB is likely to be primarily mediated by altered endothelial cell function. Inhibition of NAPDH oxidase with apocynin was effective in maintaining early BBB function. Genetic deletion of a NADPH subunit was also effective in preventing early BBB dysfunction and offered protection from brain swelling after stroke, suggesting an important contribution from NADPH oxidase to early BBB disruption [59]. [ii] Late disruption is also mediated by ROS through the activation of matrix metalloproteinases [MMPs] that degrade collagen and laminin in the basal lamina and disrupt the integrity of the vascular wall, increasing BBB permeability. Indeed, late disruption is a consequence of a complex scenario that includes inflammation, cell necrosis, the production of tissue-degrading enzymes such as MMPs and alterations in gene expression [60, 61]. NO seems to be the most important molecule in terms of MMPs activation and has been suggested to regulate MMPs via several routes. NO can directly activate MMP-9 by reacting with cysteine residues in the propeptide domain of MMP-9 to form S-nitrosylated derivatives [62] and by up-regulating MMP-9 through an increase of MMP-9 mRNA turnover [63].

Early after brain ischemia [in the first 2 hours], NO produced by eNOS exerts beneficial effects by promoting vasodilation and decreasing both platelet aggregation and leukocyte adhesion to the endothelium. However, NO produced during later stages can rapidly interact with superoxide to form peroxynitrite, the key RNS in ischemic

It is important to note that BBB disruption results in tissue swelling and thus, increases regional tissue hydrostatic pressure. Therefore, it is possible that the primary disruptionin the BBB that results in tissue swellingcan lead to constriction of vascular structures [i.e., the veins] (Fig. 1), resulting in reduced cerebral perfusion pressure [CPP] [64].

Four to six hours after ischemia, astrocytes become hypertrophic. This is followed by activation of microglial cells that develop into amoeboid shapes with enlarged cell bodies and shortened cellular processes [43]. Within hours after the ischemic insult, increased levels of cytokines and chemokines enhance the expression of adhesion molecules such as intercellular adhesion molecule 1 [ICAM-1]. The increased expression of adhesion molecules reaches a peak between six and twelve hours after the onset of ischemia [44] and can promote the adhesion and transendothelial migration of circulating monocytes and neutrophils.


3.4. Oedema Formation Brain oedema is a leading cause of death after stroke. ROS exert their deleterious actions in both cytotoxic and vasogenic oedema as described below. Cytotoxic oedema is the primary mechanism responsible for brain oedema during the earliest phase of ischemic injury. Cytotoxic oedema depends primarily on the duration and depth of ischemia and is an important indicator of ultimate infarct size and stroke severity. Impaired ion transport may play amajor role in the pathophysiology of cellular swelling. It has been proposed that ion transporters can be modulated by ROS, alterations which can include the peroxidation of membrane phospholipids, the oxidation of sulfhydryl groups in ion transporters and protein modification [65]. Indeed, irongenerated ROS in vitro inhibit sodium-potassium pump [[Na + K]-ATPase], Ca2+-ATPase, calmodulin-associated Ca2+ATPase, and Na+ –Ca2+-exchanger activities [66]. Oxidative stress also supports the formation of cytotoxic oedema through interactions with other mediators of cellular swelling. For example, lactic acidosis is known to cause swelling of neurons and astrocytes, triggering the release of iron from its binding site. This enhances hydroxyl radical formation by increasing intracellular H2O2levels and by supplying protons during the chain reaction between superoxide and NO [67]. In contrast, vasogenic oedema is caused by uncontrolled fluid leakage from the blood to the brain parenchyma through a weakened BBB and contributes to an actual net volume increase of the brain. As discussed above, reactive speciessuch as superoxide and NO exert direct effects on mediators of BBB disruption such asleukocytes, inflammatory response, MMPs and microvascular integrity. ROS/RNS also contribute to vascular endothelial growth factor [VEGF] expression, leading to abnormally permeable vasculature and vasogenic oedema [68]. 3.5. Apoptosis Apoptosis is caused by an imbalance between proapoptotic and anti-apoptotic signals. Specific ROS such as H2O2 or superoxide have been implicated as crucial mediators of apoptotic cell death [69, 70]. Indeed, several apoptotic signalling pathways are modulated by cellular redox status [71]. One model ofH2O2-induced apoptosis is the up-regulation of the Fas–Fas ligand [FasL] system, leading to the activation of caspase-8 and downstream caspases [72]. Extracellular FasL first binds to a receptor and an adaptor molecule, Fas-associated death domain [FADD protein], activates procaspase-8. Next, caspase-8 activates caspase-3, and this effector caspase cleaves poly [ADP-ribose] polymerase [PARP] and activates caspase-activated deoxyribonuclease [CAD], leading to DNA damage and cell death. Caspase-8 is also able to truncate and activate one of the B-cell lymphoma 2 [Bcl-2] family proteins, Bid, and initiate the mitochondrial pathway of apoptosis [73]. Alternatively, cerebral ischemia and reperfusion generates ROS within mitochondria, which then signal the release of cytochrome c by mechanisms that may be related


5

to the Bcl-2 family of proteins [69, 74] or GSH depletion [75]. The binding of released cytochrome c to Apoptotic protease activating factor 1 [Apaf-1] in the cytosol is a critical step in the formation of the apoptosome, which activates caspase-9, which then activates caspase-3 [73]. Activated caspase-3 is known to cleave many nuclear DNA repair enzymes, which then leads to nuclear DNA damage without repair, resulting in apoptosis. Additionally, H2O2 may activate nuclear transcription factors such as NF-B, activator protein [AP-1], and p53, which can up-regulate death proteins or produce inhibitors of survival proteins [76]. Indeed, accumulating evidence demonstrates that p53 is involved in neuronal death incerebral ischemic animal models [77-79]. The role of ROS in apoptosis, particularly peroxynitrite, has also been emphasised to cause single-strand breaks in DNA, which activate DNA-dependent kinases and ataxia telangiecta protein, leading to the activation, stabilisation, and upregulation of p53 [80, 81]. Moreover, levels of p53 and its products increase during the early hours after ischemia onset and are especially high in the infarct border zone in focal cerebral ischemia models [82, 83], suggesting a key role in cell death at the penumbra area. Oxidative stress may regulate p53-dependent transcription, p53 translocation, and pro-survival Akt signalling partially through phosphorylation. Decreasing oxidative stress by SOD1 overexpression results in neuroprotection. Mitochondrial dysfunction and oxidative stress may determine neuronal death/survival after stroke and neurodegeneration [84]. ROS-induced cell injury has been attributed in part to activation of mitogen-activated protein kinase [MAPK] pathways [85]. Members of the NADPH oxidase family that produce ROS, have been shown to be involved in cellular functions related to innate immunity, signal transduction, proliferation, and/or apoptosis [86]. Indeed, the 28-kDa NOX4 isoform plays a role in the induction of toll-like receptor 4 [TLR4]-mediated apoptosis caused by ischemiareperfusion injury [87]. ROS are also known to contribute to the activation of the extracellular signal-regulated kinases [ERK] 1/2 pathway. Although it is generally thought that activation of ERK 1/2 can confer a survival advantage to cells, there is growing evidence suggesting that persistent activation of ERK leads to cell death [88]. 3.6. Autophagy Unlike apoptosis, which is a caspase-dependent process, autophagy is a caspase-independent process in which cellular proteins and organelles are sequestered in double-membrane vesicles knownas autophagosomes, delivered to lysosomes, and ultimately digested by lysosomal hydrolases [89-91]. Although considered primarily to be a homeostatic response [92], the presence of autophagic structures in dying cells has implicated autophagy inthe cell death process, as excessive autophagic activity may destroy much of the cytosol and organelles, leading to the total collapse of all cellular functions. Protein levels of autophagy regulators Beclin 1 and microtubule-associated protein 1 light chain 3 [LC3] are reported to be up-regulated during cerebral ischemia [93]. In a rat model, the earliest changes of Beclin 1 levels in the

6


Rodrigo et al.

penumbra occurred at 6 h, peaked at 24 h and lasted for at least 2 days post-ischemia, and the observed Beclin 1 upregulation occurred in neuronsand astrocytes [93, 94].

activity [114] in erythrocytes. Previous findings relating stroke and oxidative stress biomarkers are summarized in Table 1.

Cellular oxidative stress and increased generation of ROS have been reported to serve as important autophagy stimuli during periods of nutrient deprivation, ischemia/reperfusion (Fig. 1), hypoxia, and in response to cell stress [95-98]. During cellular starvation and nutrient deprivation, there is an increased generation of mitochondrial-derived H2O2 through a Phosphoinositide 3-kinase [PI3K]/beclin-1 dependent pathway [95]. This leads to oxidation and consequent inhibition of autophagy-related protein 4 [ATG4], ultimately promoting autophagosome maturation. Beclin-1 is negatively regulated by its interaction with the anti-apoptotic protein Bcl-2 under normal conditions [99]. However, increased ROS activates the ubiquitin-proteosome system, which degrades Bcl-2 [100]. This allows for beclin-1 activation, subsequently resulting in autophagic cell death [101]. Additionally, oxidised low-density lipoproteins may enhance autophagy by up-regulating beclin-1 gene expression [102].

Table 1.

During reperfusion, ROS also damages organelles and cytosolic proteins and causes lipid peroxidation in the mitochondria, all of which exacerbate autophagy [103]. Additionally, antioxidant enzymes such as CAT and SOD are targeted by autophagosomes, thereby increasing the presence of ROS and creating a positive autophagic feedback loop leading to cell death [95]. Furthermore, adenosine monophosphate-activated protein kinase [AMPK] activity decreases during reperfusion, thus increasing autophagic death and beclin-1 up-regulation [104-106] 3.7. Oxidative Stress-Related Biomarkers Finally, it must be emphasized that stroke is an important factor in the overall antioxidant capacity and ROS production [107, 108]. Several studies have described increased levels of lipid peroxidation biomarkers in stroke patients, such as thiobarbituric acid reactive substances [TBARS] [109-112], MDA [113, 114], and lipid peroxides [111, 115], in plasma and erythrocytes. Plasma levels of various non-enzymatic endogenous antioxidants, such as vitamin C [107, 110, 113], vitamin E [109], vitamin A and uric acid [107], were also reported to be lower in patients with ischemic stroke than in a healthy control population. It has also been reported a decreased total antioxidant capacity [TAOC] in stroke patients, compared to 1 month after the ischemic episode [112]. These data suggest that the aforementioned antioxidants are oxidized during the cerebral ischemia-reperfusion event. These oxidative stress biomarkers also seem to be related to stroke outcome. Indeed, several studies have shown that brain damage and post-ischemic neurological deficit are correlated with increased SOD 1 and 3 activity in cerebrospinal fluid [CSF] [116], lipid peroxides in plasma [112, 115], MDA and TBARS in erythrocytes [111, 114], and also the ROS production by peripheral phagocytes [111]. In addition, histological and neurological parameters have been demonstrated to be negatively correlated with plasma levels of vitamins C and E [117], uric acid [118], TAOC [117, 112], SOD activity [119] or with SOD and GSH-Px

Previous Findings Relating Stroke and Oxidative Stress

Finding

Increased levels of lipid peroxidation biomarkers

Lower plasma levels of non-enzymatic endogenous antioxidant systems

Antioxidant or Prooxidant System Assessed

References

MDA by TBARS

[109-111]

MDA by HPLC

[113, 114]

Lipid peroxides in plasma and erytrocytes

[111, 115]

Vitamin C

[107, 110, 113]

Vitamin E

[109]

Vitamin A

[107]

Uric Acid

[107]

4. ANTIOXIDANT SUPPLEMENTATION IN STROKE TREATMENT AND CURRENT CLINICAL TRIALS Perhaps one of the most dramatic acute oxidative stresses in the CNS is the ischemia–reperfusion injury that occurs with ischemic stroke. The currently applied measures to protect the brain against the severe damage caused by stroke have yielded insufficient improvement for the outcome of these patients. Therefore, there is substantial need for further research on agents able to significantly reduce the cerebral damage related to both ischemia and reperfusion. These agents might be of particular importance not only in those patients who cannot receive thrombolysis but also in those who, undergoing this type of treatment, are at risk of the socalled reperfusion injury. Since substantial evidence supports the occurrence of oxidative stress during brain ischemiareperfusion, acute antioxidant enhancement has been evaluated as a neuroprotective strategy in stroke [120]. Furthermore, a significant positive association between plasma lipid hydroperoxide concentration and the National Institute of Health Stroke Scale score has been reported, as well as a significant negative correlation with the Glasgow Coma Scale score [115]. The therapeutic use of most studied antioxidants against stroke-induced brain damage is discussed below. A summary of the previous antioxidant strategies can be found on Table 2, with the most significative results. 4.1. Inhibition of ROS-Producing Enzymes 4.1.1. NADPH Oxidase Inhibition Superoxide producing NADPH oxidase has been shown to contribute to oxidative stress-mediated brain injury during both ischemia and reperfusion. Apocynin [4-hydroxy-3methoxyacetophenone] has been used as an efficient inhibitor of the NADPH oxidase complex in many experimental models [121,122], though the mechanism of inhibition has not yet been fully elucidated. It has been demonstrated that treatment with apocynin can attenuate brain injury after experimental ischemic stroke in the hippocampus after global cerebral ischemia [123-126].


