Why have Ionotropic and Metabotropic Glutamate
Antagonists Failed in Stroke Therapy?
Gianfranco Di Renzo, Giuseppe Pignataro, and Lucio Annunziato
1 Introduction
The concept of ‘‘excitotoxicity’’ was introduced in 1969 when Olney and Sharpe
first demonstrated that neurons exposed to their own neurotransmitter glutamate were destined to die [49]. Later on, in 1985, glutamate toxicity was
associated with anoxic cell death, since anoxic depolarization resulted in the
release of glutamate into the extracellular compartments [42, 54]. Similarly, in
1987, Choi indicated that glutamate was a remarkably potent and rapidly acting
neurotoxin able to mediate neurotoxic effects by inducing Ca2þ influx through
glutamate receptor activation, and he supported the theory that glutamate can
be considered a key neurotransmitter in developing many neurological diseases
[13, 14, 15]. Since then, glutamate receptors have been the most studied channels
involved in ischemic stroke pathophysiology. Glutamate can exert its effects by
interacting with both ionotropic glutamate receptors (iGluRs), also referred to
as ligand-gated ion channels, and metabotropic glutamate G-protein-coupled
receptors. The group of iGluRs comprises three major classes, the a-amino-3hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, kainate (KA)
receptors, and N-methyl-D-aspartate (NMDA) receptors, named according
to their selective agonists [62]. Glutamatergic synapses frequently harbor both
AMPA and NMDA receptors. Characteristically, both classes of receptors
differ in their response kinetics upon presynaptic glutamate release. Indeed,
AMPA receptors mediate fast glutamate-gated postsynaptic responses, even at
very negative potentials or in the absence of action potentials. The fast desensitization of AMPA receptors leads to short excitatory postsynaptic currents
(EPSCs). In contrast, NMDA receptors contain an agonist-binding site, i.e.,
a glycine modulatory site, and other binding sites within the ion channel, where
magnesium exerts a voltage-dependent block [21]. Acting as detectors of
G. Di Renzo (*)
Division Pharmacology, Department of Neuroscience, School of Medicine, ‘‘Federico II’’
University of Naples, Via Pansini 5, 80131, Naples, Italy
e-mail: [email protected]
L. Annunziato (ed.), New Strategies in Stroke Intervention,
Contemporary Neuroscience, DOI 10.1007/978-1-60761-280-3_2,
Ó Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009
13
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membrane depolarization and ligand-gated channel activation, NMDA
receptors require the removal of the Mg2þ block by an increase in membrane
potential to allow cation permeation through the receptor pore [62].
The excitotoxicity theory encountered a great favor for almost 40 years,
because of a discrete success of glutamate receptors antagonists in treating
experimentally induced stroke. Unfortunately, however, over the last two decades, all stroke clinical trials with agents acting on these receptors have been
disappointing [16, 17, 45, 46, 48]. The disappointing experience derived from the
use of pharmacological agents interfering with glutamate-mediated mechanisms in stroke pathophysiology has been paid by many scientists working in the
field with the reluctance of government and private associations to grant
projects on the development of new drugs to treat stroke. Thus, the discouraging results have redirected scientists’ attention to other channels and membrane transporters able to control neuronal ionic homeostasis.
The purpose of this chapter is to review some of the evidence in the field in an
attempt to explain why drugs acting on glutamate receptors have resulted
ineffective in treating stroke.
2 Glutamate and Stroke
It is widely accepted that a critical factor in determining neuronal and glial
death during cerebral ischemia is the progressive accumulation of intracellular
Naþ([Naþ]i) and Ca2þ([Ca2þ]i) ions, which can precipitate necrosis and apoptosis of vulnerable neurons. Whereas the detrimental action of [Naþ]i increase is
attributable to both cell swelling and microtubular disorganization – two
phenomena that lead to cell necrosis [59] – a rise in [Ca2þ]i has been shown to
be a key factor in ischemic brain damage, for it modulates several death pathways, including oxidative and nitrosative stress, mitochondrial dysfunction,
and protease activation. Since Olney’s seminal work first suggested that excitatory amino acids could elicit neurotoxicity [49], a vast amount of data have
demonstrated that glutamate extracellular concentrations briskly rise during
acute brain injury, thus triggering an influx of Ca2þ and Naþ ions into neurons
through glutamate receptors [13]. This evidence has led to the elaboration of the
paradigm of glutamate excitotoxicity, a theory that explained ischemic neuronal cell death as a mere consequence of Naþ and Ca2þ influx through glutamate
receptors [13]. Although this paradigm has been guiding basic research in the
field of neurodegeneration for almost three decades, more recently it has
become the object of serious criticism and re-assessment. What has aroused
such skepticism among researchers has been the fact that although first-,
second-, and third-generation glutamate receptor antagonists have long yielded
promising results in animal models of brain ischemia, they have failed to elicit a
neuroprotective action in stroke and traumatic brain injury in humans. Therefore, the theory of excitotoxicity, though a fascinating paradigm, can only
Failure of Ionotropic and Metabotropic Glutamate Antagonists
15
explain some of the events occurring in the acute phase of anoxic insult but
cannot be seen as a major target for developing new therapeutic avenues for
brain ischemia. In fact, the energy failure occurring during the course of a
stroke episode triggers many events that are strictly dependent on several
factors:
1. the type of ischemic event, i.e., focal vs global ischemia
2. the region of the central nervous system (CNS) involved in the mechanism of
ischemic damage
3. the distance from the area primarily affected by the ischemic event, i.e.,
ischemic core vs ischemic penumbra
4. the duration of the ischemic event, and
5. the time elapsing between the onset of the ischemic event and the evaluation
of the severity of the insult.
