Neur CL in Pract 2013005496

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Neuron. Author manuscript; available in PMC 2011 July 29.
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Neuron. 2010 July 29; 67(2): 181–198. doi:10.1016/j.neuron.2010.07.002.
The Science of Stroke: Mechanisms in Search of Treatments

Michael A. Moskowitz1, Eng H. Lo1, and Costantino Iadecola2
1 Departments of Radiology and Neurology, Massachusetts General Hospital, Charlestown, MA
02129 USA
2 Division of Neurobiology, Weill Cornell Medical College, New York, NY 10065 USA
Summary
This review focuses on mechanisms and emerging concepts that drive the science of stroke in a
therapeutic direction. Once considered exclusively a disorder of blood vessels, growing evidence
has led to the realization that the biological processes underlying stroke are driven by the
interaction of neurons, glia, vascular cells and matrix components, which actively participate in
mechanisms of tissue injury and repair. As new targets are identified, new opportunities emerge
that build on an appreciation of acute cellular events acting in a broader context of ongoing
destructive, protective and reparative processes. The burden of disease is great and its magnitude
widens as a role for blood vessels and stroke in vascular and non-vascular dementias becomes
more clearly established. This review then poses a number of fundamental questions, the answers
to which may generate new directions for research and possibly new treatments that could reduce
the impact of this enormous economic and societal burden.
Introduction
Few neurological conditions are as complex and devastating as stroke, the second leading
cause of death worldwide. Also called a brain attack, victims may suddenly experience
paralysis, impaired speech or loss of vision due to interruption of blood flow (ischemia)
caused by thrombosis or embolism. Less frequently (<15%), strokes are caused by
hemorrhage or cardiac arrest. On average, strokes in the USA strike once every 40 seconds
and cause death every 4 minutes, with an estimated 41.6% death rate in 2007 (Lloyd-Jones
et al., 2009). With an aging population, the absolute numbers are likely to rise. Among
survivors, work capacity is compromised in 70% of victims, and 30% need assistance with
self-care. Hence, the disease burden is great. The estimated cost for stroke is 73.7 billion
dollars in 2010 (USA) and projected to be 1.52 trillion dollars in 2050 (in 2005 dollars)
(Lloyd-Jones et al., 2009). No racial or ethnic groups are immune and the problem is global.
For example, in the Russian Federation and China, the estimated death rates per 100,000
population are 5–10 times higher than in the USA (Lloyd-Jones et al., 2009). Hence, stroke
is an affliction of mankind.
For the above considerations and more, there is a compelling need to accelerate efforts to
interrogate the stroke process and to define the links that exist with other conditions such as
vascular and neurodegenerative dementia. It is also crucial to expand the narrow repertoire
Corresponding author: Michael A. Moskowitz, M.D., Massachusetts General Hospital, 149 13th Street, Room 6403, Charlestown, MA
02129 USA, [email protected], 617-726-8442.
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Moskowitz et al. Page 2
of therapeutic opportunities for these devastating conditions. To accomplish this, novel

approaches are required that expand upon our evolving mechanistic understanding of the
fundamentals of cell survival and death processes as well as tissue repair. The future
depends upon how successful we are in deciphering these mechanisms and bringing clarity
to the complex interactions between the multiplicity of cell and tissue types within brain (Lo
et al., 2003). Armed with this knowledge and its successful therapeutic application, the field
of stroke could be transformed.
In this spirit then, this brief review addresses selected issues fundamental to the science of
ischemic stroke and vascular dementia. It begins with posing questions about stroke risk
factors followed by a discussion of key cell and tissue mechanisms that render brain
susceptible as well as tolerant to ischemic injury, including those promoting tissue
protection and repair. The review ends by highlighting promising treatment strategies,
inspired by these endogenous mechanisms, which present the opportunity to open new
avenues in stroke therapy.
Stroke risk factors and triggers

A stroke risk factor is a characteristic of an individual that increases the risk for stroke
compared to someone without that characteristic (Hankey, 2006). Some risk factors cannot
be modified, such as a family history of cerebrovascular diseases, older age, male sex and
Hispanic or Black race (Allen and Bayraktutan, 2008) (Hankey, 2006),. Other risk factors
are modifiable and their correction reduces the chance of having a stroke (Table 1). These
factors, which often coexist, have been estimated to account for 60–80% of stroke risk in the
general population (Allen and Bayraktutan, 2008) (Hankey, 2006). Genome-wide
association studies are increasingly being employed to identify susceptibility genes for
stroke (Hegele and Dichgans, 2010). Although several loci have been identified, the need for
independent replication and the modest effect sizes have precluded the full assessment of the
clinical relevance of these findings (Hegele and Dichgans, 2010).
How do risk factors increase the propensity to stroke?

Risk factors have profound effects on the structure and function of blood vessels, and on
their interface with circulating blood. Many of the established risk factors alter vascular
structure by promoting atherosclerosis and stiffening of arteries, and by inducing narrowing,
thickening and tortuosity of arterioles and capillaries (Allen and Bayraktutan, 2008)
(Iadecola and Davisson, 2008). In brain, these morphological changes are often associated
with reductions in resting cerebral blood flow (CBF) and marked alterations in CBF
regulation. Thus, aging, hypertension, diabetes and hypercholesterolemia impair vital
adaptive mechanisms that assure that the brain is adequately perfused (Arrick et al., 2007;
Iadecola and Davisson, 2008; Iadecola et al., 2009; Kitayama et al., 2007). The ability of the
endothelium to regulate microvascular flow is compromised, while the increase in blood
flow evoked by neural activity is suppressed, resulting in a mismatch between the brain’s
energy supply and demand (Arrick et al., 2007) (Iadecola and Davisson, 2008), (Iadecola et
al., 2009) (Zou et al., 2004a)
Some risk factors, like hypertension and diabetes, impair protective vascular mechanisms
that keep CBF stable during reductions in blood pressure (cerebrovascular autoregulation),
facilitating the occurrence of ischemia if intravascular pressure drops (Immink et al., 2004)
(Kim et al., 2008). These vascular alterations increase the brain’s vulnerability to ischemia
after arterial occlusion because they compromise the development of collateral flow arising
from adjacent non-ischemic vascular territories, which is vital to the survival of the ischemic
perinfarct zone (see Parenchymal Failure section). In addition to their vascular effects,
some risk factors, like aging and diabetes, may enhance the intrinsic susceptibility of brain

cells to injury, amplifying the tissue damage produced by ischemia (Biessels et al., 2002),
but the biological bases of this effect are not well understood. Little is known about the
interaction among the different stroke risk factors and whether their vascular effects are
additive or synergistic. Furthermore, the relative contribution of parenchymal and vascular

factors to stroke risk remains to be determined.
How do risk factors alter cerebral blood vessels?

Many cardiovascular risk factors increase production of reactive oxygen species (ROS) and
promote inflammation in systemic and cerebral blood vessels. The predominant vascular
sources of ROS are the superoxide-producing enzyme NADPH oxidase, xanthine oxidase,
mitochondrial enzymes and uncoupling of nitric oxide synthase (NOS), a state in which this
enzyme generates superoxide instead of NO (Faraci, 2006) (fig. 1). Many of the damaging
effects of oxidative stress on blood vessels are related to the biological inactivation of NO
by the free radical superoxide, which reduces NO bioavailability and prevents its beneficial
effects (Pacher et al., 2007) (Schulz et al., 2008). Thus, loss of the vasoregulatory effects of
endothelial NO leads to vasoconstriction and reduced NO-dependent vascular responses,
with a negative impact on the regulation of microvascular flow (Schulz et al., 2008). Loss of
the antiaggregant, anti-proliferative, and anti-cell adhesion effects of NO promotes platelet
aggregation, leukocyte adhesion to endothelial cells, and smooth muscle proliferation, key
steps in vascular inflammation (Pepine, 2009) (Zou et al., 2004b). In addition, ROS can
directly promote inflammation by increasing blood brain barrier (BBB) permeability
through upregulation of vascular endothelial growth factor (VEGF), and by inducing the
expression of cytokines, matrix remodeling enzymes, including metalloproteases, and
proinflammatory genes through NF-kb activation (Marchesi et al., 2008). One such NFκb-
dependent gene product, inducible nitric oxide synthase, produces large amounts of NO,
which alter vascular structure and function through nitration and nitrosylation of critical
proteins (Gunnett et al., 2005) (Guzik et al., 2003) (Lima et al., 2010). Peroxynitrite, a
potent nitrating agent produced by the reaction of NO with superoxide, alters vasomotor
reactivity by inactivating critical endothelial and smooth muscle enzymes, and by activating
the DNA repair enzyme poly-ADP-ribose polymerase (PARP), which leads to ATP
depletion and vascular ion channel dysfunction (Pacher et al., 2007). Whereas ROS may set
the stage for inflammation, vascular inflammation, in turn, leads to ROS production creating
a vicious circle that enhances vascular damage. Activation of the plasminogen system by
oxidative stress and inflammation promotes matrix remodeling, smooth muscle cell
migration, and intimal hyperplasia (Nicholl et al., 2006), factors that promote atherosclerosis
and alterations in vascular structure. Thus, oxidative stress and vascular inflammation are
major pathways through which risk factors exert their deleterious effects on blood vessels.
However, it remains to be determined how individual risk factors trigger the activation of
one or both of these processes. This is a critical question for targeting preventive strategies
in patients with specific risk factors.
Why now? Role of stroke triggers