Table 2.


7

Main Antioxidant Strategies to Prevent Stroke-Induced Cerebral Damage

Compound

Mechanism

Outcomes

References

Vitamin C

Water-soluble antioxidant, enzyme modulator

Reduced lipid peroxidation, increased plasma total antioxidant capacity

[151, 158, 159]

Vitamin E

Lipid-soluble antioxidant, enzyme modulator

Reduced lipid peroxidation, increased plasma total antioxidant capacity, anti-inflammatory effects

[159, 165]

Resveratrol

Antioxidant, anti-apoptotic, antiinflammatory

Reduced lipid peroxidation and oxidative stress, neuron death reduction, infarct size reduction

[169, 170, 172, 173]

Allopurinol

ROS production inhibition due to xanthine oxidase inhibition

35% infarct size reduction, neurological deficit attenuation

[140-145]

N-Acetylcysteine

Antioxidant

29-49% infarct size reduction, reduced oxidative stress

[181, 182]

Recent studies show that treatment with apocynin 5 minutes before reperfusion attenuated the cerebral injury in 75-minute and 24-hour ischemia models in rats [127, 128]. Administration of apocynin showed a marked reduction in infarct size compared with that of control rats. Medial carotid artery occlusion-induced cerebral ischemia was also associated with an increase in lipid and DNA peroxidation and nitrotyrosine formation, as well as IL-1 expression, inhibitor B [IB] degradation, ICAM expression and neutrophil infiltration in ischemic regions. ROS induce redox changes in the cell that result in phosphorylation of the I-B subunit of NF-B complex. After I-B is phosphorylated, a process of the proteolytic digestion of this subunit is activated. When the inhibitor subunit is dislodged from the P50/P65 heterodimer the activator NF-B can migrate to the nucleus and bind to DNA, thereby initiating transcription activating inflammatory cascade. These changes were markedly inhibited by apocynin treatments [127, 128]. Apocynin produces neuroprotection via decreasing ROS production; however, apocynin-induced increase in cellular stress may activate pro-apoptotic pathways [129]. It was also demonstrated that apocynin reduces the levels of apoptosis-inducing enzymes [Terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL], Bax and Bcl2 expression] resulting in a reduction in the infarct volume in ischemia–reperfusion brain injury [128]. Pharmacological inhibition of NADPH oxidases using the specific NADPH oxidase inhibitor VAS2870 [Vasopharm] [130] protects mice from brain ischemia within a clinically meaningful 2-hours time window [35]. This protection resulted in a decrease in infarct volume, oxidative stress, neuronal apoptosisand BBB leakage, andan increase in neurological score, motor function and survival. In contrast, the more commonly used inhibitor, apocynin, may not be specific for NADPH oxidase in vascular cells but rather functions as a nonspecific antioxidant [131], also inhibiting Rho kinase inhibitor [132], an activity that increases its nonspecific actions. If apocynin inhibits NADPH oxidases to any extent, it supposedly blocks the migration of the cellular NADPH oxidase complex subunit p47phox to the membrane, thus interfering with assembly of the functional NADPH oxidase complex [133]. Therefore, it is unlikely to inhibit the NOX4, which acts independently of any cytosolic subunits [134]. Indeed, application of apocynin had no effect on the formation of ROS or on the functional

outcome after experimental strokein a NOX4 knockout model [35]. 4.1.2. XO Inhibition Ischemia-induced depletion of intracellular ATP leads to the accumulation of hypoxanthine and xanthine, substrates for ROS-producing XO, which are the main sources of ROS in the setting of ischemia-reperfusion [135]. Allopurinol is an inhibitor of XO, andboth allopurinol and its metabolite, oxypurinol, have been suggested as hydroxyl radical scavengers [136]. During ischemia, xanthine dehydrogenase undergoes irreversible proteolytic conversion to XO, producing superoxide and H2O2 in the presence of O2[137]. In vitro studies in rodents demonstrate that hypoxia and reoxygenation increase the activity of XO and xanthine dehydrogenase [138]. Allopurinol was found to be protective against mortality and neurological impairment in a stroke model in spontaneously hypertensive rats [139]. In a permanent middle cerebral artery occlusion [MCAO] model in Sprague-Dawley rats, allopurinol pre-treatment reduced the infarct size by approximately 35% [140]. Allopurinol was also effective in a rabbit model of focal cerebral ischemia [141] and was effective in transient MCAO models at relatively high doses [142]. Oxypurinol reduced ischemic cerebral damage and attenuated neurological deficits [143, 144]. In a relatively mild model of stroke [20 min of ischemia and 10–90 min of reperfusion], oxypurinol produced improvements in cellular ATP levels [145]. To date, the human therapeutic experience with allopurinol is limited to one study conducted in 22 severely asphyxiated infants [146]. In this study, allopurinol tended to improve survival and exerted beneficial effects on ROS formation, cerebral blood flow, infarct volume, and electrical brain activity. Larger follow-up studies are required to expand on these pilot findings [147]. 4.2. Antioxidant Supplementation to Scavenge ROS 4.2.1. Vitamin C Vitamin C [ascorbic acid, ascorbate] is a potent watersoluble antioxidant in humans. Its role as a ROS scavenger and its redox relationship with other antioxidants have been of continuing interest [148]. Ascorbate directly scavenges oxygen- or nitrogen-based radical species generated during normal cellular metabolism [149]. This antioxidant vitamin

8


also behaves as an enzyme modulator, causing the upregulation of eNOS and down-regulation of NADPH oxidase [150]. Vitamin C antioxidant properties are enhanced when combined with other antioxidant agents, such as vitamin E [151]. Consumption of 5 to 9 servings of fruits and vegetables daily achieves steady-state vitamin C plasma concentrations of 80 μmol/L or less, and the peak values do not exceed 220 μmol/L, even after maximum oral administration of 3 grams 6 times daily [18 grams total] [152]. In contrast, the use of intravenous vitamin C is able to sustain plasma concentrations at levels as high as 15000 μmol/L [153]. Therefore, vitamin C plasma concentrations high enough to efficiently scavenge ROS can only be achieved by intravenous supplementation. Indeed, superoxide reacts with NO at a rate 105-fold greater than the rate at which superoxide reacts with ascorbic acid [154], and10 mmol/L ascorbate is needed to support its competition with NO for superoxide. The number of expressed sodium-dependent vitamin C transporter 2 [SVCT2] will limit ascorbate uptake and content in neurons, and the affinity only becomes relevant when a deficiency is present [149]. Indeed, vitamin C is accumulated mainly in the brain and adrenal gland [155] in part by the sodium-dependent transporter with saturable kinetics [153]. Although glia may not normally express the SVCT2, oxidative stress due to ischemia-reperfusion injury has been shown to increase SVCT2 mRNA expression in both neurons and glia [156] and its activity in brain capillary endothelial cells [157]. To our knowledge, there are only three studies concerning the effect of oral supplementation of vitamin C on acute ischemic stroke outcomes. In 59 stroke patients admitted to the hospital within 24 hours, Polidori and colleagues demonstrated that those randomly treated with 300 mg aspirin with200 mg vitamin C per day had slightly lower levels of lipid peroxidation markers [F2-isoprostanes] compared with those taking aspirin alone [158]. In this pilot study, no difference in clinical outcomes was observed between the two groups. Ullegaddi and colleagues found that antioxidant supplementation [500 mg of vitamin C plus 800 IU of -tocopherol] administered in acute ischemic stroke patients within 12 hours of symptom onset and with continued treatment up to 14 days was able to enhance antioxidant capacity and mitigate the oxidative damage [159]. A more recent study showed that 500 mg/day IV vitamin C alone resulted in increased plasma total antioxidant capacity but did not substantially improve the clinical and functional status of patients after 3 months [151]. Recently, the selective hydroxyl radical scavenger EPC-K1, a phosphate diester of vitamins C and E able to penetrate the BBB, has been reported to reduce infarct size and lipid peroxidation, as evaluated by TBARS levels, after transient ischemia [160]. 4.2.2. Vitamin E Vitamin E [-tocopherol], a fat-soluble vitamin known to be one of the most potent antioxidants, acts by preventing the propagation of the ROS chain reaction in biological membranelipids. Vitamin E is likely the most studied antioxidant due to its potential use in the prevention and

Rodrigo et al.

treatment of ischemic cerebrovascular disease in randomised controlled trials. Vitamin E has been reported to reduce infarct volume by 45–55% in permanent and transient models of cerebral ischemia [161]. Recent studies in humans have reported an association between the plasma concentration of ascorbic acid and tocopherol and the early clinical outcome after acute ischemic stroke [107, 117]. Indeed, ascorbate has been shown to spare/recycle -tocopherol in lipid bilayers [162] and in erythrocytes [163], thereby enhancing the antioxidant properties of vitamin E antioxidant. It was also reported that vitamins C and E are associated with decreased activation of NADPH oxidase [150]. Given the lipid-rich environment of the brain, the sparing or recycling of -tocopherol may be a very important role for ascorbate. Accordingly, when ascorbate is added to cultured cells, brain slices, and brain microsomes, it prevents lipid peroxidation, especially in combination with -tocopherol [164]. According to studies in stroke patients, the supplementation of vitamin E [727 mg/day] together with vitamin C [500 mg/day] within 12 hours post-infarct reduced the peroxidation of lipids and exerted anti-inflammatory effects [159, 165]. The differences in TAOC and MDA were significant during the first week. This occurred in association with a predicted increase in the plasma concentration of tocopherol and ascorbic acid. Plasma C reactive proteinwas also lower in those stroke patients who received antioxidant supplements compared with controls at 7 and 14 days [165], accounting for reduced systemic inflammation. Another uncontrolled study of 22 ischemic stroke patients reported an association between the plasma concentrations of ascorbic acid and -tocopherol and the degree of neurological impairment after ischemic stroke [117]. They demonstrated an association between the total plasma antioxidant activity, the volume of ischemic cerebral infarction and the degree of subsequent neurological impairment. 4.2.3. Resveratrol Resveratrol [3,5,4V-trihydroxystilbene], one of the major components of red wine and a powerful polyphenol, is a representative of this group and was found to scavenge ROS and prevent lipid peroxidation [166, 167]. In addition to antioxidant activity, resveratrol activates the peroxisome proliferator activated receptor alpha [PPAR-] and PPAR- receptors, which are recognised to have anti-inflammatory actions. To our knowledge, two studies have demonstrated that this compound can reduce the size of cerebral infarction, and the neuroprotective effect of resveratrol has been associated with an inhibition of the elevation in MDA and a reduction in GSH levels induced after ischemia in rats [168]. Resveratrol [20 mg/Kg, 3 days] reduced the infarct volume by 36% at 24 hours after MCAO in wild-type mice. This effect has been partially confirmed in PPAR- knockout mice, where resveratrol does not reduce the infarct volume after MCAO [169]. In mice, a single 20 mg/kg dose of resveratrol given orally 1 hour before permanent MCAO does not protect against ischemic damage. However, when given 20 mg/kg/day for 3 days before ischemia, resveratrol