Furthermore, the fact that after stroke onset Ca2þ concentrations follow a
triphasic response that is not equally sensitive to NMDA receptor antagonists
[52, 66] should be taken into account. In fact, during exposure to excitatory
neurotransmitters, intracellular calcium increase is followed by a transient
return to basal levels. After a free interval of a few hours, a gradually progressing secondary increase occurs. The initial Ca2þ influx can be blocked by
NMDA receptor antagonists or by the removal of Ca2þ from the extracellular
medium but not by antagonists of non-NMDA receptors [52, 66]. The second
delayed Ca2þ increase can be counteracted by extracellular Ca2þ removal but
not by the application of an NMDA or a non-NMDA receptor antagonist.
Interestingly, this second Ca2þ increase can be accelerated by increasing extracellular Ca2þ or by blocking the plasma membrane of Naþ/Ca2þ exchanger [2,
66]. The changes in [Ca2þ]i probably produce other leak currents that result in
irreversible disturbances in ion homeostasis and, eventually, in cell death.
Although the dominant mediator of the delayed injury has not yet been established, it is likely that a complex chain of events, including the activation of the
Ca2þ-dependent catabolic process, the early damage by Naþ overload that
induces oncotic cell swelling and ionic edema, and the dysregulation of the
ionic homeostasis of other ions, such as zinc and magnesium, may act to destroy
cellular integrity. This indicates that not only glutamate antagonists but also a
great variety of other drugs that interfere with the other processes might
improve neuronal survival after ischemic damage.
In addition, when the features of global and focal ischemia are analyzed in
association with glutamate levels, several substantial findings challenge the
excitotoxic hypothesis of ischemic injury.
First, global ischemia induces a damage that is confined to well-defined
vulnerable areas although glutamate is released in the same amount throughout
the brain [6, 57]. Second, ischemic damage after global ischemia is delayed for
days whereas glutamate levels increase immediately after the onset of the
ischemic event. Third, since neuronal death in the core region is mainly due to
energy depletion, glutamate toxicity should be considered important only for
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G. Di Renzo et al.
the damage occurring in penumbra, the peripheral region of the ischemic lesion.
However, in focal ischemia extracellular glutamate increases up to 100 times
above the basal level in the ischemic core, reaches peak values 1–2 hours later,
and declines slowly thereafter. By contrast, in the ischemic penumbra, it
increases only up to 25 times above the basal level and may return to normal
within 30 min even if blood flow does not improve [20].
Fourth, the hypothesis that the possible excitotoxicity is induced by glutamate release is also challenged by the fact that the release of excitatory amino
acids can be counteracted by other ischemia-related factors such as acidosis and
by the release of hyperpolarizing inhibitory neurotransmitters, which during
stroke are released at concentrations higher than those of glutamate [40].
Finally, microdialysis studies of glutamate levels after stroke have clearly
demonstrated that although the extracellular glutamate concentration increases
sharply and rapidly, reaching concentrations 10–100 times higher than preinsult values [7], this increase lasts only for 10–30 min [7]. Therefore, although
glutamate may be involved in the acute neurodestructive phase that occurs
immediately after ischemic injury, its normal physiological functions, including
the promotion of neuronal survival, are resumed directly after this phase [37]. In
addition, delayed treatment with NMDA antagonists suppresses neurogenesis,
triggered by focal cerebral ischemia, in the hippocampus [4]. These findings
suggest that in addition to damaging neurons immediately after the injury,
glutamate may also facilitate repair shortly thereafter. Interestingly, whereas
excitotoxic effects of glutamate are short lasting, repair mechanisms appear to
be long lasting.
The interference with neuronal survival means that NMDA antagonists are
unsuitable neuroprotective drugs for stroke therapy. The only way to provide
pharmacological neuroprotection with NMDA antagonists would be to administer them before the insult and for a very short period (even minutes) after the
injury, circumstances that would be virtually impossible in a clinical emergency
setting.