While vascular risk factors increase the likelihood of stroke in affected individuals,
precipitating factors that act as “stroke triggers” must also be postulated. In some patients,
the specific event responsible for the arterial occlusion can be identified, e.g., neck trauma
leading to dissection and occlusion of the internal carotid artery (Kim and Schulman, 2009).
But in most instances the factors precipitating the ischemic event cannot be established.
Although systemic infections, pregnancy and puerperium, use of illicit drugs, and mental
stress often trigger a stroke (Elkind, 2007), precisely how these factors exert their effect
remains unclear. Exacerbation of vascular inflammation and activation of the coagulation
cascade are likely to play a role (Welsh et al., 2009). For example, the added vascular
dysfunction and blood clotting abnormalities, superimposed on those induced by stroke risk

factors, could precipitate vascular occlusion or hemodynamic insufficiency. This view is

supported by the fact that acute stroke often occurs in the setting of increased circulating
leukocytes, and elevated plasma markers of systemic inflammation and vascular activation,
which also predict a poor outcome (Elkind, 2007) (Welsh et al., 2009). Thus, our
understanding of stroke triggers is limited, and mechanistic studies addressing these issues
would be valuable in the identification of high-risk cases.
Parenchymal Failure: Why and how does the brain die during ischemia?
Because of its high intrinsic metabolic activity activity and large concentrations of the
neurotransmitter-excitotoxin glutamate (Choi, 1992), the brain is especially vulnerable to
ischemic insult. This can develop either as a consequence of thrombosis in situ or following
embolic occlusion of a cerebral blood vessel, the latter usually arising from the heart or
atherosclerotic plaques in the carotid artery and aortic arch. Although neurological
dysfunction occurs within seconds to minutes of vessel occlusion, the evolution of ischemic
injury and cell death continues in stages for minutes, hours and even days, depending, in
part, upon the vulnerability of the particular brain region, its cellular constituents, and the
extent of residual perfusion.
The responses of blood vessels and their perfusion are important to outcome as well. For
example, a deficiency of vasodilating molecules such as endothelial NO enlarges stroke size
(Huang et al., 1996). Early restoration of blood flow by clot lysis and reperfusion decreases
ischemic injury, and this may be achievable by giving recombinant tissue plasminogen
activator (tPA), the only FDA-approved treatment for reestablishing flow and salvaging
brain tissue. However, tPA is used in <10% of patients and even less frequently after 3 hrs
because of the risk of hemorrhage into ischemic tissue (NINDS TPA Study) (1995).
Moreover, tPA may have other unintended risks (Kaur et a., 2002) such as injury to the
blood brain barrier by activating matrix metalloproteinases (Wang et al., 2003), or
excitotoxicity in experimental models (Nicole et al., 2001). Combination therapies that
ameliorate these effects may extend tPA’s therapeutic window while mitigating the
untoward effects of reperfusion and plasminogen activation (Liu et al., 2004) (Cheng et al.,
2006) (Murata et al., 2008). Theoretically, that could prolong tissue viability for hours or
even longer, depending upon the rapid delivery of drug to compromised brain (Ginsberg,
2008).
Whether or not vulnerable brain tissue can be rescued or protected after a protracted time
period in the absence of partial or complete restoration of blood flow is an unanswered
question. In all likelihood, areas of vulnerable brain do remain viable for hours after vessel
occlusion, at least in some instances. For example, the hemiparesis that follows middle
cerebral artery occlusion in the baboon was reversible in 73% of animals with reperfusion at
2–4 hours, compared to 33% and 17% in animals with reperfusion at 8 and 16–24 hours,
respectively (Marcoux et al., 1982) (Crowell et al., 1981) (Jones et al., 1981). The challenge
then is to restore adequate blood flow quickly after vessel occlusion and to protect viable
tissue from unleashed mechanisms that lead to cell and tissue demise.
Salvageable vs Non-Salvageable Tissue: Salvageable tissue is the target for therapy

Tissue distal to an occluded blood vessel is often heterogeneous, meta-stable and
differentially susceptible to ischemic injury. This meta-stable zone, termed the ischemic
penumbra or perinfarct zone (Astrup et al., 1981), is characterized by significantly depressed
tissue perfusion that is barely sufficient to support basal ATP levels and oxygen metabolism
as well as normal ionic gradients in the presence of electrical silence and suppressed protein
synthesis. The ischemic penumbra then denotes an “at risk” region that is functionally
impaired, but potentially salvageable. However, unless perfusion is improved or cells made

relatively more resistant to injury, the tissue at risk dies within a few hours (Lo, 2008a, b).
The ischemic core by contrast, refers to the irreversibly damaged tissue distal to an occluded
blood vessel characterized by <20% of baseline blood flow levels, depleted ATP stores and
irreversible failure of energy metabolism (Lo, 2008b). With time, the infarct core expands
into the ischemic penumbra and therapeutic opportunity is lost. Therefore, detecting a
penumbra in patients can help to identify those who might benefit most from acute
treatments that restore blood flow (thrombolysis or endovascular clot retrieval) or treatments
for the future that render viable brain more resistant to ischemic injury. For this purpose,
magnetic resonance imaging methods such as perfusion-weighted and diffusion-weighted
imaging are most often used. Perfusion-weighted imaging measures the spatial extent of
blood flow compromise whereas diffusion weighted imaging is thought to detect the region
of attenuated diffusion of water, a putative surrogate marker for severely damaged brain
tissue. The difference is thought to reflect the ischemic penumbra (fig. 2) (Ebinger et al.,
2009). Although the predictive value of this method is still debated, perfusion-diffusion
mismatch provides a useful first attempt to define the tissue at risk.
What are the prominent mechanisms leading to cell and tissue demise?
Within the ischemic penumbra, multiple mechanisms have been identified over the past few
decades that irreversibly damage brain tissue (fig 2). Understanding the contribution of each
informs us about potential therapeutic opportunities and treatment targets. In the area of
severely limited blood supply, ATP utilization continues in the presence of minimal
synthesis and levels drop, leading to acidosis and loss of ionic homeostasis. As a
consequence, cells swell and membranes rupture. However, ischemic cell death cannot be
equated with limited ATP availability; rather, tissue death develops as a consequence of
numerous ionic, biochemical and cellular events that impose overwhelming stresses upon
already compromised tissue (see for review (Dirnagl et al., 1999) (Lo et al., 2003).
Excitotoxicity—Excitotoxicity and calcium overload are major factors contributing to the

early stages of ischemic cell death. The canonical pathway asserts that glutamate, the most
abundant neurotransmitter, accumulates into the extracellular space as a consequence of
energy and ion pump failure, as well as failure of reuptake mechanisms (Choi and Rothman,
1990). The glutamate overload leads to prolonged stimulation of AMPA and NMDA
ionotropic receptor subtypes to dramatically enhance the influx of calcium, sodium and
water into neurons. Massive calcium influx activates catabolic processes mediated by
proteases, lipases, and nucleases (Ankarcrona et al., 1995). In addition, activation of nNOS,
PLA2 and other Ca2+ dependent enzymes, leads to production of NO, arachidonic acid
metabolites and superoxide, which act as additional triggers of cell death (Dirnagl et al.,
1999) (Lo et al., 2003). For these and other reasons, oxidative phosphorylation becomes
uncoupled, leading to further ATP depletion, ROS production and release of stored Ca2+
from mitochondria, further accelerating a series of catastrophic events that lead to acute cell
death.
Despite the validation of glutamate toxicity in the laboratory setting and its enormously
positive impact on the quest for new therapeutics in neuroscience, numerous clinical trials
targeting NMDA and AMPA receptors have failed to improve outcome in stroke patients
(Ginsberg, 2009). Although replete with shortcomings in trial design and implementation,
the failure to translate has reinforced the notion that the glutamate hypothesis may have
oversimplified the complexity of the cell death process, and underestimated the diversity of
expressing cell types as well as the heterogeneity of glutamate responses following receptor
over stimulation in stroke. Moreover, it is likely that NMDA-AMPA pathways comprise
only a subset of routines disrupting ionic imbalance following glutamate receptor activation.
As originally conceived, the NMDA-AMPA model did not consider the delayed and

