J. Physiol. Biochem., 63 (3), 261-278, 2007
Stroke pathophysiology: management challenges
and new treatment advances
J. Jordán1,5, I. Ikuta2, J. García-García3, S. Calleja4 and T. Segura1,3
1Departamento
de Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La
Mancha, Spain, 2SUNY Upstate Medical University, College of Medicine, Syracuse,
New York, U.S.A., 3Departamento de Neurología, Hospital General Universitario de
Albacete, Spain, 4Departamento de Neurología, Hospital Universitario Central de
Asturias, Oviedo, Spain, 5Grupo de Neurofarmacología, Centro Regional de Investigaciones Biomédicas, Universidad Castilla-La Mancha, Spain
(Received on October, 2007)
J. JORDÁN, I. IKUTA, J. GARCÍA-GARCÍA, S. CALLEJA and T. SEGURA.
Stroke pathophysiology: Management challenges and new treatment advances
(minireview). J. Physiol. Biochem., 63 (3), 261-278, 2007.
Stroke is the second leading cause of death and the first cause of lost disabilityadjusted years in developed countries. During the past decade, new developments in
thrombolytic therapy have led to the implementation of emergency intervention protocols for the treatment of ischemic stroke, replacing the widespread sense of therapeutic nihilism in the past. Treatment with rtPA has shown to be effective within the
first 3 hours following stroke onset and the FDA and the European Medical Agency
(EMEA) have approved its use. Acknowledging the urgency and intricacies of stroke,
Stroke Units allow the monitoring of physiological parameters in the acute phase of
stroke and are considered an important management tool that can significantly
improve the quality of care provided to the patient. The concept of neuroprotective
therapy for acute ischemic stroke to salvage tissue at risk and improve functional outcome is based on sound scientific principles and extensive preclinical animal studies
demonstrating efficacy. However, most neuroprotective drugs in clinical trials have
failed, possibly due to inadequate preclinical testing or flawed clinical development
programs. Several new treatment strategies are under development and are being tested. This review is directed at understanding the management of acute ischemic stroke
pathophysiology. We address the management challenges and new treatment
advances by integrating the knowledge of possible pharmacological targets for acute
ischemic stroke. We hope to shed new light upon the controversy surrounding the
management of acute ischemic stroke in an attempt to elucidate why failed clinical
trials continue to occur despite promising neuroprotective preclinical studies.
Key words: Apoptosis, Neuroprotection, Neurodegeneration, Ischemia, Pharmacology.
Correspondence to J. Jordán (Tel.: +34 967 599 200; Fax. +34 967 599 327; e-mail: [email protected]).
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J. JORDÁN, I. IKUTA, J. GARCÍA-GARCÍA, S. CALLEJA, T. SEGURA
Stroke is the main cause of lost disability-adjusted life years and ranks second
after ischemic heart disease as a cause of
death in developed countries. The incidence of stroke increases exponentially
with age but varies among countries.
Approximately 15 million people suffer a
stroke every year, and in spite of the medical advances, 5 million patients will die
and at least 5 million will be left disabled.
Of the 3 million Americans who have survived a stroke, 2 million will sustain some
permanent disability, with an overall cost
to the nation of $40 billion per year.
Stroke is not a simple disease. It can be
ischemic or hemorrhagic. The former is
quite more common. Embolic blockage of
a blood vessel is the most frequent cause
of ischemic stroke, wich can involve small
or large arteries, intracranial or extracranially and other mechanisms as local
thrombosis or hypoperfusion can occur.
Stroke evolution is quite variable and can
be influenced by various factors such as
age, sex racial background hypotension,
fever, or hyperglycemia. It can be influenced by comorbidities and concurrent
medications, responsible for about 80%
of the approximately 700,000 strokes in
the United States each year. Probably due
to this enormous variability, despite many
years of intensive research, treatment of
ischemic stroke continues to be one of the
major challenges in current medicine. In
this review we are going to present the
accepted drugs as well as some of the latest trials that try to add new therapeutic
alternatives. Herein, we have divided the
text in the two main pharmacologic
approaches: restoration of cerebral perfusion and neuroprotection.
Pharmacology of restoration
of cerebral perfusion
Ischemic stroke is a dynamic process
where a series of excitotoxic, inflammatoJ. Physiol. Biochem., 63 (3), 2007
ry and microvascular mechanisms take
place that lead to tissue necrosis. However, some ischemic brain tissue may be salvageable if reperfusion occurs before
damage becomes irreversible. Initially
after arterial occlusion, a poorly perfused
central core area is surrounded by an area
of dysfunction caused by metabolic and
ionic disturbances but with preserved
structural integrity (the ischemic penumbra). The ischemic penumbra (Fig. 1) has
been documented in the laboratory animal
as still viable brain tissue surrounding the
irreversibly damaged ischemic core. Thus,
current efforts of all acute stroke therapy
are aimed at that portion of ischemic
region which is potentially salvageable.
Saving as much of the penumbra as possible is the main target of acute stroke therapy, and is the theoretical basis behind the
reperfusion concept. The goal of reperfusion treatment is to restore cerebral blood
flow, and the main clinical tools to achieve
this objective are thrombolytic drugs.
These agents enhance the endogenous formation of plasmin from plasminogen,
what in turn can potentially dissolve the
blood clot occluding a cerebral vessel by
disrupting fibrin. rt-PA is the only thrombolytic agent currently licensed to treat
selected patients with acute ischemic
stroke in the first three hours after stroke
onset. In 1996, rt-PA was granted FDA
approval largely on the basis of the
National Institute of Neurological Disorders and Stroke (NINDS) rt-PA study
(77). In this pivotal study, 624 patients
were randomly assigned treatment with
rt-PA or placebo. The outcome at 3
months was significantly better in the
treated group. The study only included
patients presenting within 3 hours of
symptom onset and approximately one
half was treated within 90 minutes. In the
European Union, however, regulatory
ADVANCES IN STROKE THERAPY
263
Fig. 1. Pharmacologic approaches in stroke: restoration of cerebral perfusion, neuroprotection and others.
authorities did not approve the use of
rt-PA until 2002, and at the moment it is
still limited to a restricted license at specialist centers. The situation in Europe is
probably due to the unsuccessful result of
the two European Cooperative Acute
Stroke Studies (ECASS and ECASS 2) (37,
32). In these two large trials, IV rt-PA was
not more effective than placebo in
improving neurological outcomes 3
months after stroke. It appears quite likely that time was a limiting factor in the
ECASS studies since these trials treated
patients up to 6 hours after stroke. However, a post-hoc analysis concluded that
patients treated within 3 hours appeared
to benefit from rt-PA treatment. In fact,
community studies have confirmed the
safety and usefulness of the drug in routine clinical practice when administered
within the first 3 hours after stroke symptom onset (2, 30, 74). Recently finished is
the multinational Safe Implementation of
Thrombolysis in Stroke Monitoring
Study (SITS-MOST) which was designed
J. Physiol. Biochem., 63 (3), 2007
to evaluate the safety and efficacy of IV rtPA under routine clinical use when
administered within 3 hours of symptom
onset in acute ischemic stroke patients.
