Excitotoxic mechanisms in stroke: An update of concepts and treatment strategies

Neurochemistry International 50 (2007) 941–953 www.elsevier.com/locate/neuint

Excitotoxic mechanisms in stroke: An update of concepts and treatment strategies Alan S. Hazell * Department of Medicine, University of Montreal, Montreal, Quebec, Canada Accepted 18 April 2007 Available online 10 May 2007

Abstract Cerebral damage as a consequence of glutamate-mediated excitotoxicity represents a major consequence of stroke. However, the development of effective clinical treatments for this potentially devastating condition has been largely unsuccessful to date, despite promising basic research. This review will focus on the latest advances in our understanding of the excitotoxic process including the release of glutamate as a neurotransmitter and the potential contribution of complexins, the important role of astrocytes, including its involvement in glutamate uptake, alterations in glutamate transporter levels, reversed glutamate uptake, and the vesicular release of glutamate. Recent progress in our understanding of the involvement of excitotoxicity in white matter injury following ischemic insults is also discussed, as is oxidative stress and ischemic tolerance, along with an update on the use of treatment strategies with potential therapeutic benefit including stimulation of neurogenesis. Such key issues are at the heart of future interventions directed at limiting the extent of the excitotoxic process, and remain a viable consideration for effective stroke management. # 2007 Elsevier Ltd. All rights reserved. Keywords: Glutamate transporter; Cerebral ischemia; Excitotoxicity; Therapeutic; Complexin; Astrocyte; Glutamate; Neurogenesis

1. Introduction A complex series of events underlie the pathophysiology of stroke, a leading cause of death and disability worldwide, and which can be either ischemic or hemorrhagic in nature. Most commonly, the phenomenon arises as a consequence of permanent or prolonged occlusion of a cerebral artery (Ameriso and Sahai, 1997). Immediately following interruption of blood flow to brain tissue the injury process is initiated. Without recovery of normal blood flow within a short period of time, death of all cells within the ischemic territory is typically the final outcome (Pulsinelli, 1997). While complete details of this pathway of destruction remain unclear, considerable advances in our knowledge of this process have been made over the last 20 years. Substantial evidence indicates that glutamate-mediated excitotoxicity is a major contributor to the resulting neuropathology. Survival of the affected area is then dependent on its

* Correspondence address: NeuroRescue Laboratory, Hoˆpital Saint-Luc (CHUM), 1058 St-Denis, Montreal, Quebec, Canada H2X 3J4. Tel.: +1 514 890 8310x35740/35758; fax: +1 514 412 7314. E-mail address: [email protected]. 0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2007.04.026

ability to respond to this insult. This review will discuss major events associated with the process of excitotoxicity, therapeutic strategies, and current limitations in the treatment of stroke. 2. Complexins and neurotransmitter release Rapidly following cessation of blood flow, energy metabolism is compromised in the affected territory of a stroke, quickly leading to large increases in neuronal activity and a resulting enhancement in glutamate release (Benveniste et al., 1984). Recent studies indicate that complexins are important regulators of neurotransmitter release. Complexin I and complexin II are two genes with a high degree of homology which are differentially expressed in human brain (Harrison and Eastwood, 1998). The products of these two genes are 15–16 kDa cytosolic proteins that each contain an a-helical middle domain of approximately 58 amino acids (Pabst et al., 2000). Complexin I is expressed in axosomatic (inhibitory) synapses, while complexin II is localized in axodendritic and axospinous synapses, of which the majority are excitatory (Harrison and Eastwood, 1998; Yamada et al., 1999). At the presynaptic terminal, complexins compete with the chaperone protein a-SNAP

942

A.S. Hazell / Neurochemistry International 50 (2007) 941–953

(soluble N-ethylmaleimide-sensitive factor-attachment protein) for binding to SNAP receptors (SNAREs) (Pabst et al., 2000). These SNAREs consist of the synaptic vesicle protein synaptobrevin as well as the synaptic membrane proteins SNAP-25 and syntaxin 1 (McMahon et al., 1995; Pabst et al., 2000). Complexins have been shown to rapidly bind to the SNARE complex in an anti-parallel configuration and with high affinity (Pabst et al., 2002). Prior to vesicle release into the synaptic cleft, these membrane proteins form a stable core complex; interaction of complexins with the SNARE complex influence its stability (Pabst et al., 2000) by promoting the direct assembly of all three SNARE proteins, and which involves an interaction of the transmembrane regions of both syntaxin and synaptobrevin (Hu et al., 2002). Recently, studies indicate that release of complexins from the SNARE complex by its competition with another synaptic vesicle protein, synaptotagmin 1, triggers fast exocytosis and may explain the speed and efficiency of this process (Tang et al., 2006). Current evidence therefore support an important role for complexins in the modulation of the neurotransmitter release process and the maintenance of normal synaptic function, with alternations in their levels being associated with brain injury and psychiatric illness (Eastwood and Harrison, 2005; Hazell and Wang, 2005; Yi et al., 2006). However, the effects of alterations in complexin levels on neurotransmitter release remain unclear. Increased expression following injection of recombinant complexin II inhibits neurotransmitter release while decreased activity following anti-complexin II antibody treatment results in increased release (Ono et al., 1998), suggesting that the protein may inhibit neurotransmitter release. On the other hand, reports have also demonstrated that loss of complexins leads to reduced neurotransmitter release efficiency (Reim et al., 2001) and loss of hippocampal longterm potentiation (Takahashi et al., 1995), evidence of a facilitatory role. Thus, although the exact role of complexins in synaptic vesicle exocytosis is still unresolved, current evidence suggest that loss of complexin II is likely to destabilize synaptic terminal release of glutamate, contributing to dysfunction of circuitry with possible pathological consequences. Alterations in the function and/or expression of complexins may therefore be a complicating factor contributing to the excessive sustained release of glutamate following acute stroke. 3. Glutamate receptors and excitotoxicity Neuronal release of glutamate following cerebral ischemia activates several types of pre- and post-synaptic glutamate receptors. The consequent rise in intracellular calcium concentration may lead to mitochondrial dysfunction, generation of reactive oxygen species, and the activation of proteases, phospholipases, and endonucleases, leading to cell death.

kainate receptor. AMPA and kainate receptors have fast kinetics and are permeable to Na+ and K+ and also to low levels of Ca2+ (Hollmann et al., 1991). NMDA receptors possess voltagedependent divalent and monovalent cation channels that are more permeable to Ca2+ ions. Because of the higher permeability towards Ca2+ and the voltage-dependent fluxes, NMDA receptors are thought to play an important role in development of excitotoxicity. In fact, glutamate receptor agonists such as NMDA, kainate, or quisqualate are shown to be far more potent neurotoxins than glutamate itself when infused into the brain (Obrenovitch et al., 1994). This is due to the presence of efficient uptake mechanisms for glutamate but not for these agents. The neuroprotective influence exerted by NMDA receptor antagonists, as well as by AMPA and kainate receptor antagonists following brain injury in animal models further supports the notion that excessive glutamate receptor stimulation contributes to damage via an excitotoxic process. It is now generally accepted that Ca2+ overload and the activation of Ca2+-dependent enzymes following excessive overstimulation of ionotropic glutamate receptors are key factors determining excitotoxicity. Studies have long established that removal of extracellular Ca2+, but not Na+, reduces cell death in response to addition of glutamate in cultured neurons (Choi, 1987). The importance of source-specific excitotoxic Ca2+ entry mediated via ionotropic receptors was emphasized when it was later shown that Ca2+ loads produced by voltage-sensitive Ca2+ channels were not damaging but a similar increase in intracellular Ca2+ via NMDA receptors was detrimental (Tymianski et al., 1993). 3.2. Metabotropic glutamate receptors Metabotropic glutamate receptors (mGluRs) are G-proteincoupled receptors that produce their effects via signalling mechanisms involving phosphoinositide-dependent processes, cyclic AMP or protein kinase C. Recent studies have identified mGluRs as a way in which neural cells regulate the release of glutamate and its uptake. Three groups of mGluRs have been characterized to date. Group I mGluR agonists have been reported to cause a downregulation of the EAAT1 transporter, while the Group II agonist DCG IV upregulates its expression (Gegelashvili et al., 2000). Group II mGluRs are found on both pre- and post-synaptic membranes as well as glial cells (Petralia et al., 1996), are negatively coupled to cyclic AMP, and regulate glutamate release via presynaptic Group II autoreceptors (Glaum and Miller, 1994). Group III mGluRs are also negatively coupled to cyclic AMP. Cerebral ischemia results in decreased ATP levels (Folbergrova´ et al., 1992), a source of cyclic AMP via activity of adenylate cyclase. Thus, it is conceivable that loss of glutamate transporter regulation occurs as a consequence of changes in activity of mGluRs due to the declining ATP status.

3.1. Ionotropic glutamate receptors 4. Astrocytes and glutamate transporters There are three known types of ionotropic glutamate receptors based on their pharmacological properties; the Nmethyl-D-aspartate (NMDA) receptor, AMPA receptor, and the

Astrocytes are responsible for many key processes in brain including buffering of K+, inactivation of released

A.S. Hazell / Neurochemistry International 50 (2007) 941–953

neurotransmitters, and brain water homeostasis (GardnerMedwin et al., 1981; Schousboe, 1981; Walz, 1987). Increasingly, more emphasis is being placed on astrocytes and their involvement in cerebral injury and neurodegenerative disease (Maragakis and Rothstein, 2006). A major function of these glial cells is the efficient removal of glutamate from the extracellular space (Drejer et al., 1983), a process that is instrumental in maintaining normal interstitial levels of this neurotransmitter (Nicholls and Attwell, 1990). In brain, five subtypes of Na+-dependent glutamate transporter have so far been cloned: EAAT1 (Storck et al., 1992), EAAT2 (Pines et al., 1992), also known as GLAST and GLT-1 respectively, EAAC1 (Kanai and Hediger, 1992), also known as EAAT3, EAAT4 (Fairman et al., 1995) and EAAT5 (Arriza et al., 1997). Of these, EAAT1and EAAT2 are predominantly localized in astrocytes (Danbolt et al., 1992; Rothstein et al., 1994; Lehre et al., 1995), and provide the major spatial buffering of extracellular glutamate levels in brain (Danbolt, 2001), while EAAT3, EAAT4, and EAAT5 are mainly neuronal. Because astrocytes maintain a high outwardly directed glutamate gradient across the cell membrane, activity of glutamate transporters in the cell membrane is highly dependent on cellular energy status to ensure efficient uptake of glutamate. These glutamate transporters are driven by Na+, K+, and possibly by hydroxide ion gradients. Uptake of glutamate by the transporters is coupled to the co-transport of three Na+ and one H+ with each glutamate ion into the cell, while one K+ is transported out of the cell (Phillis et al., 1994; Levy et al., 1998; Anderson and Swanson, 2000). 5. The glutamate transport response of astrocytes to stroke 5.1. Downregulation of glutamate transporters Following cerebral ischemia, a loss of EAAT1 and EAAT2 occurs (Fig. 1), contributing to increased extracellular glutamate levels and ultimately neuronal cell death (Rothstein et al., 1996; Rao et al., 2001; Yeh et al., 2005). Indeed, mutant mice with a knockout of the EAAT2 gene show more damage following cerebral injury than their wild-type counterparts (Tanaka et al., 1997). Recently, focus has been placed on EAAT2v (GLT-1v), a splice-variant form of EAAT2 (Chen et al., 2002; Schmitt et al., 2002). EAAT2v shows high amino acid sequence homology with the predominant form of the transporter, EAAT2a, differing only at its C-terminal region (Utsunomiya-Tate et al., 1997; Schmitt et al., 2002). Following brain injury, EAAT2v levels are decreased while EAAT4 is upregulated (Yi et al., 2005, 2007); involvement of these transporters in cerebral ischemia is therefore also a possibility. 5.2. Glutamate efflux via transporter reversal Previously, astrocytes were considered to be resistant to extended periods of ischemia because of their stores of glycogen providing glucose (Swanson, 1992). However, prolonged cessation of blood flow leads to anerobic conditions, resulting in lactic acid accumulation with continued glucose