invariably fatal glutamate-independent calcium influx following oxygen-glucose deprivation

or the failure of glutamate receptor blockade to halt cell death after very intense oxygen-
glucose deprivation (Aarts et al., 2003). Equally importantly, it could not account in an
obvious way for the death of non-neuronal cell types such as, astrocytes, vascular cells or
microglia in ischemic tissue. An alternative viewpoint is that the drugs tested in clinical
trials were poorly chosen, because many of them block all receptor functions (Lipton,
2007a). As a result, the dosing may have been suboptimal, due to the development of
untoward side effects that prevented dose escalation to neuroprotective levels. A more
suitable approach would be to use therapeutic agents that selectively target excessive
channel opening (e.g., uncompetitive inhibitors with a relatively fast off-rate, such as
memantine (Orgogozo et al., 2002; Mobius and Stoffler, 2002; Lipton, 2006; Kaviragan and
Schneider 2007) thereby retaining normal receptor functions (Lipton, 2007a).
To add to the complexity, it is now known that NMDA receptor location and subunit
composition differentially regulate neuronal survival or death, although the strength of the
calcium signal may specify the fate of neurons best following NMDA over activation
(Stanika et al., 2009). With notable exceptions, selective enhancement of synaptic receptors
(in which NR2A peptide subunits predominate over NR2B) promotes neuronal survival
whereas activating extrasynaptic receptors (in which NR2B peptide subunits predominate
over NR2A) promote cell death (Chen et al., 2007), in part, by activating death-associated
protein kinase 1 (Tu et al., 2010). Turning CREB-dependent signaling and BDNF expression
up or down is critical for cell fate determination during NMDA receptor activation. The
MAP kinase family members P38 and ERK1/2 participate, most likely by phosphorylation-
dependent regulation of submembrane scaffolding proteins and NR2A and NR2B receptor
subunits (Hardingham et al., 2002). Under ischemic conditions, the vulnerability to cell
death is reduced when postsynaptic scaffolding proteins are modified and PSD-95 binding is
prevented (Cui et al., 2007). Moreover, successfully targeting the glutamate receptor
complex early on would also diminish the opening of downstream channels, e.g., transient
receptor potential channels (TRP) and acid sensing ion channels, as well as suppress the
frequency of cortical spreading depolarizations (see below), ongoing within the ischemic
penumbra. Hence, understanding and leveraging the repertoire of diverse responses and
mediators at the receptor and second messenger level can only enhance possibilities for
successfully targeting excitotoxicity for the treatment of ischemic injury.
Calcium dysregulation—It is also known that increased calcium influx from glutamate
receptor overactivation combined with release of Ca2+ from mitochondria and other stores
(e.g., endoplasmic reticulum) may not fully account for the irreversible buildup of
intracellular Ca2+ after excitotoxic stimulation. Other channels and ion pumps activated
during ischemia in addition to glutamate receptors have been implicated in the Ca2+
accumulation. These include the Na/Ca2+ exchanger (Bano et al., 2007) hemichannels
(Contreras et al., 2004), acid sensing ion channels (Xiong et al., 2004), volume regulated
anion channels (Kimelberg et al., 2006; Simard et al., 2007), and TRP channels (Aarts and
Tymianski, 2005). In particular, ASIC1a, activated by ischemia-induced acidosis, is
involved in the Ca2+ influx and its inhibition is neuroprotective (Simon, 2006). ASICs are
stimulated within the pH range commonly found in ischemic brain tissue, thus explaining
the well established link between acidosis and worsening of ischemic outcome in animals
and humans. Failure of Ca2+ efflux mechanisms, especially the Na/Ca2+ exchanger also
contributes to the Ca2+ accumulation. Prostaglandin E2 EP1 receptors have a role in the
failure of the Na/Ca2+ exchanger during ischemia and their inhibition is markedly
neuroprotective (Abe et al., 2009); (Kawano et al., 2006). Therefore, targeting Ca2+ influx-
efflux mechanisms may be a valuable approach to counteract Ca2+ dysregulation and
associated injury in conjunction with novel drugs targeting glutamate receptors.

Oxidative and Nitrosative Stress—Oxidative and nitrosative stress are also powerful
mediators of ischemic injury. In one scenario, the redox environment of cells modulates
signal transduction cascades that tip the balance between pro-death and pro-survival
pathways (Crack and Taylor, 2005). In the second scenario, ROS and perhaps reactive
nitrogen species, including peroxynitrite, act directly as executioners of cell death (Chan,
2001). The brain appears particularly vulnerable to radical-mediated attack because of its
limited antioxidant defenses (Adibhatia and Hatcher, 2010). Until recently, it was accepted
that during ischemia ROS were generated principally by mitochondria, a well-known ROS
source during electron transport and oxidative phosphorylation. However, it was recently
shown that NADPH oxidase generates the majority of superoxide anion produced in vivo
and in vitro during NMDA receptor activation and ischemia (Brennan et al., 2009a;
Girouard et al., 2009). Thus, the mitochondrion does not appear to be the major source of
radicals following NMDA receptor activation. However, the close proximity of NADPH
oxidase to neuronal mitochondria (Girouard et al., 2009) raises that possibility that this
enzyme generates “kindling” ROS that promote mitochondrial uncoupling, triggering a
secondary ROS surge from mitochondria (fig. 1). These observations point to NADPH
oxidase as a potentially important therapeutic target in stroke, but also highlight the need for
a better understanding of the interaction among the different ROS sources and the relative
contribution of vascular vs. neuronal sources. In this regard, a surprising finding has been
that activation of NMDA receptors increases ROS both in neurons and vascular cells
(Girouard et al., 2006), suggesting a role of vascular ROS in excitotoxic brain damage.
Although this observation emphasizes the close relationships between vessels and neurons
(see Protecting the Neurovascular Unit), it has not been established how vascular oxidative
stress may contribute to tissue outcome in excitotoxic brain injury.
NO and related oxidation products are key players in excitotoxicity. Reactive nitrogen
species have important cellular effects, such as inhibition of key mitochondrial enzymes,
facilitating mitochondrial transition pore formation, DNA damage, PARP activation, and
activation of Ca2+ permeable TRPM7 channels (Aarts and Tymianski, 2005) (Pacher et al.,
2007). As a potential second mechanism, NO modifies protein groups by covalently
attaching to cysteine residues forming S-nitrosothiol derivatives. This posttranslational
modification significantly impacts cell survival by altering for example, the function of
critical regulatory proteins such as caspases, metalloproteases (Gu et al., 2002), and the
glycolytic enzyme GAPDH (Nakamura and Lipton, 2009). As NO has the potential to target
a number of specific cysteine residues to alter protein function, the impact of S-nitrosylation
is [largely] underexplored in the ischemic process.
Taken together, the experimental evidence implicating oxidative and nitrosative stress in
stroke is strong. But the translation of these fundamental concepts into clinical applications
has proven to be challenging. Most recently, a nitrone-based radical spin trap failed in the
final Phase 3 clinical trial for acute ischemic stroke (Shuaib et al., 2007). And more broadly,
the history of anti-oxidant therapies in clinical trials for neurodegeneration has been littered
with many disappointments (Kamat et al., 2008). Over-production of reactive nitrogen and
oxygen species are surely damaging to brain cells. But at lower, homeostatic levels, radicals
are critical signaling molecules participating in normal neuronal and vascular function
(Faraci, 2006). Finding novel ways to scavenge or suppress deleterious radicals without
interfering with endogenous signaling will be important to design effective therapies. As
discussed above for excitotoxicity, a desirable strategy would be to develop drugs that only
become activated by the pathological state intended for inhibition, i.e., during oxidative
stress (Lipton, 2007b). Another approach would be to test antioxidant molecules with
pleiotropic properties, such as activated protein-C (APC). APC blocks ROS generation,
suppresses post-ischemic inflammation and blocks apoptosis in neurons and endothelium,
thereby providing both parenchymal and vascular protection (Yamaji et al., 2005) (Cheng et

al., 2006) (Liu et al., 2004). APC, as well as other pleiotropic agents with a broad spectrum
of activity in experimental stroke, are undergoing clinical trial testing (Ginsberg, 2009).
Cortical Spreading Depolarizations—In addition to excitotoxic events at the molecular