Thus, when designing novel therapies, researchers need to consider and
respect the physiological role of glutamate in the brain. By focusing on the
destructive effects of glutamate after injury and by ignoring its physiological
functions, many patients were unnecessarily exposed to glutamate NMDA
antagonists. In addition, when we consider that overlooking the potential
detrimental role of glutamate has resulted in years of long painstaking but
unpromising research, as well as in unprofitable investments from pharmaceutical companies, the need to re-evaluate the physiological role of glutamate and
thus to invest human and financial resources in this or other related lines of
research becomes apparent.
In the last few years, several seminal experimental works have markedly
changed the scenario in this field. In fact, it has been shown that some integral
plasma membrane proteins, involved in the control of Ca2þ, Naþ, Kþ, and Cl–
ion influx or efflux and, therefore, responsible for maintaining the homeostasis
of these cations and anions, might function as crucial players in the brain
Failure of Ionotropic and Metabotropic Glutamate Antagonists
17
ischemic process [1, 43, 50, 64]. Indeed, these proteins may provide the molecular basis underlying glutamate-independent ionic homeostasis dysregulation
in neuronal ischemic cell death and, most important, may represent more
suitable molecular targets for therapeutic intervention.
3 The Reasons of the Failure of Excitotoxicity Theory
In the last decade, several lines of evidence have accumulated against the
concept that high levels of extracellular glutamate are associated with neurological disorders and thus may contribute to neuronal death. The reasons for that
can be summarized in the following six ‘‘Hossmann postulates’’ [32]:
1. The type of metabolic and biochemical response induced by glutamate
exposure is considerably different from that evoked by ischemic injury. In
fact, in vitro and in vivo studies clearly demonstrated that a transient
suppression of energy metabolism, as well as changes in protein synthesis,
occurs after experimental ischemia [19]. In contrast, exposure to a high dose
of glutamate changes neither energy state nor protein synthesis rate.
Changes in protein synthesis are a consistent and probably fatal event
occurring in all kinds of ischemic cell death [31]. Preservation of protein
synthesis after glutamate exposure is, consequently, a strong indication that
glutamate-induced damage is different from ischemic-induced damage.
2. Microdialysis and other techniques, which allow to measure glutamate levels
in the brain after experimentally induced cerebral ischemia, clearly indicate
that increases in glutamate after stroke are not necessarily required for
induction of the pathological process. In fact, in models mimicking focal
cerebral ischemia, threshold determinations of glutamate release clearly
show that glutamate rises to high levels in the ischemic core but not in the
penumbra [47, 56]. However, the only reason for cell necrosis in the core is
energy depletion; therefore, although glutamate can contribute to cell death,
it cannot be considered the main player in this mechanism. Furthermore,
although the increase in extracellular glutamate is much lower in the penumbra than in the infarct core, a neurotoxic effect cannot be excluded. However, the probability of such an effect is rather small. On the other hand, in
global ischemia, the measurement of glutamate in the different brain regions
of rats subjected to 4-Vessel Occlusion (4-VO) have revealed that during
ischemia the increases in glutamate levels are almost identical to those found
in the hippocampus, striatum, cortex, and thalamus [23]. In addition, even
within the hippocampus, glutamate release is the same in all affected regions.
By contrast, global ischemia induces a region-specific damage and the different hippocampal regions show a selective vulnerability to the global
ischemic insult. The difference is even more evident when different durations
of ischemia are compared. In fact, despite the varying concentrations of
glutamate in the different regions, the pattern of vulnerability does not
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4.
5.
6.
G. Di Renzo et al.
change [24]. This hypothesis is further supported by the fact that ischemia
induces glutamate release in regions spared from histopathological damage
in the brain [24].
Since neuronal death occurring in the core region is mainly due to energy
depletion, glutamate toxicity should be important only for the damage
occurring in penumbra. If so, it is possible to hypothesize that glutamate
neurotoxicity in vivo is less toxic than in vitro. However, owing to the
activation of anaerobic glycolysis, the penumbral region suffers from a
substantial degree of tissue acidosis, a condition that has been shown to
alleviate glutamate toxicity in vitro [38].
The ischemic penumbra is not affected by its trophic environment because
the structural integrity of this region is fully preserved. Therefore, excitotoxicity induced by glutamate is not plausible in this ischemic area.
Glutamate toxicity in vitro is a delayed phenomenon that requires 24 hours
to for complete evolution [15]. By contrast, the ischemic penumbra survives
no longer than 6 hours. At this time point, in fact, the ischemic penumbra
becomes the ischemic core [5]
Besides glutamate, other ischemia-related factors such as inhibitory neurotransmitters are released and can be expected to reduce excitotoxicity [38, 40,
66]. For instance, GABA is released at the same blood flow level as glutamate and adenosine even at higher flow thresholds. Thus, the relative
increase in GABA is more pronounced than that of glutamate [40].