943

utilisation. The relative sensitivity of astrocytes to extended periods of lactic acidosis (Giffard et al., 1990) leads to an inability of these cells to maintain ATP production below a pH of 6.6 (Swanson et al., 1997). The resulting decrease in ATP levels then causes a collapse of the ionic gradients across the astrocyte cell membrane due to suppression of Na+–K+– ATPase activity, leading to movement of Na+ into the cell along its concentration gradient, elevated intracellular Na+ concentration, and transport of glutamate from the intracellular compartment of the astrocyte to the extracellular space as a consequence of movement of transporter-mediated Na+ movement back out of the astrocyte (Lees, 1991), effectively a reversal of glutamate transporter function (Szatkowski et al., 1990; Longuemare and Swanson, 1995; Seki et al., 1999) (Fig. 1). The major consequence of this transporter reversal is an increase in extracellular glutamate concentration, thus contributing to the excitotoxicity process. Therefore, while astrocytes perform a neuroprotective role by way of glutamate transporter-mediated uptake under conditions of moderately increased levels of extracellular glutamate, severe ischemia can have the opposite effect in which the damage is amplified (Dronne et al., 2007). Interestingly, evidence suggest that excessive influx of Na+ into astrocytes under conditions of ischemia can also lead to death of these cells due to Ca2+ overload (Takahashi et al., 2000), most likely a consequence of a reversed activity of the cell membrane Na+–Ca2+ exchanger. In a recent in vitro study, simulated ischemia led to considerable astrocytic cell death which was attenuated following upregulation of EAAT2 levels, and this attenuation antagonized by treatment with dihydrokainate (DHK), a glutamate uptake inhibitor (Kosugi and Kawahara, 2006). It was suggested that the increased levels of EAAT2 resulted in greater reversal of glutamate transporter activity due to the increased removal of internal Na+ by co-transport on the EAAT2 transporter, leading to less Ca2+ overload and more astrocyte survival. Antagonism of this effect with DHK suggests that increased EAAT2 activity was responsible for this astrocyte protection. Thus, reversal of glutamate transporter activity, while damaging to neurons, is also important for the survival of astrocytes under conditions of ischemia. 5.3. Glutamate efflux via vesicular release Despite the important role of astrocytes in glutamate clearance from the extracellular space, studies have established these cells also have the ability to release glutamate in a calcium-dependent manner similar to that found at the synaptic terminal (Parpura et al., 1994; Bezzi et al., 1998). Evidence also suggest this glutamate release occurs via a vesicular-mediated process since clostridial toxins known to cleave specific proteins recognized as essential for vesicle fusion block the astrocyte release of transmitter (Bezzi et al., 1998; Araque et al., 2000) (Fig. 1). Recently, studies identified this vesicular compartment in astrocytes capable of releasing glutamate which is similar to that found at the synaptic terminal (Bezzi et al., 2004; Montana et al., 2006). In addition, evidence suggest that astrocytes contain the chemical machinery for release of

944

A.S. Hazell / Neurochemistry International 50 (2007) 941–953

Fig. 1. Glutamate transport function and dysfunction in the astrocyte. A number of processes contribute to excitotoxic conditions under conditions of cerebral ischemia. Development of oxidative stress due to impairment of energy metabolism following cessation of blood flow can affect normal glutamate transport in astrocytes (1) leading to nitrosylation of glutamate transporters and a decrease in the uptake of glutamate (2). Lack of blood flow also decreases glutamate uptake as a consequence of glutamate transporter downregulation (3). Prolonged ischemia leads to decreased ATP levels in the astrocyte, resulting in failure of the cell membrane Na+–K+–ATPase and leading to glutamate efflux due to a reversal of ionic gradients that drive transporter function (reversed uptake) (4). Although astrocytes modulate synaptic transmission via the vesicular release of glutamate, under ischemic conditions, dysregulation of this process may lead to enhanced glutamate release (5). In addition, following a stroke, astrocytic swelling due to glutamate uptake or excessive K+ spatial buffering can result in depolarization of these cells, leading to release of glutamate via transporter reversal (6). Together, these processes lead to increased extracellular glutamate concentation and excitotoxic-mediated cell death. Glu, glutamate.

glutamate including SNARE proteins and the vesicular glutamate transporters VGLUT1 and VGLUT2, consistent with a functional role in the vesicular release of glutamate and vesicle refilling (Zhang et al., 2004; Montana et al., 2004). Studies have established that a major function of this calciumdependent glutamate release is to modulate synaptic transmission since the release of glutamate increases the spontaneous frequency of minature postsynaptic currents and is mediated by presynaptic NMDA receptors (Araque et al., 1998a; Angulo et al., 2004). In addition, electrical or mechanical activation of astrocytes resulting in increased calcium wave propagation reduced postsynaptic currents evoked by electrical stimulation of presynaptic neurons (Araque et al., 1998b). This depression of evoked synaptic activity was shown to be mediated by glutamate release from the astrocytes and could be blocked by mGluR antagonists. Thus, astrocytes can directly regulate both spontaneous and evoked neuronal activity in an excitatory or inhibitory manner via glutamate release, which has important implications in terms of our understanding of the control of synaptic transmission, and the role of astrocytes in this process (Newman, 2003). Since astrocytes can release glutamate using

a vesicular mechanism similar to that at the presynaptic terminal, it is possible that ischemic conditions may lead to a loss of control of this process, and increased excitotoxic conditions (Fig. 1). 6. White matter injury following stroke Evidence indicate that cellular elements of white matter are significantly affected significantly during cerebral ischemia. For example, oligodendrocytes and the product of their activities, myelin, are known to be damaged by glutamate (Alberdi et al., 2002; Li et al., 1999; Matute et al., 1997; McDonald et al., 1998; Rosenberg et al., 1999; Sanchez-Gomez and Matute, 1999), while axons are injured by ionic mechanisms leading to damaging accumulation of intracellular Ca2+ (Fern et al., 1995; Stys et al., 1990; Wolf et al., 2001). In a recent study, treatment of cultured mouse optic nerve with oxygen glucose deprivation that mimics conditions following cerebral ischemia led to the demonstration that excitotoxic white matter injury is correlated with glutamate release (Tekko¨k et al., 2007). Furthermore, this glutamate release,

A.S. Hazell / Neurochemistry International 50 (2007) 941–953

most likely brought about by disruption of glial and axonal ion gradients, was mediated by reversed Na+-dependent glutamate transport since both DL-TBOA, a glutamate transporter inhibitor, and PDC, a glutamate antagonist that inhibits glutamate release at the cytoplasmic surface, protected against the injury. Other previous studies have demonstrated that glutamate receptor blockade protects against white matter injury (Agrawal and Fehlings, 1997; Li et al., 1999; Li and Stys, 2000; Tekko¨k and Goldberg, 2001), thus providing additional evidence that glutamate-mediated excitotoxicity is a valid mechanism contributing to the overall cerebral damage that results following stroke. Current findings have identified the presence of AMPA and kainate receptors on oligodendrocytes (Gallo et al., 1994), suggesting these receptors likely mediate injury and death of these cells following stroke. 7. Role of oxidative stress Brain tissue possesses a number of important endogenous defences against ischemic injury, including glutathione, and the enzymatic Mn2+ superoxide dismutase and Cu2+/Zn2+ superoxide dismutase. However, during the injury process, these natural antioxidant defences can be quickly overwhelmed following energy impairment, leading to increased production of superoxide radicals, nitric oxide, and hydrogen peroxide (Warner et al., 2004). Development of this oxidative stress can rapidly lead to serious disturbances in cerebral function, including an inhibition of glutamate transport due to transporter protein nitrosylation by peroxynitrite formation (Volterra et al., 1994; Agostinho et al., 1997; Trotti et al., 1996,1998) (Fig. 1). When administered following cerebral ischemia, antioxidants have shown benefits in minimizing the extent of injury and neuronal loss. Treatment of rats with the antioxidant Nacetylcysteine (NAC) which protects against reactive oxygen species through restoration of intracellular glutathione (Ratan et al., 1994; Juurlink and Paterson, 1998) has decreased the extent of injury in different models of brain ischemia (Knuckey et al., 1995; Sekhon et al., 2003; Jatana et al., 2006). On the other hand, some studies have shown no neuroprotective effect with this antioxidant (Silbergleit et al., 1999). NAC has previously been used successfully in the treatment of several disease states including acetaminophen-induced liver failure (Oh and Shenfield, 1980), pulmonary oxygen toxicity (Wagner et al., 1989; Sa¨rnstrand et al., 1995), and in experimental studies on HIV infection (Sung et al., 2001). It reacts with the hydroxyl radical and hypochloric acid but reacts poorly with hydrogen peroxide and the superoxide radical. Thus, it is possible that certain antioxidants with a particular oxidant treatment profile may be beneficial in treating some types of cerebral injury, while others may have a greater impact in combating other types of damage. Further studies on the effects of different antioxidants in stroke are warranted at this time. 8. Hemorrhagic stroke Spontaneous intracerebral hemorrhage (ICH) accounts for 15–20% of all strokes (Ribo and Grotta, 2006) and has a