significantly reduces the infarct size [169]. Another study using 30 mg/kg/day resveratrol for 7 days prior to ischemia significantly attenuated neuronal death and decreased the generation of ROS, lipid peroxidation and NO content in addition torestoring the antioxidant enzyme activity and [Na + K]-ATPaseactivity [170]. Furthermore, resveratrol brought antioxidant and [Na + K]-ATPaseactivity in the cortex and hippocampus back to normal levels. It is noteworthy that these findings are not clinically translatable, as in clinical practice resveratrol would be given after ischemia is induced. Gerbils injected intraperitoneally with resveratrol experienced decreased neuronal death in the hippocampus and hada lower glial cell activation either during or shortly after common carotid artery ligation and 24 hours afterwards [171]. In mice, resveratrol prevents the elevation of MMP-9, which is induced by cerebral ischemia-reperfusion [172]. By using in vitro neuronal cultures, resveratrol has been shown to induce a neuroprotective enzyme, HO-1, which is believed to exert a protective effect by counteracting the intracellular increase of heme, a pro-oxidant agent [173, 174]. The HO system is the rate-limiting step in the conversion of heme into biliverdin, carbon monoxide [CO] and free iron. Three HO isoforms have been identified: inducible HO-1, constitutively expressed HO-2, and the less well characterized HO-3. In brain, HO-2 is the major HO isoform under physiological conditions. The HO-1system exerts three major functions in ischemia/reperfusion injury: [i] antioxidant effects; [ii] maintenance of microcirculation; and [iii] modulatory effects upon the cell cycle. The antioxidant functions depend on heme degradation. The production of CO, which has vasodilatory and anti-platelet aggregative properties, can maintain tissue microcirculation. Strikingly, CO may also be instrumental in anti-apoptotic and cell arrest mechanisms. The HO-1 system prevents early injury in the re-perfused organ, and inhibits the function of immune reactive cells, such as neutrophils, macrophages and lymphocytes [175]. The HO/CO system serves as a neurovascular regulator. Thus, HO-2 can function as an O2 sensor in the brain and an O2–CO–Sulfhydric acid [H2S] cascade rapidly mediates hypoxia-induced cerebral vasodilation. In this cascade, hypoxia elicits vasodilation via the coordinate actions of CO generated by HO-2 and H2S generated by cystathionine synthase, a generating system of H2S that mediates the vasodilation of small arteries. Since CO tonically inhibits this enzyme, hypoxia releases the tonic inhibition leading to increased levels of H2S [14]. Therefore, constitutive gas CO plays physiologic roles for maintaining homeostasis of cell and organ function. Up-regulation of HO-1 may be among of the most critical cytoprotective mechanisms that are activated during times of cellular stress, and is thought to play a key role in maintaining antioxidant/oxidant homeostasis during times of cellular injury. Resveratrol neuroprotective effect is likely exerted by up-regulated expression of transcription factor Nrf2 and HO-1 to ameliorate oxidative damage, decreased the protein expression of caspase-3 [176]. Finally, recent studies show that resveratrol may be beneficial due to activation of the Silent Information Regulator T1 [SIRT1] pathway. SIRT is a nicotinamide


9

adenine dinucleotide [NAD]-dependent histone deacetylase, which has been shown to inhibit stress-induced apoptosis and to increase cellular tolerance to stress [177]. During ischemic/reperfusion damage, resveratrol-induced activation of the SIRT1 molecular pathway increases neuronal cell survival in an otherwise unfavourable environment [178]. 4.2.4. N-Acetylcysteine [NAC] NAC has been reported to effectively scavenge ROS, such as hydroxyl radicals, H2O2, and peroxyl radicals, and can also increase the rate of endogenous GSH synthesis [179]. GSH, a central component in the antioxidant defence of cells, acts either directly or indirectly as a substrate for various GSH-Pxs to detoxify ROS [180]. Thus, NAC may be able to reduce the volume of postischemic lesions. For example, Carroll et al. [181] showed that NAC administered prior to or after reperfusion in a transient MCAO in rats resulted in a 49% and 29% reduction of the lesion size, respectively. Another study demonstrated that NAC administration following a 30 min ischemic MCAO in rats protected the brain from ROS injury with aneffective therapeutic window of up to 6 hours after reperfusion, and this neuroprotection was associated with increased GSH levels [182]. Moreover, repeated administration of NAC is more neuroprotective than a single dose, perhaps because of the short half-life of this antioxidant [167]. 4.3. Other Strategies B-group vitamin supplementation immediately postinfarct may have antioxidant and anti-inflammatory effects independent of their homocysteine-lowering properties [183]. The important results of this study were that supplementary antioxidants with or without B-group vitamins, when given postinfarct, enhanced antioxidant capacity and mitigated oxidative damage in stroke disease. No additive or synergistic effects of antioxidants and Bgroup vitamins were reported for any measured outcomes and all groups receiving vitamin supplementation demonstrated reduced tissue inflammation [165]. Lee et al. examined the neuroprotective action of a standardized extract of Ginkgo biloba leaves in permanent and transient MCAO models in Sprague-Dawley rats. They concluded that Ginkgo biloba protected against transient and permanent focal cerebral ischemia and was effective after a prolonged reperfusion period even when therapy is delayed up to 2 hours [184]. The significant therapeutic effects Ginkgo biloba on ischemic stroke perhaps work through activating the Akt-CREB-BDNF pathway [185]. It has been reported that red wine polyphenols compounds also significantly improved blood flow during reperfusion and brain tissue preservation as observed 24 h after MCAO in treated animals. These findings strongly suggest that polyphenols are agents able to fight against the excitotoxic, oxidative pathways and metabolic dysfunction induced by cerebral ischemia [186]. Pharmacological studies in animals have shown that antioxidant molecules capable of crossing the BBB, such as lazaroids [187], polyethylene glycol-conjugated SOD and CAT [188] reduce ischemic cerebral damage. It has been reported that the flavonoid scutellarin exhibits

10


neuroprotective properties, increasing SOD and CAT activities and GSH levels in ischemic brain tissues, thereby enhancing endogenous antioxidant activity. This protects neurons against lethal stimuli by decreasing the percentage of apoptotic cells and reducing ROS generation in OGDinduced primary cortical neurons in vitro [189]. Edaravone, a ROS scavenger, is the first clinical drug for neuroprotection in the world, which has been used from 2001 in most ischemic stroke patients in Japan. It scavenges hydroxyl radicals both in hydrophilic and hydrophobic conditions, and is especially useful in thrombolytic therapy with tissue plasminogen activator [tPA], a thrombolytic agent [190]. In a MCAO rat model, edaravone administration ameliorated neurological and histological outcomes, reduced the induction of apoptotic responses, and was associated with elevated endogenous antioxidants [191]. Possible mechanismsto explain this result include a decrease in oxidative stress, the protection of neurovascular units, and a reduction in activation of microglia after ischemic stress [191, 31]. As a neuroprotective agent, edaravone can effectively reduce infarct size and improve the clinical outcome. In experimental models, combination therapy of edaravone with tPA, greatly increased survival of stroke animals, reduced infarct size, and reduced the levels of molecular markers of lipid peroxidation, protein carbonylation and DNA damage. In human stroke patients the use of edaravone greatly reduced haemorrhagic transformation accompanied by tPA treatment, and may also extend therapeutic time window with tPA therapy for more than 3 hours [190]. Quercetin, a natural flavonoid, is well known as a strong antioxidant and ROS scavenger and is found in high quantities in fruits and vegetables [192]. Quercetin has been shown to have anti-inflammatory, anti-blood coagulation, anti-ischemic, and anticancer effects [193]. Quercetin can also prevent oxidative damage and morphological changes in the MCAO and may have a therapeutic value for the treatment of stroke. The administration of quercetin showed marked reduction in infarct size, reduced the neurological deficits in terms of behaviors, suppressed neuronal loss and diminished the p53 expression in MCAO rats [194]. Finally, Squina DNA, a nucleic acid–based health product derived from salmon milt, is also claimed to protect DNA from oxidation [195]. Squina DNA treatment can enhance mitochondrial antioxidant status, presumably by eliciting a GSH antioxidant response, and preserve the mitochondrial structural integrity, thereby protecting against brain and neuronal injury induced by acute and chronic oxidative stress [196]. 5. PARTICULAR CONSIDERATIONS ANTIOXIDANT TREATMENT

IN

Consideration should be applied when using acute antioxidant supplementation in the course of ischemic stroke. Antioxidants can become pro-oxidative in the presence of free iron, as when iron is detached from hemoglobin during haemorrhage; this may also occur to some degree during ischemic stroke. Allopurinol constitutes a secure therapy, for it utilizes a low adverse reaction rate medication, being the majority of

Rodrigo et al.

them minor adverse effects, such as allergic dermal reactions. The spectrum considers among the severe adverse reactions, the Stevens-Johnson syndrome. Even though this is a hypersensitivity reaction, it is documented to occur at doses 200 mg / day or more [197]. Moreover, its incidence has been estimated to be one case of allopurinol-induced Stevens-Johnson syndrome or toxic epidermal necrolysis per 3.76 million in the whole population per year [197], as well as 1.5 cases per million allopurinol takers per week—ie, 0.008% of allopurinol takers per year [198]. Patients with pre-existing renal insufficiency/failure or glucose 6-phosphate dehydrogenase [G6PD] deficiency, are predisposed to vitamin C toxicity [199]. Other than the known complications of IV vitamin C in those with renal impairment or G6PD deficiency, high dose intravenous vitamin C appears to be remarkably safe [200]. There have been no reported resveratrol adverse effects derived from intakes currently associated with its consumption from a natural source. However, dietary supplementation for health-protection should be cautiously used as no definition has been given for the levels or limits for a safe dosage. At dosages of 1,200 mg twice daily or lower, NAC is well tolerated. At these dosages, side effects are unusual, but may include nausea, vomiting, diarrhea, transient skin rash, flushing, epigastric pain, and constipation [201]. 6. CONCLUDING REMARKS AND PERSPECTIVES Stroke is a major cause of morbidity and mortality among western people. Because the majority of stroke cases are non-fatal, it is a major burden in long-term disability. ROS is implicated in brain injury following ischemic stroke, and the occurrence and consequences of vessel occlusion initiate a vicious cycle that results in increased ROS production and neuronal injury. Most patients do not reach medical facilities early enough for conventional reperfusion strategies, which are the current cornerstone of stroke management. Therefore, therapies targeting patients who cannot undergo thrombolysis must be considered to reduce the consequences ofstroke. The potential use of unconventional therapies, such as antioxidant defence system enhancement, might play a key role in the management of this disease. A combined therapy aimed to diminish brain injury and improve patient outcome could be suggested. This therapy should be based on the time course of ROS production, the inhibition of ROS-producing enzymes and increased non-enzymatic antioxidant delivery to neurons. A schematic approach to antioxidant enhancement is proposed in Fig. (2). As can be seen in the figure, this type of therapy is effective over a longer time frame compared to reperfusion strategies, increasing the pool of eligible patients. OGD cause the first ROS increase. Since this phase occurs in few minutes it is difficult to achieve a successful intervention in it. XO activation is an important source of ROS early in the time course of the ischemic stroke. Therefore, XO inhibitors [such as allopurinol] could be beneficial in mitigating the initial ROS damage. This ROS generation together with the ischemia lead to BBB disruption, oedema and inflammation, all causing neutrophils infiltration to the infarct zone. In turn, another ROS



11

Fig. (2). Therapeutic opportunities throughout the time-course of oxidative stress development in ischemic stroke. Early intervention with Xanthine oxidase inhibitors [such as Allopurinol] could be beneficial in mitigating the initial ROS-damage. Vitamin C or L-NA could be used to mitigate the next ROS increase due to the iNOS activation. ROS scavenger such as resveratrol, vitamin C [massive infusion] or Nacetylcysteine and NADPH inhibitors such as vitamin C, vitamin E or apocynin could be used in the reperfusion phase. BBB, blood brain barrier; L-NA, L-Nitro Arginine; iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase; NADPH oxidase, reduced nicotine adenine dinucleotide phosphate oxidase, inhibition.

generation due to the iNOS activation occurs. iNOS inhibitors such as vitamin C or L-Nitro Arginine [L-NA] could be use at this level. Finally, at the time of reperfusion, a major burst of ROS is generated. Taking into account that NADPH oxidase is the main responsible for ROS generation during reperfusion, vitamin C, vitamin E or apocynin could be used in order to inhibit this enzyme. In addition, ROS scavengers such as resveratrol or NAC could be administrated. Massive infusion of vitamin C is also an attractive and safe alternative in order to scavenge ROS [200]. Antioxidant defence system enhancement constitutes a safe, low-cost and widely applicable therapy for stroke patients. Taking into account the promising beneficial effects of this therapy as well as its safety and lowcost, it is emerging as a highly cost-effective alternative for the 97% of ischemic stroke patients that cannot benefit from reperfusion therapy. However, randomised, double-blind, and placebo-controlled clinical trials are still lacking to fully establish the effectiveness of this treatment.