To these six postulates it should be added that, in the whole brain, the level of
glutamate can be maintained at a low threshold by all those systems, which
being localized on the membrane of neurons and glial cells are able to induce
glutamate re-uptake. More important, it must not be forgotten that recent
experimental evidence has suggested a protective role for glutamate in the
pathophysiology of the ischemic event [37]. In fact, whereas synaptic transmission mediated by NMDA receptors is essential for neuronal survival, blockade
of NMDA receptors triggers apoptosis in the developing brain [35, 51]. Environmental enrichment, which stimulates synaptic activity, inhibits spontaneous
apoptosis in the hippocampus and is neuroprotective [65]. Accordingly, when
NMDA receptor antagonists are administered during slowly progressing neurodegeneration, they markedly exacerbate damage in the adult brain [36]. In
addition, NMDA receptor antagonists cause apoptosis in primary hippocampal cultures and can exacerbate apoptosis induced by staurosporine [29]. By
contrast, NMDA-receptor-mediated synaptic activation is neuroprotective in
vitro and diminishes apoptosis induced by staurosporine [29]. Activation of
pro-survival transcription factors, such as cAMP response element-binding
protein (CREB), accompanies NMDA-mediated neuroprotection in vitro
[29]. In particular, the Ca2þ pool in the immediate vicinity of synaptic
NMDA receptors is able to trigger signaling from the synapse to the nucleus
via the extracellular signal-regulated kinase (ERK1/2) (Fig. 1) [27]. One important function of this Ca2þ microdomain, which is located near NMDA
Failure of Ionotropic and Metabotropic Glutamate Antagonists
Fig. 1 Activation of prosurvival factors by NMDA
receptor agonists
19
NMDA
Channel Activation
Ca2+
CaMK
ERK
P
CBF
P
CREB
Nucleus
Activation of
prosurvival
factors
NEUROPROTECTION
receptors, is to prolong CREB phosphorylation induced by synaptic stimulation, thereby enhancing CREB-mediated gene expression. CREB controls transcription of pro-survival genes such as the brain-derived neurotrophic factor
(BDNF), the vasoactive intestinal peptide (VIP), bcl-2, and mcl-1 [53, 61]. Thus,
the survival-promoting properties of NMDA receptor could derive from the
transcription of such pro-survival genes. In addition, NMDA activation
induces an increase of cerebral blood flow NO-dependent [22], thus ameliorating brain ischemic conditions.
Indeed, neurons in the penumbra that survive ischemic insults are characterized by high concentrations of BDNF, and bcl-2, and activated CREB [60],
hence suggesting a sustained induction of pro-survival signals. These findings
lead to the logical conclusion that suppression of survival signals promoted by
NMDA receptor activation may facilitate cell death.
Taken together, there is sufficient evidence to suggest that synaptic activity
mediated by NMDA receptors might promote neuronal survival. Blockade of
NMDA-mediated synaptic transmission could therefore be detrimental in situations when support by endogenous measures is required, as occurs after stroke or
in chronic neurodegenerative disorders. From all the above-mentioned considerations it is possible to state that it is time to conclude that NMDA antagonists
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have failed and the attention of researchers working in the field has to be
redirected to other cation channels, pumps, and ionic transporters.
4 Conclusion: Beyond NMDA Receptors
The NMDA receptor has long been viewed as a major player in inducing
excitotoxic cell death. For instance, Simon et al. [58] proposed that the inhibition of the receptor would protect neurons from this death. However, further
animal trials demonstrated that the neuroprotective effect of NMDA receptor
blockers, such as MK-801, was in fact due to a hypothermia induced by the
agent and not by a specific action of the drug [9, 10, 11]. From a number of
clinical trials, the surfacing of certain adverse effects has deeply discouraged
scientists from fulfilling further research goals in the field. Indeed, NMDA
antagonists cause a number of physiological perturbations, including an
increase in blood pressure, as well as tachycardia [12]. In particular, achieving
serum concentrations high enough to equal those neuroprotective ones
obtained in rodents has been challenging, for competitive NMDA receptor
antagonists cause many toxic side effects when given at the recommended
doses [11]. As suggested by Hoyte [34], there is actually ‘‘a loss in translation’’
when moving from animal models to clinical trials. Intriguingly, the latest
literature has highlighted that the activation of NMDA receptors can result in
either cell survival or cell death depending on whether the receptor is synaptic or
extrasynaptic [22, 26, 28, 30]. The activation of synaptic NMDA receptors
promotes cell survival by activation of the CaM kinase and Ras–ERK1/2
(extracellular signal-regulated kinase) pathways and subsequent expression of
BDNF [28]. In contrast, the activation of extrasynaptic NMDA receptors
inactivates the CREB pathway and downregulates BDNF [28]. Beyond the
synapse, different glutamate mechanisms might also operate in other parts of
the neuron. In particular, in the axon, the release of glutamate and the subsequent activation of AMPA are both initiated by a large Naþ influx and the
reverse of Naþ-dependent glutamate transporters [39]. Under some experimental conditions, imbalances in sodium might even be more important than
calcium in axonal compartments. In neuronal dendrites, overactivation of
NMDA receptors is damaging. However, activation of kainate-type receptors,
which are closely related to AMPA receptors, might actually promote growth
and remodeling [44]. Ultimately, glutamergic signaling is mediated not just by
NMDA and AMPA currents. Many other glutamate receptors and transporters exist in the brain. Careful targeting of other neuronal glutamate receptors
and transporters, including the five metabotropic glutamate (mGlu) receptor
subtypes and the excitatory amino acid transporter 2 (EAAT2), could also
prove fruitful, on the basis of similar variations in their capacity to trigger
death or survival [8, 55]. It should be underlined that beyond the glutamateassociated channels, many other ionic channels also carry large ionic currents in
Failure of Ionotropic and Metabotropic Glutamate Antagonists
21
damaged neurons [25]. Altogether, it is important to realize that
NMDA–AMPA pathways comprise only a subset of the multiple routes of
ionic imbalance that are induced in brain injury and neurodegeneration.