This large, international, multi-center,
open study, has confirmed the safety and
efficacy of rt-PA within this 3-hour time
window (79). Furthermore, a combined
analysis of six randomized control trials
(RCTs) of IV rt-PA (31) showed that the
sooner patients received thrombolytic
therapy, the greater the benefit. This same
analysis found that the benefit of IV rt-PA
in acute ischemic stroke seems to disappear 4.5 hours after the onset. While the
efficacy of thrombolytic agents is very
much under the influence of time, other
factors will modify the potential expected
efficacy. Although it is probable that part
of the efficacy of desmoteplase lies in its
pharmaceutical properties (higher fibrin
specificity and absence of NMDA-mediated neurotoxicity), the DIAS results suggested that thrombolytic therapy may be
safe beyond 3 hours if the proper patients
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J. JORDÁN, I. IKUTA, J. GARCÍA-GARCÍA, S. CALLEJA, T. SEGURA
were selected. This finding gave hope to
stroke investigators that in the near future
it will be possible to extend the therapeutic window far beyond 3 hours. However,
it has been recently announced that the
larger prospective clinical trial DIAS-II
does not reproduce the positive effects of
the previous study. Other thrombolytic
agents, including reteplase, anistreplase,
and tenecteplase, in conjunction with new
strategies based on evolving multimodal
MRI or CT techniques, soon will likely
allow for the selection of patients for late
IV reperfusion therapy.
Another possibility is ultrasoundenhanced thrombolysis. Ultrasound can
induce microstreams within the clot to
increase the distribution of rt-PA for
improved thrombolysis. Transcranial
ultrasound–enhanced intravenous thrombolysis leads to an absolute 20% increase
in the rate of middle cerebral artery
(MCA) recanalization according to
CLOTBUST results (5). Moreover, transcranial ultrasound has been shown to
accelerate clot dissolution in some
patients with contraindications for rt-PA,
although these preliminary data should be
confirmed in the setting of a large trial.
Finally, ultrasound in combination with
novel microparticles offers further potential for targeted thrombolytic drugs.
Intra-arterial (IA) thrombolysis has
been shown effective up to 6 hours after
symptom onset in MCA occlusions. IA
thrombolysis significantly increases the
probability of a favorable outcome 2.3fold (44). Although no RCTs have compared IA versus IV thrombolysis, some
indirect data suggest a higher rate of
recanalization via IA administration.
Given that the IA administration takes
longer than IV, a combined IV and rescue
IA approach has been proposed (23). This
way begins immediately with IV rt-PA
J. Physiol. Biochem., 63 (3), 2007
and ultrasound monitoring of the MCA
artery. If partial or completed recanalization has not been achieved after thirteen
minutes, then the patient is moved to
angiography and IA thrombolysis is
administered. This strategy allows more
time to prepare patients for IA thrombolysis without increasing any delay in
potential treatment.
Although endovascular interventions
of acute ischemic stroke show great
promise, available data has been limited.
There are some successful reports of
angioplasty combined with thrombolysis
in patients with occlusions in the vertebrobasilar circulation, but until now both
angioplasty and stenting of intracranial
vessels remain to be demonstrated as an
emergency treatment of stroke. The FDA
has recently approved the use of the
MERCI device (based in the results of the
Mechanical Embolus Removal in Cerebral
Embolism trial) for reopening intracranial
arteries, although its clinical utility has not
yet been established (70). This option may
be particularly important in proximal
artery occlusions, nonresponders to
thrombolytic agents, and for patients otherwise ineligible for thrombolytic therapy.
Pharmacology of neuroprotection
“Neuroprotection” is a term used to
describe the putative effect of interventions protecting the brain from pathological damage. Hundreds of neuroprotective
strategies have been shown to improve
outcome in animal models of focal cerebral ischemia, but thus far none of them
have been shown to be clearly efficacious
in patients. However, our increasing
knowledge concerning the ischemic cascade is leading to considerable development of pharmacological tools suggesting
ADVANCES IN STROKE THERAPY
that each step of this cascade might be a
target for cytoprotection. In stroke, the
concept of neuroprotection involves inhibition of a cascade of pathological molecular events which occur under ischemia
and lead to calcium influx, activation of
free radical reactions, and cell death. Following a vessel occlusion, critical decrease
in focal cerebral blood flow promote a
complex biochemical cascade within
ischemic tissue. This ischemia-induced
biochemical cascade interferes with glucose metabolism and causes cell Na+
pump failure. These events lead to neuronal depolarization and the release of
excitotoxic neurotransmitters which are
able to cause abrupt necrotic cell death
within the infarct core. In the surrounding
greater volume of the ischemic tissue (the
penumbra area), a dynamic process is initiated (Fig. 1). This process also involves
intracellular calcium influx, multiple free
radical production, and expression of
adhesion molecules leading to inflammation and cell death.
It is now clear that a stroke causes different types of cell death. Much like an
earthquake, a stroke wreaks havoc around
its epicenter, destroying cells in a devastating process called necrosis. But farther
away from the epicenter, the stroke’s ripple effects cause cells within the penumbra
to kill themselves by apoptosis. This
slower process can take days, and it ultimately determines the extent of the ensuing spread of brain damage. Programmed
cell death is probably the critical process
in the evolution of the penumbra area
given that it is the main cause of neuronal
death in this region. Besides the morphological and biochemical differences
between necrosis and apoptosis, the main
differential characteristic is that apoptotic
pathways allow pharmacological intervention at the three established stages of
J. Physiol. Biochem., 63 (3), 2007
265
programmed cell death: activation, commitment and execution. In the first stage,
excitotoxic processes include rising calcium ion concentrations and reactive oxygen species formation which promote
apoptotic gene expression. Mitochondria,
through a process mainly regulated by
changes in the permeability of their outer
membrane, play a central role in the commitment stage, being considered the
point-of-no-return. Finally, in the execution stage, degradation enzyme complexes
such as caspases, calpains and endonucleases assume an active role. It is typically
considered that only by preventing the
first two stages it is possible to avoid neuronal damage, since acting on the execution stage can delay but not prevent
impending cell death. So, we are going to
review the different strategies that could
potentially abort apoptosis in ischemic
brain tissue.
The apoptosis activation process
involved in stroke includes the alteration
of calcium and sodium ion homeostasis
due to reversal of the glutamate transporter (Fig. 2). The subsequent increase of
this neurotransmitter at the synapse overactivates the receptors. This increased
agonism of receptors results in an increase
of cytoplasmic calcium that might depolarize the mitochondria, which will
increase the amount of free radicals and
lipid peroxidation (Fig. 3). Stroke excitotoxicity is primarily mediated by several
glutamate receptor subtypes, including
the NMDA and AMPA receptors (10).
NMDA receptor antagonists were the
first neuroprotective agents tested in
stroke clinical trials. Despite the lack of
unacceptable side effects, no efficacy was
found with competitive NMDA antagonists (selfotel) or non-competitive
NMDA antagonists (dextrorphan, aptiganel and eliprodil). Activation of the
NMDA receptor requires the co-agonist
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J. JORDÁN, I. IKUTA, J. GARCÍA-GARCÍA, S. CALLEJA, T. SEGURA
Fig. 2. Molecular and cellular mechanisms related to the evolution of oxygen and glucose deprivation.
glycine. Studies of the glycine antagonist,
gavestinel, found the medication to be
safe, but favorable outcomes were not
found. Another glycine antagonist,
licostinel, was associated with numerous
side effects.