and cellular level, massive release of glutamate and ionic imbalance negatively impact the
evolution of ischemic injury at the tissue level. Cortical spreading depolarization (CSD), a
high energy consuming phenomenon akin to the cortical spreading depression of Leao, is
triggered by high levels of extracellular glutamate and K+ and is characterized by slowly
propagating massive depolarization of neurons and astrocytes along with drastic disruption
of ionic gradients (Somjen, 2001). Electrophysiological and imaging data convincingly
demonstrate CSD in patients with ischemic stroke (Dohmen et al., 2008). In experimental
animals, a single cortical spreading depolarization expands the area of severe hypoperfusion
by more than 20% (Shin et al., 2006). In fact, it appears that a greater frequency but more
importantly, duration of DC shifts correspond to accelerated infarct maturation and core
expansion (Mies et al., 1993) (Back et al., 1996). The infarct expansion is probably due to
mismatch between high metabolic demand to support membrane repolarization and marginal
tissue perfusion due to constrained penumbral blood flow (Back et al., 1996). Hence,
suppressing CSD may prove worthwhile for reducing infarct maturation. Notably, the non-
competitive NMDA receptor antagonist ketamine markedly suppressed CSD frequency in
head injury patients (Sakowitz et al., 2009), but it is unclear whether it improved clinical
outcome. By contrast, irreversible depolarizations resistant to NMDA receptor antagonists
(anoxic depolarization) develop in the ischemic core territory (Nellgard and Wieloch, 1992)
and are probably terminal events contributing to severe ionic imbalance and tissue failure.
Inflammation—Focal ischemia evokes a robust inflammatory response that begins within

a few hrs of onset and typifies the secondary or delayed response to ischemia. It involves
activation of microglia and astrocytes as well as influx of hematogenous cells recruited by
cytokines, adhesion molecules and chemokines across the activated blood vessel wall.
Stimulated brain cells and blood vessels then communicate with one another through a
complex network of paracrine and autocrine signaling. The highly choreographed response
evolves over many days, with multiple signals and targets triggering expression of novel
proteins and secondary infarct expansion (Kleinig and Vink, 2009). Innate and adaptive
immune systems participate as well. At least early on, inflammation amplifies the ischemic
lesion, but, as discussed later, it may it may set the stage for tissue repair in the late post-
ischemic period. A number of therapeutic trials designed to target the inflammatory response
have failed to show clinical benefit (Sughrue et al., 2004), underscoring the need to clarify
further the complexities of acute and delayed post-ischemic inflammatory signaling within
brain.
As noted above, cerebral blood vessels are the first to be exposed to the ischemic insult and
their reaction to injury sets the stage for the inflammatory response. Early on, production of
cytokines, such as TNFα and IL1β, in vascular cells and perivascular microglia-
macrophages upregulates adhesion molecule expression (e.g, ICAM-1, P and E-selectin)
and, along with integrins, promote leukocyte rolling and sticking to the vessel surfaces
(Zhang et al., 1998). Neutrophils are the first blood borne cells to be recruited into the brain,
followed by monocytes and, starting on days 1–2, lymphocytes. As the brain’s primary
immune cell, activated microglia transform into macrophages and accumulate at the border
zone along with blood-borne macrophages to clear debris and dead cells, and produce
proinflammatory mediators and toxic molecules (Schilling et al., 2003).
Molecules released from injured or dying brain cells also contribute to the inflammatory
response. In particular, products from cells undergoing oxidative stress and necrosis, for
example lipopeptides, advanced glycation end-products (AGE), modified lipids, heat shock

proteins, hyaluronic acid, and the nuclear protein HMGB1, lead to activation of the innate
immune system by engaging toll like receptors (TLRs). TLRs, are expressed in multiple
cells and, in conjunction with CD36, receptors for AGE (RAGE) and other scavenger
receptors, may serve to detect “danger signals” triggering NFκb-dependent inflammatory

signaling (Oppenheim and Yang, 2005) (Wang et al., 2007). PARP activity appears to
function as an NFκb co-activator (Chiarugi and Moskowitz, 2003), with therapeutic
potential for delayed administration of PARP-1 inhibitors (Kauppinen et al., 2009). Beside
NF-kb, other transcriptional activators including activator protein-1, STAT-3, interferon
regulatory factor 1, and C/EBP beta, exert a proinflammatory effect (Kapadia et al., 2006)
(Iadecola et al., 1999), while PPARgamma acts as an a negative modulator of post-ischemic
inflammation (Kapadia et al., 2008). In addition to neutrophils and monocytes, T cell subsets
have recently been implicated as modulators of secondary infarct progression. Treg cells
express the antinflammatory cytokines IL10 and TGFβ, and normally dampen the immune
response by suppressing T cell effector activity (Liesz et al., 2009). Secondary infarct
progression is impacted by a second subset of invading T lymphocytes, gamma-delta T cells,
a source of the proinflammatory cytokine IL-17. After deletion of IL-17, infarct size
decreases (Shichita et al., 2009).
These findings support the notion that taming post-ischemic inflammation may block
secondary events that extend brain injury. However, as noted below, during the later stages
in the injury process, inflammation promotes critical events necessary for tissue repair.
Therefore, therapeutic interventions targeting post-ischemic inflammation should be mindful

of temporal considerations by minimizing the destructive potential of inflammation in the
acute phase while enhancing its beneficial contributions to tissue repair in the late stages of
cerebral ischemia.
Necrosis, necroptosis and autophagy: The execution

Necrosis and apoptosis are the principal mechanisms of cell death after ischemic injury, and
the details of both have been reviewed (Yuan, 2006, 2009). Depending in part upon the
magnitude, type of stimulus, and cell type, a number of cell death triggers promote necrosis
or apoptosis, although cellular features of apoptosis and necrosis are sometimes found
together in dying ischemic cells. Probably due to its ATP dependence, apoptotic cell death is
prominent after milder injury and is more delayed (Bonfoco et al., 1995), although severe
ischemia is associated with early-onset caspase cleavage, enzyme activation and acute
cleavage of substrate proteins (Namura et al., 1998). Apoptosis and necrosis can be induced
by multiple triggers such as Ca2+ overload or oxidative stress. Mitochondria play an
essential role in both (Schinzel et al., 2005), serving as a reservoir for pro-apoptotic and
anti-apoptotic proteins and cytochrome C. One such protein apoptosis inducing factor (AIF)
is released from the outer membrane of mitochondria in response to activation of the nuclear
protein PARP and generation of a PAR polymer, its toxic product (Yu et al., 2006). AIF
then translocates to the nucleus to promote a caspase independent form of cell death in part
by promoting DNA fragmentation and chromatin condensation. Release of mitochondrial
proteins sets in motion (or suppresses) multiple caspase-dependent and independent
processes to dismantle critical homeostatic and repair mechanisms. Despite the expression
of executioner pro-caspases, activated caspases and caspase cleavage products, classic
apoptotic morphology is rarely found in humans within adult ischemic brain. However, it
does appear more prominently in the ischemic brains of neonates (Ferriero, 2004).
Until now, necrotic cell death was defined as unregulated, unprogrammed and lacking cell
signaling events. It is the predominant form of ischemic cell death. It was recently shown
that certain cells including those within brain possess a molecular switch that determines
whether cells die by necrosis or apoptosis in a TNF-dependent way (Holler et al., 2000). In
this cell death variation, necrosis is triggered by two kinases, RIP 1 and RIP 3, reciprocally

interacting, often in the presence of an apoptosis inhibitor (Cho et al., 2009). Activated RIP3
is capable of stimulating enzymes such as the glycogen degrading enzyme, glycogen
phosphorylase to steer TNF-induced apoptosis towards necrosis in part, through bursting
energy metabolism and ROS generation (Zhang et al., 2009). Nec-1, a small molecule
inhibitor of RIP-1, decreases ischemic cell injury in models of ischemia-reperfusion and
improves neurological outcome, suggesting RIP-1 as a potential therapeutic target (Degterev
et al., 2005).
Autophagy, an energy-dependent process implicated as a cell death mechanism, is used by

eukaryotes to degrade and recycle subcellular organelles. Its precise role in the death process
is controversial because autophagic activity maybe cytoprotective, at least in some disease
models, e.g., polyglutamine expansions or in aging models (Levine and Kroemer, 2009). In
other disease models such as in neonatal hypoxia-ischemia, autophagy reportedly promotes
cell death (Puyal et al., 2009). Precisely how this is accomplished remains for further study.
Stroke and dementia