Another potential limitation of the standard NMDA–AMPA model is the
focus on neurons alone. Indeed, some types of glial cells, including astrocytes
and oligodendrocytes, play crucial roles in glutamate regulation, and, thus their
roles ought also to be considered alongside neurons. More specifically, glutamate uptake by astrocytes via GLAST and GLT-1, the rodent form of the
EAAT2, normally keeps extracellular glutamate below toxic levels. If these
mechanisms are impaired by ischemia, neuronal excitotoxicity can be amplified
[41]. As both astrocytes and oligodendrocytes express NMDA and AMPA/
kainate receptors, they are also vulnerable to high levels of glutamate [18, 41].
But as already seen in neurons, the processes of oligodendrocytes and astrocytes
might respond differently from the cell body. It has been proposed that NMDA
receptors comprising NR2C and NR3A subunits mediate injury in the glial
processes whereas damages to the glial cell bodies are mediated by AMPA/
kainate receptors [63]. Additionally, glia express many different subtypes of
mGlu receptors that exhibit a variety of modulatory functions [18]. Because of
all these complexities in glial glutamate regulation and signaling, it might
perhaps be difficult to design a single NMDA or AMPA antagonist that
would protect all neurons and glial cells after stroke.
In the final analysis, the standard NMDA–AMPA model of excitotoxicity is
perhaps oversimplistic and does not take into account the complex interactions
with other parallel routes of ionic entry and imbalance within the injured cell.
Accordingly, several other emerging mechanisms of ionic imbalance deserve
further attention as we continue searching for neuroprotectants against stroke
and brain injury. Some of the potentially promising ones could include sodium/
potassium ATPase, sodium–calcium exchangers (NCXs), sodium–proton
exchangers (NHEs), sodium–potassium–chloride (NKCC), Kþ channels, Naþ
channels, acid-sensing ion channels (ASICs), transient receptor potential channels (TRPs), and other non-selective cation channels [3]. Finally, we hope that
future research projects have to prize glutamate lesson highly in order to be on the
straight and narrow path for setting up new effective strategies in stroke therapy.
5 Future Perspectives
Two obvious factors emerge from the discussion presented above:
(a) Virtually, all preclinical studies have suggested that NMDA antagonists
are effectively protective in focal ischemia only if administered immediately
after the insult. This property obviously constitutes a clear drawback in using
NMDA antagonists in humans, for as highlighted in clinical trials, longer
periods of time necessarily elapse between the onset of the insult and the actual
administration of the first treatment.
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G. Di Renzo et al.
(b) The possibility exists that NMDA antagonism, though potentially protective in focal ischemia, is also deleterious in that it adversely affects endogenous NMDA-receptor-mediated neuronal-survival mechanisms [33].
However, intriguing futuristic possibilities are receiving attention and new signs
of hope appear over the horizon. In fact, although the disappointment regarding
the failure of NMDA antagonists is high, the neuroprotective potential of NMDA
isoform-specific antagonists acting extrasynaptically remains to be explored.
Furthermore, by combining glutamate, non-glutamatergic receptors, pump, and
ionic transporter modulators with antioxidants, as well as anti-apoptotic and antiinflammatory agents, it will be possible to cover the time course of stroke development and eventually develop effective treatments for stroke.
References
1. Aarts M, Iihara K, Wei WL, Xiong ZG, Arundine M, Cerwinski W, MacDonald JF, and
Tymianski M. A key role for TRPM7 channels in anoxic neuronal death. Cell 115:
863–877, 2003.