The AMPA receptor is the principal
mediator of fast excitatory neurotransmission. This ligand-gated cation channel is
primarily permeable to sodium rather
than calcium. In a variety of animal models, a reduction of infarct volume has been
demonstrated with AMPA blockade. First
generation AMPA receptor blockers were
competitive antagonists, like NBQX,
which showed robust neuroprotection in
a variety of disease-related animal models.
Its clinical use, however, was restricted by
very low solubility, inducing kidney precipitation in vivo. Second generation
competitive antagonists are available,
which do not possess this property. However, none of these second generation
competitive antagonists are currently in
J. Physiol. Biochem., 63 (3), 2007
clinical use. The AMPA antagonist ZK
200775 led to neurological deterioration
in a preliminary study. A novel, highly
water-soluble, competitive AMPA receptor antagonist, YM872 (Zonampanel), has
been identified. Phase I studies showed
YM872 was well tolerated at 1.25 mg/kg/h
when given as a 24-hour infusion to
healthy subjects. Sedation and other
CNS-associated adverse events determine
the ceiling dose and become more problematic with infusion times exceeding 24
hours. Phase II trial of YM872 in acute
ischemic stroke is ongoing. Awaiting the
final results of the last studies, a systematic review of trials testing excitatory amino
acid antagonists in stroke (51) found no
improvements in rates of either death or
favorable outcomes with treatment.
Another agent that modulates the glutamate receptor family includes magnesium. Magnesium blocks NMDA receptors, inhibits release of presynaptic excitatory neurotransmitters, and antagonizes
ADVANCES IN STROKE THERAPY
267
Fig. 3. Ischemia results in rapid loss of high-energy phosphate compounds and generalized depolarization, which
induces the reversal of glutamate transporters and the opening of both voltage-dependent and glutamate-regulated calcium channels. These changes lead to a large increase in cytosolic Ca2+ associated with an alteration of
mitochondrial stability. Mitochondrial membrane inestability eventually activates proteases with consequent
proteolysis of substrates.
voltage-sensitive calcium channels. Due to
all of these actions, it was thought that this
agent could be useful in the reduction of
ischemic brain injury. However, an international phase III trial investigating the
efficacy of magnesium in stroke, the
IMAGES (Intravenous Magnesium Efficacy in Stroke) trial did not find such benefit (50). Most patients in this study
received magnesium beyond 3 hours. As
such, the investigators of the Pilot Study
Field Administration of Stroke TherapyMagnesium (FAST-MAG) planned a
scaled-up randomized control trial to test
magnesium efficacy when the drug was
administered in a hyperacute stage (63).
Patients received paramedic-administered
magnesium on average within 26 minutes
of symptom onset, with safe results. Clinicians noted patient improvement in 20%
of the cases, deterioration in 7%, and no
change in 73%. A FAST-MAG Phase III
J. Physiol. Biochem., 63 (3), 2007
Trial is ongoing to evaluate the efficacy of
hyperacute, paramedic-initiated magnesium sulfate administration in improving
the long-term functional outcome of
patients with acute stroke. This
adamatane derivate preferentially blocks
excessive NDMA receptor activity without disrupting normal activity. Memantine does this through its action as a lowaffinity, noncompetitive open-channel
blocker with a relatively rapid off-rate
from the channel. The unique and subtle
difference of the site of memantine action
at the channel pore may explain many
advantageous properties of memantine
administration. Memantine binds at the
“intracellular” magnesium site in the
channel pore. In a rat model of stroke,
memantine reduces the amount of brain
damage by approximately 50% when
given as long as 2 hours after the ischemic
event (43).
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On the other hand, enhancing the
activity of the GABA-ergic system may
be neuroprotective in acute stroke, as it
inhibits presynaptic glutamate release.
The neuroprotective effect of diazepam
has been demonstrated in global ischemia
models in vivo and in vitro (65). Clomethiazole, a GABA agonist with the property of increasing cellular resistance to
glutamate toxicity, was tested in the clinical trial CLASS (Clomethiazole Acute
Stroke Study). This trial was unable to
show significant efficacy and a subsequent
study restricted to patients with total
anterior circulation syndrome also failed
to show benefit (80).
Another possibility is blocking the
intracellular increase in calcium. Voltagedependent Ca2+ channels (L-, N-, T-, Pand Q-type) have been widely recognized
as an important regulator of the nervous
system. Interestingly, several reports have
suggested that a blockade or lack of Ntype Ca2+ channels can suppress the neuronal pathologic processes of ischemic
brain injury in animal models. Cilnidipine
is a Ca2+ channel blocker with suppressive
effects on L- and N-type Ca2+ channels
that reduced neuronal damage in ischemic
rat brains. TAKAHARA et al. proposed that
this drug may be suitable for hypertensive
patients with a risk of brain attack (72). As
of January 2006, 110 patients had been
enrolled in a multi-center, randomized,
double-blind, active control, titrated dose,
non-inferiority trial, the Cilnidipine
Effect on High Blood Pressure and Cerebral Perfusion in Ischemic Stroke Patients
with Hypertension (CHERISH). They
are going to compare the effect of cilnidipine and losartan (angiotensin II receptor blocker) on cerebral blood flow and
blood pressure in hypertensive patients
with a previous history of ischemic stroke.
J. Physiol. Biochem., 63 (3), 2007
A second approach to block rising
intracellular calcium is by buffering intracellular calcium concentration. We can do
so by using a calcium chelator such as
DP-b99. DP-b99 is a newly developed
lipophilic, cell permeable derivative of
BAPTA
(1,2-bis(2-aminophenoxy)
ethane-N,N,N’,N’-tetraacetic
acid),
which selectively modulates the distribution of metal ions in hydrophobic milieu,
and is in clinical development as a neuroprotectant for cerebral ischaemic stroke.
Final results in Phase IIB showed a 90-day
median change in NIHSS scores of 6.0 and
5.0 points (nonsignificant) in the placebo
(n=72) and treatment (n=75) groups,
respectively, from the baseline scores of 720; and of 5.0 and 8.0 points in the placebo and treatment groups, respectively,
(p=0.03) from the baseline scores of 10 16. Recovery rates for the DP-b99 and
placebo groups per modified Rankin scale
was 30.6% and 16.0% (p=0.05), per
NIHSS was 23.6% and 12.0% (p=0.08),
and by either scale was 37.5% and 18.7%
(p=0.02), respectively. In Phase IIa, an
efficacy evaluation demonstrated significant improvements in clinical stroke outcome assessed with the NIH Stroke Scale
(NIHSS) at 2, 7 and 30 days after stroke in
patients treated with DP-b99 within 12
hours of the onset of stroke symptoms.
There were no significant differences in
the number of serious adverse events,
cases of death, or causes of death between
the DP-b99 and placebo groups (61).
Sodium channel blockers have also
been used in stroke therapy. Lubeluzole is
a sodium channel blocker with additional
effects on nitric oxide. Although a pilot
study suggested that this agent was safe
and might reduce the death rate in stroke,
subsequent clinical trials found no significant effects in reducing deaths or improving outcomes (15). Fosphenytoin is a well
ADVANCES IN STROKE THERAPY
known anticonvulsant agent that blocks
voltage-dependent sodium and calcium
channels and prevents glutamate release.