ABBREVIATIONS ATP

= Adenosine-5’-triphosphate

BBB

= Blood brain barrier

Bcl

= B-cell lymphoma 2

Ca2+

= Calcium

CAT

= Catalase

CNS

= Central nervous system

CO

= Carbon monoxide

DNA

= Deoxyribonucleic acid

eNOS

= Endothelial nitric oxide synthase

ERK

= Extracellular signal-regulated kinases

GSH-Pxs

= Glutathione peroxidases

GSH

= Glutathione

12


H2S

= Sulfhydric acid

HO

= Heme oxygenase

iNOS

= Inducible nitric oxide synthase

MCAO

= Middle cerebral artery occlusion

MDA

= Malondialdehyde

MMPs

= Matrix metalloproteinases

mRNA

= Messenger ribonucleic acid

Rodrigo et al. [5]

[6]

[Na + K]-ATPase = Sodium-potassium pump [7]

Na+

= Sodium

NAC

= N-acetylcysteine

NADPH

= Reduced nicotinamide adenine dinucleotide phosphate

nNOS

= Neuronal nitric oxide synthase

NO

= Nitric oxide

NOX4

= NADPH oxidase subunit 4

OGD

= Oxygen-glucose deprivation

[10]

O2

= Oxygen

[11]

PPAR

= Peroxisome proliferator activated receptor

[12]

[8]

[9]

RNS

= Reactive nitrogen species

ROS

= Reactive oxygen species

SOD

= Superoxide dismutase

SVCT2

= Sodium-dependent vitamin C transporter 2

TAOC

= Total antioxidant capacity

[15]

TBARS

= Thiobarbituric acid reactive substances

[16]

tPA

= Tissue plasminogen activator

[17]

XO

= Xanthine oxidase

[13] [14]

[18]

CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS Funding: Clínica Santa María, Santiago de Chile. REFERENCES [1] [2]

[3] [4]

World Health Organization. Top ten causes of death. http://www.who.int/mediacentre/factsheets/fs310/en/index.html. (accessed August 19, 2012). Lopez, A.D.; Mathers, C.D.; Ezzati, M.; Jamison, D.T.; Murray, C.J. Global and regional burden of disease and risk factors, 2001: systematic analysis of population health data. Lancet, 2006, 367(9524), 1747-1757. Sarti, C.; Rastenyte, D.; Cepaitis, Z.; Tuomilehto, J. International trends in mortality from stroke, 1968 to 1994. Stroke, 2000, 31(7), 1588-1601. Ayala, C.; Greenlund, K.J.; Croft, J.B.; Keenan N.L.; Donehoo R.S.; Giles W.H.; Kittner S.J.; Marks J.S. Racial/ethnic disparities in mortality by stroke subtype in the United States, 1995-1998. Am. J. Epidemiol., 2001, 154(11), 1057-1063.

[19]

[20] [21]

[22]

[23]

The Global burden of disease: 2004 Upddate. World Health Organization 2008.http://www.who.int/healthinfo/global_burden_disease/GBD_r eport_2004update_full.pdf (accessed December 9, 2012). Rosamond, W.; Flegal, K.; Friday, G.; Furie, K.; Go, A.; Greenlund, K.; Haase, N.; Ho, M.; Howard, V.; Kissela, B.; Kittner, S.; Lloyd-Jones, D.; McDermott, M.; Meigs, J.; Moy, C.; Nichol, G.; O'Donnell, C.J.; Roger, V.; Rumsfeld, J.; Sorlie, P.; Steinberger, J.; Thom, T.; Wasserthiel-Smoller, S.; Hong, Y.; American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics--2007 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee.Circulation, 2007, 115(5), 69-171. Cherubini, A.; Ruggiero, C.; Polidori, M.C.; Mecocci, P. Potential markers of oxidative stress in stroke. Free Radic. Biol. Med., 2005, 39(7), 841-852. Xavier, A.R.; Siddiqui, A.M.; Kirmani, J.F.; Hanel, R.A.; Yahia, A.M.; Qureshi, A.I. Clinical potential of intra-arterial thrombolytic therapy in patients with acute ischemic stroke. CNS Drugs, 2003, 17(4), 213-224. Hacke, W.; Kaste, M.; Bluhmki, E.; Brozman, M.; Dávalos, A.; Guidetti, D.; Larrue, V.; Lees, K.R.; Medeghri, Z.; Machnig, T.; Schneider, D.; von Kummer, R.; Wahlgren, N.; Toni, D.; ECASS Investigators. Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke. N. Engl. J. Med., 2008, 359(13), 1317-1329. McCulloch, J.; Dewar, D. A radical approach to stroke therapy. Proc. Natl. Acad. Sci. USA, 2001, 98(20), 10989-10991. Rodrigo, R.; González, J.; Paoletto, F. The role of oxidative stress in the pathophysiology of hypertension. Hypertens. Res., 2011, 34(4), 431-440. Pacher, P.; Beckman, J.S.; Liaudet, L. Nitric oxide and peroxynitrite in health and disease. Physiol. Rev., 2007, 87(1), 315424. Tsuchihashi, S.; Fondevila, C.; Kupiec-Weglinski, J.W. Heme oxygenase system in ischemia and reperfusion injury. Ann. Transplant., 2004, 9, 84-87. Hishiki, T.; Yamamoto, T.; Morikawa, T.; Kubo, A.; Kajimura, M.; Suematsu, M. Carbon monoxide: impact on remethylation/transsulfuration metabolism and its pathophysiologic implications. J. Mol. Med. (Berl.), 2012, 90(3), 245-254. Zimmerman, J.J. Defining the role of oxyradicals in the pathogenesis of sepsis. Crit. Care Med., 1995, 23(4), 616-617. Adibhatla, R.M.; Hatcher, J.F. Altered lipid metabolism in brain injury and disorders. Subcell. Biochem., 2008, 49, 241-268. Lu, S.P.; Lin Feng, M.H; Huang, H.L.; Huang, Y.C.; Tsou, W.I.; Lai, M.Z. Reactive oxygen species promote raft formation in T lymphocytes. Free Radic. Biol. Med., 2007, 42(7), 936-944. Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M.T.; Mazur, M.; Telser, J. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol., 2007, 39(1), 44-84. Hu, D.; Serrano, F.; Oury, T.D.; Klann, E. Aging-dependent alterations in synaptic plasticity and memory in mice that overexpress extracellular superoxide dismutase. J. Neurosci., 2006, 26(15), 3933-3941. Saeed, S.A.; Shad, K.F.; Saleem, T.; Javed, F.; Khan, M.U. Some new prospects in the understanding of the molecular basis of the pathogenesis of stroke. Exp. Brain Res., 2007, 182(1), 1-10. Li, F.; Silva, M.D.; Liu, K.F.; Helmer, K.G.; Omae, T.; Fenstermacher, J.D.; Sotak, C.H.; Fisher, M. Secondary decline in apparent diffusion coefficient and neurological outcome after a short period of focal brain ischemia in rats. Ann. Neurol., 2000, 48(2), 236-244. Kidwell, C.S.; Saver, J.L.; Mattiello, J.; Starkman, S.; Vinuela, F.; Duckwiler, G.; Gobin, Y.P.; Jahan, R.; Vespa, P.; Kalafut, M.; Alger, J.R. Thrombolytic reversal of acute human cerebral ischemic injury shown by diffusion/perfusion magnetic resonance imaging. Ann. Neurol., 2000, 47(4), 462-469. Yoo, A.J.; Hu, R.; Hakimelahi, R.; Lev, M.H.; Nogueira, R.G.; Hirsch, J.A.; González, R.G.; Schaefer, P.W.CT Angiography Source Images Acquired with a Fast-Acquisition Protocol Overestimate Infarct Core on Diffusion Weighted Images in Acute Ischemic Stroke. J. Neuroimaging, 2012, 22(4), 329-335.

Antioxidants in Ischemic Stroke Treatment [24] [25] [26] [27] [28]

[29] [30] [31] [32] [33]

[34]

[35]

[36] [37]

[38]

[39]

[40]

[41]

[42]

[43]

Lo, E.H.; Dalkara, T.; Moskowitz, M.A. Mechanisms, challenges and opportunities in stroke. Nat. Rev. Neurosci., 2003, 4(5), 399415. Warner, D.S.; Sheng, H.; Batini-Haberle, I. Oxidants, antioxidants and the ischemic brain. J. Exp. Biol., 2004, 207(Pt 18), 3221-3231. Kaushal, V.; Schlichter, L.C. Mechanisms of microglia-mediated neurotoxicity in a new model of the stroke penumbra. J. Neurosci., 2008, 28(9), 2221-2230 Moustafa, R.R.; Baron, J.C. Pathophysiology of ischaemic stroke: insights from imaging, and implications for therapy and drug discovery. Br. J. Pharmacol., 2008, 153(Suppl 1), S44-S54. Gleichmann, M.; Collis, L.P.; Smith, P.J.; Mattson, M.P. Simultaneous single neuron recording of O2 consumption, [Ca2+]i and mitochondrial membrane potential in glutamate toxicity. J. Neurochem., 2009, 109(2), 644-655. Chao, X.D.; Fei, F.; Fei, Z. The role of excitatory amino acid transporters in cerebral ischemia. Neurochem. Res., 2010, 35(8), 1224-1230. Gorren, A.C.; Mayer, B. Nitric-oxide synthase: a cytochrome P450 family foster child. Biochim. Biophys. Acta, 2007, 1770(3), 432445. Balaban, R.S.; Nemoto, S.; Finkel, T. Mitochondria, oxidants, and aging. Cell, 2005, 120(4), 483-495. Murphy, M.P. How mitochondria produce reactive oxygen species. Biochem. J., 2009, 417(1), 1-13. Abramov, A.Y.; Scorziello, A.; Duchen, M.R. Three distinct mechanisms generate oxygen free radicals in neurons and contribute to cell death during anoxia and reoxygenation. J. Neurosci., 2007, 27(5), 1129-1138. Kontos, C.D.; Wei, E.P.; Williams, J.I.; Kontos, H.A.; Povlishock, J.T. Cytochemical detection of superoxide in cerebral inflammation and ischemia in vivo. Am. J. Physiol., 1992, 263(4 Pt 2), H1234H1242. Kleinschnitz, C.; Grund, H.; Wingler, K.; Armitage, M.E.; Jones, E.; Mittal, M.; Barit, D.; Schwarz, T.; Geis, C.; Kraft, P.; Barthel, K.; Schuhmann, M.K.; Herrmann, A.M.; Meuth, S.G.; Stoll, G.; Meurer, S.; Schrewe, A.; Becker, L.; Gailus-Durner, V.; Fuchs, H.; Klopstock, T.; de Angelis, M.H.; Jandeleit-Dahm, K.; Shah, A.M.; Weissmann, N.; Schmidt, H.H. Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration. PLoS Biol., 2010, 8(9), pii: e1000479. Kent, T.A.; Soukup, V.M.; Fabian, R.H. Heterogeneity affecting outcome fromacute stroke therapy: making reperfusion worse. Stroke, 2001, 32(10), 2318-2327. Miller, A.A.; Drummond, G.R.; Schmidt, H.H.; Sobey, C.G. NADPH oxidase activity and function are profoundly greater in cerebral versus systemic arteries. Circ. Res., 2005, 97(10), 10551062. McCann, S.K.; Dusting, G.J.; Roulston, C.L. Early increase of Nox4 NADPH oxidase and superoxide generation following endothelin-1-induced stroke in conscious rats. J. Neurosci. Res., 2008, 86(11), 2524-2534. Schilling, M.; Besselmann, M.; Leonhard, C.; Mueller, M.; Ringelstein, E.B.; Kiefer, R. Microglial activation precedes and predominates over macrophage infiltration in transient focal cerebral ischemia: a study in green fluorescent protein transgenic bone marrow chimeric mice. Exp. Neurol., 2003, 183(1), 25-33. Tanaka, R.; Komine-Kobayashi, M.; Mochizuki, H.; Yamada, M.; Furuya, T.; Migita, M.; Shimada, T.; Mizuno, Y.; Urabe, T. Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia. Neuroscience, 2003, 117(3), 531-539. Gerhard, A.; Neumaier, B.; Elitok, E.; Glatting, G.; Ries, V.; Tomczak, R.; Ludolph, A.C.; Reske, S.N. In vivo imaging of activated microglia using [11C]PK11195 and positron emission tomography in patients after ischemic stroke. Neuroreport, 2000, 11(13), 2957-2960. Price, C.J.; Menon, D.K.; Peters, A.M.; Ballinger, J.R.; Barber, R.W.; Balan, K.K.; Lynch, A.; Xuereb, J.H.; Fryer, T.; Guadagno, J.V.; Warburton, E.A. Cerebral neutrophil recruitment, histology, and outcome in acute ischemic stroke: an imaging-based study. Stroke, 2004, 35(7), 1659-1664. Schilling, M.; Besselmann, M.; Müller, M.; Strecker, J.K.; Ringelstein, E.B.; Kiefer, R. Predominant phagocytic activity of resident microglia over hematogenous macrophages following


