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Pathogenic mechanisms following ischemic stroke

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DOI: 10.1007/s10072-017-2938-1

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Neurol Sci (2017) 38:1167–1186
DOI 10.1007/s10072-017-2938-1

REVIEW ARTICLE

Pathogenic mechanisms following ischemic stroke

Seyed Esmaeil Khoshnam 1 & William Winlow 2,3 & Maryam Farzaneh 4 &
Yaghoob Farbood 1 & Hadi Fathi Moghaddam 1

Received: 28 November 2016 / Accepted: 25 March 2017 / Published online: 17 April 2017
# Springer-Verlag Italia 2017

Abstract Stroke is the second most common cause of death
and the leading cause of disability worldwide. Brain injury
following stroke results from a complex series of pathophys-
iological events including excitotoxicity, oxidative and
nitrative stress, inflammation, and apoptosis. Moreover, there
is a mechanistic link between brain ischemia, innate and adap-
tive immune cells, intracranial atherosclerosis, and also the gut
microbiota in modifying the cerebral responses to ischemic
insult. There are very few treatments for stroke injuries, partly
owing to an incomplete understanding of the diverse cellular
and molecular changes that occur following ischemic stroke
and that are responsible for neuronal death. Experimental dis-
coveries have begun to define the cellular and molecular
mechanisms involved in stroke injury, leading to the develop-
ment of numerous agents that target various injury pathways.
In the present article, we review the underlying
* Seyed Esmaeil Khoshnam
Esmaeil.khoshnam1392@gmail.com
* William Winlow
bill.winlow@gmail.com
1
Department of Physiology, Faculty of Medicine, Physiology
Research Center, Ahvaz Jundishapur University of Medical
Sciences, Ahvaz, Iran
2
Dipartimento di Biologia, Università degli Studi di Napoli, Federico
II, Via Cintia 26, 80126 Napoli, Italy
3
Honorary Research Fellow, Institute of Ageing and Chronic
Diseases, The APEX building, 6 West Derby Street, University of
Liverpool, Liverpool L7 8TX, UK
4
Department of Stem Cells and Developmental Biology, Cell Science
Research Center, Royan Institute for Stem Cell Biology and
Technology, ACECR, Tehran, Iran
pathophysiology of ischemic stroke and reveal the intertwined
pathways that are promising therapeutic targets.
Keywords Ischemia . Stroke . Oxidative stress .
Inflammation . Apoptosis
Introduction
Stroke occurs due to disruption in the blood supply to a brain
region, resulting in death or permanent neurological deficits
[1]. It is the second leading cause of death and the major cause
of adult physical disability in the world [2]. Neurological def-
icits which occur following stroke include balance problems,
hemiplegia, loss of sensory and vibratory sensation, numb-
ness, decreased reflexes, ptosis (of the eyelid), visual field
defects, aphasia, and apraxia [3]. Stroke accounts for 44 mil-
lion physical disabilities annually, with 5.5 million deaths
worldwide. There are very few treatments for stroke, and the
development of new therapeutic agents or combined therapies
is imperative. At present the only effective treatment for stroke
is limited to recombinant tissue plasminogen activator (tPA)
[4]. tPA is the only appropriate thrombolytic agent available
for stroke treatment [5, 6]. However, due to a narrow time
window, this treatment is only suitable for a minority
(<10%) of stroke patients [7].
Ischemic stroke pathogenesis
Stroke can be classified as ischemic or hemorrhagic stroke,
depending on the underlying pathology [8]. Ischemic stroke
accounts for 85% of all strokes, while hemorrhagic stroke is
responsible for 15% of them [9]. Cerebral ischemia results
from cerebral artery occlusion obstructing blood flow to a
1168
portion of the brain [10]. According to the etiologies, ischemic
stroke can be further classified into four main categories as
follows: small vessel disease, athero–thrombotic, cardio–em-
bolic, and undetermined causes [8].
Following ischemic stroke, neurons are deprived of oxygen
and energy with detrimental effects on energy-dependent pro-
cesses in neuronal cells [11]. Immediately after ischemia, neu-
rons are unable to sustain their normal transmembrane ionic