However, its clinical efficacy in ischemic
stroke has not been demonstrated. Sipatrigine (619C89), a sodium and calcium
channel blocker, also failed to have any
favorable effect on outcome measures in a
phase II clinical trial using a continuous
intravenous infusion in acute stroke (49).
Besides calcium and sodium, reactive
oxygen species participate in stroke by
activating apoptotic pathways since brain
ischemia initiates a complex cascade of
metabolic events, several of which involve
the generation of nitrogen and oxygen
free radicals. In fact, reactive oxygen
species activate Bax to induce mitochondrial cytochrome c release and apoptosis
in response to chemical ischemia (29).
And reactive oxygen species induce
swelling and cytochrome c release but not
transmembrane depolarization in isolated
rat brain mitochondria (25). Antioxidant
drugs including compounds with free radical scavenging activity (Ebselen, tirilazad), iron chelators (deferoxamine) and
antioxidants with free radical trapping
properties (NXY-059), and albumin have
been examined in experimental models of
stroke and evaluated clinically as potential
neuroprotective agents. We will now discuss the results of clinical evaluations of
these potential stroke modifiers.
Ebselen, a selene-organic compound
with antioxidant activity, improves the
outcome of acute ischemic stroke. A randomized, double-blind, placebo-controlled trial of ebselen was conducted in
patients with complete occlusion of the
middle cerebral artery. There was a corresponding significant reduction in the volume of cerebral infarct and an improvement in the outcome of patients who
started treatment within 6 hours of onset.
J. Physiol. Biochem., 63 (3), 2007
269
Ebselen might be safe and effective in
improving outcomes after stroke, and
another clinical trial is under way (82). On
the other hand, preclinical data on tirilazad in animal models of acute ischemic
stroke were neither comprehensive nor
consistent. So, when studied in man, tirilazad not only did not improve outcome
after stroke, but potentially appeared to
marginally worsen it (7).
Iron is generally believed to participate
in neuronal dysfunction and death
through its ability to catalyze (via electron
donation) the generation of highly reactive hydroxyl radicals via Fenton reaction
chemistry. In this model, hydroxyl radicals modify lipid, protein, and DNA targets to induce cell dysfunction of these
cellular constituents. Iron chelators can
inhibit Fenton reaction chemistry and
prevent neuronal cell injury by sequestering accumulated free iron. However, this
pathway of iron chelator-mediated protection remains controversial. The neuroprotective potential of the iron chelator
deferoxamine comes from its antioxidant
effect as well as its capacity to stabilize the
hypoxia-inducible factor-1 (HIF-1) protein expression (discussed later). The safety of deferoxamine in acute stroke
patients over 50 years old without known
iron overload is currently being tested.
The NXY-059 compound has been
purposed to act as a free radical scavenger
and was effective in reducing lesion
volume and neurological deficits. Recently, the positive results from the first
Stroke-Acute-Ischemic-NXY-Treatment
(SAINT-I) trial, which followed many of
the Stroke Therapy Academic Industry
Roundtable (STAIR) guidelines, reinvigorated the enthusiasm for neuroprotection
(41). The STAIR recommendations seek
to improve the quality of preclinical
research and to ensure that the data gener-
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ated will enable the selection of those
agents most suitable for progression from
the laboratory to clinical trials. However,
the SAINT-II trial did not reproduce the
positive effects on the same primary prespecified outcome measure. In this trial
the investigators failed to confirm the efficacy of NXY-059 for the treatment of
acute ischemic stroke within 6 hours after
the onset of symptoms (69).
Preclinical studies have suggested that
albumin has potentially neuroprotective
effects (26, 27). Recent data from a phase I
dose-escalation study provide evidence
that human serum albumin is safe after
stroke, despite a mild-to-moderate
increase in pulmonary edema, even when
given with thrombolytic therapy (26). The
study provided preliminary evidence of
efficacy, with patients in the highest dose
tiers having about an 80% greater chance
of good outcome at 3 months than the
lower dose tiers (57). There also seemed to
be a synergistic effect between albumin
and thrombolytic therapy. A recently
published phase I/II clinical trial confirmed the clinical benefit of albumin
administered within 24 hours from symptom onset, showing that these effects were
dose- and time-related (68). A more definitive phase-III trial (Albumin-in-Acute
Stroke) is underway.
Protein synthesis inhibition occurs in
neurons during reperfusion after
ischemia, highlighting the likely role that
these pathways play in prosurvival versus
proapoptotic processes that may be differentially expressed in vulnerable and resistant regions of the reperfused brain tissue.
The activation of some transcription factors has been detected in neurons and
microglia in the penumbra area. These
include the hypoxia-inducible factor-1
(HIF-1), p53, peroxisome proliferatoractivated receptors (PPARs) and S-100β.
J. Physiol. Biochem., 63 (3), 2007
The HIF-1 is of the main transcriptional
factors regulated by oxygen level. HIF-1
increases the expression of several beneficial genes such as erythropoietin, glucose
transporter-1, and vascular endothelial
growth factor. As previously commented,
the iron chelator deferoxamine stabilizes
HIF-1α protein expression. The p53 protein is activated upon detection of stress
signals, such as hypoxia and DNA damage. The regulation of p53 functions is
tightly controlled through several mechanisms including p53 transcription and
translation, protein stability, post-translational modifications, and subcellular
localization (28). So, p53 can be considered a pharmacological target. Blocking
p53 expression demonstrably and efficiently protects against cerebral ischemia
in experimental models, and this protection includes reduction in rates of apoptosis (36). Indeed, preclinical studies
demonstrate the efficacy of a p53 inhibitor
in models of stroke and neurodegenerative disorders, and suggest that drugs that
inhibit p53 may reduce the extent of brain
damage in related human neurodegenerative conditions. PPARs are endogenous
protective factors in cerebral ischemia.
Activation of PPAR-γ attenuates the
expression of ICAM-1, matrix metalloproteinase MMP-9, and various cytokines
in ischemic brain tissue. Activation of
PPAR-γ with pioglitazone or other thiazolidinediones might reduce immune
reactions, and at the same time release a
powerful anti-inflammatory potential in
ischemic brains. PPAR agonists decrease
the expression of COX-2, an enzyme
involved in the production of reactive
oxygen species (ROS). PPAR agonists
also increase in the expression level of the
ROS scavenger Cu-Zn-superoxide dismutase. The PPAR-γ agonist rosiglitazone
could be a potential novel therapeutic
271
ADVANCES IN STROKE THERAPY
agent for stroke (46). Arundic acid (AA;
ONO-2506), a novel modulator of astrocyte activation, may improve neuronal
survival after stroke by suppressing
S-100β‚ protein production in astrocytes.
In the presence of ischemia or other biochemical insults, S-100β‚ protein is overexpressed in astrocytes, and in turn
induces mRNA expression of iNOS,
NGFβ‚ and COX-2. AA reduces cerebral
infarct volumes and improves neurological function in rodent surgical models of
acute ischemic stroke. AA is active in a
wide time window after the onset of neurological injury since astrocyte activation
occurs over many hours in both human
stroke patients and preclinical stroke
models. In seven completed phase I
healthy volunteer studies and two previous stroke patient safety studies, AA was
well-tolerated. The efficacy of AA in
acute stroke treatment should be confirmed in future clinical trials (58).