The major mechanisms of tissue dysfunction and death reviewed above develop as a
consequence of acute vessel occlusion and the attendant activation of endogenous
mechanisms of cell injury and tissue demise. More subtle and less well understood
mechanisms are beginning to define a new research frontier linking cerebrovascular
dysfunction and small strokes to the development of vascular cognitive impairment (VCI)
and Alzheimer’s disease (AD), the most common causes of dementia (Fotuhi et al., 2009).
AD and “vascular” dementia have traditionally been considered distinct entities (Iadecola,
2004). Thus, AD, the most prevalent of the two, is characterized pathologically by
deposition of the amyloid beta peptide (Abeta) in the brain parenchyma (amyloid plaques)
and blood vessels (amyloid angiopathy), and by neurofibrillary tangles (Querfurth and
LaFerla, 2010). On the other hand, cerebrovascular diseases can lead to cognitive
impairment in different ways. For example, a single stroke can cause dementia by damaging
brain regions critical for cognition (strategic infarct dementia), while multiple strokes can
cause stepwise cognitive deterioration through cumulative brain damage (multi infarct
dementia) (Leys et al., 2005) (Pinkston et al., 2009). Most often VCI is due to small white
matter lesions (leukoaraiosis) that are thought to interrupt neural pathways involved in
cognition (Pinkston et al., 2009). A recent realization has been that AD and cerebrovascular
diseases coexist in up to 60% of cases (Leys et al., 2005) (Pinkston et al., 2009). Thus,
although cases of “pure” AD and vascular dementia can exist, in most cases the cognitive
decline can be attributed to overlapping cerebrovascular and AD pathologies (Querfurth and
LaFerla, 2010). The causes of leukoaraiosis and the overlap between AD and
cerebrovascular diseases are discussed in the next sections.
What causes the white matter damage underlying VCI?

In the cerebral autosomal dominant arteriopathy with subcortical infarcts and
leukoencephalopathy (CADASIL syndrome), a monogenic disease associated with
dementia, extensive white matter damage is caused by notch-3 mutations in vascular smooth
muscle leading to vascular insufficiency (Chabriat et al., 2009). In common forms of
leukoaraiosis, however, the mechanism of the white matter damage remains unclear,
although family history, hypertension, diabetes, and chronic smoking are well-established
risk factors (Selnes and Vinters, 2006). The white matter lesions consist of demyelination,
axonal loss, enlarged perivascular spaces, astrogliosis and microglial activation (Fernando et
al., 2006) (Pantoni and Garcia, 1995). The wall of arterioles is thickened by accumulation of
hyaline material (lipoialinosis) or completely disrupted (fibrinoid necrosis) leading to
microhemorrhages (Fisher, 1968) (Pantoni and Garcia, 1995). Capillary density, resting

CBF and cerebrovascular reactivity are reduced both in normal and affected white matter
(Brown et al., 2007) (Mandell et al., 2008) (O’Sullivan et al., 2002).
Disruption of the arteriolar wall by amyloid, as observed in genetic or sporadic forms of

cerebral amyloid angiopathy and in AD, is also associated with white matter lesions (Weller
et al., 2009). Hypoxia-inducible genes and markers of endothelial activation are upregulated,
suggesting hypoxia-ischemia (Fernando et al., 2006). A causative role for hypoxia-ischemia
is also suggested by the fact that the blood supply to the subcortical and basal ganglia white
matter is provided by terminal arterioles located at the watershed between two vascular
territories: perforating arteries from the neocortex and penetrating branches arising from
larger arteries at the base of the brain (Iadecola et al., 2009). Therefore, these areas may be
particularly susceptible to vascular insufficiency, especially if the structure of the arterioles
and their vasomotor function are altered. On the other hand, energy and blood flow
requirements of white matter are much lower than those of gray matter, rendering the tissue,
in theory, less vulnerable to anoxia-ischemia (Hertz, 2008). Therefore, alternative
mechanisms leading to an increased intrinsic vulnerability to injury may also play a role. For
example, risk factors for dementia could enhance white matter susceptibility to anoxia, as
aging has been described to do (Baltan et al., 2008). Disruption of the BBB, an early event
in leukoaraiosis, could lead to injury by producing perivascular edema, micro-hemorrhages
and inflammation, contributing to demyelination (Farrall and Wardlaw, 2009). Demylinated
axons may be more susceptible to ischemic injury due to their heightened metabolic
requirements caused by loss of energy-efficient saltatory conduction and leaky sodium

channels (Trapp and Stys, 2009). Furthermore, dysfunction within the endothelium may
compromise the trophic effect that these cells exert on neurons and glia, contributing to
brain dysfunction and damage (see section on repair mechanisms). Aging, inflammation,
hypoxia and, possibly, other risk factors can also reduce the repair potential of the damaged
white matter by impairing oligodendrocyte progenitors recruitment and differentiation (Back
et al., 2002) (Pang et al., 2010) (Sim et al., 2002). Therefore, the mechanisms underlying
leukoaraiosis might be distinct from those of ischemic cell death and are a fertile ground for
new investigations.
Why does stroke increase the risk of dementia?

Stroke doubles the risk of developing dementia (Leys et al., 2005). Although, as discussed
above, location, size, and number of ischemic lesions, as well as leukoaraiosis can account
for the cognitive impairment in some cases, epidemiological and pathological evidence
indicates that stroke also increases the risk for AD. Alzheimer pathology can facilitate the
development of dementia in patients with ischemic injury and, vice-versa, ischemic injury
can aggravate the cognitive deficits induced by AD (Fotuhi et al., 2009). These findings
have suggested a previously unrecognized interaction between amyloid pathology and
ischemic injury. This is not surprising considering that Abeta has powerful cerebrovascular
effects. Abeta constricts cerebral vessels, suppresses vascular reactivity and impairs
autoregulation, leading to vascular insufficiency and increased susceptibility to ischemic
injury (Iadecola, 2004) (Zhang et al., 1997). Conversely, ischemia and hypoxia can induce
Abeta accumulation by promoting its cleavage from APP (Tesco et al., 2007) and by
downregulating the lipoprotein related receptor protein-1, a critical receptor for the vascular
clearance of Abeta (Bell et al., 2009) (Tesco et al., 2007) (Wu et al., 2005). Therefore, in a
vicious cycle, the vascular effects of Abeta aggravate ischemia, and, in turn, brain ischemia
enhances Abeta accumulation (Iadecola, 2004). Recent studies have also linked plasma
Abeta to the white matter damage underlying leukoaraiosis (Gomis et al., 2009) (Gurol et
al., 2006). The significance of this finding and its underlying mechanisms remain to be
defined and placed in context with other findings showing that Abeta could promote axonal
injury directly through its cytotoxic effects (Querfurth and LaFerla, 2010) or indirectly by

inducing vasoconstriction and ischemia (Iadecola, 2004). Therefore, the vascular

dysfunction induced by circulating and parenchymal Abeta may act in concert with the
structural and functional vascular changes induced by risk factors to amplify the vascular
insufficiency in small white matter arterioles leading to white matter injury. However,
factors other than Abeta could also contribute to the interplay between AD pathology and
cerebrovascular diseases. For example, cerebral arterioles of AD patients exhibit a tendency
to constrict due to increased activity of transcription factors (serum response factor/
myocardin) that control the expression of contractile proteins in smooth muscle cells (Chow
et al., 2007). This finding may play a role in the increased vasoconstrictor tone and reduced
CBF observed in AD (Iadecola, 2004). It remains to be established whether these changes in
gene expression are related to Abeta or to upstream molecular events independent of it.
How does the brain repair itself after stroke?

A well-known fact in clinical stroke is that most patients show some degree of recovery over
time. As the brain recovers, considerable remapping of cortical areas take place. Functional
MRI studies demonstrate that peri-infarct areas are highly dynamic (Dijkhuizen et al., 2003)
(Chopp et al., 2007). Representational areas shift as latent networks are unmasked and
parallel circuits are recruited adjacent to damaged regions (Murphy and Corbett, 2009).
More recently, advanced optical imaging techniques combined with electrophysiology
demonstrated that this remodeling at a circuit level can be correlated with specific processes
of dendritic and synaptic plasticity at a cellular level (Li and Murphy, 2008). Imaging of
mitochondrial function (Liu and Murphy, 2009) and vascular dynamics (Schaffer et al.,
2006) have provided new insight into how the brain responds to microvascular perturbations
following ischemia. These powerful tools may eventually allow one to interrogate
mechanisms of stroke recovery at a molecular and cellular level while testing pro-recovery
therapies and strategies (Murphy and Corbett, 2009; Zhang and Chopp, 2009).
Repair and remodeling processes after stroke