945

poor prognosis. Since hemorrhaged blood leads to exposure of brain tissue to hemoglobin that can liberate free heme and other hemoproteins (Paoli et al., 2002; Wagner et al., 2003) and which can cause cell death (Dennery et al., 2003; Ostrow et al., 2003), efficient and rapid metabolism of this heme is crucial for the resolution of ICH. Although heme oxygenase-1 (HO-1) is an inducible form of the heme oxygenases which metabolizes heme molecules to carbon monoxide, iron and biliverdin, interestingly, studies involving HO-1 knockout mice have demonstrated that HO-1 exerts damaging effects at early times following ICH (Wang and Dore´, 2007). This is contrary to its expected protective function as a member of the heat shock family of chaperone proteins (Hsp32). Further studies on the effects of HO-1 regulation in a clinical setting may therefore lead to improved treatment of this type of stroke. Other studies have reported that levels of EAAT1 and EAAT4 are considerably reduced in neonatal infants with sub-arachnoid hemorrhage relative to age-matched control patients, possibly leading to cell death and olivocerebellar degeneration (Inage et al., 2000), suggesting a role for excitotoxicity in hemorrhagic stroke. 9. Brain edema Raised intracranial pressure (ICP) is often a major feature of stroke with serious, sometimes fatal consequences in patients, particularly following ICH. The cause of increased ICP in such cases is brain swelling associated with the development of edema, and astrocytic swelling is a wellknown factor contributing to the morbidity and mortality of brain injured patients (Kimelberg, 1992). Although pharmacological and surgical interventions are important treatments for elevated ICP following hemorrhagic stroke, understanding the precise cellular mechanisms that lead to raised ICP is an important step towards designing future therapeutic strategies. Astrocytic glutamate transporters contribute to brain edema following cerebral ischemia (Namura et al., 2002), with initial attempts by these cells to clear the elevated glutamate leading to their swelling (Hansson et al., 1994), partly due to the accumulation of glutamine, an important osmolyte, as a consequence of increased activity of the astrocyte-specific enzyme glutamine synthetase (Norenberg and Martinez-Hernandez, 1979). This swelling can cause astrocytic depolarization (Kimelberg and O’Connor, 1988), resulting in reversal of glutamate transport activity (swelling-induced release) that contributes to the increased extracellular glutamate concentration (Fig. 1). Previous studies have indicated that cerebral ischemia leads to increased expression of the water channel aquaporin-4 (AQP-4) (Taniguchi et al., 2000). This finding suggests the protein may be involved in the process of edema observed following stroke. Also, AQP-4 knockout mice exhibit decreased brain edema following an ischemic insult (Manley et al., 2000), adding credence to this possibility and supporting an important role for this gene in the pathophysiology of stroke.

946

A.S. Hazell / Neurochemistry International 50 (2007) 941–953

10. Ischemic tolerance and glutamate transporters The protective function afforded by ischemic tolerance (‘‘pre-conditioning’’), discovered by Kitagawa et al. (1990), and subsequently confirmed by other studies (e.g., Chen et al., 1996; Kirino, 2002) has been shown to be related to many factors including heat shock proteins (Nishino and Nowak, 2004), immediate-early genes (Tomimoto et al., 1999), nitric oxide (Atochin et al., 2003; Liu et al., 2006) and the release of glutamate (Kawahara et al., 2005; Kosugi et al., 2005). In fact, recent evidence indicate that upregulation of astrocytic glutamate transporters including EAAT2 plays an important role in the induction of ischemic tolerance (Romera et al., 2004; Kawahara et al., 2005; Kosugi et al., 2005; Chu et al., 2007; Zhang et al., 2007), emphasizing the significance of glutamate transporters in the protection of cerebral tissue to various insults such as stroke. Further studies that investigate whether preconditioning can specifically influence excitotoxicity are necessary in order to determine its effect on this process. 11. K+ spatial buffering and ischemic damage In addition to the uptake of glutamate, astrocytes perform another important role of clearance of K+ from the extracellular space (Hertz, 1965). Although this astrocytic spatial buffering of K+ is carried out mainly by entry of K+ into the cells via passive currents with subsequent propagation through the glial syncytium via gap junctional communication (Kuffler et al., 1966; Karwoski et al., 1989), higher extracellular K+ levels due to increased neuronal activity following ischemia leads to uptake of K+ via activity of the astrocytic Na+–K+–ATPase (Walz and Hertz, 1982). Such spatial buffering of external K+ by astrocytes can also cause their swelling (Hansson et al., 1994), possibly contributing to swelling-induced release of glutamate (Fig. 1). In addition, while removal of K+ from the extracellular space is a major neuroprotective function of these cells, excessive buffering of K+ can also contribute to spreading depression under conditions of ischemia (Nedergaard and Hansen, 1993), which has the ability to increase the extent of damage (Busch et al., 1996; Mies et al., 1993). Mathematical modeling of the response of astrocytes to development of a stroke reveals changes in these cells that are consistent with a loss of K+ homeostasis (Dronne et al., 2007). 12. The future of therapeutic strategies in stroke 12.1. Tissue-type plasminogen activator (tPA) Although tPA remains the only approved acute therapy for stroke patients in which thrombolysis is an urgent requirement, important clinical limitations have been described that include a high risk of hemorrhage and the narrow therapeutic window (Lebeurrier et al., 2004). Because tPA is present not only in the vascular compartment but also within neurons and glial cells, this points to the possibility of unexpected functions for this serum protease, particularly as it crosses the blood-brain barrier (BBB) (Benchenane et al., 2005). Indeed, tPA knockout mice

exhibit impressive resistance to both excitotoxic and ischemic damage (Tsirka et al., 1995; Wang et al., 1998; Nagai et al., 1999), an indicator of the potential deleterious consequences of its use in brain. This is reflected in studies demonstrating that tPA increases NMDA receptor activity, possibly leading to excessive calcium entry and cell death due to its proteolytic effects on this receptor (Fernandez-Monreal et al., 2004). Thus, development of new, safer thrombolytic strategies are essential for the future successful treatment of stroke. 12.2. Cytidine-50 -diphosphocholine (citicoline) In humans, citicoline is the only agent with proven neuroprotection efficacy in patients with moderate to severe stroke, having shown positive results in randomized, doubleblind trials and in a meta-analysis with similar safety to placebo (Tazaki et al., 1988; Da´valos et al., 2002). On the other hand, a few studies have reported the drug to be safe but ineffective (Clark et al., 1999, 2001). Experimentally, studies have demonstrated a clear neuroprotective effect of citicoline in cerebral ischemia (Andersen et al., 1999; Aronowski et al., 1996; Grieb et al., 2001; Kakihana et al., 1988; Onal et al., 1997; Schabitz et al., 1996; Shuaib et al., 2000) along with a variety of CNS injury models. Studies performed under in vivo and in vitro conditions aimed at determining how citicoline is able to afford such neuroprotection in stroke have demonstrated this drug improves glutamatergic neurotransmission by decreasing glutamate release by neurons and improving its uptake into astrocytes by increasing both the activity of glutamate transporters and their expression (Hurtado et al., 2005). In addition, citicoline inhibited the decrease in ATP levels normally accompanying this type of insult. Since neurological progression of patients with acute ischemic stroke is accompanied by elevated levels of glutamate in blood and cerebrospinal fluid (CSF) (Castillo et al., 1997), this impressive and specific protection afforded by citicoline points to its possible further use in the future management of patients at risk of ischemic events. 12.3. Enhancement of glutamate transporter function Enhancement of glutamate transport function represents a logical approach towards the treatment of excitotoxicity following stroke. Beta-lactam antibiotics represent an exciting group of compounds that could prove beneficial in this regard. Ceftriaxone, a member of this group, was shown to selectively upregulate EAAT2 (Rothstein et al., 2005), and was recently reported to reduce injury volume by over 50% in focal ischemia when administered days in advance (Chu et al., 2007). On the other hand, ceftriaxone lacked neuroprotective efficacy when used shortly after the ischemic event, thus suggesting it may be more effective when used in a preconditioning capacity. This compound has also shown protective benefits when studied in in vitro models of stroke (Lipski et al., 2007). MS-153 is an agent that also provides benefit against stroke, acting via a kinaserelated mechanism to potentiate glutamate uptake (Umemura et al., 1996; Shimada et al., 1999), while recently, we

A.S. Hazell / Neurochemistry International 50 (2007) 941–953

demonstrated that under conditions of impaired oxidative metabolism loss of EAAT1 transporter activity can be prevented by the group II mGluR agonist DCG IV (Hazell et al., 2003). DCG IV has also been shown to have neuroprotective efficacy in cultured cortical neurons (Bruno et al., 1994; Miyamoto et al., 1994) and in a model of traumatic brain injury (Zwienenberg et al., 2001). It is therefore possible this ligand or related mGluR compounds may be useful as a treatment strategy for excitotoxicity in stroke. From a clinical standpoint, one of the safest methods of overexpressing a potentially neuroprotective gene in vivo may be to use an ex vivo gene therapy approach (Aebischer et al., 1996) that involves the use of encapsulated, genetically engineered cell lines, which avoids immune rejection and tumour formation. Recently, it has been demonstrated that cells engineered to overexpress EAAT2 exhibit neuroprotective efficacy against excitotoxic death under in vitro conditions (Wisman et al., 2003), suggesting this type of approach may provide benefit in minimizing glutamate-mediated cell death in the future. 12.4. NMDA receptor antagonists Studies identifying the potential benefits of glutamate receptor antagonists in treating ischemic stroke have not translated into a clinical benefit over the years. Indeed, early attempts to overcome excitotoxic injury in patients with stroke by application of glutamate receptor antagonists in clinical trials have suffered serious drawbacks (Ikonomidou and Turski, 2002). In fact, evidence suggest that administration of NMDA receptor antagonists within 24 h following the ischemic event produces a trend towards worse functional outcome or mortality (Muir and Lees, 2003). The basis of this finding is unclear. However, many of these antagonists at beneficial dosages, produce major side effects and are potentially dangerous (Goldberg, 2002). In addition, at lower dosages synaptic tortuosity can hinder efficient drug delivery, making the half-life of these compounds a significant factor. Furthermore, the fact that many patients arrive at hospital for treatment several hours after the insult, limits any potential benefits of these agents. Recently, Mallolas et al. (2006) reported that analysis of the EAAT2 promotor identified a polymorphism that leads to enhanced repression of this region, resulting in decreased expression of EAAT2, increased levels of plasma glutamate and early neurologial worsening in human stroke. This mutation may also contribute to the lack of effectiveness of glutamate receptor antagonists in stroke patients in cases where this mutant genotype is present. 12.5. Mg2+ as a potential therapy Certain properties of Mg2+ that include its ability to inhibit NMDA receptors in a non-competitive manner, inhibition of neurotransmitter release, suppression of cortical spreading depression and anoxia-induced depolarization, have made it a candidate for study in a clinical setting. However, a randomized control trial of intravenous Mg2+ for acute ischemic stroke