Mitochondria are considered the main
link between cellular stress signals and the
execution of neuronal programmed cell
death (34). Conditions during reperfusion
are likely to be conducive to the induction
of the permeability transition in mitochondria. Translocation of cytochrome c
(Cyt c) from the mitochondria to the
cytoplasm is crucial to the commitment
step of apoptosis. Calcium-induced Cyt c
release, as occurs in neurons during stroke
and ischemia, involves rupture of the
mitochondrial outer membrane and can
be blocked by inhibitors of the mitochondrial permeability transition pore (mPT).
Induction of the mPT has been linked to
cytotoxicity after pathological stimuli
such as stroke. mPT blockers, such as
cyclosporin A and bongkrekic acid, have
shown neuroprotective effects in animal
models of ischemia. Several inhibitors of
Cyt c release have shown promise in modJ. Physiol. Biochem., 63 (3), 2007
els of neuronal apoptosis. STRAVROSKAYA
et al. (71) identified several drugs, including antidepressants and antipsychotics,
capable of delaying the mPT. Interestingly, promethazine at clinically achievable
doses inhibits mPT, and was protective
both in vitro as well as in mouse models of
stroke.
In the last portion of the apoptotic
pathway, referred to as the execution
phase, the participation of proteases and
endonucleases has been described. A
number of studies already indicated the
prominent role of the cysteine proteases
of the calpain and caspase families in
apoptosis following brain ischemia. Proteolytic activities of these proteases
degrade various cytoskeletal proteins and
integral membrane proteins, destabilizing
cellular structural integrity and leaving
neurons within the ischemic penumbra to
an inevitable death (60). Some current
studies have unequivocally confirmed the
neuronal apoptosis in ischemia and
showed that administration of calpain and
caspase inhibitors alone or in combination
can provide functional neuroprotection in
various animal models of cerebral
ischemia. However, further investigations
are necessary for improvements in the
structural design of calpain and caspase
inhibitors for persistent therapeutic efficacy in animal models of stroke and for clinical trials in the future.
Other treatments
The potential for neuroprotection of
hypothermia has long been suspected. In
fact hypothermia is an essential method of
cerebral protection during circulatory
arrest. Most likely, hypothermia exerts
multiple and synergistic effects on brain
metabolism. Hypothermia reduces cerebral metabolic activity and the oxygen
272
J. JORDÁN, I. IKUTA, J. GARCÍA-GARCÍA, S. CALLEJA, T. SEGURA
demand. Victims of near-drowning in icecold waters can have a remarkable neurological recovery even after prolonged
cerebral ischemia (84). Hypothermia
decreases the cerebral metabolic rates of
glucose and oxygen and slows ATP breakdown (21). In the range of 22°C to 37°C,
brain oxygen consumption is reduced by
5% for every degree fall in body temperature (33). However, the most important
mechanisms that cause the strong neuroprotection
provided
by
induced
hypothermia are still a matter of debate
and are probably multiple. Some of the
suggested mechanisms reported in the literature include the reduction of neurotransmitter release during ischemia
(including glutamate) (52), reduced
inflammation, and reduced free radical
generation. Hypothermia lowers metabolic rate, limits edema formation, and interrupts necrosis/apoptosis (73, 81). In addition, hypothermia reduces intracellular
calcium rises after ischemia due to
impaired glutamate-mediated calcium
influx (73).
Advantages of surface cooling are that
it requires neither advanced equipment
nor expertise in catheter placement and
averts the risks associated with central
venous catheter placement. Cooling
through external methods, however,
requires many hours to reach and maintain a temperature below 35 °C, and in
most cases demands the use of sedatives
and paralytics to prevent discomfort and
shivering. In addition, cooling of the skin
leads to vasoconstriction and reduces the
heat exchange in cooled patients, which
makes temperature control very difficult.
This may lead to target temperature overshoot and lack of control during passive
rewarming, which may be associated with
reactive brain edema. In patients who are
awake, surface cooling is only achieved as
J. Physiol. Biochem., 63 (3), 2007
an approach for mild hypothermia and for
fever control. New surface cooling
devices, such as energy-transferring skin
pads (48), may demonstrate a reduction in
the time to target temperature and allow
better temperature control. In a pilot trial
(17), treatment with paracetamol at 6
grams daily, compared with placebo, led
to a 0.3 ºC reduction in body temperature
in normothermic patients. This temperature reduction is of potential but
unproven clinical benefit. In another
study (14, 46) 40 patients were randomly
assigned either standard care or endovascular intravenous cooling to 33 ºC for 24
hours within 12 hours of symptom onset.
Hypothermia was well tolerated, but clinical and MRI measures did not differ
between treatment groups. Further studies are needed to determine whether
hypothermia improves outcome. The use
of hypothermia might also be limited by
its substantial side-effects which include
shivering and infections. The application
of hypothermia is also limited by practical
considerations such as the need for treatment in an intensive care unit.
Despite the absence of evidence,
heparin and heparinoids are still frequently used. According to the American and
European Guidelines, the use of anticoagulants should be restricted to special cases,
such as cerebral venous sinus thrombosis
and possibly arterial dissections (2, 13, 19,
20, 54, 62, 66). Several studies have shown
no benefit of anticoagulation in acute
ischemic stroke (1, 3, 11, 12, 67). Therefore, anticoagulants such as heparin have
no role in the routine management of
acute ischemic stroke.
Acute aspirin has been tested in two
large trials. The International Stroke Trial
(76) compared aspirin alone with two different doses of heparin within 48 hours of
symptom onset. Aspirin was associated
ADVANCES IN STROKE THERAPY
with significantly fewer recurrent
ischemic strokes and no significant
increase in hemorrhagic strokes at 14
days. The rate of death or dependency did
not differ between the two groups at six
months. The Chinese Acute Stroke Trial
(9, 73) also compared aspirin with placebo
within 48 hours of symptom onset. Significantly fewer recurrent ischemic strokes
occurred in the aspirin group, but the
number of hemorrhagic strokes increased.
Aspirin given within 48 hours of acute
ischemic stroke seems to reduce death and
disability. However, the use of aspirin in
conjunction with alteplase might increase
the risk of bleeding (39, 75). Acute aspirin
is recommended for patients with acute
ischemic stroke who are ineligible for
thrombolytic therapy.
Minocycline, a semisynthetic second
generation derivate of tetracycline, was
show to have clear beneficial neuroprotective effects in animal models of stroke
(85). The proposed mechanism of
minocycline include its anti-inflammatory
effect, reduction of microglial activation,
matrix metalloproteinase reduction, nitric
oxide production, and inhibition of apoptotic cell death (for review see 35). Recently in an open-label, evaluator-blinded
study, minocycline at a dosage of 200 mg
was administrated orally for 5 days. The
therapeutic window of time was 6 to 24 h
after onset of stroke. Data from NIH
Stroke Scale (NIHSS), modified Rankin
Scale (mRS), and Barthel Index (BI) were
evaluated. Patients with acute stroke had
significantly better outcome with minocycline treatment compared with placebo
(38).