2. Andreeva N, Khodorov B, Stelmashook E, Cragoe E Jr., and Victorov I. Inhibition of
Na+/Ca2+ exchange enhances delayed neuronal death elicited by glutamate in cerebellar
granule cell cultures. Brain Res 548: 322–325, 1991.
3. Annunziato L, Cataldi M, Pignataro G, Secondo A, and Molinaro P. Glutamate-independent calcium toxicity: introduction. Stroke 38: 661–664, 2007.
4. Arvidsson A, Kokaia Z, and Lindvall O. N-methyl-D-aspartate receptor-mediated
increase of neurogenesis in adult rat dentate gyrus following stroke. Eur J Neurosci 14:
10–18, 2001.
5. Back T, Kohno K, and Hossmann KA. Cortical negative DC deflections following
middle cerebral artery occlusion and KCl-induced spreading depression: effect on
blood flow, tissue oxygenation, and electroencephalogram. J Cereb Blood Flow Metab
14: 12–19, 1994.
6. Benveniste H. Brain microdialysis. J Neurochem 52: 1667–1679, 1989.
7. Benveniste H, Drejer J, Schousboe A, and Diemer NH. 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, 1984.
8. Besancon E, Guo S, Lok J, Tymianski M, and Lo EH. Beyond NMDA and AMPA
glutamate receptors: emerging mechanisms for ionic imbalance and cell death in stroke.
Trends Pharmacol Sci 29: 268–275, 2008.
9. Buchan A, Li H, and Pulsinelli WA. The N-methyl-D-aspartate antagonist, MK-801,
fails to protect against neuronal damage caused by transient, severe forebrain ischemia in
adult rats. J Neurosci 11: 1049–1056, 1991.
10. Buchan A and Pulsinelli WA. Hypothermia but not the N-methyl-D-aspartate antagonist, MK-801, attenuates neuronal damage in gerbils subjected to transient global ischemia. J Neurosci 10: 311–316, 1990.
11. Buchan AM. Do NMDA antagonists protect against cerebral ischemia: are clinical trials
warranted? Cerebrovasc Brain Metab Rev 2: 1–26, 1990.
12. Buchan AM, Slivka A, and Xue D. The effect of the NMDA receptor antagonist MK-801
on cerebral blood flow and infarct volume in experimental focal stroke. Brain Res 574:
171–177, 1992.
13. Choi DW. Glutamate neurotoxicity and diseases of the nervous system. Neuron 1:
623–634, 1988.
Failure of Ionotropic and Metabotropic Glutamate Antagonists
23
14. Choi DW. Ionic dependence of glutamate neurotoxicity. J Neurosci 7: 369–379, 1987.
15. Choi DW, Maulucci-Gedde M, and Kriegstein AR. Glutamate neurotoxicity in cortical
cell culture. J Neurosci 7: 357–368, 1987.
16. Davis SM, Albers GW, Diener HC, Lees KR, and Norris J. Termination of acute stroke
studies involving selfotel treatment. ASSIST Steering Committed. Lancet 349: 32, 1997.
17. Davis SM, Lees KR, Albers GW, Diener HC, Markabi S, Karlsson G, and Norris J.
Selfotel in acute ischemic stroke: possible neurotoxic effects of an NMDA antagonist.
Stroke 31: 347–354, 2000.
18. Deng W, Wang H, Rosenberg PA, Volpe JJ, and Jensen FE. Role of metabotropic
glutamate receptors in oligodendrocyte excitotoxicity and oxidative stress. Proc Natl
Acad Sci USA 101: 7751–7756, 2004.
19. Djuricic B, Rohn G, Paschen W, and Hossmann KA. Protein synthesis in the hippocampal slice: transient inhibition by glutamate and lasting inhibition by ischemia. Metab
Brain Dis 9: 235–247, 1994.
20. Fabricius M, Jensen LH, and Lauritzen M. Microdialysis of interstitial amino acids
during spreading depression and anoxic depolarization in rat neocortex. Brain Res 612:
61–69, 1993.
21. Ginsberg MD. Neuroprotection for ischemic stroke: past, present and future. Neuropharmacology 55: 363–389, 2008.
22. Girouard H, Wang G, Gallo EF, Anrather J, Zhou P, Pickel VM, and Iadecola C.
NMDA receptor activation increases free radical production through nitric oxide and
NOX2. J Neurosci 29: 2545–2552, 2009.
23. Globus MY, Busto R, Dietrich WD, Martinez E, Valdes I, and Ginsberg MD. Effect of
ischemia on the in vivo release of striatal dopamine, glutamate, and gamma-aminobutyric
acid studied by intracerebral microdialysis. J Neurochem 51: 1455–1464, 1988.