947

showed no protective efficacy in terms of death or disability at 90 days (Muir et al., 2004). On the other hand, acute treatment with Mg2+ within 12 h of presentation has yielded a benefit to patients with subcortical infarcts, which may have relevance where on site paramedic-initiated treatment is concerned (Saver et al., 2004). In addition, studies have demonstrated that Mg2+ deficiency in experimental ischemic stroke is associated with more severe damage (Demougeot et al., 2004), suggesting that Mg2+ does play a role in limiting the extent of cerebral injury. 12.6. Oxaloacetate In a recent study, Zlotnik et al. (2007) have reported that administration of oxaloacetate to rats provided neuroprotection following traumatic brain injury (TBI). This effect is believed to occur due to oxaloacetate being able to transform glutamate to 2-ketoglutarate in the presence of glutamate-oxaloacetate transaminase, a blood-resident enzyme, in effect acting as a blood glutamate scavenger (Gottlieb et al., 2003). The resulting decrease in blood glutamate then increases potential brain-toblood glutamate efflux by the resulting creation of a larger glutamate concentration gradient between the CSF/capillary endothelial cell and blood plasma. Previous in vitro and in vivo studies have demonstrated that abluminal glutamate transporters have the ability to export glutamate from brain into blood across the BBB, thus facilitating the process of lowering increased extracellular levels of glutamate. Since both TBI and stroke are associated with the excessive release of glutamate into the extracellular space and CSF, use of blood glutamate scavengers such as oxaloacetate may provide a novel way to reduce the extracellular glutamate load at early times following an ischemic insult with therapeutic implications. 12.7. Peroxisome proliferator-activated receptor (PPAR) agonists PPARs belong to the nuclear receptor superfamily and function as transcription factors in many important biological processes. Agonists of PPAR subtypes, e.g. thiazolidinediones such as rosiglitazone and pioglitazone which are exogenous agonists of PPAR-gamma have been shown to prevent inflammation and neuronal death following stroke and spinal cord injury (Luo et al., 2006; Tureyen et al., 2007; Park et al., 2007). Agonists of PPAR-alpha such as fenofibrate (Deplanque et al., 2003), and the PPAR-delta agonists L-165041 and GW501516 have also been reported to be protective following cerebral ischemia (Iwashita et al., 2007). Such studies are particularly significant as PPAR-gamma agonist-induced neuroprotection may be mediated by an upregulation of glutamate transporters (Romera et al., 2007). 12.8. Other agents with neuroprotective potential Several other agents that have shown promise in terms of neuroprotective efficacy in experimental models of stroke remain untested in a clinical setting. These include vitamin E (Yamagata et al., 2004), sodium channel blockers (Callaway

948

A.S. Hazell / Neurochemistry International 50 (2007) 941–953

et al., 2004), activated protein C (Griffin et al., 2004), and heat shock proteins (DeFranco et al., 2004). In addition, the serum protease inhibitor neuroserpin has been shown to protect neurons against excitotoxicity following cerebral ischemia (Yepes et al., 2000; Lebeurrier et al., 2005). 12.9. Post-ischemic neurogenesis and stem cell treatment Recent evidence of some degree of replacement of neurons following injury to the brain has been reported. Administration of erythropoietin that has the ability to increase the expression of vascular-endothelial and brain-derived neurotrophic growth factors (VEGF and BDNF) has been shown to increase neurogenesis following experimental ischemic stroke (Leist et al., 2004; Wang et al., 2004). In addition, a preliminary trial of erythropoietin in humans showed beneficial and safe effects in terms of functional and radiological outcome (Ehrenreich et al., 2002). Treatment with epidermal or fibroblast growth factors (EGF, FGF) has also shown therapeutic potential in terms of enhanced neurogenesis (Nakatomi et al., 2002; Manoonkitiwongsa et al., 2004), while delivery of glial-derived neuronal neurotrophic factor (GDNF) using a lentiviral approach has proven successful in protecting against excitotoxicity in the hippocampus (Wong et al., 2005), thus demonstrating the usefulness of viral vectors in applying putative neuroprotective genes with or without neurogenic potential to target brain areas susceptible to ischemic injury. In considering adult neurogenesis, two key aspects of this process are regulation and function. At the present time, we know next to nothing about the situation in humans. Although animal studies have been promising thus far (Wiltrout et al., 2007), there is some evidence that adult neurogenesis in humans may be considerably different to that in rodents. Indeed, the architectural layout of the subventricular zone and rostral migratory stream, two principal locations of neurogenesis in the adult brain, are substantially different in the two cases (Rakic, 2004; Sanai et al., 2004). On the other hand, neurogenesis in the adult hippocampus has been shown to occur in humans (Eriksson et al., 1998), thus providing at least for now, a proof of principle, and the possibility that its exploration in terms of ischemic stroke may prove beneficial in the future. Despite a few clinical trials in which bone marrow cells have been infused into stroke patients, thus far, there is no clear-cut experimental evidence showing that these stem cells integrate into an ischemic area of the brain and lead to structural restoration. 13. Conclusions Effective treatment of stroke remains a major problem worldwide. While the existence of a glutamate-mediated excitotoxic process in this condition is undeniable, development of viable therapeutic strategies is a major limiting factor at the present time. Since excitotoxicity is a multifaceted event that is rapid in its onset, successful treatments will most likely require a multifactorial approach that takes this into account. Thus, attempts to rapidly increase cerebral perfusion in order to

prevent significant reperfusion injury is an important consideration, as is the ability to quickly eliminate the rise in interstitial glutamate levels in order to minimize the extent of cerebral damage. The ability of the astrocyte to respond under these circumstances will be of paramount importance to the success of such interventions. Recent increased focus on these cells and their central position in the phenomenon of excitotoxicity provides hope for future breakthroughs in the clinical management of stroke. Acknowledgements The author’s laboratory is supported by the Canadian Institutes of Health Research and the Marie Robert Head Trauma Foundation (Quebec). References Aebischer, P., Schluep, M., Deglon, N., Joseph, J.M., Hirt, L., Heyd, B., Goddard, M., Hammang, J.P., Zurn, A.D., Kato, A.C., Regli, F., Baetge, E.E., 1996. Intrathecal delivery of CNTF using encapsulated genetically modified xenogeneic cells in amyotrophic lateral sclerosis patients. Nat. Med. 2, 696–699. Agrawal, S.K., Fehlings, M.G., 1997. Role of NMDA and non-NMDA ionotropic glutamate receptors in traumatic spinal cord axonal injury. J. Neurosci. 17, 1055–1063. Agostinho, P., Duarte, C.B., Oliveira, C.R., 1997. Impairment of excitatory amino acid transporter activity by oxidative stress conditions in retinal cells: effect of antioxidants. FASEB J. 11, 154–163. Alberdi, E., Sanchez-Gomez, M.V., Marino, A., Matute, C., 2002. Ca2+ influx through AMPA or kainate receptors alone is sufficient to initiate excitotoxicity in cultured oligodendrocytes. Neurobiol. Dis. 9, 234–243. Ameriso, S.F., Sahai, S., 1997. Mechanisms of ischemia in in situ vascular occlusive disease. In: Welch, K.M.A., Caplan, L.R., Reis, D.J., Siesjo, B.K., Weir, B. (Eds.), Primer on Cerebrovascular Diseases. Academic Press, San Diego, pp. 104–107. Anderson, C.M., Swanson, R.A., 2000. Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia 32, 1–14. Andersen, M., Overgaard, K., Meden, P., Boysen, G., Choi, S.C., 1999. Effects of citicoline combined with thrombolytic therapy in a rat embolic stroke model. Stroke 30, 1464–1471. Angulo, M.C., Kozlov, A.S., Charpak, S., Audinat, E., 2004. Glutamate released from glial cells synchronizes neuronal activity in the hippocampus. J. Neurosci. 24, 6920–6927. Araque, A., Sanzgiri, R.P., Parpura, V., Haydon, P.G., 1998a. Calcium elevation in astrocytes causes an NMDA receptor-dependent increase in the frequency of miniature synaptic currents in cultured hippocampal neurons. J. Neurosci. 18, 6822–6829. Araque, A., Parpura, V., Sanzgiri, R.P., Haydon, P.G., 1998b. Glutamatedependent astrocyte modulation of synaptic transmission between cultured hippocampal neurons. Eur. J. Neurosci. 10, 2129–2142. Araque, A., Li, N., Doyle, R.T., Haydon, P.G., 2000. SNARE protein-dependent glutamate release from astrocytes. J. Neurosci. 20, 666–673. Aronowski, J., Strong, R., Grotta, J.C., 1996. Citicoline for treatment of experimental focal ischemia: histologic and behavioral outcome. Neurol. Res. 18, 570–574. Arriza, J.L., Eliasof, S., Kavanaugh, M.P., Amara, S.G., 1997. Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance. Proc. Natl. Acad. Sci. U.S.A. 94, 4155–4160. Atochin, D.N., Clark, J., Demchenko, I.T., Moskowitz, M.A., Huang, P.L., 2003. Rapid cerebral ischemic preconditioning in mice deficient in endothelial and neuronal nitric oxide synthases. Stroke 34, 1299–1303. Benchenane, K., Berezowski, V., Ali, C., Fernandez-Monreal, M., LopezAtalaya, J.P., Brillault, J., Chuquet, J., Nouvelot, A., MacKenzie, E.T., Bu,