Besides the effect of statin treatment in
stroke prevention, it has also been shown
that statins (HMG-CoA reductase
inhibitors) have other beneficial effects
and these may be independent of the subJ. Physiol. Biochem., 63 (3), 2007
273
ject’s basal lipid levels (47, 86). In fact,
recent studies suggest a neuroprotective
power in the acute phase of stroke. It is
well known that statins have direct influences on endothelial function by up-regulating endothelial nitric oxide synthase,
inhibiting inducible nitric oxide synthase,
reducing plaque stability due to an antithrombotic and anti-inflammatory mechanism, modulating the immune system,
and enhancing vasomotor reactivity.
These properties, also called pleiotropic
effects, are responsible for the potentially
beneficial use of statins in acute ischemic
stroke. A recent study demonstrated the
beneficial effects of statins (atorvastatin,
20 mg) in the acute phase of stroke by
attenuating
the
pro-inflammatory
cytokine responses as well as the expression of adhesion molecules that accompany cerebral ischemia. These protective
effects are also related with less infarct
volume and a more favorable outcome at
three months from stroke onset (53). In
the National Institutes of Health Suburban Hospital Stroke Program Study, 22%
of patients were taking a statin when they
were admitted. In this observational
study, 51% of patients taking these drugs
had a good outcome compared with 38%
of those not taking statins (83). Another
study (18) included patients with stroke of
which 18% were using statins when
admitted. Better prognosis at 3 months
was significantly more frequent in the
statin group, indicating that these drugs
may provide benefits for the long-term
functional outcome when administered
before the onset of cerebral ischemia.
Additionally, the preliminary results of a
pilot clinical trial have been communicated, suggesting that simvastatin therapy
initiated in the acute phase of ischemic
stroke might improve neurological outcomes as well. Statins have also been
274
J. JORDÁN, I. IKUTA, J. GARCÍA-GARCÍA, S. CALLEJA, T. SEGURA
found to be useful when given in combination with thrombolytic therapy. In an
embolic model of cerebral ischemia in
rats, the combination treatment with atorvastatin and t-PA exerts a neuroprotective
effect when administered 4 hours after
stroke (14). In clinical practice, a recent
study has evaluated 145 patients with a
stroke involving the middle cerebral
artery who received tissue plasminogen
activator treatment (6). Statistically significant differences were found among
27.3% of patients who were using statins
at the time of the index stroke and were
functionally independent at 3 months, as
compared with 13.6% among the group of
patients who were dependent or dead.
Systemic hypertension in the acute
phase of ischemic stroke is common and is
believed to be a physiologic response that
attempts to maintain an adequate cerebral
perfusion pressure within the ischemic
penumbra (59). While appropriate in primary and secondary stroke prevention,
the lowering of elevated blood pressure
(BP) in the acute setting of ischemic
stroke has been a matter of debate for several years. Some studies have suggested
that it could result in the extension and
worsening of stroke symptoms, resulting
in unfavorable outcomes (8, 22, 40, 45, 55,
56, 78). In contrast, other experimental
and clinical studies demonstrated that a
cautious reduction of BP may even
improve the prognosis in acute cerebral
ischemia (24, 45). The current recommendation to tolerate acute hypertension in
cerebral ischemia is based on the concept
of disturbed autoregulation of cerebral
blood flow in the penumbra surrounding
the zone of necrosis. However, the
ACCESS study (a multicenter phase II
study) (31, 64) showed that candesartan in
the acute phase of ischemic stroke was
safe, with no cerebrovascular events
J. Physiol. Biochem., 63 (3), 2007
occurring as a result of hypotension.
Encouraged by the success of ACCESS,
several studies dealing with antihypertensive drugs for acute ischemic stroke are
ongoing, including COSSACS (to determine whether antihypertensive therapy
should be continued in the acute stroke
setting), CHHIPS (to determine the
extent to which BP should be lowered in
the acute stroke setting), and SCAST (to
assess whether candesartan given to
patients with elevated BP in the acute
phase of stroke reduces the cardiovascular
risk).
Elevated blood glucose is common in
the early phase of stroke. Hyperglycemia,
defined as blood glucose level
>108mg/dL, may influence neuronal
damage through accentuated tissue acidosis and lactate generation. Moderately and
severely increased blood glucose has been
found to further deteriorate the metabolic
state and mitochondrial function in the
area of the ischemic penumbra. Moreover,
the blood-brain barrier is vulnerable to
hyperglycemia presumably through the
liberation of lactic acid and free radicals
(42). However, although diabetes and
hyperglycemia are associated with worse
outcomes in acute ischemic stroke, the
threshold blood sugar levels to initiate
treatment with insulin are arbitrary and
the efficacy and safety of aggressive
glycemic control is unknown. Furthermore, the level of target glucose concentrations is not the same for the different
current target values in the published
guidelines: EUSI: <10nmol/L; ASA: <16.6
nmol/L. A reasonable target in most cases
of lower blood glucose levels is between
100 and 200 mg/dl, although levels of
glycemia >150 mg/dL should probably be
avoided and insulin therapy should be initiated (16). Most recently, The United
Kingdom Glucose Insulin in Stroke Trial
ADVANCES IN STROKE THERAPY
(GIST-UK), a multicenter randomized
trial, demonstrates the safety of administering a glucose-potassium-insulin infusion in acute phase stroke patients with
the aim of keeping glucose level between
72-126 mg/dL. However, until further
results prove the effectiveness of this
approach, it cannot be regarded as standard practice.
Conclusion
After the NINDS rt-PA study publication in 1995, the classical approach to
ischemic stroke started to change. However, more than ten years later, less than
4% of patients receive treatment with
alteplase. Moreover, IV thrombolysis has
many limitations and there are no broadly
accepted alternatives for nonresponders.
So, neurologists are currently involved in
new investigations to fight against these
limitations. In the coming years, medicine
will have to find effective neuroprotection
with the ability to prolong the window of
opportunity for time-sensitive ischemic
tissue. It will probably become necessary
to improve our current methodology for
investigations and assume the STAIR recommendations. Finding effective neuroprotection will require identifying strategies to protect not only neurons, but also
vascular and glial cells. Furthermore, in
spite of the development of new drugs
capable of prolonging the efficacy and
improving the safety of clot lysis, reperfusion therapy in ischemic stroke incorporates pharmaceutical and mechanical therapies. Endovascular devices and acute
angioplasty will likely have a prominent
role, as has occurred in acute coronary
syndrome management. However, the
paramount advance in acute stroke therapy may be the use of multi-modal imaging
techniques and serum biomarkers to preJ. Physiol. Biochem., 63 (3), 2007
275
dict with reasonable accuracy the location
and amount of irreversible ischemic damage. This fact will change the idea of a general timetable in thrombolysis to an individualized timetable in ischemic stroke.
Acknowledgments
This work was funded by SAF2005-07919-C0201 from CICYT and 04005-00 Consejería de Sanidad
from Junta de Comunidades de Castilla La Mancha
to J.J.
J. JORDÁN, I. IKUTA, J. GARCÍAGARCÍA, S. CALLEJA y T. SEGURA. Fisiopatología del ictus: clásicos y nuevos tratamientos. J. Physiol. Biochem., 63 (3), 261-278, 2007.