24. Globus MY, Busto R, Martinez E, Valdes I, and Dietrich WD. Ischemia induces release
of glutamate in regions spared from histopathologic damage in the rat. Stroke 21:
III43–46, 1990.
25. Gribkoff VK and Winquist RJ. Voltage-gated cation channel modulators for the treatment of stroke. Expert Opin Investig Drugs 14: 579–592, 2005.
26. Habas A, Kharebava G, Szatmari E, and Hetman M. NMDA neuroprotection against a
phosphatidylinositol-3 kinase inhibitor, LY294002 by NR2B-mediated suppression of
glycogen synthase kinase-3beta-induced apoptosis. J Neurochem 96: 335–348, 2006.
27. Hardingham GE, Arnold FJ, and Bading H. A calcium microdomain near NMDA
receptors: on switch for ERK-dependent synapse-to-nucleus communication. Nat Neurosci 4: 565–566, 2001.
28. Hardingham GE and Bading H. The Yin and Yang of NMDA receptor signalling. Trends
Neurosci 26: 81–89, 2003.
29. Hardingham GE, Fukunaga Y, and Bading H. Extrasynaptic NMDARs oppose synaptic
NMDARs by triggering CREB shut-off and cell death pathways. Nat Neurosci 5:
405–414, 2002.
30. Hetman M and Kharebava G. Survival signaling pathways activated by NMDA receptors. Curr Top Med Chem 6: 787–799, 2006.
31. Hossmann KA. Disturbances of cerebral protein synthesis and ischemic cell death. Prog
Brain Res 96: 161–177, 1993.
32. Hossmann KA. Excitotoxic mechanisms in focal ischemia. Adv Neurol 71: 69–74, 1996.
33. Hoyte L, Barber PA, Buchan AM, and Hill MD. The rise and fall of NMDA antagonists
for ischemic stroke. Curr Mol Med 4: 131–136, 2004.
34. Hoyte L, Kaur J, and Buchan AM. Lost in translation: taking neuroprotection from
animal models to clinical trials. Exp Neurol 188: 200–204, 2004.
35. Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J, Dikranian K, Tenkova TI,
Stefovska V, Turski L, and Olney JW. Blockade of NMDA receptors and apoptotic
neurodegeneration in the developing brain. Science 283: 70–74, 1999.
24
G. Di Renzo et al.
36. Ikonomidou C, Stefovska V, and Turski L. Neuronal death enhanced by N-methyl-Daspartate antagonists. Proc Natl Acad Sci USA 97: 12885–12890, 2000.
37. Ikonomidou C and Turski L. Why did NMDA receptor antagonists fail clinical trials for
stroke and traumatic brain injury? Lancet Neurol 1: 383–386, 2002.
38. Kaku DA, Giffard RG, and Choi DW. Neuroprotective effects of glutamate antagonists
and extracellular acidity. Science 260: 1516–1518, 1993.
39. Li S and Stys PK. Mechanisms of ionotropic glutamate receptor-mediated excitotoxicity
in isolated spinal cord white matter. J Neurosci 20: 1190–1198, 2000.
40. Matsumoto K, Graf R, Rosner G, Taguchi J, and Heiss WD. Elevation of neuroactive
substances in the cortex of cats during prolonged focal ischemia. J Cereb Blood Flow
Metab 13: 586–594, 1993.
41. Matute C, Alberdi E, Ibarretxe G, and Sanchez-Gomez MV. Excitotoxicity in glial cells.
Eur J Pharmacol 447: 239–246, 2002.
42. Meldrum B. Possible therapeutic applications of antagonists of excitatory amino acid
neurotransmitters. Clin Sci (Lond) 68: 113–122, 1985.
43. Molinaro P, Cuomo O, Pignataro G, Boscia F, Sirabella R, Pannaccione A, Secondo A,
Scorziello A, Adornetto A, Gala R, Viggiano D, Sokolow S, Herchuelz A, Schurmans S,
Di Renzo G, and Annunziato L. Targeted disruption of Na+/Ca2+ exchanger 3 (NCX3)
gene leads to a worsening of ischemic brain damage. J Neurosci 28: 1179–1184, 2008.
44. Monnerie H and Le Roux PD. Glutamate receptor agonist kainate enhances primary
dendrite number and length from immature mouse cortical neurons in vitro. J Neurosci
Res 83: 944–956, 2006.
45. Obrenovitch TP. High extracellular glutamate and neuronal death in neurological disorders. Cause, contribution or consequence? Ann N Y Acad Sci 890: 273–286, 1999.
46. Obrenovitch TP. Neuroprotective strategies: voltage-gated Na+-channel down-modulation versus presynaptic glutamate release inhibition. Rev Neurosci 9: 203–211, 1998.