A major advance within this past decade has been the realization that adult mammalian
brains also exhibit focal areas of ongoing neurogenesis in the subventricular zone and
subgranular zone. Under normal conditions, these newborn neurons migrate to olfactory
regions and hippocampus. But after cerebral ischemia or hemorrhage, the birth of new cells
seems to increase and neuroblasts re-route towards damaged brain tissue (Arvidsson et al.,
2002); (Parent et al., 2002), perhaps as part of an endogenous attempt by the brain to repair
itself. However, whether these new neurons significantly contribute to functional recovery
remains unclear. Neurogenic responses may persist for surprisingly long periods of time, but
the majority of these newborn cells survives only for a few days (Arvidsson et al., 2002).
Whether these cells develop normally, express neurotransmitters or even integrate into
existing neural networks remain to be fully elucidated. Future attempts to promote

endogenous neurogenesis for therapeutic purposes will have to address ways to modify
parenchymal micro-environments to enhance neuronal survival and integrate newly born
cells into viable circuitry.
In concert with neurogenesis and neural plasticity, the brain recovering from stroke exhibits
complex patterns of vascular remodeling. Angiogenesis and vasculogenesis in peri-infarct
regions have been detected in rodent models of cerebral ischemia (Ding et al., 2008) as well
as in human stroke (Krupinski et al., 1996). Indeed, it is now recognized that angiogenic and
neurogenic responses are tightly co-regulated after stroke and brain injury (Arai et al.,
2009). This might not be surprising since molecular mechanisms of neurogenesis and
angiogenesis have been evolutionarily conserved so that similar mediators and pathways are
involved in both phenomena (Carmeliet and Tessier-Lavigne, 2005). In the normal brain, the
neurovascular niche defines these complex mechanisms of cell-cell signaling between

cerebral endothelium and neural precursors in the subventricular and subgranular zones of
ongoing neurogenesis. In the context of post-stroke recovery, these close relationships
between neurogenesis and angiogenesis are maintained. Neuroblasts migrate along
perivascular routes (Thored et al., 2007). Promotion of neurogenesis enhances vascular

regrowth and conversely, angiogenic stimulation enhances neurogenesis (Ohab et al., 2006;
Taguchi et al., 2004). Hence, it seems likely that brain recovery after stroke comprises inter-
dependent neurovascular plasticity and remodeling processes that recruit multiple common
mediators and signals (Snapyan et al., 2009). The new frontier of neurovascular repair may
require a detailed exploration of how these multi-cell and multi-signal phenomena are
regulated in the context of stroke. Recent experimental studies have suggested that
pharmacologic interventions such as EPO, statins, activated protein C, and
phosphodiesterase inhibitors may promote functional recovery after cerebral ischemia
(Wang et al., 2004) (Zhang et al., 2006) (Thiyagarajan et al., 2008) (Chen et al., 2009).
Ultimately, therapies that can augment these endogenous signals and substrates of
neurovascular remodeling may have long therapeutic time windows in stroke (Zhang and
Chopp, 2009).
Moving forward: charting a course toward new stroke therapies

Tremendous progress has been made in the understanding of fundmental mechanisms of
neuronal cell death. However, the translation of these powerful molecular and cellular
principles into clinically effective neuroprotective therapies in stroke has been challenging.
More recently, emerging data from both experimental and clinical studies now suggest that
all cell types in the brain participate in the complex pathophysiology of brain injury
following stroke. Consequently, therapeutic approaches should target multiple cell types in
an attempt to protect their structural and functional integrity, and their reciprocal
interactions.
Protecting the Neurovascular Unit

The so-called “neurovascular unit” provides a conceptual framework that integrates
responses in all cell types, including neuronal, glial and vascular elements (del Zoppo, 2009)
(Iadecola, 2004) (Zacchigna et al., 2008) (fig. 3). Cell-cell interactions in the neurovascular
unit form the basis for brain function. Dysfunctional signaling in the neurovascular unit
underlies the basis for disease. Whereas the intricate molecular pathways of cell death in the
neuron have been dissected in detail, the mechanisms of how the entire neurovascular unit
responds to stroke are not completely understood. As discussed in the previous sections,
cerebral ischemia can rapidly induce perturbations in cell-cell signaling and function within
the neurovascular unit. How each cell type succumbs or adapts to injury is dependent on
responses in other cells. For example, genomic and proteomic responses in neurons
subjected to metabolic stress have been fairly well described. But how each cell type might
alter its response to vascular risk factors and ischemia in the context of other elements in the
neurovascular unit remains to be elucidated. As the nexus of cell-cell interactions grows, a
systems biology approach may eventually be required to rigorously define mechanisms as
well as targets for complete neurovascular protection. The importance of cell-cell signaling
in the neurovascular unit has been underscored by many studies (Arai and Lo, 2009b)
(Dugas et al., 2008) (Eroglu et al., 2009), (Guo et al., 2008). Hence, under normal
conditions, trophic coupling within the neurovascular unit helps maintain homeostasis. In
contrast, under diseased conditions, it is likely that loss of coupling would lead to
pathophysiology (Grammas et al., 1999) (Nagai et al., 2007). Loss of trophic coupling is
also likely to play a role in the way vascular risk factors render neurons and glia more
vulnerable, such as in the link between leukoaraiosis and brain dysfunction underlying
cognitive impairment. Recently, BDNF has been implicated as a candidate factor that

mediates trophic coupling between vascular and neuronal compartments (Arai and Lo,
2009a) (Guo et al., 2008). Insofar as BDNF polymorphisms have been implicated in
modulating synaptic plasticity (Kleim et al., 2006), these neurovascular trophic signals may
also influence mechanisms of recovery after stroke.
The concept of the neurovascular unit may provide an integrated framework for
investigating mechanisms and therapies. Salvaging neurons alone may not be sufficient. One
has to protect glial and vascular elements as well. Future investigations might be better
served by pursuing targets that are expressed on multiple cell types in brain. Furthermore,
prevention of cell death alone may also not be sufficient. In order for therapies to be
clinically meaningful, cellular function must be preserved and/or restored. Neurons that are
alive but lack proper connectivity or do not express the correct array of transmitter release-
reuptake and synaptic activity may not be functional. Astrocytes that survive but cannot
provide hemodynamic coupling would be unable to link neuronal activation with the
required vascular responses. Oligodendrocytes that are respiring but metabolically impaired
might not provide the necessary myelination for axonal conduction. Endothelial cells that
survive but do not maintain blood-brain barrier properties would facilitate damage to
adjacent parenchyma. Ultimately, any stroke therapy must include both prevention of cell
death as well as rescue of integrated neurovascular function. Ischemic tolerance and post-
ischemic hypothermia are some of the most potent protective strategies for ischemic brain
injury and represent examples of multimodal therapies targeting the neurovascular unit as a
whole. These are described in the next sections.
How does the brain protect itself? Lessons from ischemic tolerance
Some of the very same mechanisms implicated in the tissue damaging responses after stroke
can also be engaged to protect the brain after threatened injury and to induce ischemic
tolerance (IT). IT is a concerted organ response to protect itself against tissue injury and has
been widely studied over the past 3 decades in an effort to identify endogenous mechanisms
of protection and to exploit them for therapeutic purposes (Obrenovitch, 2008). In its
delayed form, it is induced after challenge by a subthreshold noxious stimulus, developing
within 1–3 days and lasting for several additional days. Experimentally it can be induced by
exposure to preconditioning stimuli such as neurotoxins (e.g., glutamate) substrate
deprivation (e.g., ischemia, hypoxia), or inflammation (e.g., cytokines and immune
responses) and engages mechanisms that detect, transduce and promote a complex tissue
reaction that reflects a fundamental genomic reprogramming after threatened injury (Dirnagl
et al., 2009) (Gidday, 2006) (Stenzel-Poore et al., 2004). In this altered physiological state,
tissue damage is mitigated when challenged by exposure to a wide range of deleterious
stimuli, even if unrelated to the initial challenge.
IT is mediated by highly redundant cellular, molecular and physiological mechanisms

involving neurons, astrocytes and vascular cells (Gidday, 2006). Upstream sensors have
been implicated including adenosine and its A1 receptor, the NR2A NMDA receptor
subunit, and the transcriptional regulator hypoxia inducible factor (HIF). HIF is an attractive
candidate because it regulates a constellation of genes including those whose protein
products facilitate oxygen transport (erythropoietin), regulate glycolytic metabolism (e.g.,
glucose transporters), and promote angiogenesis (e.g., VEGF), by way of example.
Unexpectedly, genetic deletion of HIF1α improves infarct volume after middle cerebral
artery occlusion, implicating as yet unknown pathogenic mechanisms activated by ischemia
(Helton et al., 2005). Erythropoietin, one of the gene products of HIF1α, promotes PI3
kinase/AKT activity and the expression of NFkB-dependent protective genes (Digicaylioglu
and Lipton, 2001). Erythropoietin also downregulates hypoxia-induced pro-apoptotic
mechanism (Noguchi et al., 2007) (Prass et al., 2003) (Ruscher et al., 2002), and
erythropoietin-based therapies have been investigated in the clinical arena (Ehrenreich et al.,