[44] [45]

[46]

[47] [48] [49] [50]

[51] [52]

[53] [54]

[55]

[56] [57]

[58]

[59]

[60] [61] [62]

[63]

[64]

13

transient focal cerebral ischemia: an investigation using green fluorescent protein transgenic bone marrow chimeric mice. Exp. Neurol., 2005, 196(2), 290-297. Wang, X.; Feuerstein, G.Z. Induced expression of adhesion molecules following focal brain ischemia. J. Neurotrauma, 1995, 12(5), 825-832. Nakashima, M.N.; Yamashita, K.; Kataoka, Y.; Yamashita, Y.S.; Niwa M. Time course of nitric oxide synthase activity in neuronal, glial, and endothelial cells of rat striatum following focal cerebral ischemia. Cell. Mol. Neurobiol., 1995, 15(3), 341-349. Iadecola, C.; Zhang, F.; Casey, R.; Clark, H.B.; Ross, M.E. Inducible nitric oxide synthase gene expression in vascular cells after transient focal cerebral ischemia. Stroke, 1996, 27(8), 13731380. Forster, C.; Clark, H.B.; Ross, M.E.; Iadecola, C. Inducible nitric oxide synthase expression in human cerebral infarcts. Acta Neuropathol., 1999, 97(3), 215-220. Moro, M.A.; Cárdenas, A.; Hurtado, O.; Leza, J.C.; Lizasoain, I. Role of nitric oxide after brain ischaemia. Cell. Calcium, 2004, 36(3-4), 265-275. Pawate, S.; Shen, Q.; Fan, F.; Bhat, N.R. Redox regulation of glial inflammatory response to lipopolysaccharide and interferon gamma. J. Neurosci. Res., 2004, 77(4), 540-551. Nogawa, S.; Forster, C.; Zhang, F.; Nagayama, M.; Ross, M.E.; Iadecola, C. Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia. Proc. Natl. Acad. Sci. USA, 1998, 95(18), 10966-10971. Minghetti, L. Role of COX-2 in inflammatory and degenerative brain diseases. Subcell. Biochem., 2007, 42, 127-141. Ahmad, M.; Zhang, Y.; Liu, H.; Rose, M.E.; Graham, S.H. Prolonged opportunity for neuroprotection in experimental stroke with selective blockade of cyclooxygenase-2 activity. Brain Res., 2009, 1279, 168-173. Candelario-Jalil, E.; Fiebich, B.L. Cyclooxygenase inhibition in ischemic brain injury. Curr. Pharm. Des., 2008, 14(14), 14011418. Iadecola, C.; Niwa, K.; Nogawa, S.; Zhao, X.; Nagayama, M.; Araki, E.; Morham, S.; Ross, M.E. Reduced susceptibility to ischemic brain injury and N-methyl-d-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice. Proc. Natl. Acad. Sci. USA, 2001, 98(3), 1294-1299. Doré, S.; Otsuka, T.; Mito, T.; Sugo, N.; Hand, T.; Wu, L.; Hurn, P.D.; Traystman, R.J.; Andreasson, K. Neuronal overexpression of cyclooxygenase-2 increases cerebral infarction. Ann. Neurol., 2003, 54(2), 155-162. Mattson, M.P. NF-kappaB in the survival and plasticity of neurons. Neurochem. Res., 2005, 30(6-7), 883-893. van der Goes, A.; Wouters, D.; van der Pol, S.M.; Huizinga, R.; Ronken, E.; Adamson, P.;Greenwood, J.; Dijkstra, C.D.; De Vries, H.E. Reactive oxygen species enhance the migration of monocytes across the blood-brain barrier in vitro. FASEB J., 2001, 15(10), 1852-1854. Lee, H.S.; Namkoong, K.; Kim, D.H.; Kim, K.J.; Cheong, Y.H.; Kim, S.S.; Lee, W.B.; Kim, K.Y. Hydrogen peroxide-induced alterations of tight junction proteins in bovine brain microvascular endothelial cells. Microvasc. Res., 2004, 68(3), 231-238. Kahles, T.; Luedike, P.; Endres, M.; Galla, H.J.; Steinmetz, H.; Busse, R.; Neumann-Haefelin, T.; Brandes, R.P. NADPH oxidase plays a central role in blood-brain barrier damage in experimental stroke. Stroke, 2007, 38(11), 3000-3006. Rosell, A.; Lo, E.H. Multiphasic roles for matrix metalloproteinases after stroke. Curr. Opin. Pharmacol., 2008, 8(1), 82-89. Rosenberg, G.A. Matrix metalloproteinases in neuroinflammation. Glia, 2002, 39(3), 279-291. Gu, Z.; Kaul, M.; Yan, B.; Kridel, S.J.; Cui, J.; Strongin, A.; Smith, J.W.; Liddington, R.C.; Lipton, S.A. S-nitrosylation of matrix metalloproteinases: signaling pathway to neuronal cell death. Science, 2002, 297(5584), 1186-1190. Akool, El.-S.; Kleinert, H.; Hamada, F.M.; Abdelwahab, M.H.; Förstermann, U.; Pfeilschifter, J.; Eberhardt, W. Nitric oxide increases the decay of matrix metalloproteinase 9 mRNA by inhibiting the expression of mRNA-stabilizing factor HuR. Mol. Cell. Biol., 2003, 23(14), 4901-4916. Bektas, H.; Wu, T.C.; Kasam, M.; Harun, N.; Sitton, C.W.; Grotta, J.C.; Savitz, S.I. Increased blood-brain barrier permeability on

14

[65] [66]

[67] [68]

[69] [70] [71]

[72]

[73]

[74]

[75] [76] [77]

[78] [79]

[80]

[81]

[82]

[83]

CNS & Neurological Disorders - Drug Targets, 2013, Vol. 12, No. 2 perfusion CT might predict malignant middle cerebral artery infarction. Stroke, 2010, 41(11), 2539-2544. Kourie, J.I. Interaction of reactive oxygen species with ion transports mechanisms. Am. J. Physiol., 1998, 275(1 Pt 1), C1-C24. Rohn, T.T.; Hinds, T.R.; Vincenzi, F.F. Inhibition of Ca2+-pump ATPase and the Na+/K+-pump ATPase by iron-generated free radicals. Protection by 6, 7-dimethyl-2, 4-DI-1- pyrrolidinyl-7Hpyrrolo [2, 3-d] pyrimidine sulfate (U-89843D), a potent, novel, antioxidant/free radical scavenger. Biochem. Pharmacol., 1996, 51(4), 471-476. Siesjö, B.K.; Katsura, K.I.; Kristián, T.; Li, P.A.; Siesjö, P. Molecular mechanisms of acidosis-mediated damage. Acta Neurochir. Suppl., 1996, 66, 8-14. Brandes, R.P.; Miller, F.J.; Beer, S.; Haendeler, J.; Hoffmann, J.; Ha, T.; Holland, S.M.; Görlach, A.; Busse, R. The vascular NADPH oxidase subunit p47phox is involved in redox-mediated gene expression. Free Radic. Biol. Med., 2002, 32(11), 1116-1122. Circu, M.L.; Aw, T.Y. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic. Biol. Med.2010, 48(6), 749762. Carmona-Gutierrez, D.; Eisenberg, T.; Buttner, S.; Meisinger, C.; Kroemer, G.; Madeo, F. Apoptosis in yeast: triggers, pathways, subroutines. Cell Death Differ., 2010, 17(5), 763-773 Chan, H.L.; Chou, H.C.; Duran, M. Major role of epidermal growth factor receptor and Src kinases in promoting oxidative stressdependent loss of adhesion and apoptosis in epithelial cells. J. Biol. Chem., 2010, 285(7), 4307-4318. Denning, T.L.; Takaishi, H.; Crowe, S.E.; Boldogh, I.; Jevnikar, A.; Ernst, P.B. Oxidative stress induces the expression of Fas and Fas ligand and apoptosis in murine intestinal epithelial cells.Free Radic. Biol. Med., 2002, 33(12), 1641-1650. Sugawara, T.; Noshita, N.; Lewen, A.; Gasche, Y.; Ferrand-Drake, M.; Fujimura, M.; Morita-Fujimura, Y.; Chan, P.H. Overexpression of copper/zinc superoxide dismutase in transgenic rats protects vulnerable neurons against ischemic damage by blocking the mitochondrial pathway of caspase activation. J. Neurosci., 2002, 22(1), 209-217. Matés, J.M.; Segura, J.A.; Alonso, F.J.; Marquez, J. Intracellular redox status and oxidative stress: implications for cell proliferation, apoptosis, and carcinogenesis. Arch. Toxicol., 2008, 82(5), 273299. Franco, R.; Cidlowski, J.A. Apoptosis and glutathione: beyond an antioxidant. Cell Death Differ., 2009, 16(10), 1303-1314. Chandra, J.; Samali, A.; Orrenius, S. Triggering and modulation of apoptosis by oxidative stress. Free Radic. Biol. Med., 2000, 29(34), 323-333. Culmsee, C.; Siewe, J.; Junker, V.; Retiounskaia, M.; Schwarz, S.; Camandola, S.; El-Metainy, S.; Behnke, H.; Mattson, M.P.; Krieglstein, J. Reciprocal inhibition of p53 and nuclear factorkappaB transcriptional activities determines cell survival or death in neurons. J. Neurosci., 2003, 23(24), 8586-8595. Yonekura, I.; Takai, K.; Asai, A.; Kawahara, N.; Kirino, T. p53 potentiates hippocampal neuronal death caused by global ischemia. J. Cereb. Blood Flow Metab., 2006, 26(10), 1332-1340. Endo, H.; Kamada, H.; Nito, C.; Nishi, T.; Chan, P.H. Mitochondrial translocation of p53 mediates release of cytochrome c and hippocampal CA1 neuronal death after transient global cerebral ischemia in rats. J. Neurosci., 2006, 26(30), 7974-7983. Han, E.S.; Muller, F.L.; Pérez, V.I.; Qi, W.; Liang, H.; Xi, L.; Fu, C.; Doyle, E.; Hickey, M.; Cornell, J.; Epstein, C.J.; Roberts, L.J.; Van Remmen, H.; Richardson, A. The in vivo gene expression signature of oxidative stress. Physiol. Genom., 2008, 34(1), 112126. Shangary, S.; Brown, K.D.; Adamson, A.W.; Edmonson, S.; Ng, B.; Pandita, T.K.; Yalowich, J.; Taccioli, G.E.; Baskaran, R. Regulation of DNA-dependent protein kinase activity by ionizing radiation-activated Abl kinase is an ATM-dependent process. J. Biol. Chem., 2000, 275(39), 30163-30168. Chopp, M.; Zhang, R.L.; Chen, H.; Li, Y.; Jiang, N.; Rusche, J.R. Postischemic administration of an anti-Mac-1 antibody reduces ischemic cell damage after transient middle cerebral artery occlusion in rats. Stroke, 1994, 25(4), 869-875. van Lookeren Campagne, M.; Gill, R. Increased expression of cyclin G1 and p21WAF1/CIP1 in neurons following transient forebrain ischemia: comparison with early DNA damage. J. Neurosci. Res., 1998, 53(3), 279-296.