A.S. Hazell / Neurochemistry International 50 (2007) 941–953 G., Cecchelli, R., Touzani, O., Vivien, D., 2005. Tissue-type plasminogen activator crosses the intact blood-brain barrier by low-density lipoprotein receptor-related protein-mediated transcytosis. Circulation 111, 2241– 2249. Benveniste, H., Drejer, J., Schousboe, A., Diemer, N.H., 1984. Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J. Neurochem. 43, 1369–1374. Bezzi, P., Carmignoto, G., Pasti, L., Vesce, S., Rossi, D., Rizzini, B.L., Pozzan, T., Volterra, A., 1998. Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature 391, 281–285. Bezzi, P., Gundersen, V., Galbete, J.L., Seifert, G., Steinhauser, C., Pilati, E., Volterra, A., 2004. Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate. Nat. Neurosci. 7, 613–620. Bruno, V., Copani, A., Battaglia, G., Raffaele, R., Shinozaki, H., Nicoletti, F., 1994. Protective effect of the metabotropic glutamate receptor agonist, DCG-IV, against excitotoxic neuronal death. Eur. J. Pharmacol. 256, 109– 112. Busch, E., Gyngell, M.L., Eis, M., Hoehn-Berlage, M., Hossmann, K.A., 1996. Potassium-induced cortical spreading depressions during focal cerebral ischemia in rats: contribution to lesion growth assessed by diffusionweighted NMR and biochemical imaging. J. Cereb. Blood Flow Metab. 16, 1090–1099. Callaway, J.K., Castillo-Melendez, M., Giardina, S.F., Krstew, E.K., Beart, P.M., Jarrott, B., 2004. Sodium channel blocking activity of AM-36 and sipatrigine (BW619C89): in vitro and in vivo evidence. Neuropharmacology 47, 146–155. Castillo, J., Davalos, A., Noya, M., 1997. Progression of ischaemic stroke and excitotoxic aminoacids. Lancet 349, 79–83. Chen, J., Graham, S.H., Zhu, R.L., Simon, R.P., 1996. Stress proteins and tolerance to focal cerebral ischemia. J. Cereb. Blood Flow Metab. 16, 566– 577. Chen, W., Aoki, C., Mahadomrongkul, V., Gruber, C.E., Wang, G.J., Blitzblau, R., Irwin, N., Rosenberg, P.A., 2002. Expression of a variant form of the glutamate transporter GLT1 in neuronal cultures and in neurons and astrocytes in the rat brain. J. Neurosci. 22, 2142–2152. Choi, D.W., 1987. Ionic dependence of glutamate neurotoxicity. J. Neurosci. 7, 369–379. Chu, K., Lee, S.T., Sinn, D.I., Ko, S.Y., Kim, E.H., Kim, J.M., Kim, S.J., Park, D.K., Jung, K.H., Song, E.C., Lee, S.K., Kim, M., Roh, J.K., 2007. Pharmacological induction of ischemic tolerance by glutamate transporter-1 (EAAT2) upregulation. Stroke 38, 177–182. Clark, W.M., Williams, B.J., Selzer, K.A., Zweifler, R.M., Sabounjian, L.A., Gammans, R.E., 1999. A randomized efficacy trial of citicoline in patients with acute ischemic stroke. Stroke 30, 2592–2597. Clark, W.M., Wechsler, L.R., Sabounjian, L.A., Schwiderski, U.E., 2001. A phase III randomized efficacy trial of 2000 mg citicoline in acute ischemic stroke patients. Neurology 57, 1595–1602. Da´valos, A., Castillo, J., Alvarez-Sabin, J., Secades, J.J., Mercadal, J., Lopez, S., Cobo, E., Warach, S., Sherman, D., Clark, W.M., Lozano, R., 2002. Oral citicoline in acute ischemic stroke: an individual patient data pooling analysis of clinical trials. Stroke 33, 2850–2857. Danbolt, N.C., 2001. Glutamate uptake. Prog. Neurobiol. 65, 1–105. Danbolt, N.C., Storm-Mathisen, J., Kanner, B.I., 1992. An [Na+ + K+]coupled L-glutamate transporter purified from rat brain is located in glial cell processes. Neuroscience 51, 295–310. DeFranco, D.B., Ho, L., Falke, E., Callaway, C.W., 2004. Small molecule activators of the heat shock response and neuroprotection from stroke. Curr. Atheroscler. Rep. 6, 295–300. Demougeot, C., Bobillier-Chaumont, S., Mossiat, C., Marie, C., Berthelot, A., 2004. Effect of diets with different magnesium content in ischemic stroke rats. Neurosci. Lett. 362, 17–20. Dennery, P.A., Visner, G., Weng, Y.H., Nguyen, X., Lu, F., Zander, D., Yang, G., 2003. Resistance to hyperoxia with heme oxygenase-1 disruption: role of iron. Free Radic. Biol. Med. 34, 124–133. Deplanque, D., Gele, P., Petrault, O., Six, I., Furman, C., Bouly, M., Nion, S., Dupuis, B., Leys, D., Fruchart, J.C., Cecchelli, R., Staels, B., Duriez, P., Bordet, R., 2003. Peroxisome proliferator-activated receptor-alpha activation

949

as a mechanism of preventive neuroprotection induced by chronic fenofibrate treatment. J. Neurosci. 23, 6264–6271. Drejer, J., Larsson, O.M., Schousboe, A., 1983. Characterization of uptake and release processes for D- and L-aspartate in primary cultures of astrocytes and cerebellar granule cells. Neurochem. Res. 8, 231–243. Dronne, M.A., Grenier, E., Dumont, T., Hommel, M., Boissel, J.P., 2007. Role of astrocytes in grey matter during stroke: a modelling approach. Brain Res. 1138, 231–242. Eastwood, S.L., Harrison, P.J., 2005. Decreased expression of vesicular glutamate transporter 1 and complexin II mRNAs in schizophrenia: further evidence for a synaptic pathology affecting glutamate neurons. Schizophr. Res. 73, 159–172. Ehrenreich, H., Hasselblatt, M., Dembowski, C., Cepek, L., Lewczuk, P., Stiefel, M., Rustenbeck, H.H., Breiter, N., Jacob, S., Knerlich, F., Bohn, M., Poser, W., Ruther, E., Kochen, M., Gefeller, O., Gleiter, C., Wessel, T.C., De Ryck, M., Itri, L., Prange, H., Cerami, A., Brines, M., Siren, A.L., 2002. Erythropoietin therapy for acute stroke is both safe and beneficial. Mol. Med. 8, 495–505. Eriksson, P.S., Perfilieva, E., Bjork-Eriksson, T., Alborn, A.M., Nordborg, C., Peterson, D.A., Gage, F.H., 1998. Neurogenesis in the adult human hippocampus. Nat. Med. 4, 1313–1317. Fairman, W.A., Vandenberg, R.J., Arriza, J.L., Kavanaugh, M.P., Amara, S.G., 1995. An excitatory amino-acid transporter with properties of a ligandgated chloride channel. Nature 375, 599–603. Fern, R., Ransom, B.R., Waxman, S.G., 1995. Voltage-gated calcium channels in CNS white matter: role in anoxic injury. J. Neurophysiol. 74, 369–377. Fernandez-Monreal, M., Lopez-Atalaya, J.P., Benchenane, K., Cacquevel, M., Dulin, F., Le Caer, J.P., Rossier, J., Jarrige, A.C., Mackenzie, E.T., Colloc’h, N., Ali, C., Vivien, D., 2004. Arginine 260 of the amino-terminal domain of NR1 subunit is critical for tissue-type plasminogen activator-mediated enhancement of N-methyl-D-aspartate receptor signaling. J. Biol. Chem. 279, 50850–50856. Folbergrova´, J., Memezawa, H., Smith, M.L., Siesjo, B.K., 1992. Focal and perifocal changes in tissue energy state during middle cerebral artery occlusion in normo- and hyperglycemic rats. J. Cereb. Blood Flow Metab. 12, 25–33. Gallo, V., Wright, P., McKinnon, R.D., 1994. Expression and regulation of a glutamate receptor subunit by bFGF in oligodendrocyte progenitors. Glia 10, 149–153. Gardner-Medwin, A.R., Coles, J.A., Tsacopoulos, M., 1981. Clearance of extracellular potassium: evidence for spatial buffering by glial cells in the retina of the drone. Brain Res. 209, 452–457. Gegelashvili, G., Dehnes, Y., Danbolt, N.C., Schousboe, A., 2000. The highaffinity glutamate transporters GLT1, GLAST, and EAAT4 are regulated via different signalling mechanisms. Neurochem. Int. 37, 163–170. Giffard, R.G., Monyer, H., Choi, D.W., 1990. Selective vulnerability of cultured cortical glia to injury by extracellular acidosis. Brain Res. 530, 138–141. Glaum, S.R., Miller, R.J., 1994. Inhibition of phosphoprotein phosphatases blocks metabotropic glutamate receptor effects in the rat nucleus tractus solitarii. Mol. Pharmacol. 45, 1221–1226. Goldberg, M.P., 2002. Stroke Trials Directory. Internet Stroke Center. Web site, URL: www.strokecenter.org/trials. Gottlieb, M., Wang, Y., Teichberg, V.I., 2003. Blood-mediated scavenging of cerebrospinal fluid glutamate. J. Neurochem. 87, 119–126. Grieb, P., Gadamski, R., Wojda, R., Janisz, M., 2001. CDP-choline, but not cytidine, protects hippocampal CA1 neurones in the gerbil following transient forebrain ischaemia. Folia Neuropathol. 39, 141–145. Griffin, J.H., Fernandez, J.A., Liu, D., Cheng, T., Guo, H., Zlokovic, B.V., 2004. Activated protein C and ischemic stroke. Crit. Care Med. 32 (5 Suppl), S247–S253. Hansson, E., Johansson, B.B., Westergren, I., Ronnback, L., 1994. Glutamateinduced swelling of single astroglial cells in primary culture. Neuroscience 63, 1057–1066. Harrison, P.J., Eastwood, S.L., 1998. Preferential involvement of excitatory neurons in medial temporal lobe in schizophrenia. Lancet 352, 1669–1673. Hazell, A.S., Wang, C., 2005. Downregulation of complexin I and complexin II in the medial thalamus is blocked by N-acetylcysteine in experimental Wernicke’s encephalopathy. J. Neurosci. Res. 79, 200–207.