47. Obrenovitch TP, Urenjak J, Richards DA, Ueda Y, Curzon G, and Symon L. Extracellular neuroactive amino acids in the rat striatum during ischaemia: comparison
between penumbral conditions and ischaemia with sustained anoxic depolarisation.
J Neurochem 61: 178–186, 1993.
48. Obrenovitch TP, Urenjak J, Zilkha E, and Jay TM. Excitotoxicity in neurological
disorders – the glutamate paradox. Int J Dev Neurosci 18: 281–287, 2000.
49. Olney JW and Sharpe LG. Brain lesions in an infant rhesus monkey treated with
monsodium glutamate. Science 166: 386–388, 1969.
50. Pignataro G, Gala R, Cuomo O, Tortiglione A, Giaccio L, Castaldo P, Sirabella R,
Matrone C, Canitano A, Amoroso S, Di Renzo G, and Annunziato L. Two sodium/
calcium exchanger gene products, NCX1 and NCX3, play a major role in the development of permanent focal cerebral ischemia. Stroke 35: 2566–2570, 2004.
51. Pohl D, Bittigau P, Ishimaru MJ, Stadthaus D, Hubner C, Olney JW, Turski L, and
Ikonomidou C. N-Methyl-D-aspartate antagonists and apoptotic cell death triggered by
head trauma in developing rat brain. Proc Natl Acad Sci USA 96: 2508–2513, 1999.
52. Randall RD and Thayer SA. Glutamate-induced calcium transient triggers delayed
calcium overload and neurotoxicity in rat hippocampal neurons. J Neurosci 12:
1882–1895, 1992.
53. Riccio A, Ahn S, Davenport CM, Blendy JA, and Ginty DD. Mediation by a CREB
family transcription factor of NGF-dependent survival of sympathetic neurons. Science
286: 2358–2361, 1999.
54. Rothman SM and Olney JW. Glutamate and the pathophysiology of hypoxic – ischemic
brain damage. Ann Neurol 19: 105–111, 1986.
55. Rothstein JD, Patel S, Regan MR, Haenggeli C, Huang YH, Bergles DE, Jin L, DykesHoberg M, Vidensky S, Chung DS, Toan SV, Bruijn LI, Su ZZ, Gupta P, and Fisher PB.
Beta-lactam antibiotics offer neuroprotection by increasing glutamate transporter
expression. Nature 433: 73–77, 2005.
Failure of Ionotropic and Metabotropic Glutamate Antagonists
25
56. Shimada N, Graf R, Rosner G, Wakayama A, George CP, and Heiss WD. Ischemic flow
threshold for extracellular glutamate increase in cat cortex. J Cereb Blood Flow Metab 9:
603–606, 1989.
57. Shimizu H, Graham SH, Chang LH, Mintorovitch J, James TL, Faden AI, and Weinstein
PR. Relationship between extracellular neurotransmitter amino acids and energy metabolism during cerebral ischemia in rats monitored by microdialysis and in vivo magnetic
resonance spectroscopy. Brain Res 605: 33–42, 1993.
58. Simon RP, Swan JH, Griffiths T, and Meldrum BS. Blockade of N-methyl-D-aspartate
receptors may protect against ischemic damage in the brain. Science 226: 850–852, 1984.
59. Syntichaki P and Tavernarakis N. The biochemistry of neuronal necrosis: rogue biology?
Nat Rev Neurosci 4: 672–684, 2003.
60. Walton MR and Dragunow I. Is CREB a key to neuronal survival? Trends Neurosci 23:
48–53, 2000.
61. Wang JM, Chao JR, Chen W, Kuo ML, Yen JJ, and Yang-Yen HF. The antiapoptotic
gene mcl-1 is up-regulated by the phosphatidylinositol 3-kinase/Akt signaling pathway
through a transcription factor complex containing CREB. Mol Cell Biol 19: 6195–6206,
1999.
62. Wollmuth LP and Sobolevsky AI. Structure and gating of the glutamate receptor ion
channel. Trends Neurosci 27: 321–328, 2004.
63. Wong R. NMDA receptors expressed in oligodendrocytes. Bioessays 28: 460–464, 2006.
64. Xiong ZG, Pignataro G, Li M, Chang SY, and Simon RP. Acid-sensing ion channels
(ASICs) as pharmacological targets for neurodegenerative diseases. Curr Opin Pharmacol
8: 25–32, 2008.
65. Young D, Lawlor PA, Leone P, Dragunow M, and During MJ. Environmental enrichment inhibits spontaneous apoptosis, prevents seizures and is neuroprotective. Nat Med
5: 448–453, 1999.
66. Zinkand WC, Thompson C, Salama AI, and Patel J. Excitatory amino acid-evoked
calcium influx and calcium-dependent neurotoxicity in rat cortical cultures. Ann N Y
Acad Sci 648: 355–357, 1992.