gradient and homoeostasis. This elicits several processes that
lead to cell death: excitotoxicity, oxidative and nitrative stress,
inflammation, and apoptosis. These pathophysiological pro-
cesses are seriously injurious to neurons, glia, and endothelial
cells [12–15] and are interlinked, triggering each other in a
positive feedback loop that terminates in neuronal destruction
[16].
A spectrum of severity is observed in the affected region of
the brain, owing to differential lessening of blood supply to
different zones. Thus, part of the brain tissue (core) undergoes
irreversible neuronal damage due to necrotic cell death, while
the surrounding tissues contain salvageable and metabolically
active cells (penumbra), in which cell death occurs less rapid-
ly. Most therapeutic methods established in the laboratory
have focused on safeguarding neurons from the main patho-
physiological processes, such as inflammation and apoptosis,
that might otherwise occur in the penumbra, and this area is
the target for therapeutic agents [17–20].
Postischemic excitotoxicity
The brain oxygen consumption relative to its weight is very
high (approximately 20% of the body’s oxygen) and must
generate sufficient ATP through mitochondrial electron trans-
port chain to maintain and restore ionic gradients [21]. At a
cellular level, ischemia causes failure of oxidative phosphor-
ylation and ATP synthesis, which leads to ATP being con-
sumed within 2 min. This adversely affects Na+/K+ ATPase
pump, resulting in plasma membrane depolarization, release
of potassium into the extracellular space, and entry of sodium
into cells [22]. In addition, the Ca2+ pump also fails [23] caus-
ing a dramatic rise in intracellular calcium concentration dur-
ing ischemia. This activates several death-signaling proteins
such as calcium-dependent proteases, lipases, and DNases
resulting in cell death in the ischemic core [21].
One reason for the particular susceptibility of the brain to
ischemic injury is that brain tissue contains high levels of
glutamate, and many neurons in the brain are activated by
glutamate receptors. Following a stroke, glutamate is critical
for neuron degeneration [24] when it acts as a neurotoxic
excitatory neurotransmitter playing a pivotal role in ischemia
via excitotoxic pathogenesis [25, 26]. In vivo, it has been
shown that the rapid neuronal swelling that occurs after glu-
tamate treatment is entirely abolished by removal of external
Neurol Sci (2017) 38:1167–1186
sodium ions [27], but is not affected by removal of calcium
ions [28]. Furthermore, delayed neuronal cell death that oc-
curs following glutamate treatment is abolished by calcium
removal and potentiated by increased calcium concentrations
in the bathing medium [29]. Moreover, the initial calcium
influx is known to trigger a secondary intracellular, toxic cal-
cium overload, which is strongly correlated with neuronal
death [30]. Thus, calcium ions are essential to glutamate
neurotoxicity.
Glutamate receptors: the gateway to excitotoxicity
Several types of ionotropic and metabotropic glutamate recep-
tors have been identified in the central nervous system
[31–33]. Activation of the N-methyl-D-aspartate (NMDA)
and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA) type of glutamate receptors leads to membrane de-
polarization and facilitates influx of calcium ions into neurons,
which may lead to excitotoxicity [34–36]. There is good evi-
dence that NMDA receptors (NMDARs) are responsible for
the neuronal damage after ischemic stroke, and these receptors
are the most calcium-permeable ionotropic glutamate recep-
tors [24]. Several studies have revealed that inhibition of the
NMDARs and AMPA receptors (AMPARs) prevent calcium
entry, resulting in neuro-protective effects in models of focal
ischemia [37].
Expression of the sodium–calcium exchanger (NCX) gene
is influenced by cerebral ischemia, which is an important reg-
ulator of intracellular calcium and sodium homeostasis in the
brain [38]. Following ischemia, activation of the NCX could
ameliorate the consequences of ischemic brain injury [39]. It
has been demonstrated that NCX dysfunction is mediated by