950

A.S. Hazell / Neurochemistry International 50 (2007) 941–953

Hazell, A.S., Pannunzio, P., Rama Rao, K.V., Pow, D.V., Rambaldi, A., 2003. Thiamine deficiency results in downregulation of the GLAST glutamate transporter in cultured astrocytes. Glia 43, 175–184. Hertz, L., 1965. Possible role of neuroglia: a potassium-mediated neuronalneuroglial-neuronal impulse transmission system. Nature 206, 1091–1094. Hollmann, M., Hartley, M., Heinemann, S., 1991. Ca2+ permeability of KAAMPA-gated glutamate receptor channels depends on subunit composition. Science 252, 851–853. Hu, K., Carroll, J., Rickman, C., Davletov, B., 2002. Action of complexin on SNARE complex. J. Biol. Chem. 277, 41652–41656. Hurtado, O., Moro, M.A., Cardenas, A., Sanchez, V., Fernandez-Tome, P., Leza, J.C., Lorenzo, P., Secades, J.J., Lozano, R., Davalos, A., Castillo, J., Lizasoain, I., 2005. Neuroprotection afforded by prior citicoline administration in experimental brain ischemia: effects on glutamate transport. Neurobiol. Dis. 18, 336–345. Ikonomidou, C., Turski, L., 2002. Why did NMDA receptor antagonists fail clinical trials for stroke and traumatic brain injury? Lancet Neurol. 1, 383– 386. Inage, Y.W., Itoh, M., Wada, K., Hoshika, A., Takashima, S., 2000. Glutamate transporters in neonatal cerebellar subarachnoid hemorrhage. Pediatr. Neurol. 23, 42–48. Iwashita, A., Muramatsu, Y., Yamazaki, T., Muramoto, M., Kita, Y., Yamazaki, S., Mihara, K., Moriguchi, A., Matsuoka, N., 2007. Neuroprotective efficacy of the peroxisome proliferator-activated receptor delta-selective agonists in vitro and in vivo. J. Pharmacol. Exp. Ther. 320, 1087–1096. Jatana, M., Singh, I., Singh, A.K., Jenkins, D., 2006. Combination of systemic hypothermia and N-acetylcysteine attenuates hypoxic-ischemic brain injury in neonatal rats. Pediatr. Res. 59, 684–689. Juurlink, B.H., Paterson, P.G., 1998. Review of oxidative stress in brain and spinal cord injury: suggestions for pharmacological and nutritional management strategies. J. Spinal Cord Med. 21, 309–334. Kakihana, M., Fukuda, N., Suno, M., Nagaoka, A., 1988. Effects of CDPcholine on neurologic deficits and cerebral glucose metabolism in a rat model of cerebral ischemia. Stroke 19, 217–222. Karwoski, C.J., Lu, H.K., Newman, E.A., 1989. Spatial buffering of lightevoked potassium increases by retinal Muller (glial) cells. Science 244, 578–580. Kirino, T., 2002. Ischemic tolerance. J. Cereb. Blood Flow Metab. 22, 1283– 1296. Kanai, Y., Hediger, M.A., 1992. Primary structure and functional characterization of a high-affinity glutamate transporter. Nature 360, 467–471. Kawahara, K., Kosugi, T., Tanaka, M., Nakajima, T., Yamada, T., 2005. Reversed operation of glutamate transporter GLT-1 is crucial to the development of preconditioning-induced ischemic tolerance of neurons in neuron/astrocyte co-cultures. Glia 49, 349–359. Kimelberg, H.K., 1992. Astrocytic edema in CNS trauma. J. Neurotrauma 1 (Suppl.), S71–S81. Kimelberg, H.K., O’Connor, E., 1988. Swelling of astrocytes causes membrane potential depolarization. Glia 1, 219–224. Kitagawa, K., Matsumoto, M., Tagaya, M., Hata, R., Ueda, H., Niinobe, M., Handa, N., Fukunaga, R., Kimura, K., Mikoshiba, K., Kamada, T., 1990. ‘Ischemic tolerance’ phenomenon found in the brain. Brain Res. 528, 21–24. Knuckey, N.W., Palm, D., Primiano, M., Epstein, M.H., Johanson, C.E., 1995. N-Acetylcysteine enhances hippocampal neuronal survival after transient forebrain ischemia in rats. Stroke 26, 305–310. Kosugi, T., Kawahara, K., 2006. Reversed astrocytic GLT-1 during ischemia is crucial to excitotoxic death of neurons, but contributes to the survival of astrocytes themselves. Neurochem. Res. 31, 933–943. Kosugi, T., Kawahara, K., Yamada, T., Nakajima, T., Tanaka, M., 2005. Functional significance of the preconditioning-induced down-regulation of glutamate transporter GLT-1 in neuron/astrocyte co-cultures. Neurochem. Res. 30, 1109–1116. Kuffler, S.W., Nicholls, J.G., Orkand, R.K., 1966. Physiological properties of glial cells in the central nervous system of amphibia. J. Neurophysiol. 29, 768–787. Lebeurrier, N., Vivien, D., Ali, C., 2004. The complexity of tissue-type plasminogen activator: can serine protease inhibitors help in stroke management? Expert Opin. Ther. Targets 8, 309–320.

Lebeurrier, N., Liot, G., Lopez-Atalaya, J.P., Orset, C., Fernandez-Monreal, M., Sonderegger, P., Ali, C., Vivien, D., 2005. The brain-specific tissuetype plasminogen activator inhibitor, neuroserpin, protects neurons against excitotoxicity both in vitro and in vivo. Mol. Cell. Neurosci. 30, 552–558. Lees, G.J., 1991. Inhibition of sodium–potassium–ATPase: a potentially ubiquitous mechanism contributing to central nervous system neuropathology. Brain Res. Rev. 16, 283–300. Lehre, K.P., Levy, L.M., Ottersen, O.P., Storm-Mathisen, J., Danbolt, N.C., 1995. Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J. Neurosci. 15, 1835–1853. Leist, M., Ghezzi, P., Grasso, G., Bianchi, R., Villa, P., Fratelli, M., Savino, C., Bianchi, M., Nielsen, J., Gerwien, J., Kallunki, P., Larsen, A.K., Helboe, L., Christensen, S., Pedersen, L.O., Nielsen, M., Torup, L., Sager, T., Sfacteria, A., Erbayraktar, S., Erbayraktar, Z., Gokmen, N., Yilmaz, O., Cerami-Hand, C., Xie, Q.W., Coleman, T., Cerami, A., Brines, M., 2004. Derivatives of erythropoietin that are tissue protective but not erythropoietic. Science 305, 239–242. Levy, L.M., Warr, O., Attwell, D., 1998. Stoichiometry of the glial glutamate transporter GLT-1 expressed inducibly in a Chinese hamster ovary cell line selected for low endogenous Na+-dependent glutamate uptake. J. Neurosci. 18, 9620–9628. Li, S., Stys, P.K., 2000. Mechanisms of ionotropic glutamate receptor-mediated excitotoxicity in isolated spinal cord white matter. J. Neurosci. 20, 1190– 1198. Li, S., Mealing, G.A., Morley, P., Stys, P.K., 1999. Novel injury mechanism in anoxia and trauma of spinal cord white matter: glutamate release via reverse Na+-dependent glutamate transport. J. Neurosci. 19, RC16. Lipski, J., Wan, C.K., Bai, J.Z., Pi, R., Li, D., Donnelly, D., 2007. Neuroprotective potential of ceftriaxone in in vitro models of stroke. Neuroscience [Epub ahead of print]. Liu, H.Q., Li, W.B., Li, Q.J., Zhang, M., Sun, X.C., Feng, R.F., Xian, X.H., Li, S.Q., Qi, J., Zhao, H.G., 2006. Nitric oxide participates in the induction of brain ischemic tolerance via activating ERK1/2 signaling pathways. Neurochem. Res. 31, 967–974. Longuemare, M.C., Swanson, R.A., 1995. Excitatory amino acid release from astrocytes during energy failure by reversal of sodium-dependent uptake. J. Neurosci. Res. 40, 379–386. Luo, Y., Yin, W., Signore, A.P., Zhang, F., Hong, Z., Wang, S., Graham, S.H., Chen, J., 2006. Neuroprotection against focal ischemic brain injury by the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone. J. Neurochem. 97, 435–448. Mallolas, J., Hurtado, O., Castellanos, M., Blanco, M., Sobrino, T., Serena, J., Vivancos, J., Castillo, J., Lizasoain, I., Moro, M.A., Davalos, A., 2006. A polymorphism in the EAAT2 promoter is associated with higher glutamate concentrations and higher frequency of progressing stroke. J. Exp. Med. 203, 711–717. Manley, G.T., Fujimura, M., Ma, T., et al., 2000. Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke. Nat. Med. 6, 159–163. Manoonkitiwongsa, P.S., Schultz, R.L., McCreery, D.B., Whitter, E.F., Lyden, P.D., 2004. Neuroprotection of ischemic brain by vascular endothelial growth factor is critically dependent on proper dosage and may be compromised by angiogenesis. J. Cereb. Blood Flow Metab. 24, 693–702. Maragakis, N.J., Rothstein, J.D., 2006. Mechanisms of disease: astrocytes in neurodegenerative disease. Nat. Clin. Pract. Neurol. 2, 679–689. Matute, C., Sanchez-Gomez, M.V., Martinez-Millan, L., Miledi, R., 1997. Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes. Proc. Natl. Acad. Sci. U.S.A. 94, 8830–8835. McDonald, J.W., Althomsons, S.P., Hyrc, K.L., Choi, D.W., Goldberg, M.P., 1998. Oligodendrocytes from forebrain are highly vulnerable to AMPA/ kainate receptor-mediated excitotoxicity. Nat. Med. 4, 291–297. McMahon, H.T., Missler, M., Li, C., Sudhof, T.C., 1995. Complexins: cytosolic proteins that regulate SNAP receptor function. Cell 83, 111–119. Mies, G., Iijima, T., Hossmann, K.A., 1993. Correlation between peri-infarct DC shifts and ischaemic neuronal damage in rat. Neuroreport 4, 709–711.