2009). Heat shock protein family members are upregulated in ischemic tolerance and, based
on their multiple cytoprotective actions, may promote survival. Preconditioning stimuli
preserve connexin 43 hemichannels in astrocytes facilitating ATP released from these cells
and increasing the extracellular concentration of the beneficial nucleoside adenosine (Lin et
al., 2008). At the same time, preconditioning safeguards the function of cerebral blood
vessels from the deleterious effects of cerebral ischemia resulting in improved penumbral
flow and reduced brain damage (Kunz et al., 2007b). These astrocytic and vascular changes
are coupled with mechanisms that reduce energy demands, lessen inflammatory responses,
and ion channel activity, and decrease cascades promoting blood clotting and apoptotic cell
death (Dirnagl et al., 2009) (Obrenovitch, 2008) (Gidday, 2006). Factors involving
mitochondria as well as its uncoupling proteins have also been suggested and include
improved efficiency of ATP synthesis, preservation of the mitochondrial membrane
potential and delayed opening of mitochondrial transition pore formation, as well as opening
of mitochondrial ATP-dependent K+ channels (Dirnagl and Meisel, 2008). One of the key
questions defying explanation at the moment is how and why diverse preconditioning
stimuli targeting single genes or key steps in seemingly unrelated cascades all impact infarct
injury in genetically engineered and wild type mice. It may be telling us that the
mechanisms through which the brain protects itself involve a highly integrated network
response that emphasizes multiple cells within the neurovascular unit. If true, comparable
multicellular approaches are needed that capitalize on this integrated response in the design
of successful treatments (see next section).
Therapeutic Hypothermia: Engaging Pleiotropic Mechanisms in Multiple Cell Types

The therapeutic hypothermia field has advanced significantly during the past decade,
inspired by positive outcomes from clinical trials in cardiac arrest victims (Bernard et al.,
2002). Satisfactory neurological recovery was achieved in about half of the treated patients
when treatment was begun within 2 hrs of cardiac arrest. Promising results were reported in
small feasibility studies in neonatal ischemia (Shankaran et al., 2005). Current efforts are
directed towards translating these successes in cardiac arrest and neonatal ischemia to
patients with ischemic stroke, but there remain unresolved technical issues surrounding the
timing and method for inducing hypothermia, and the deleterious effect of re-warming
(Wagner and Zuccarello, 2005). It is now well accepted that hypothermia reduces ischemia
in animal models of stroke, and so far, preclinical results have helped guide the design of
clinical trials in human subjects. Combining hypothermia with thrombolysis is one example.
Experimental evidence suggests that hypothermia protects the brain through pleiotropic
effects acting on multiple cell types within the neurovascular unit. Hypothermia reduces
brain metabolism, thereby significantly decreasing oxygen consumption and the work load
of the entire organ (Sakoh and Gjedde, 2003). Hypothermia also reduces glutamate release,
ROS production, and peri-infarct depolarizations. Inflammatory markers decrease, as well as

microglial activation, leucocyte infiltration, MMP activation and BBB disruption. In
addition, hypothermia suppresses apoptotic cell death, decreases mitochondrial release of
cytochrome C and apoptosis inducing factor (see for review (Tang and Yenari, 2009)). Cell
survival pathways are upregulated and appear to contribute to the multiplicity of
neuroprotective mechanisms. To what extent each of these promotes tissue protection or
interacts synergistically to enhance tissue survival is not known and remains for further
investigation. Nevertheless, therapeutic hypothermia does illustrate the potential power of
approaching stroke therapy using combinations of modalities that engage multiple cell types
to enhance protection and recovery.
Additional strategies for repair and recovery: trophic factors and cell based therapies
No matter how successful acute neuroprotective strategies become, many stroke patients
may still fall outside of clinical time windows for effective treatment. Hence, approaches

that promote repair and recovery will be essential for an integrated stroke armamentarium.
Logically, these approaches can be divided into two categories – trophic factor treatments
that seek to amplify and augment endogenous processes of neurovascular plasticity and
recovery, and cell-based therapies that seek to replace damaged or lost brain cells. Many
approaches that augment endogenous recovery have been explored. Growth factors such as
NGF, FGF, GDNF and BDNF have all met with variable degrees of success in animal
models (Semkova and Krieglstein, 1999). But a limiting factor in many of these studies is
their relatively large molecular weight, making transport into brain difficult. Some
alternatives may yet exist such as smaller peptides, transnasal delivery of growth factors to
bypass the blood brain barrier (Fletcher et al., 2009), peptidomimetics and compounds that
can upregulate endogenous trophic factor production but do not require direct infusion into
brain (Zhang and Chopp, 2009). Additionally, one may also target inhibitory molecules that
accumulate in the area surrounding the injury and block neuronal repair, such as chondroitin
sulfate proteoglycans, myelin associated proteins, NOGO and semaphorins (Silver and
Miller, 2004).
Although stroke-damaged brain usually exhibits varying degrees of spontaneous recovery,

there will always be significant areas where endogenous repair is insufficient. In this regard,
cell-based therapies may represent a new frontier. To date, a variety of cell sources have
been tested including neural stem cells, bone marrow-derived mesenchymal cells as well
endothelial progenitor cells (Koch et al., 2009). Treatments have typically involved direct
transplantations into brain parenchyma, catheter infusions into cerebral ventricles, as well as
systemic injections into circulating blood. The latter approach is especially intriguing, as
complex mechanisms of cell targeting seem to be operational in brain. Infarcted brain tissue
upregulates and releases various chemokines such as SDF-1, to establish gradients that
broadly serve to attract stem and precursor cell populations (Madri, 2009). Induced
pluripotent stem cells may also provide an alternative cell source to embryonic stem cells
(Takahashi and Yamanaka, 2006). One advantage is the theoretical possibility that cell
repair can be custom-designed to suit specific diseases or individual needs, as described in a
flurry of recent studies showing that neurons can be successfully derived from pluripotent
cells taken from patients with Huntingtons disease, ALS and spinal muscular atrophy
(Dimos et al., 2008) (Ebert et al., 2009) (Park et al., 2008). Whether similar methods can be
applied to stroke victims remains to be tested.
Overall, cell-based therapies in models of experimental stroke suggest that delayed repair is
a viable treatment approach. But more work is needed to translate these promising results
into effective stroke therapies. Many questions remain and the precise mechanisms of cell-
induced repair are still unclear. For example, do the reported benefits occur because cells are
being explicitly replaced or because transplanted cells serve as a source of trophic factor
production? How well do transplanted cells survive? Will adjunctive therapies to decrease
apoptotic dropout be necessary? If cells survive, how well do they integrate into existing
neural or vascular networks? What is the optimum timing for such therapy? What types of
cells should be used? And finally, what subtypes of stroke patients should be selected for
robust clinical trial results?
As the basic mechanisms of trophic and cell-based repair continue to be defined, an

interesting underlying theme has emerged that links the survival of newborn or transplanted
cells to the host environment. As discussed in the previous sections, activated vascular cells
and microglia may be detrimental to cell survival in the early stages of cerebral ischemia,
due to production of cytokines, ROS and toxic amount of NO. In the recovery phase,
however, chronically activated microglia may now secrete beneficial factors such as BDNF
and GDNF. Hence, prolonged inflammation can serve biphasic roles – dampening cellular
recovery acutely but then enhancing tissue repair later on. In fact, this principle of biphasic

mediators may be broadly applicable in stroke pathophysiology. There are many examples.
Over-activation of NMDA receptors induces acute excitotoxicity (Besancon et al., 2008),
but without NMDA signaling, chronic neuronal remodeling cannot take place (Young et al.,
1999). Matrix metalloproteinases degrade and damage neurovascular substrates in the acute
phase (Rosenberg, 2009), but the same proteases are critically important for neurovascular
remodeling during the recovery phase (Zhao et al., 2006) (Lee et al., 2006). The intracellular
mediator HMGB1 promotes necrosis and expands the core infarct during acute cerebral
ischemia (Qiu et al., 2008) but during the repair phase, HMGB1 release from reactive
astrocytes promotes angiogenesis and synaptic plasticity (Hayakawa et al., 2010a)
(Hayakawa et al., 2010b) Altogether, the boundaries between cell death and cell repair may
be blurred (Lo, 2008b). Future studies that dissect these biphasic mechanisms should prove
fruitful if we are to develop clinically meaningful strategies for both neurovascular
protection as well as repair.
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Figure 1. Major sources of ROS in brain and blood vessels

Stroke risk factors and ischemia lead to the activation of several ROS-generating enzymatic
systems. In ischemia, the increase in cytosolic Ca2+ activates the superoxide-producing
enzyme NADPH oxidase (NOX), through PKC and NO derived from neuronal NOS
(Brennan et al., 2009b; Girouard et al., 2009). Mitochondria become depolarized by the
Ca2+ overload (Nicholls, 2008) and, possibly, by ROS derived from NADPH located near
mitochondria (Girouard et al., 2009) producing large amounts of superoxide. The Ca2+-
induced activation of proteases and/or the pro-oxidant environment convert xanthine
dehydrogenase (XDH) to xanthine oxidase (XO) (Abramov et al., 2007), a superoxide-
generating enzyme involved in purine degradation. Finally, the substrate deprivation
induced by ischemia and the oxidative inactivation of essential co-factors like
tetrahydrobiopterin (BH4) uncouple L-arginine oxidation from NO formation by NOS,
resulting in superoxide production (NOS uncoupling) (Forstermann, 2010). Other potential
sources of ROS include enzymes for arachidonic acid or catecholamine metabolism, but
their participation in stroke-induced oxidative stress remains in question (Adibhatla and
Hatcher, 2010; Kunz et al., 2007a).