El ictus representa la segunda causa de
muerte y la primera de incapacidad en los países desarrollados. Pese a su frecuencia y gravedad, hasta hace pocos años no se disponía de
tratamientos eficaces en la fase aguda, lo que
condicionaba una actitud nihilista en todo el
proceso terapéutico. En la última década se ha
conseguido demostrar la efectividad de la
administración precoz de tratamiento intravenoso trombolítico con rtPA en el ictus isquémico, por lo que tanto la FDA americana como
la agencia europea del medicamento han autorizado su uso, si bien con restricciones diferentes. Además, la monitorización de los parámetros biológicos y estandarización de los cuidados médicos y de enfermería en los primeros
días, realizados en las Unidades de Ictus, han
demostrado también su eficacia terapéutica. En
la actualidad el esfuerzo investigador en el ictus
intenta encontrar moléculas que solas o en
combinación trasladen a la práctica clínica la
capacidad de neuroprotección que muchas de
ellas ya han demostrado en el laboratorio o en
los modelos animales. Sin embargo, hasta la
fecha este esfuerzo no ha sido recompensado
plenamente y científicos básicos y clínicos tendrán que intensificar su trabajo y comunicación para lograr al fin estrategias que permitan
mantener viable el tejido cerebral sometido a
agresión tras el ictus.
276
J. JORDÁN, I. IKUTA, J. GARCÍA-GARCÍA, S. CALLEJA, T. SEGURA
Palabras clave: Apoptosis, Neuroprotección,
Neurodegeneración, Isquemia, Farmacología.
References
1. Adams, H.P., Jr. (2002): Stroke, 33, 856-861.
2. Albers G.W., Bates, V.E., Clark W.M., Bell, R.,
Verro, P. and Hamilton, S.A. (2000): JAMA, 283,
1145–1150
3. Albers, G.W., Amarenco, P., Easton, J.D., Sacco,
R.L. and Teal, P. (2001): Chest., 119, 300s-320s.
4. Albers, G.W., Amarenco, P., Easton, J.D., Sacco,
R.L. and Teal, P. (2004): Chest., 126, 483s-512s.
5. Alexandrov, A.V., Molina, C.A., Grotta, J.C., et
al. (2004): N. Engl. J. Med., 351, 2170–2178.
6. Alvarez-Sabin, J., Huertas, R., Quintana, M.,
Rubiera, M., Delgado, P., Ribo, M. et al. (2007):
Stroke, 38, 1076-1078.
7. Bath, P.M., Iddenden, R., Bath, F.J. and Orgogozo, J.M. (2001): Cochrane Database Syst. Rev.
CD002087
8. Britton, M., De F.U. and Helmers, C. (1980):
Acta Med. Scand., 207, 253-257.
9. CAST (1997): Lancet, 349, 1641-1649.
10. Choi, D.W. and Rothman, S.M. (1990): Annu.
Rev. Neurosci., 13, 171-182.
11. Coull, B.M., Williams, L.S., Goldstein, L.B.,
Meschia, J.F., Heitzman, D., Chaturvedi, S. et al.
(2002): Stroke, 33, 1934-1942.
12. Coull, B.M., Williams, L.S., Goldstein, L.B.,
Meschia, J.F., Heitzman, D., Chaturvedi, S. et al.
(2002): Neurology, 59, 13-22.
13. De Bruijn, S.F. and Stam, J. (1999): Stroke, 30,
484-8.
14. De Georgia, M.A., Krieger, D.W., Abou-Chebl,
A., Devlin, T.G., Jauss, M., Davis, S.M. et al.
(2004): Neurology, 63, 312-317.
15. Diener, H.C., Hacke, W., Hennerici, M., Radberg, J., Hantson, L. and De Keyser, J (1996):
Stroke 27, 76–81.
16. Díez-Tejedor, E. and Fuentes, B. (2004): Cerebrovasc. Dis., 17, 130-137.
17. Dippel, D.W., Van Breda, E.J., Van Gemert,
H.M. et al. (2001): Stroke, 32, 1607-1612.
18. Dippel, D.W., Van Breda, E.J., Van Gemert,
H.M., Van Der Worp, H.B., Meijer, R.J., Kappelle, L.J. et al. (2001): Stroke, 32, 1607-1612.
19. Einhaupl, K.M., Villringer, A., Meister, W.,
Mehraein, S., Garner, C., Pellkofer, M. et al.
(1991): Lancet, 338, 597-600.
20. Einhaupl, K., Bousser, M.G., De Bruijn, S.F.,
Ferro, J.M., Martinelli, I., Masuhr, F. et al.
(2006): Eur. J. Neurol., 13, 553-9.
21. Erecinska, M., Thoresen, M. and Silver, I.A.
(2003): J. Cereb. Blood Flow Metab., 23, 513-530.
J. Physiol. Biochem., 63 (3), 2007
22. Fischberg, G.M., Lozano, E., Rajamani, K.,
Ameriso, S. and Fisher, M.J. (2000): J. Emerg.
Med., 19, 339-346.
23. Flaherty, M.I. et al. (2005): Neurology, 64, 386388.
24. Fujii, K., Weno, B.L., Baumbach, G.L. and Heistad, D.D. (1992): Hypertension, 19, 713-716.
25. Galindo, M.F., Jordan, J., Gonzalez-Garcia, C.
and Cena V. (2003) Br. J. Pharmacol., 139,797804.
26. Ginsberg, M.D, Hill, M.D., Palesch, Y.Y., Ryckborst, K.J. and Tamariz, D. (2006): Stroke, 37,
2100-6.
27. Ginsberg, M.D. (2003): Stroke, 34, 214-223.
28. Gomez-Lazaro M, Fernandez-Gomez, F.J. and
Jordan J. (2004): J. Physiol. Biochem., 60,287-307.
29. Gomez-Lazaro, M., Galindo, M.F., Melero-Fernandez de Mera, R.M., Fernandez-Gomez, F.J.,
Concannon, C.G., Segura, M.F., Comella, J.X.,
Prehn, J.H. and Jordan J. (2007): Mol. Pharmacol., 71, 736-743.
30. Grond, M., Stenzel, C., Schmulling, S. et al.
(1998): Stroke, 29, 1544–1549.
31. Hacke, W., Donnan, G., Fieschi, C. et al. (2004):
Lancet, 363,768-774.
32. Hacke W., Kaste, M., Fieschi, C., et al. (1998)
Lancet, 352, 1245–1251
33. Hagerdal, M., Harp, J. and Siesjo, B.K. (1975): J.
Neurochem., 24, 743-748.
34. Jordan, J., Cena, V. and Prehn, J.H. (2003): J.
Physiol. Biochem., 59, 129-141.
35. Jordan, J., Fernandez-Gomez, F.J., Ramos, M.,
Ikuta, I., Aguirre, N. and Galindo M.F. (2007):
Curr. Drug. Deliv., 4, 225-231.
36. Jordan, J., Galindo, M.F, Gonzalez-Garcia, C.
and Cena V. (2003) Neuroscience, 122, 707-715.
37. Kaste, M., Hacke, W., Fieschi, C., et al. (1995):
Cerebrovasc. Dis., 5, 225.
38. Lampl, Y., Boaz, M., Gilad, R., Lorberboym, M.,
Dabby, R., Rapoport, A., Anca-Hershkowitz,
M. and Sadeh, M. (2007): Neurology, 69,14041410.
39. Larrue, V., Von Kummer, R.R., Muller, A. and
Bluhmki, E. (2001): Stroke, 32, 438-441.