Rodrigo et al. [84]

[85]

[86] [87] [88]

[89]

[90]

[91]

Niizuma, K.; Endo, H.; Nito, C.; Myer, D.J.; Chan, P.H. The potential role of PUMA in delayed death of hippocampal CA1 neurons after transient global cerebral ischemia. Stroke, 2009, 40(2), 618-625. Nakano, H.; Nakajima, A.; Sakon-Komazawa, S.; Piao, J.H.; Xue, X.; Okumura, K. Reactive oxygen species mediate crosstalk between NF-kappaB and JNK. Cell Death Differ., 2006, 13(5), 730-737. Bedard, K.; Krause, K.H. The NOX Family of ROS-Generating NADPH Oxidases: Physiology and Pathophysiology. Physiol. Rev., 2007, 87(1), 245-313. Mkaddem, S.B.; Bens, M.; Vandewalle, A. Differential activation of Toll-like receptor-mediated apoptosis induced by hipoxia. Oncotarget, 2010, 1(8), 741-750. Kauppinen, T.M.; Chan, W.Y.; Suh, S.W.; Wiggins, A.K.; Huang, E.J.; Swanson, R.A. Direct phosphorylation and regulation of poly(ADP-ribose) polymerase-1 by extracellular signal-regulated kinases 1/2. Proc. Natl. Acad. Sci. USA, 2006, 103(18), 7136-7141. Klionsky, D.J.; Abeliovich, H.; Agostinis, P.; Agrawal, D.K.; Aliev, G.; Askew, D.S.; Baba, M.; Baehrecke, E.H.; Bahr, B.A.; Ballabio, A.; Bamber, B.A.; Bassham, D.C.; Bergamini, E.; Bi, X.; Biard-Piechaczyk, M.; Blum, J.S.; Bredesen, D.E.; Brodsky, J.L.; Brumell, J.H.; Brunk, U.T.; Bursch, W.; Camougrand, N.; Cebollero, E.; Cecconi, F.; Chen, Y.; Chin, L.S.; Choi, A.; Chu, C.T.; Chung, J.; Clarke, P.G.; Clark, R.S.; Clarke, S.G.; Clavé, C.; Cleveland, J.L.; Codogno, P.; Colombo, M.I.; Coto-Montes, A.; Cregg, J.M.; Cuervo, A.M.; Debnath, J.; Demarchi, F.; Dennis, P.B.; Dennis, P.A.; Deretic, V.; Devenish, R.J.; Di Sano, F.; Dice, J.F.; Difiglia, M.; Dinesh-Kumar, S.; Distelhorst, C.W.; DjavaheriMergny, M.; Dorsey, F.C.; Dröge, W.; Dron, M.; Dunn, W.A., Jr.; Duszenko, M.; Eissa, N.T.; Elazar, Z.; Esclatine, A.; Eskelinen, E.L.; Fésüs, L.; Finley, K.D.; Fuentes, J.M.; Fueyo, J.; Fujisaki, K.; Galliot, B.; Gao, F.B.; Gewirtz, D.A.; Gibson, S.B.; Gohla, A.; Goldberg, A.L.; Gonzalez, R.; González-Estévez, C.; Gorski, S.; Gottlieb, R.A.; Häussinger, D.; He, Y.W.; Heidenreich, K.; Hill, J.A.; Høyer-Hansen, M.; Hu, X.; Huang, W.P.; Iwasaki, A.; Jäättelä, M.; Jackson, W.T.; Jiang, X.; Jin, S.; Johansen, T.; Jung, J.U.; Kadowaki, M.; Kang, C.; Kelekar, A.; Kessel, D.H.; Kiel, J.A.; Kim, H.P.; Kimchi, A.; Kinsella, T.J.; Kiselyov, K.; Kitamoto, K.; Knecht, E.; Komatsu, M.; Kominami, E.; Kondo, S.; Kovács, A.L.; Kroemer, G.; Kuan, C.Y.; Kumar, R.; Kundu, M.; Landry, J.; Laporte, M.; Le, W.; Lei, H.Y.; Lenardo, M.J.; Levine, B.; Lieberman, A.; Lim, K.L.; Lin, F.C.; Liou, W.; Liu, L.F.; Lopez-Berestein, G.; López-Otín, C.; Lu, B.; Macleod, K.F.; Malorni, W.; Martinet, W.; Matsuoka, K.; Mautner, J.; Meijer, A.J.; Meléndez, A.; Michels, P.; Miotto, G.; Mistiaen, W.P.; Mizushima, N.; Mograbi, B.; Monastyrska, I.; Moore, M.N.; Moreira, P.I.; Moriyasu, Y.; Motyl, T.; Münz, C.; Murphy, L.O.; Naqvi, N.I.; Neufeld, T.P.; Nishino, I.; Nixon, R.A.; Noda, T.; Nürnberg, B.; Ogawa, M.; Oleinick, N.L.; Olsen, L.J.; Ozpolat, B.; Paglin, S.; Palmer, G.E.; Papassideri, I.; Parkes, M.; Perlmutter, D.H.; Perry, G.; Piacentini, M.; Pinkas-Kramarski, R.; Prescott, M.; ProikasCezanne, T.; Raben, N.; Rami, A.; Reggiori, F.; Rohrer, B.; Rubinsztein, D.C.; Ryan, K.M.; Sadoshima, J.; Sakagami, H.; Sakai, Y.; Sandri, M.; Sasakawa, C.; Sass, M.; Schneider, C.; Seglen, P.O.; Seleverstov, O.; Settleman, J.; Shacka, J.J.; Shapiro, I.M.; Sibirny, A.; Silva-Zacarin, E.C.; Simon, H.U.; Simone, C.; Simonsen, A.; Smith, M.A.; Spanel-Borowski, K.; Srinivas, V.; Steeves, M.; Stenmark, H.; Stromhaug, P.E.; Subauste, C.S.; Sugimoto, S.; Sulzer, D.; Suzuki, T.; Swanson, M.S.; Tabas, I.; Takeshita, F.; Talbot, N.J.; Tallóczy, Z.; Tanaka, K.; Tanaka, K.; Tanida, I.; Taylor, G.S.; Taylor, J.P.; Terman, A.; Tettamanti, G.; Thompson, C.B.; Thumm, M.; Tolkovsky, A.M.; Tooze, S.A.; Truant, R.; Tumanovska, L.V.; Uchiyama, Y.; Ueno, T.; Uzcátegui, N.L.; van der Klei, I.; Vaquero, E.C.; Vellai, T.; Vogel, M.W.; Wang, H.G.; Webster, P.; Wiley, J.W.; Xi, Z.; Xiao, G.; Yahalom, J.; Yang, J.M.; Yap, G.; Yin, X.M.; Yoshimori, T.; Yu, L.; Yue, Z.; Yuzaki, M.; Zabirnyk, O.; Zheng, X.; Zhu, X.; Deter, R.L. Guidelines for use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy, 2008, 4(2), 1-25. Komatsu, M.; Waguri, S.; Chiba, T.; Murata, S.; Iwata, J.; Tanida, I.; Ueno, T.; Koike, M.; Uchiyama, Y.; Kominami, E.; Tanaka, K. Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature, 2006, 441(7095), 880-884. Wang, Q.J.; Ding, Y.; Kohtz, S.; Mizushima, N.; Cristea, I.M.; Rout, M.P.; Chait, B.T.; Zhong, Y.; Heintz, N.; Yue, Z.


[92] [93] [94]

[95] [96]

[97] [98] [99]

[100]

[101] [102]

[103]

[104]

[105]

[106]

[107]

[108]

[109]

[110] [111]

Induction of autophagy in axonal dystrophy and degeneration. J. Neurosci., 2006, 26(31), 8057-8068. Webb, J.L.; Ravikumar, B.; Atkins, J. Alpha-Synuclein is degraded by both autophagy and the proteasome. J. Biol. Chem., 2003, 278(27), 25009-25013. Rami, A. Upregulation of Beclin 1 in the ischemic penumbra. Autophagy, 2008, 4(2), 1-3. Qin, A.P.; Liu, C.F.; Qin, Y.Y.; Hong, L.Z.; Xu, M.; Yang, L.; Liu, J.; Qin, Z.H.; Zhang, H.L. Autophagy was activated in injured astrocytes and mildly decreased cell survival following glucose and oxygen deprivation and focal cerebral ischemia. Autophagy, 2010, 6(6), 738-753. Scherz-Shouval, R.; Elazar, Z. ROS, mitochondria and the regulation of autophagy. Trends Cell. Biol., 2007, 17(9), 422-427. Wu, H.H.; Hsiao, T.Y.; Chien, C.T.; Lai, M.K. Ischemic conditioning by short periods of reperfusion attenuates renal ischemia/reperfusion induced apoptosis and autophagy in the rat. J. Biomed. Sci., 2009, 16, 19. Azad, M.B.; Chen, Y.; Gibson, S.B. Regulation of autophagy by reactive oxygen species (ROS): implications for cancer progression and treatment. Antioxid. Redox. Signal., 2009, 11(4), 777-790. Moore, M.N.; Viarengo, A.; Donkin, P.; Hawkins, A.J. Autophagic and lysosomal reactions to stress in the hepatopancreas of blue mussels. Aquat. Toxicol., 2007, 84(1), 80-91. Pattingre, S.; Tassa, A.; Qu, X.; Garuti, R.; Liang, X.H.; Mizushima, N.; Packer, M.; Schneider, M.D.; Levine, B. Bcl2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell, 2005, 122(6), 927-939. Breitschopf, K.; Haendeler, J.; Malchow, P.; Zeiher, A.M.; Dimmeler, S. Posttranslational modification of Bcl-2 facilitates its proteasome-dependent degradation: molecular characterization of the involved signaling pathway. Mol. Cell Biol., 2000, 20(5), 18861896. De Meyer, G.R.; Martinet, W. Autophagy in the cardiovascular system. Biochim. Biophys. Acta, 2009, 1793(9), 1485-1495. Zabirnyk, O.; Liu, W.; Khalil, S.; Sharma, A.; Phang, J.M. Oxidized low-density lipoproteins upregulate proline oxidase to initiate ROS-dependent autophagy. Carcinogenesis, 2010, 31(3), 446-454. Juhaszova, M.; Zorov, D.B.; Kim, Pepe, S.H.; Fu, Q.; Fishbein, K.W.; Ziman, B.D.; Wang, S.; Ytrehus, K.; Antos, C.L.; Olson, E.N.; Sollott, S.J.; Glycogen synthase kinase-3+ mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J. Clin. Invest., 2004, 113(11), 15351549. Matsui, Y.; Takagi, H.; Qu, X.; Abdellatif, M.; Sakoda, H.; Asano, T.; Levine, B.; Sadoshima, J. Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMPactivated protein kinase and beclin 1 in mediating autophagy. Circ. Res., 2007, 100(6), 914-922. Matsui, Y.; Kyoi, S.; Takagi, H.; Hsu, C.P.; Hariharan, N.; Ago, T.; Vatner, S.F.; Sadoshima, J. Molecular mechanisms and physiological significance of autophagy during myocardial ischemia and reperfusion. Autophagy, 2008, 4(4), 409-415. Zhu, H.; Tannous, P.; Johnstone, J.L.; Kong, Y.; Shelton, J.M.; Richardson, J.A.; Le, V.; Levine, B.; Rothermel, B.A.; Hill, J.A. Cardiac autophagy is a maladaptive response to hemodynamic stress. J. Clin. Invest., 2007, 117(7), 1782-1793. Cherubini, A.; Polidori, M.C.; Bregnocchi, M.; Pezzuto, S.; Cecchetti, R.; Ingegni, T.; di Iorio, A.; Senin, U.; Mecocci, P. Antioxidant Profile and Early Outcome in Stroke Patients. Stroke, 2000, 31(10), 2295-2300. Chang, C.Y.; Chen, J.Y.; Ke, D.; Hu, M.L. Plasma levels of lipophilic antioxidant vitamins in acute ischemic stroke patients: correlation to inflammation markers and neurological deficits. Nutrition, 2005, 21(10), 987-993. Chang, C.Y.; Lai, Y.C.; Cheng, T.J.; Lau, M.T.; Hu, M.L. Plasma levels of antioxidant vitamins, selenium, total sulfhydryl groups and oxidative products in ischemic-stroke patients compared to matched controls in Taiwan. Free Radic. Res., 1998, 28(1), 15-24. El Kossi, M.M.; Zakhary, M.M. Oxidative stress in the context of acute cerebrovascular stroke. Stroke, 2000, 31(8), 1889-1892. Alexandrova, M.; Bochev, P.; Markova, V.; Bechev, B.; Popova, M.; Danovska, M.; Simeonova, V. Dynamics of free radical processes in acute ischemic stroke: influence on neurological status and outcome. J. Clin. Neurosci., 2004, 11(5), 501-506.