A.S. Hazell / Neurochemistry International 50 (2007) 941–953 Miyamoto, M., Ishida, M., Shinozaki, H., 1994. Protective effect of the metabotropic glutamate receptor agonist, DCG-IV, against excitotoxic neuronal death. Eur. J. Pharmacol. 256, 109–112. Montana, V., Ni, Y., Sunjara, V., Hua, X., Parpura, V., 2004. Vesicular glutamate transporter-dependent glutamate release from astrocytes. J. Neurosci. 24, 2633–2642. Montana, V., Malarkey, E.B., Verderio, C., Matteoli, M., Parpura, V., 2006. Vesicular transmitter release from astrocytes. Glia 54, 700–715. Muir, K.W., Lees, K.R., 2003. Excitatory amino acid antagonists for acute stroke. Cochrane Database Syst. Rev. (3), CD001244. Muir, K.W., Lees, K.R., Ford, I., Davis, S., 2004. Intravenous Magnesium Efficacy in Stroke (IMAGES) Study Investigators. Magnesium for acute stroke (Intravenous Magnesium Efficacy in Stroke trial): randomised controlled trial. Lancet 363, 439–445. Nagai, N., De Mol, M., Lijnen, H.R., Carmeliet, P., Collen, D., 1999. Role of plasminogen system components in focal cerebral ischemic infarction: a gene targeting and gene transfer study in mice. Circulation 99, 2440–2444. Nakatomi, H., Kuriu, T., Okabe, S., Yamamoto, S., Hatano, O., Kawahara, N., Tamura, A., Kirino, T., Nakafuku, M., 2002. Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell 110, 429–441. Namura, S., Maeno, H., Takami, S., Jiang, X.F., Kamichi, S., Wada, K., Nagata, I., 2002. Inhibition of glial glutamate transporter GLT-1 augments brain edema after transient focal cerebral ischemia in mice. Neurosci. Lett. 324, 117–120. Newman, E.A., 2003. New roles for astrocytes: regulation of synaptic transmission. Trends Neurosci. 26, 536–542. Nicholls, D., Attwell, D., 1990. The release and uptake of excitatory amino acids. Trends Pharmacol. Sci. 11, 462–468. Nedergaard, M., Hansen, A.J., 1993. Characterization of cortical depolarizations evoked in focal cerebral ischemia. J. Cereb. Blood Flow Metab. 13, 568–574. Nishino, K., Nowak Jr., T.S., 2004. Time course and cellular distribution of hsp27 and hsp72 stress protein expression in a quantitative gerbil model of ischemic injury and tolerance: thresholds for hsp72 induction and hilar lesioning in the context of ischemic preconditioning. J. Cereb. Blood Flow Metab. 24, 167–178. Norenberg, M.D., Martinez-Hernandez, A., 1979. Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res. 161, 303–310. Obrenovitch, T.P., Urenjak, J., Zilkha, E., 1994. Intracerebral microdialysis combined with recording of extracellular field potential: a novel method for investigation of depolarizing drugs in vivo. Br. J. Pharmacol. 113, 1295– 1302. Oh, T.E., Shenfield, G.M., 1980. Intravenous N-acetylcysteine for paracetamol poisoning. Med. J. Aust. 1, 664–665. Onal, M.Z., Li, F., Tatlisumak, T., Locke, K.W., Sandage Jr., B.W., Fisher, M., 1997. Synergistic effects of citicoline and MK-801 in temporary experimental focal ischemia in rats. Stroke 28, 1060–1065. Ono, S., Baux, G., Sekiguchi, M., Fossier, P., Morel, N.F., Nihonmatsu, I., Hirata, K., Awaji, T., Takahashi, S., Takahashi, M., 1998. Regulatory roles of complexins in neurotransmitter release from mature presynaptic nerve terminals. Eur. J. Neurosci. 10, 2143–2152. Ostrow, J.D., Pascolo, L., Shapiro, S.M., Tiribelli, C., 2003. New concepts in bilirubin encephalopathy. Eur. J. Clin. Invest. 33, 988–997. Pabst, S., Hazzard, J.W., Antonin, W., Sudhof, T.C., Jahn, R., Rizo, J., Fasshauer, D., 2000. Selective interaction of complexin with the neuronal SNARE complex. Determination of the binding regions. J. Biol. Chem. 275, 19808–19818. Pabst, S., Margittai, M., Vainius, D., Langen, R., Jahn, R., Fasshauer, D., 2002. Rapid and selective binding to the synaptic SNARE complex suggests a modulatory role of complexins in neuroexocytosis. J. Biol. Chem. 277, 7838–7848. Paoli, M., Marles-Wright, J., Smith, A., 2002. Structure–function relationships in heme-proteins. DNA Cell Biol. 21, 271–280. Park, S.W., Yi, J.H., Miranpuri, G., Satriotomo, I., Bowen, K., Resnick, D.K., Vemuganti, R., 2007. Thiazolidinedione class of peroxisome proliferatoractivated receptor gamma agonists prevents neuronal damage, motor dysfunction, myelin loss, neuropathic pain, and inflammation after spinal cord injury in adult rats. J. Pharmacol. Exp. Ther. 320, 1002–1012.

951

Parpura, V., Basarsky, T.A., Liu, F., Jeftinija, K., Jeftinija, S., Haydon, P.G., 1994. Nature 369, 744–747. Petralia, R.S., Wang, Y.X., Niedzielski, A.S., Wenthold, R.J., 1996. The metabotropic glutamate receptors, mGluR2 and mGluR3, show unique postsynaptic, presynaptic and glial localizations. Neuroscience 71, 949–976. Phillis, J.W., Smith-Barbour, M., Perkins, L.M., O’Regan, M.H., 1994. Characterization of glutamate, aspartate, and GABA release from ischemic rat cerebral cortex. Brain Res. Bull. 34, 457–466. Pines, G., Danbolt, N.C., Bjoras, M., Zhang, Y., Bendahan, A., Eide, L., Koepsell, H., Storm-Mathisen, J., Seeberg, E., Kanner, B.I., 1992. Cloning and expression of a rat brain L-glutamate transporter. Nature 360, 464–467. Pulsinelli, W.A., 1997. Selective neuronal vulnerability and infarction in cerebrovascular disease. In: Welch, K.M.A., Caplan, L.R., Reis, D.J., Siesjo, B.K., Weir, B. (Eds.), Primer on Cerebrovascular Diseases. Academic Press, San Diego, pp. 104–107. Rakic, P., 2004. Neuroscience: immigration denied. Nature 427, 685–686. Rao, V.L., Bowen, K.K., Dempsey, R.J., 2001. Transient focal cerebral ischemia down-regulates glutamate transporters GLT-1 and EAAC1 expression in rat brain. Neurochem. Res. 26, 497–502. Ratan, R.R., Murphy, T.H., Baraban, J.M., 1994. Macromolecular synthesis inhibitors prevent oxidative stress-induced apoptosis in embryonic cortical neurons by shunting cysteine from protein synthesis to glutathione. J. Neurosci. 14, 4385–4392. Reim, K., Mansour, M., Varoqueaux, F., McMahon, H.T., Sudhof, T.C., Brose, N., Rosenmund, C., 2001. Complexins regulate a late step in Ca2+-dependent neurotransmitter release. Cell 104, 71–81. Ribo, M., Grotta, J.C., 2006. Latest advances in intracerebral hemorrhage. Curr. Neurol. Neurosci. Rep. 6, 17–22. Romera, C., Hurtado, O., Mallolas, J., Pereira, M.P., Morales, J.R., Romera, A., Serena, J., Vivancos, J., Nombela, F., Lorenzo, P., Lizasoain, I., Moro, M.A., 2007. Ischemic preconditioning reveals that GLT1/EAAT2 glutamate transporter is a novel PPARgamma target gene involved in neuroprotection. J. Cereb. Blood Flow Metab. [Epub ahead of print]. Romera, C., Hurtado, O., Botella, S.H., Lizasoain, I., Cardenas, A., FernandezTome, P., Leza, J.C., Lorenzo, P., Moro, M.A., 2004. In vitro ischemic tolerance involves upregulation of glutamate transport partly mediated by the TACE/ADAM17-tumor necrosis factor-alpha pathway. J. Neurosci. 24, 1350–1357. Rosenberg, L.J., Teng, Y.D., Wrathall, J.R., 1999. 2,3-Dihydroxy-6-nitro-7sulfamoyl-benzo(f)quinoxaline reduces glial loss and acute white matter pathology after experimental spinal cord contusion. J. Neurosci. 19, 464–475. Rothstein, J.D., Martin, L., Levey, A.I., Dykes-Hoberg, M., Jin, L., Wu, D., Nash, N., Kuncl, R.W., 1994. Localization of neuronal and glial glutamate transporters. Neuron 13, 713–725. Rothstein, J.D., Dykes-Hoberg, M., Pardo, C.A., Bristol, L.A., Jin, L., Kuncl, R.W., Kanai, Y., Hediger, M.A., Wang, Y., Schielke, J.P., Welty, D.F., 1996. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 16, 675–686. Rothstein, J.D., Patel, S., Regan, M.R., Haenggeli, C., Huang, Y.H., Bergles, D.E., Jin, L., Dykes Hoberg, M., Vidensky, S., Chung, D.S., Toan, S.V., Bruijn, L.I., Su, Z.Z., Gupta, P., Fisher, P.B., 2005. Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter expression. Nature 433, 73–77. Sanchez-Gomez, M.V., Matute, C., 1999. AMPA and kainate receptors each mediate excitotoxicity in oligodendroglial cultures. Neurobiol. Dis. 6, 475–485. Sanai, N., Tramontin, A.D., Quinones-Hinojosa, A., Barbaro, N.M., Gupta, N., Kunwar, S., Lawton, M.T., McDermott, M.W., Parsa, A.T., Manuel-Garcia Verdugo, J., Berger, M.S., Alvarez-Buylla, A., 2004. Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks chain migration. Nature 427, 740–744. Sa¨rnstrand, B., Tunek, A., Sjodin, K., Hallberg, A., 1995. Effects of Nacetylcysteine stereoisomers on oxygen-induced lung injury in rats. Chem. Biol. Interact. 94, 157–164. Saver, J.L., Kidwell, C., Eckstein, M., Starkman, S., 2004. FAST-MAG Pilot Trial Investigators. Prehospital neuroprotective therapy for acute stroke:

952

A.S. Hazell / Neurochemistry International 50 (2007) 941–953

results of the Field Administration of Stroke Therapy-Magnesium (FASTMAG) pilot trial. Stroke 35, e106–e108. Schabitz, W.R., Weber, J., Takano, K., Sandage, B.W., Locke, K.W., Fisher, M., 1996. The effects of prolonged treatment with citicoline in temporary experimental focal ischemia. J. Neurol. Sci. 138, 21–25. Schmitt, A., Asan, E., Lesch, K.P., Kugler, P., 2002. A splice variant of glutamate transporter GLT1/EAAT2 expressed in neurons: cloning and localization in rat nervous system. Neuroscience 109, 45–61. Schousboe, A., 1981. Transport and metabolism of glutamate and GABA in neurons are glial cells. Int. Rev. Neurobiol. 22, 1–45. Sekhon, B., Sekhon, C., Khan, M., Patel, S.J., Singh, I., Singh, A.K., 2003. NAcetyl cysteine protects against injury in a rat model of focal cerebral ischemia. Brain Res. 971, 1–8. Seki, Y., Feustel, P.J., Keller Jr., R.W., Tranmer, B.I., Kimelberg, H.K., 1999. Inhibition of ischemia-induced glutamate release in rat striatum by dihydrokinate and an anion channel blocker. Stroke 30, 433–440. Shimada, F., Shiga, Y., Morikawa, M., Kawazura, H., Morikawa, O., Matsuoka, T., Nishizaki, T., Saito, N., 1999. The neuroprotective agent MS-153 stimulates glutamate uptake. Eur. J. Pharmacol. 386, 263–270. Shuaib, A., Yang, Y., Li, Q., 2000. Evaluating the efficacy of citicoline in embolic ischemic stroke in rats: neuroprotective effects when used alone or in combination with urokinase. Exp. Neurol. 161, 733–739. Silbergleit, R., Haywood, Y., Fiskum, G., Rosenthal, R.E., 1999. Lack of a neuroprotective effect from N-acetylcysteine after cardiac arrest and resuscitation in a canine model. Resuscitation 40, 181–186. Storck, T., Schulte, S., Hofmann, K., Stoffel, W., 1992. Structure, expression, and functional analysis of a Na+-dependent glutamate/aspartate transporter from rat brain. Proc. Natl. Acad. Sci. U.S.A. 89, 10955–10959. Stys, P.K., Ransom, B.R., Waxman, S.G., Davis, P.K., 1990. Role of extracellular calcium in anoxic injury of mammalian central white matter. Proc. Natl. Acad. Sci. U.S.A. 87, 4212–4216. Sung, J.H., Shin, S.A., Park, H.K., Montelaro, R.C., Chong, Y.H., 2001. Protective effect of glutathione in HIV-1 lytic peptide 1-induced cell death in human neuronal cells. J. Neurovirol. 7, 454–465. Swanson, R.A., 1992. Astrocyte glutamate uptake during chemical hypoxia in vitro. Neurosci. Lett. 147, 143–146. Swanson, R.A., Farrell, K., Stein, B.A., 1997. Astrocyte energetics, function, and death under conditions of incomplete ischemia: a mechanism of glial death in the penumbra. Glia 21, 142–153. Szatkowski, M., Barbour, B., Attwell, D., 1990. Non-vesicular release of glutamate from glial cells by reversed electrogenic glutamate uptake. Nature 348, 443–446. Takahashi, S., Yamamoto, H., Matsuda, Z., Ogawa, M., Yagyu, K., Taniguchi, T., Miyata, T., Kaba, H., Higuchi, T., Okutani, F., Fujimoto, S., 1995. Identification of two highly homologous presynaptic proteins distinctly localized at the dendritic and somatic synapses. FEBS Lett. 368, 455–460. Takahashi, S., Shibata, M., Gotoh, J., Fukuuchi, Y., 2000. Astroglial cell death induced by excessive influx of sodium ions. Eur. J. Pharmacol. 408, 127–135. Tanaka, K., Watase, K., Manabe, T., Yamada, K., Watanabe, M., Takahashi, K., Iwama, H., Nishikawa, T., Ichihara, N., Kikuchi, T., Okuyama, S., Kawashima, N., Hori, S., Takimoto, M., Wada, K., 1997. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276, 1699–1702. Tang, J., Maximov, A., Shin, O.H., Dai, H., Rizo, J., Sudhof, T.C., 2006. A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis. Cell 126, 1175–1187. Taniguchi, M., Yamashita, T., Kumura, E., Tamatani, M., Kobayashi, A., Yokawa, T., Maruno, M., Kato, A., Ohnishi, T., Kohmura, E., Tohyama, M., Yoshimine, T., 2000. Induction of aquaporin-4 water channel mRNA after focal cerebral ischemia in rat. Mol. Brain Res. 78, 131–137. Tazaki, Y., Sakai, F., Otomo, E., Kutsuzawa, T., Kameyama, M., Omae, T., Fujishima, M., Sakuma, A., 1988. Treatment of acute cerebral infarction with a choline precursor in a multicenter double-blind placebo-controlled study. Stroke 19, 211–216. Tekko¨k, S.B., Goldberg, M.P., 2001. Ampa/kainate receptor activation mediates hypoxic oligodendrocyte death and axonal injury in cerebral white matter. J. Neurosci. 21, 4237–4248.

Tekko¨k, S.B., Ye, Z., Ransom, B.R., 2007. Excitotoxic mechanisms of ischemic injury in myelinated white matter. J Cereb. Blood Flow Metab. [Epub ahead of print]. Tomimoto, H., Takemoto, O., Akiguchi, I., Yanagihara, T., 1999. Immunoelectron microscopic study of c-Fos, c-Jun and heat shock protein after transient cerebral ischemia in gerbils. Acta Neuropathol. (Berl.) 97, 22–30. Trotti, D., Rossi, D., Gjesdal, O., Levy, L.M., Racagni, G., Danbolt, N.C., Volterra, A., 1996. Peroxynitrite inhibits glutamate transporter subtypes. J. Biol. Chem. 271, 5976–5979. Trotti, D., Danbolt, N.C., Volterra, A., 1998. Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration? Trends Pharmacol. Sci. 19, 328–334. Tsirka, S.E., Gualandris, A., Amaral, D.G., Strickland, S., 1995. Excitotoxininduced neuronal degeneration and seizure are mediated by tissue plasminogen activator. Nature 377, 340–344. Tureyen, K., Kapadia, R., Bowen, K.K., Satriotomo, I., Liang, J., Feinstein, D.L., Vemuganti, R., 2007. Peroxisome proliferator-activated receptorgamma agonists induce neuroprotection following transient focal ischemia in normotensive, normoglycemic as well as hypertensive and type-2 diabetic rodents. J. Neurochem. 101, 41–56. Tymianski, M., Charlton, M.P., Carlen, P.L., Tator, C.H., 1993. Source specificity of early calcium neurotoxicity in cultured embryonic spinal neurons. J. Neurosci. 13, 2085–2104. Umemura, K., Gemba, T., Mizuno, A., Nakashima, M., 1996. Inhibitory effect of MS-153 on elevated brain glutamate level induced by rat middle cerebral artery occlusion. Stroke 27, 1624–1628. Utsunomiya-Tate, N., Endou, H., Kanai, Y., 1997. Tissue specific variants of glutamate transporter GLT-1. FEBS Lett. 416, 312–316. Wagner, P.D., Mathieu-Costello, O., Bebout, D.E., Gray, A.T., Natterson, P.D., Glennow, C., 1989. Protection against pulmonary O2 toxicity by N-acetylcysteine. Eur. Respir. J. 2, 116–126. Wagner, K.R., Sharp, F.R., Ardizzone, T.D., Lu, A., Clark, J.F., 2003. Heme and iron metabolism: role in cerebral hemorrhage. J. Cereb. Blood Flow Metab. 23, 629–652. Walz, W., 1987. Swelling and potassium uptake in cultured astrocytes. Can. J. Physiol. Pharmacol. 65, 1051–1057. Walz, W., Hertz, L., 1982. Ouabain-sensitive and ouabain-resistant net uptake of potassium into astrocytes and neurons in primary cultures. J. Neurochem. 39, 70–77. Wang, J., Dore´, S., 2007. Heme oxygenase-1 exacerbates early brain injury after intracerebral hemorrhage. Brain 130, 1643–1652. Wang, Y.F., Tsirka, S.E., Strickland, S., Stieg, P.E., Soriano, S.G., Lipton, S.A., 1998. Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice. Nat. Med. 4, 228–231. Wang, L., Zhang, Z., Wang, Y., Zhang, R., Chopp, M., 2004. Treatment of stroke with erythropoietin enhances neurogenesis and angiogenesis and improves neurological function in rats. Stroke 35, 1732–1737. Warner, D.S., Sheng, H., Batinic-Haberle, I., 2004. Oxidants, antioxidants and the ischemic brain. J. Exp. Biol. 207, 3221–3231. Wiltrout, C., Lang, B., Yan, Y., Dempsey, R.J., Vemuganti, R., 2007. Repairing brain after stroke: a review on post-ischemic neurogenesis. Neurochem. Int. 50, 1028–1041. Wisman, L.A., van Muiswinkel, F.L., de Graan, P.N., Hol, E.M., Bar, P.R., 2003. Cells over-expressing EAAT2 protect motoneurons from excitotoxic death in vitro. Neuroreport 14, 1967–1970. Volterra, A., Trotti, D., Tromba, C., Floridi, S., Racagni, G., 1994. Glutamate uptake inhibition by oxygen free radicals in rat cortical astrocytes. J. Neurosci. 14, 2924–2932. Wolf, J.A., Stys, P.K., Lusardi, T., Meaney, D., Smith, D.H., 2001. Traumatic axonal injury induces calcium influx modulated by tetrodotoxin-sensitive sodium channels. J. Neurosci. 21, 1923–1930. Wong, L.F., Ralph, G.S., Walmsley, L.E., Bienemann, A.S., Parham, S., Kingsman, S.M., Uney, J.B., Mazarakis, N.D., 2005. Lentiviral-mediated delivery of Bcl-2 or GDNF protects against excitotoxicity in the rat hippocampus. Mol. Ther. 11, 89–95. Yamada, M., Saisu, H., Ishizuka, T., Takahashi, H., Abe, T., 1999. Immunohistochemical distribution of the two isoforms of synaphin/complexin

A.S. Hazell / Neurochemistry International 50 (2007) 941–953 involved in neurotransmitter release: localization at the distinct central nervous system regions and synaptic types. Neuroscience 93, 7–18. Yamagata, K., Ichinose, S., Tagami, M., 2004. Amlodipine and carvedilol prevent cytotoxicity in cortical neurons isolated from stroke-prone spontaneously hypertensive rats. Hypertens. Res. 27, 271–282. Yeh, T.H., Hwang, H.M., Chen, J.J., Wu, T., Li, A.H., Wang, H.L., 2005. Glutamate transporter function of rat hippocampal astrocytes is impaired following the global ischemia. Neurobiol. Dis. 18, 476–483. Yepes, M., Sandkvist, M., Wong, M.K., Coleman, T.A., Smith, E., Cohan, S.L., Lawrence, D.A., 2000. Neuroserpin reduces cerebral infarct volume and protects neurons from ischemia-induced apoptosis. Blood 96, 569–576. Yi, J.-H., Pow, D.V., Hazell, A.S., 2005. Early loss of the glutamate transporter splice-variant GLT-1v in rat cerebral cortex following lateral fluid-percussion injury. Glia 49, 121–133. Yi, J.-H., Hoover, R., McIntosh, T.K., Hazell, A.S., 2006. Early, transient increase in complexin I and complexin II in the cerebral cortex following traumatic brain injury is attenuated by N-acetylcysteine. J. Neurotrauma 23, 86–96.

953

Yi, J-H., Herrero, R., Chen, G., Hazell, A.S., 2007. Glutamate transporter EAAT4 is increased in hippocampal astrocytes following lateral fluidpercussion injury in the rat. Brain Res. April 10; [Epub ahead of print]. Zhang, Q., Pangrsic, T., Kreft, M., Krzan, M., Li, N., Sul, J.Y., Halassa, M., Van Bockstaele, E., Zorec, R., Haydon, P.G., 2004. Fusion-related release of glutamate from astrocytes. J. Biol. Chem. 279, 12724–12733. Zhang, M., Li, W.B., Geng, J.X., Li, Q.J., Sun, X.C., Xian, X.H., Qi, J., Li, S.Q., 2007. The upregulation of glial glutamate transporter-1 participates in the induction of brain ischemic tolerance in rats. J. Cereb. Blood Flow Metab. [Epub ahead of print]. Zlotnik, A., Gurevich, B., Tkachov, S., Maoz, I., Shapira, Y., Teichberg, V.I., 2007. Brain neuroprotection by scavenging blood glutamate. Exp. Neurol. 203, 213–220. Zwienenberg, M., Gong, Q.Z., Berman, R.F., Muizelaar, J.P., Lyeth, B.G., 2001. The effect of groups II and III metabotropic glutamate receptor activation on neuronal injury in a rodent model of traumatic brain injury. Neurosurgery 48, 1119–2112.