Figure 2. The Ischemic Penumbra

The ischemic penumbra represents compromised but viable brain tissue lying distal to an
occluded blood vessel. The top 3 MR images in this figure were taken in the same patient
early after occlusion of the middle cerebral artery. The images are shown in the horizontal
plane passing through the lateral ventricles. “Stunned but not dead tissue” is detected by
magnetic resonance imaging (MRI) techniques that assess (i) the spatial extent of the
perfusion or blood flow deficit that presents as red and yellow areas on perfusion weighted
imaging (PWI, top left image), and (ii) severely damaged tissue in the ischemic core that
presents as high-intensity areas on diffusion weighted imaging (DWI, top middle image)
(Ebinger et al, 2009). In this patient, the perfusion deficit in this patient is larger than
diffusion lesion. The top right image depicts the mismatch between core areas with a
diffusion lesion (dark blue) and the larger areas with low perfusion (light blue). Over hours
to days (see text), the core territory expands due to the complex cascades involving
excitotoxicity, oxidative stress, programmed cell death mechanisms and inflammation, as
the initial trigger of energy failure ultimately progresses to infarcted tissue The resulting
infarct is shown in the image at the bottom of the figure and is about the size of the initial
perfusion deficit in this untreated patient. In later stages, some patients may also partially

recover in part, due to endogenous mechanisms of repair and remodeling (Chopp et al,
2007). MRI scans courtesy of G. Albers, Stanford.

Figure 3. The Neurovascular Unit

This schematic depicts a cerebral blood vessel and surrounding brain parenchyma that
comprise the neurovascular unit. The neurovascular unit encompasses cell-cell and cell-
matrix interactions between component vascular, glial and neuronal cells. Homeostatic
signaling in the neurovascular unit sustains normal brain function. Dysfunctional
neurovascular signaling mediates injury after stroke. During the recovery phase after stroke,
neurovascular signaling may also be critically important with repair mechanisms that may
involve angiogenesis, vasculogenesis and endothelial precursor mechanisms. As the blood-
brain barrier heals, inflammation may convert from a deleterious to beneficial role. Reactive
glia (astrocytes and pericytes) may secrete trophic factors. Synaptic and dendritic plasticity
as well as axonal remyelination may provide parenchymal substrates for functional
recovery. Emerging data now suggest that these phenomenon do not occur in isolation. All
components of the NVU are likely to remodel in synchrony (Arai et al, 2009; Murphy and
Corbett, 2009; Zhang and Chopp, 2009).

Table 1
Major Modifiable Risk Factors for Ischemic Stroke
Classification Risk factor References
Causal1 Hypertension (Lawes et al., 2004a)

Diabetes (Lawes et al., 2004b)
Hypercholesterolemia (Amarenco et al., 2006)
Cigarette smoking (Bonita et al., 1999)
Atrial fibrillation (Hart et al., 1999)
Valvular heart disease (Kizer et al., 2005)
Ischemic cardiomyopathy (Loh et al., 1997)
Carotid stenosis (Rothwell et al., 2003)
Probable2 Hyperhomocysteinemia (Wald et al., 2002)

Increased inflammatory markers (WBC, CRP, infection) (Di Napoli et al., 2005)
Elevated plasma fibrinogen (Rothwell et al., 2004)
Dyslipidemia (Holme et al., 2009)
Patent foramen ovale (Nedeltchev et al., 2008)
Obstructive sleep apnea (Culebras, 2009)
Body mass index (Hu et al., 2007)

Lack of exercise (Hu et al., 2005)
Low fruit and vegetable diet (Dauchet et al., 2005)
Psychosocial stress (Simons et al., 1998)
1
Factors demonstrated in randomized clinical trials to reduce stroke risk if corrected, or for which a strong, consistent and independent association
with stroke has been established (Hankey, 2006).
2
Factors for which the association with stroke is less firm and/or no causative role has been established.
WBC, white blood cells; CRP, C-reactive protein.


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Neuron. 2010 July 29; 67(2): 181–198. doi:10.1016/j.neuron.2010.07.002.

The Science of Stroke: Mechanisms in Search of Treatments

of therapeutic opportunities for these devastating conditions. To accomplish this, novel

Stroke risk factors and triggers

How do risk factors increase the propensity to stroke?

Neuron. Author manuscript; available in PMC 2011 July 29.

additive or synergistic. Furthermore, the relative contribution of parenchymal and vascular

How do risk factors alter cerebral blood vessels?

Why now? Role of stroke triggers

Neuron. Author manuscript; available in PMC 2011 July 29.

factors, could precipitate vascular occlusion or hemodynamic insufficiency. This view is

Salvageable vs Non-Salvageable Tissue: Salvageable tissue is the target for therapy

Neuron. Author manuscript; available in PMC 2011 July 29.

Excitotoxicity—Excitotoxicity and calcium overload are major factors contributing to the

Neuron. Author manuscript; available in PMC 2011 July 29.

invariably fatal glutamate-independent calcium influx following oxygen-glucose deprivation

Neuron. Author manuscript; available in PMC 2011 July 29.

Neuron. Author manuscript; available in PMC 2011 July 29.

Cortical Spreading Depolarizations—In addition to excitotoxic events at the molecular

Inflammation—Focal ischemia evokes a robust inflammatory response that begins within

Neuron. Author manuscript; available in PMC 2011 July 29.

receptors, may serve to detect “danger signals” triggering NFκb-dependent inflammatory

Therefore, therapeutic interventions targeting post-ischemic inflammation should be mindful

Necrosis, necroptosis and autophagy: The execution

Neuron. Author manuscript; available in PMC 2011 July 29.

Autophagy, an energy-dependent process implicated as a cell death mechanism, is used by

Stroke and dementia

What causes the white matter damage underlying VCI?

Neuron. Author manuscript; available in PMC 2011 July 29.

Disruption of the arteriolar wall by amyloid, as observed in genetic or sporadic forms of

requirements caused by loss of energy-efficient saltatory conduction and leaky sodium

Why does stroke increase the risk of dementia?

Neuron. Author manuscript; available in PMC 2011 July 29.

inducing vasoconstriction and ischemia (Iadecola, 2004). Therefore, the vascular

How does the brain repair itself after stroke?

Repair and remodeling processes after stroke

existing neural networks remain to be fully elucidated. Future attempts to promote

Neuron. Author manuscript; available in PMC 2011 July 29.

perivascular routes (Thored et al., 2007). Promotion of neurogenesis enhances vascular

Moving forward: charting a course toward new stroke therapies

Protecting the Neurovascular Unit

Neuron. Author manuscript; available in PMC 2011 July 29.

also influence mechanisms of recovery after stroke.

whole. These are described in the next sections.

IT is mediated by highly redundant cellular, molecular and physiological mechanisms

Neuron. Author manuscript; available in PMC 2011 July 29.

Therapeutic Hypothermia: Engaging Pleiotropic Mechanisms in Multiple Cell Types

ROS production, and peri-infarct depolarizations. Inflammatory markers decrease, as well as

Neuron. Author manuscript; available in PMC 2011 July 29.

Although stroke-damaged brain usually exhibits varying degrees of spontaneous recovery,

As the basic mechanisms of trophic and cell-based repair continue to be defined, an

Neuron. Author manuscript; available in PMC 2011 July 29.

Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on

Neuron. Author manuscript; available in PMC 2011 July 29.

Selective vulnerability of late oligodendrocyte progenitors to hypoxia-ischemia. J Neurosci. 2002;

sciences. 2008; 29:268–275. [PubMed: 18384889]

8:643–653. [PubMed: 19539236]

Neuron. Author manuscript; available in PMC 2011 July 29.