CNS & Neurological Disorders - Drug Targets, 2013, Vol. 12, No. 2 [112] [113] [114]

[115]

[116] [117]

[118]

[119]

[120] [121]

[122]

[123]

[124] [125]

[126]

[127] [128]

[129]

[130]

[131]

15

Nanetti, L.; Raffaelli, F.; Vignini, A.; Perozzi, C.; Silvestrini, M.; Bartolini, M.; Provinciali, L.; Mazzanti, L. Oxidative stress in ischaemic stroke. Eur. J. Clin. Invest., 2011, 41(12), 1318-1322. Sharpe, P.C.; Mulholland, C.; Trinick, T. Ascorbate and malondialdehyde in stroke patients. Ir. J. Med. Sci., 1994, 163(11), 487-491. Demirkaya, S.; Topcuoglu, M.A.; Aydin, A.; Ulas, U.H.; Isimer, A.I.; Vural, O. Malondialdehyde, glutathione peroxidase and superoxide dismutase in peripheral blood erythrocytes of patients with acute cerebral ischemia. Eur. J. Neurol., 2001, 8(1), 43-51. Polidori, M.C.; Frei, B.; Cherubini, A.; Cherubini, A.; Nelles, G.; Rordorf, G.; Keaney, J.F.; Schwamm, L.; Mecocci, P.; Koroshetz, W.J.; Beal, M.F. Increased plasma levels of lipid hydroperoxides in patients with ischemic stroke. Free Radic. Biol. Med., 1998, 25(45), 561-567. Strand, T.; Marklund, S.L. Release of superoxide dismutase into cerebrospinal fluid as a marker of brain lesion in acute cerebral infarction. Stroke, 1992, 23(4), 515-518. Leinonen, J.S.; Ahonen, J.P.; Lonnrot, K.; Jehkonen, M.; Dastidar, P.; Molnár, G.; Alho, H. Low plasma antioxidant activity is associated with high lesion volume and neurological impairment in stroke. Stroke, 2000, 31(1), 33-39. Chamorro, A.; Obach, V.; Cervera, A.; Revilla, M.; Deulofeu, R.; Aponte, J.H. Prognostic significance of uric acid serum concentration in patients with acute ischemic stroke. Stroke, 2002, 33(4), 1048-1052. Spranger, M.; Krempien, S.; Schwab, S.; Donnenberg, S.; Hacke, W. Superoxide dismutase activity in serum of patients with acute cerebral ischemic injury: correlation with clinical course and infarct size. Stroke, 1997, 28(12), 2425-2428. Hickenbottom, S.L.; Grotta, J. Neuroprotective therapy. Sem. Neurol., 1998, 18(4), 485- 492. Lafeber, F.P.; Beukelman, C.J.; van den Worm, E.; van Roy, J.L.; Vianen, M.E.; van Roon, J.A.; van Dijk, H.; Bijlsma, J.W. Apocynin, a plant-derived, cartilage-saving drug, might be useful in the treatment of rheumatoid arthritis. Rheumatology, 1999, 38(11), 1088-1093. Zhang, Z.H.; Liu, S.X.; Hou, F.F.; Tian, J.W.; Wang, L.; Liu, Z.Q.; Chen, Y. Advanced oxidation protein products-induced tumor necrosis factor alpha secretion in monocytes via reactive oxygen species generation. Di. Yi. Jun. Yi. Da. Xue. Xue. Bao., 2005, 25(5), 493-497. Heumüller, S.; Wind, S.; Barbosa-Sicard, E.; Schmidt, H.H.; Busse, R.; Schröder, K.; Brandes, R.P. Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant. Hypertension, 2008, 51(2), 211-217. Tang, L.L.; Ye, K.; Yang, X.F.; Zheng, J.S. Apocynin attenuates cerebral infarction after transient focal ischaemia in rats. J. Int. Med. Res.,2007, 35(4), 517-522. Wang, Q.; Tompkins, K.D.; Simonyi, A.; Korthuis, R.J.; Sun, A.Y.; Sun, G.Y. Apocynin protects against global cerebral ischemiareperfusion-induced oxidative stress and injury in the gerbil hippocampus. Brain Res., 2006, 1090(1), 182-189. Zheng, J.S.; Zhan, R.Y.; Zheng, S.S.; Zhou, Y.Q.; Tong, Y.; Wan, S. Inhibition of NADPH oxidase attenuates vasospasm after experimental subarachnoid hemorrhage in rats. Stroke, 2005, 36(5), 1059-1064. Chen, H.; Song, Y.S.; Chan, P.H. Inhibition of NADPH oxidase is neuroprotective after ischemia-reperfusion. J. Cereb. Blood Flow Metab., 2009, 29(7), 1262-1272. Genovese, T.; Mazzon, E.; Paterniti, I.; Esposito, E.; Bramanti, P.; Cuzzocrea, S. Modulation of NADPH oxidase activation in cerebral ischemia/reperfusion injury in rats. Brain Res., 2011, 1372(1), 92-102. Connell, B.J.; Saleh, M.C.; Khan, B.V.; Saleh, T.M. Apocynin may limit total cell death following cerebral ischemia and reperfusion by enhancing apoptosis. Food Chem. Toxicol., 2011, 49(12), 30633069. ten Freyhaus, H.; Huntgeburth, M.; Wingler, K.; Schnitker, J.; Bäumer, A.T.; Vantler, M.; Bekhite, M.M.; Wartenberg, M.; Sauer, H.; Rosenkranz, S. Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation. Am. Cardiovasc. Res.2006, 71(2), 331-341. Heumüller, S.; Wind, S.; Barbosa-Sicard, E.; Schmidt, H.H.; Busse, R.; Schröder, K.; Brandes, R.P. Apocynin is not an inhibitor of

16

[132]

[133] [134]

[135] [136]

[137] [138]

[139]

[140] [141]

[142]

[143] [144] [145] [146]

[147] [148] [149] [150] [151] [152]

[153]

CNS & Neurological Disorders - Drug Targets, 2013, Vol. 12, No. 2 vascular NADPH oxidases but an antioxidant. Hypertension, 2008, 51(2), 211-217. Schlüter, T.; Steinbach, A.C.; Steffen, A.; Rettig, R.; Grisk, O. Apocynin-induced vasodilation involves Rho kinase inhibition but not NADPH oxidase inhibition. Cardiovasc. Res., 2008, 80(2), 271-279. Touyz, R.M. Apocynin, NADPH oxidase, and vascular cells: a complex matter. Hypertension, 2008, 51(2), 172-174. Opitz, N.; Drummond, G.R.; Selemidis, S.; Meurer, S.; Schmidt, H.H. The 'A's and 'O's of NADPH oxidase regulation: a commentary on "Subcellular localization and function of alternatively spliced Noxo1 isoforms". Free Radic. Biol. Med., 2007, 42(2), 175-179. Parks, D.A.; Granger, D.N. Xanthine oxidase: biochemistry, distribution and physiology. Acta Physiol. Scand. Suppl., 1986, 548, 87-99. Tsutsumi, Z.; Moriwaki, Y.; Takahashi, S.; Ka, T.; Yamamoto, T. Oxidized low-density lipoprotein autoantibodies in patients with primary gout: effect of urate-lowering therapy. Clin. Chim. Acta, 2004, 339(1-2), 117-122. Reynolds, A.; Laurie, C.; Mosley, R.L. Gendelman HE. Oxidative stress and the pathogenesis of neurodegenerative disorders. Int. Rev. Neurobiol., 2007, 82, 297-325. Sermet, A.; Tademir, N.; Deniz, B.; Atmaca, M. Time-dependent changes in superoxide dismutase, catalase, xanthine dehydrogenase and oxidase activities in focal cerebral ischaemia. Cytobios, 2000, 102(401), 157-172. Itoh, T.; Kawakami, M.; Yamauchi, Y.; Shimizu, S.; Nakamura, M. Effect of allopurinol on ischemia and reperfusion-induced cerebral injury in spontaneously hypertensive rats. Stroke, 1986, 17(6), 1284-1287. Martz, D.; Rayos, G.; Schielke, G.P.; Betz, A.L. Allopurinol and dimethylthiourea reduce brain infarction following middle cerebral artery occlusion in rats. Stroke, 1989, 20(4), 488-494. Akdemir, H.; Asik, Z.; Pasaoglu, H.; Karakucuk, I.; Oktem, I.S.; Koc, R.K. The effect of allopurinol on focal cerebral ischaemia: an experimental study in rabbits. Neurosurg. Rev., 2001, 24(2-3), 131135. Lindsay, S.; Liu, T.H.; Xu J.A.; Marshall, P.A.; Thompson, J.K., Parks, D.A.; Freeman, B.A.; Hsu, C.Y.; Beckman, J.S. Role of xanthine deshydrogenase and oxidase in focal cerebral ischemic injury to rat. Am. J. Physiol., 1991, 261(6 Pt 2), H2051-H2057. Lin, Y.; Phillis, J.W. Oxypurinol reduces focal ischemic brain injury in the rat. Neurosci. Lett., 1991, 126(2), 187-190. Lin, Y.; Phillis, J.W. Deoxycoformycin and oxypurinol: protection against focal ischemic brain injury in the rat. Brain Res., 1992, 571(2), 272-280. Phillis, J.W.; Lin, Y. Oxypurinol reduces ischemic brain injury in the gerbil and rat. Adv. Exp. Med. Biol., 1991, 309, 343-346. van Bel, F.; Shadid, M.; Moison, R.M.; Dorrepaal, C.A.; Fontijn, J.; Monteiro, L.; Van De Bor, M.; Berger, H.M. Effect of allopurinol on postasphyxial free radical formation, cerebral hemodynamics and electrical brain activity. Pediatrics, 1998, 101(2), 185-193. Pacher, P.; Nivorozhkin, A.; Szabó, C. Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after the discovery of allopurinol. Pharmacol. Rev., 2006, 58(1), 87-114. Rodrigo, R.; Miranda, A. In: Vitamin C: Nutritional Role, Supplementation in Pathophysiological States & Side Effects, Eds.; Nova Publishers: New York, 2010; pp. 20-56. Harrison, F.E.; May, J.M. Vitamin C function in the brain: vital role of the ascorbate transporter SVCT2. Free. Radic. Biol. Med., 2009, 46(6), 719-730. Ulker, S.; Mckeown, P.P.; Bayraktutan, U. Vitamins reverse endothelial dysfunction through regulation of eNOS and NAD(P)H oxidase activities. Hypertension, 2003, 41(3), 534-539. Lagowska-Lenard, M.; Stelmasiak, Z.; Bartosik-Psujek, H. Influence of vitamin C on markers of oxidative stress in the earliest period of ischemic stroke. Pharmacol. Rep., 2010, 62(4), 751-756. Padayatty, S.J.; Sun, H.; Wang, Y.; Riordan, H.D.; Hewitt, S.M.; Katz, A.; Wesley, R.A.; Levine, M. Vitamin C pharmacokinetics: implications for oral and intravenous use. Ann. Intern. Med., 2004, 140(7), 533-537. Duconge, J.; Miranda-Massari, J.R.; Gonzalez, M.J.; Jackson, J.A.; Warnock, W.; Riordan, N.H. Pharmacokinetics of vitamin C:

Rodrigo et al.