40. Lavin, P. (1986): Arch. Intern. Med., 146, 66-68.
41. Lees Kr, Zivin Ja, Ashwood T, et al. (2006): N.
Engl. J. Med., 354, 588-600.
42. Linsberg, P.J. and Roine, R.O. (2004): Stroke, 35,
363-364
43. Lipton, S.A. (2007): Curr. Drug. Targets., 8, 621632.
44. Lisboa, R.C., et al. (2002): Stroke, 33, 2866-2871.
45. Lisk, D.R., Grotta, J.C., Lamki, L.M., Tran,
H.D., Taylor, J.W., Molony, D.A. et al. (1993):
Arch. Neurol., 50, 855-862.
ADVANCES IN STROKE THERAPY
46. Luo, Y., Yin, W., Signore, A.P., Zhang, F. et al.
(2006): J. Neurochem., 97,435-448.
47. Marti-Fabregas, J., Gomis, M., Arboix, A., Aleu,
A., Pagonabarraga, J., Belvis, R. et al. (2004):
Stroke, 35, 1117-1121.
48. Mayer, S.A., Kowalski, R.G., Presciutti, M.,
Ostapkovich, N.D., Mcgann, E., Fitzsimmons,
B.F., et al. (2004): Crit. Care Med., 32, 25082515.
49. Muir, K.W., Holzapfel, L. and Lees Kr. (2000):
Cerebrovasc. Dis., 10, 431-436.
50. Muir, K.W., Lees, K.R., Ford, I. and Davis, S.
(2004): Lancet, 363, 439-445.
51. Muir K.W. and Lees, K.R. (2003): Cochrane
Database Syst. Rev., Cd001244.
52. Nakashima, K. and Todd, M.M. (1996): Stroke,
27, 913-918.
53. Nombela, F., Blanco, M., Moro, M.A.,
Rodriguez-Yañez, M., Leira, R., Lizosain, I. et
al. (2006): Neurologia, 21, 539.
54. Norris, J.W. (2005): Stroke, 36, 2041-2042.
55. O’connell, J.E. and Gray, C.S. (1996): Drugs
Aging, 8, 408-415.
56. Oliveira-Filho, J., Silva, S.C., Trabuco, C.C.,
Pedreira, B.B., Sousa, E.U. and Bacellar, A.
(2003): Neurology, 61, 1047-1051.
57. Palesch, Y.Y., Hill, M.D., Ryckborst, K.J.,
Tamariz, D. and Ginsberg, M.D. (2006): Stroke,
37, 2107-2114.
58. Pettigrew, L.C., Kasner, S.E., Albers, G.W., et al.
(2006): J. Neurol. Sci., 251, 50-56.
59. Powers, W.J. (1993): Neurology, 43, 461-467.
60. Ray, S.K. (2006): Curr. Med. Chem., 13, 34253440.
61. Rosenberg, G., Angel, I. and Kozak, A. (2005):
Br. J. Clin. Pharmacol., 60, 7-16.
62. Sacco, R.L., Adams, R., Albers, G., Alberts, M.J.,
Benavente, O., Furie, K. et al. (2006): Circulation, 113, E409-E449.
63. Saver, J.l., Kidwell, C., Eckstein, M. and Starkman, S. (2004): Stroke, 35, E106-E108.
64. Schrader, J., Luders, S., Kulschewski, A., Berger,
J., Zidek, W., Treib, J. et al. (2003): Stroke, 34,
1699-1703.
65. Schwartz-Bloom, R.D., Miller, K.A., Evenson,
D.A., Crain, B.J. and Nadler, J.V. (2000): Neuroscience, 98, 471-484.
66. Selim, M. and Caplan, L.R. (2004): Curr. Treat
Options Cardiovasc. Med., 6, 249-253.
67. Sherman, D.G. (2004): Cerebrovasc. Dis., 17,
138-143.
68. Shin, D.H., Moon, G.J. and Bang, O.Y. (2007): J.
Neurol., 254, 870-878.
J. Physiol. Biochem., 63 (3), 2007
277
69. Shuaib, A., Lees, K.R., Lyden, P., Grotta, J.,
Davalos, A., Davis, S.M., Diener, H.C., Ashwood, T., Wasiewski, W.W. and Emeribe, U:
Saint Ii Trial Investigators. (2007): N. Engl. J.
Med., 357, 562-571.
70. Smith, W.S., Sung, G., Starkman, S., et al. (2005):
Stroke, 36, 1432–1438.
71. Stavrovskaya, I.G., Narayanan, M.V., Zhang,
W., Krasnikov, B.F., Heemskerk, J., Young, S.S.,
Blass, J.P., Brown, A.M., Beal, M.F., Friedlander,
R.M. and Kristal, B.S. (2004): J. Exp. Med., 200,
211-222.
72. Takahara, A., Konda, T., Enomoto, A. and
Kondo, N. (2004): Biol. Pharm. Bull., 27, 13881391.
73. Takata, T., Nabetani, M. and Okada, Y. (1997):
Neurosci. Lett., 227, 41-44.
74. Tanne D, Bates V, Verro P, et al. (1999) : Neurology, 53, 424-427.
75. Tanne, D., Kasner, S.E., Demchuk, A.M.,
Koren-Morag, N., Hanson, S., Grond, M. et al.
(2002): Circulation, 105, 1679-1685.
76. The International Stroke Trial (Ist). (1997).
Lancet, 349, 1569-1581.
77. The National Institute of Neurological Disorders and Stroke. (1995): N. Engl. J. Med., 333,
1581-1587
78. Vlcek, M., Schillinger, M., Lang, W., Lalouschek,
W., Bur, A. and Hirschl, M.M. (2003): Ann.
Emerg. Med., 42, 619-626.
79. Wahlgren, N., Ahmed, N., Davalos, A., Ford,
G.A., Grond, M., Hacke, W., Hennerici, M.G.,
Kaste M., Kuelkens, S., Larrue, V., Lees, K.R.,
Roine, R.O. Soinne, L., Toni, D. and Vanhooren,
G., Sits-Most Investigators. (2007): Lancet, 369,
275-282. Erratum In: Lancet, 2007, 369,826.
80. Wahlgren, N.G., Bornhov, S., Sharma, A. et al.,
For The Class Study Group (1999): J. Stroke
Cerebrovasc. Dis., 8, 1-10.
81. Wang, L.M., Yan, Y., Zou, L.J., Jing, N.H. and
Xu, Z.Y. (2005): Cell. Res., 15, 387-393.
82. Yamaguchi, T., Sano, K., Takakura, K., Saito, I.,
Shinohara, Y., Asano, T. and Yasuhara H. (1998):
Stroke, 29, 12-17
83. Yoon, S.S., Dambrosia, J., Chalela, J. et al.
(2004): BMC Med., 23, 2-4.
84. Young, R.S., Zalneraitis, E.L. and Dooling, E.C.
(1980): JAMA, 244, 1233-5.
85. Yrjanheikki, J., Keinanen, R., Pellikka, M., Hokfelt, T. and Koistinaho, J. (1998): Proc. Natl.
Acad. Sci. U S A, 95, 15769-15774.
86. Zhang, L., Zhang, Z.G., Ding, G.L., Jiang, Q.,
Liu, X., Meng, H. et al. (2005): Circulation, 112,
3486-3494.