NMDARs, which explains the subsequent calcium overload
following the excitotoxic stimulus [40]. During excitotoxicity,
increasing the mitochondrial calcium concentration leads to
the production of reactive oxygen species (ROS) [41], mito-
chondrial depolarization via opening of the permeability tran-
sition pore [42], induction of calcium deregulation [43], and
finally induction of neuronal death [44]. Moreover, during
excitotoxicity, the mitochondrial NCX (mNCX) partially con-
tributes to delayed cytoplasmic calcium loading [45, 46]. The
essential role of the NMDAR in calcium-mediated death sig-
naling led to the theory that some calcium-dependent death-
signaling proteins must be closely associated with the
NMDAR [47].
Metabotropic glutamate (mGlu) receptors also contribute
to excitotoxicity. mGlu receptors belong to the G-protein
coupled receptor (GPCR) family of receptors that modulate
excitatory synaptic transmission. There are three groups of
metabotropic glutamate receptors, (groups I, II, and III
mGluRs); group I mGluR receptors comprise mGluR1 and
mGluR5, which are predominantly found at the postsynaptic
membrane of glutamatergic synapses where they increase
Neurol Sci (2017) 38:1167–1186
neuronal excitability by modulating NMDA and AMPA re-
ceptors [48]. There is substantial evidence that in vivo
mGluR1 activation is protective against NMDA-induced
excitotoxicity [40]. Several studies reported that the neuropro-
tective effects of group I mGluRs were mediated by the acti-
vation of the PI3K-Akt signaling pathway [49–51]. Activation
of other mGlu receptors (Group II and group III) by their
specific agonists demonstrates neuroprotective effects by lim-
iting induction of excitotoxicty. These mGlu receptors are
mainly found at presynaptic terminals where they inhibit the
release of glutamate [52]. Following ischemic stroke, inhibi-
tion of expression of the glutamate transporter-1 (GLT-1) pro-
motes excitotoxicity [53]. Moreover, postischemic downreg-
ulation of GLT-1 is closely associated with glutamate accumu-
lation and promotion of excitotoxicity, suggesting that
excitotoxicity could be controlled by GLT-1 expression [54].
Dual roles of NMDARs in neuronal survival and death
The mechanisms participating in stroke injury are possibly
multifactorial [55], with NMDAR-mediated excitotoxicity be-
ing a key factor [55, 56]. Many important neurological func-
tions require normal NMDAR activity, including neuronal
plasticity, brain formation, and neuronal survival [57, 58].
However, NMDAR over-activation results in excitotoxicity
and neuronal death [55, 56]. A number of studies have found
that NMDAR blockers have neuro-protective effects against
ischemic damages both in vitro [56, 59] and in vivo [59, 60].
Subfamilies of NMDAR are multimeric assemblies whose
subunits include those classed as NR1, NR2, and NR3. In the
cytoplasmic terminus, different NR2 subunits have structural
diversity that couple the receptor to different signaling ma-
chineries [57, 61]. In the stroke and traumatic models,
NR2AR and NR2BR subunits are necessary for glutamate-
mediated neuronal survival and death [59, 60, 62–64]. It has
been suggested that inhibition of NR2BR subunits protects the
ischemic brain from excitotoxic and neuronal cell death [65].
Excessive activation of NMDARs contributes to neuronal
death following stroke [66]. During basal synaptic transmis-
sion, activation of the synaptic NMDAR (predominantly
NR2A-containing) stimulates the signaling components of
the neuronal survival signaling complex (NSC) that promote
neuronal survival [59, 67]. However, under pathological con-
ditions such as stroke, elevation of the extracellular glutamate
concentration causes excitotoxic activation of extrasynaptic
NMDARs (predominantly NR2B-containing). The NR2B ac-
tivation increased Ca2+ influx and promotes aDAPK (active
death-associated protein kinase) to bind with NR2B [59, 67,
68]. aDAPK recruitment activates the neuronal death-
signaling complex (NDC) that in turn suppresses synaptic
NSC activity [67] and mediates neuronal death. It is demon-
strated that inhibition of aDAPK binding to the NR2B reduces
activation of NDC and prevents the excitotoxic neuronal