[154]

[155] [156]

[157]

[158]

[159]

[160]

[161]

[162] [163] [164] [165]

[166]

[167] [168] [169]

[170]

[171] [172]

insights into the oral and intravenous administration of ascorbate. PR Health. Sci. J., 2008, 27(1), 7-19. Jackson, T.S.; Xu, A.M.; Vita, J.A.; Keaney, J.F. Jr. Ascorbate prevents the interaction of superoxide and nitric oxide only at very high physiological concentrations. Circ. Res., 1998, 83(9), 916922. Rose, R.C.; Bode, A.M. Biology of free radical scavengers: an evaluation of ascorbate. FASEB J., 1993, 7(12), 1135-1142. Berger, U.V.; Lu, X.C.; Liu, W.; Tang, Z.; Slusher, B.S.; Hediger, M.A. Effect of middle cerebral artery occlusion on mRNA expression for the sodium-coupled vitamin C transporter SVCT2 in rat brain. J. Neurochem., 2003, 86(4), 896-906. Gess, B.; Sevimli, S.; Strecker, J.K.; Young, P.; Schäbitz, W.R. Sodium-dependent vitamin C transporter 2 (SVCT2) expression and activity in brain capillary endothelial cells after transient ischemia in mice. PLoS One, 2011, 6(2), e17139. Polidori, M.C.; Practicó, D.; Ingegni, T.; Mariani, E.; Spazzafumo, L.; Del Sindaco, P.; Cecchetti, R.; Yao, Y.; Ricci, S.; Cherubini, A.; Stahl, W.; Sies, H.; Senin, U.; Mecocci, P. AVASAS Study Group. Effects of vitamin C and aspirin in ischemic stroke-related lipid peroxidation: results of the AVASAS (Aspirin Versus Ascorbic acid plus Aspirin in Stroke) Study. Biofactors, 2005, 24(1-4), 265-274. Ullegaddi, R.; Powers, H.J.; Gariballa, S.E. Antioxidant supplementation enhances antioxidant capacity and mitigates oxidative damage following acute ischaemic stroke. Eur. J. Clin. Nutr., 2005, 59(12), 1367-1373. Kato, N.; Yanaka, K.; Nagase, S.; Hirayama, A.; Nose, T. The antioxidant EPC-K1 ameliorates brain injury by inhibiting lipid peroxidation in a rat model of transient focal cerebral ischaemia. Acta Neurochir., 2003, 145(6), 489-493. Mishima, K.; Tanaka, T.; Pu, F.; Egashira, N.; Iwasaki, K.; Hidaka, R.; Matsunaga, K.; Takata, J.; Karube, Y.; Fujiwara, M. Vitamin E isoforms alpha-tocotrienol and gamma-tocopherol prevent cerebral infarction in mice. Neurosci. Lett., 2003, 337(1), 56-60. Niki, E.; Noguchi, N.; Tsuchihashi, H.; Gotoh, N. Interaction among vitamin C, vitamin E, and -carotene. Am. J. Clin. Nutr., 1995, 62(6 Suppl), 1322S-1326S. May, J.M.; Qu, Z.C.; Mendiratta, S. Protection and recycling of tocopherol in human erythrocytes by intracellular ascorbic acid. Arch. Biochem. Biophys., 1998, 349(2), 281-289. Bano, S.; Parihar, M.S. Reduction of lipid peroxidation in different brain regions by a combination of -tocopherol and ascorbic acid. J. Neural Transm., 1997, 104(11-12), 1277-1286. Ullegaddi, R.; Powers, H.J.; Gariballa, S.E. Antioxidant supplementation with or without B-group vitamins after acute ischemic stroke: a randomized controlled trial. J. Parenter. Enteral. Nutr., 2006, 30(2), 108-114. Stivala, L.A.; Savio, M.; Carafoli, F.; Perucca, P.; Bianchi, L.; Maga, G.; Forti, L.; Pagnoni, U.M.; Albini, A.; Prosperi, E.; Vannini, V. Specific structural determinants are responsible for the antioxidant activity and the cell cycle effects of resveratrol. J. Biol. Chem., 2001, 276(25), 22586-22594. Margaill. I.; Plotkine, M.; Lerouet, D. Antioxidant strategies in the treatment of stroke. Free Radic. Biol. Med., 2005, 39(4), 429-443. Sinha, K.; Chaudhary, G.; Gupta, Y.K. Protective effect of resveratrol against oxidative stress in middle cerebral artery occlusion model of stroke in rats. Life Sci., 2002, 71(6) 655- 665. Inoue, H.; Jiang, X.F.; Katayama, T.; Osada, S.; Umesono, K.; Namura, S. Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor alpha in mice. Neurosci. Lett., 2003, 352(3), 203-206. Simão, F.; Matté, A.; Matté, C.; Soares, F.M.; Wyse, A.T.; Netto, C.A.; Salbego, C.G.; Resveratrol prevents oxidative stress and inhibition of Na(+)K(+)-ATPase activity induced by transient global cerebral ischemia in rats. J. Nutr. Biochem., 2011, 22(10), 921-928. Wang, Q.; Xu, J.; Rottinghaus, G.E.; Simonyi, A.; Lubahn, D.; Sun, G.Y.; Sun, A.Y. Resveratrol protects against global cerebral ischemic injury in gerbils. Brain Res., 2002, 958(2), 439-447. Gao, D.; Zhang, X.; Jiang, X.;Peng, Y.; Huang, W.; Cheng, G.; Song, L. Resveratrol reduces the elevated level of MMP-9 induced by cerebral ischemia-reperfusion in mice. Life Sci., 2006, 78(22), 2564-2570.



[173]

[189]

[174] [175]

[176]

[177]

[178] [179] [180] [181]

[182]

[183] [184]

[185] [186]

[187]

[188]

Zhuang, H.; Kim, Y.S.; Koehler, R.C.; Dore, S. Potential mechanism by which resveratrol, a red wine constituent, protects neurons. Ann. NY Acad. Sci., 2003, 993, 276-286. Wang, J.; Lu, S.; Moënne-Loccoz, P.; Ortiz De Montellano, P.R. Interaction of nitric oxide with human heme oxygenase-1. J. Biol. Chem., 2003, 278(4), 2341-2347. Katori, M.; Anselmo, D.M.; Busuttil, R.W.; Kupiec-Weglinski, J.W. A novel strategy against ischemia and reperfusion injury: cytoprotection with heme oxygenase system. Transpl. Immunol., 2002, 9(2-4), 227-233. Ren, J.; Fan, C.; Chen, N.; Huang, J.; Yang, Q. Resveratrol pretreatment attenuates cerebral ischemic injury by upregulating expression of transcription factor Nrf2 and HO-1 in rats. Neurochem. Res., 2011, 36(12), 2352-2362. Hisahara, S.; Chiba, S.; Matsumoto, H.; Horio, Y. Transcriptional regulation of neuronal genes and its effect on neural functions: NAD-dependent histone deacetylase SIRT1 (Sir2alpha). J. Pharmacol. Sci., 2005, 98(3), 200-204. Raval, A.P.; Dave, K.R.; Pérez-Pinzón, M.A. Resveratrol mimics ischemic preconditioning in the brain. J. Cereb. Blood Flow Metab., 2006, 26(9), 1141-1147. Cotgreave. I.A. N-acetylcysteine: pharmacological considerations and experimental and clinical applications. Adv. Pharmacol., 1997, 38, 205-227. Dringen, R. Glutathione metabolism and oxidative stress in neurodegeneration. Eur. J. Biochem., 2000, 267(16), 4903. Carroll, J.E.; Howard, E.F.; Hess, D.C.; Wakade, C.G.; Chen, Q.; Cheng, C. Nuclear factor-kappa B activation during cerebral reperfusion: effect of attenuation with N-acetylcysteine treatment. Brain. Res. Mol. Brain Res., 1998, 56(1-2), 186-191. Khan, M.; Sekhon, B.; Jatana, M.; Giri, S.; Gilg, A.G.; Sekhon, C.; Singh, I.; Singh, A.K. Administration of N-acetylcysteine after focal cerebral ischemia protects brain and reduces inflammation in a rat model of experimental stroke. J. Neurosci. Res., 2004, 76(4), 519-527. Ullegaddi, R.; Powers, H.J.; Gariballa, S.E. B-group vitamin supplementation mitigates oxidative damage after acute ischaemic stroke. Clin. Sci., 2004, 107(5), 477-484. Lee, E.J.;Chen, H.Y.; Wu, T, S.; Chen T.Y.; Ayoub, I.A.; Maynard, K.I. Acute administration of Ginkgo biloba extract (EGb 761) affords neuroprotection against permanent and transient focal cerebral ischemia in Sprague-Dawley rats. J. Neurosci. Res., 2002, 68(5), 636-645. Zhang, Z.; Peng, D.; Zhu, H.; Wang, X. Experimental evidence of Ginkgo biloba extract EGB as a neuroprotective agent in ischemia stroke rats. Brain Res. Bull., 2012, 87(2-3), 193-198. Ritz, M.F.; Curin, Y.; Mendelowitsch, A.; Andriantsitohaina R. Acute treatment with red wine polyphenols protects from ischemiainduced excitotoxicity, energy failure and oxidative stress in rats. Brain Res., 2008, 1239, 226-234. Hall, E.D.; McCall, J.M.; Means, E.D. Therapeutic potential of the lazaroids (21-aminosteroids) in acute central nervous system trauma, ischemia and subarachnoid hemorrhage. Adv. Pharmacol., 1994, 28, 221-267. Liu, T.H.; Beckman, J.S.; Freeman, B.A.; Hogan, E.L.; Hsu, C.Y. Polyethylene glycol-conjugated superoxide dismutase and catalase reduce ischemic brain injury. Am. J. Physiol., 1989, 256(2 Pt 2), H589-H593.

Received: August 24, 2012

[190]

[191]

[192] [193]

[194]

[195]

[196]

[197]

[198]

[199] [200]

[201]

17

Guo, H.; Hu, L.M.; Wang, S.X.; Wang, Y.L.; Shi, F.; Li, H.; Liu, Y.; Kang, L.Y.; Gao, X.M. Neuroprotectiveeffects of scutellarin against hypoxic-ischemicinducedcerebralinjuryviaaugmentationofantioxidantdefensecapacity . Chin. J. Physiol., 2011, 54(6), 399-405. Zhang, W.; Sato, K.; Hayashi, T.; Omori, N.; Nagano, I.; Kato, S.; Horiuchi, S.; Abe, K. Extension of ischemic therapeutic time window by a free radical scavenger, Edaravone, reperfused with tPA in rat brain. Neurol. Res., 2004, 26(3), 342-348. Ahmad, A.; Khan, M.M.; Javed, H.; Raza, S.S.; Ishrat, T.; Khan, M.B.; Safhi, M.M.; Islam, F. Edaravone ameliorates oxidative stress associated cholinergic dysfunction and limits apoptotic response following focal cerebral ischemia in rat. Mol. Cell Biochem., 2012, 367(1-2), 215-225. Hertog, M.G.; Hollman, P.C. Potential health effects of the dietary flavonol quercetin. Eur. J. Clin. Nutr., 1996, 50(2), 63-71. Moon, S.K.; Cho, G.O.; Jung, S.Y.; Gal, S.W.; Kwon, T.K.; Lee, Y.C.; Madamanchi, N.R.; Kim, C.H. Quercetin exerts multiple inhibitory effects on vascular smooth muscle cells: role of ERK1/ 2, cell-cycle regulation, and matrix metalloproteinase-9. Biochem. Biophys. Res. Commun., 2003, 301(4), 1069-1078. Ahmad, A.; Khan, M.M.;Hoda, M.N.; Raza, S.S.; Khan, M.B.; Javed, H.; Ishrat, T.; Ashafaq, M.; Ahmad, M.E.; Safhi, M.M.; Islam, F. Quercetinprotectsagainstoxidativestressassociateddamages in a ratmodel of transientfocal cerebralischemia and reperfusion. Neurochem. Res., 2011, 36(8), 1360-1371. Mitarai, M.; Hsu, T.; Wang, M.; Hirahara, H.; Sekido, H.; Hoshino, Y.; Enari, H.; Yamamoto, S. Effects of dietary DNA from chum salmon milt on memory and age-related changes in senescenceaccelerated mice. Nippon Shokuhin Kagaku Kogaku Kaishi, 2008, 55(10), 461-467. Lam, P.Y.; Chen, N.; Chiu, P.Y.; Leung, H.Y.; Ko, K.M. NeuroprotectionAgainstOxidativeInjury by a NucleicAcidBasedHealthProduct (SquinaDNA) Through EnhancingMitochondrialAntioxidantStatus and FunctionalCapacity. J. Med. Food, 2012, 15(7), 629-638. Halevy, S.; Ghislain, P.D.; Mockenhaupt, M.; Fagot, J.P.; Bouwes Bavinck, J.N.; Sidoroff, A.; Naldi, L.; Dunant, A.; Viboud, C.; Roujeau, J.C.; EuroSCAR Study Group. Allopurinol is the most common cause of Stevens-Johnson syndrome and toxic epidermal necrolysis in Europe and Israel. J. Am. Acad. Dermatol., 2008, 58(1), 25-32. Roujeau, J.C.; Kelly, J.P.; Naldi, L.; Rzany, B.; Stern, R.S.; Anderson, T.; Auquier, A.; Bastuji-Garin, S.; Correia, O.; Locati F. Medication use and the risk of Stevens-Johnson syndrome or toxic epidermal necrolysis. N. Engl. J. Med., 1995, 333(24), 1600-1607. Levine, M.; Rumsey, S.C.; Daruwala, R.; Park, J.B.; Wang, Y. Criteria and recommendations for vitamin C intake. JAMA, 1999, 281(15), 1415-1423. Padayatty, S.J.; Sun, A.Y.; Chen, Q.; Espey, M.G.; Drisko, J.; Levine, M. Vitamin C: intravenous use by complementary and alternative medicine practitioners and adverse effects. PLoS One, 2010, 5(7), e11414. Millea PJ. N-acetylcysteine: multiple clinical applications. Am. Fam. Phys., 2009, 80(3), 265-269.

Revised: December 11, 2012

Accepted: December 27, 2012