1169
injury induced by ischemic stroke [66, 68]. Thus, the NR2B
subunit is a major hub for NDC formation [68–70].
Pro-survival effects of synaptic NMDAR result in activa-
tion of the CREB (cyclic-AMP response element-binding pro-
tein) signaling pathway while pro-death effects of extra syn-
aptic NMDARs are mediated by diminishing the activity of
the CREB pathway [67]. It is interesting to note that
memantine, a low affinity uncompetitive NMDAR antagonist,
has neuroprotective effects in stroke models [71]. Also, in a
mouse model of Huntington’s disease, memantine can selec-
tively block extra synaptic NMDARs, thereby protecting
against neuronal death [72]. The NR2B–PSD95–nNOS sig-
naling complex was identified to be the primary set of death-
signaling proteins [69, 73, 74]. PSD95 is a synaptic scaffold-
ing protein that binds to carboxyl terminus of the NMDAR
NR2B subunit (NR2B-CT) and nNOS which results in the
production of the neurotoxic molecule nitric oxide (NO)
[73–75], and suppression of nNOS following stroke led to
inhibition of NMDAR-dependent neuronal death [74].
Following a stroke insult, excitotoxic neuronal death me-
diated by the NMDARs is not only limited to the NDC but
also involves further death-signaling proteins, such as the
calpains (a family of calcium-activated cysteine proteases)
which play a major role in turning the calcium influx via the
NMDAR into neuronal damage [62, 63, 76, 77]. Calpains are
only activated by extra synaptic activation of the NR2BRs
[63, 69, 77, 78], so in vitro and in vivo inhibition of these
proteins mediates strongly neuroprotective effects against
NMDAR-mediated neuronal injury [76, 78, 79].
Postischemic oxidative and nitrative stress
Oxidative and nitrative stress occurs when the production of
free radicals such as reactive oxygen/nitrogen species (ROS/
RNS) overpowers the endogenous scavenging capacity of an-
tioxidant defenses systems [80]. Substantial experimental ev-
idence indicates that in the all forms of stroke injuries free
radical formation was increased [81, 82], and both ROS and
RNS molecules have been clearly shown to be important me-
diators of tissue injury in acute ischemic stroke [83–85].
During ischemia, several mechanisms lead to the free radical
formation, including NMDAR-mediated excitotoxicity, ex-
cessive of Ca2+ influx, mitochondrial dysfunction, and neuro-
nal nitric oxide synthase (nNOS) activation [86–90].
Excessive ROS cause the devastation of cellular macromole-
cules and contribute to the signaling processes that result in
apoptotic cell death [91].
After ischemic stroke, energy depletion causes lactic acid
to accumulate and this leads to acidosis in the neurons. The
acidosis has a pro-oxidant effect via increasing H+ concentra-
tions, increasing the rate of superoxide anion (O−2) conversion
to hydrogen peroxide (H2O2), and the hydroperoxyl radical
1170
(HO2) [92]. Unlike other organs, the brain is especially vul-
nerable to free radical-mediated attack due to the highly oxy-
genated environment of neurons, coupled with highly
peroxidisable lipids and low levels of endogenous antioxi-
dants [92, 93].
Stroke risk factors and ischemia lead to the activation of
several ROS-generating enzymatic systems including
NADPH oxidase (NOX) [94, 95] mitochondrial depolariza-
tion [96], xanthine oxidase (XO) [97], and nitric oxide syn-
thase (NOS) [98]. Following ischemia and NMDAR activa-
tion, NADPH oxidase produces the majority of superoxide
anions [94, 95]. It has been shown that following activation
of NMDARs the mitochondrion ceases to be the major source
of free radicals. However, the proximity of neuronal mito-
chondria to the NADPH oxidase pathway increases the prob-
ability that this enzyme produces Bkindling^ ROS that elevate
mitochondrial uncoupling, initiating a secondary ROS surge
from mitochondria [94].
ROS have substantial cellular effects, causing tissue dev-
astation and cell death by processes including DNA damage,
protein destruction, lipid peroxidation, release of Ca2+ from
intracellular stores, cytoskeletal structural injury, and chemo-
taxis. Some of these effects lead to the activation of antioxi-
dant defense mechanisms to counterbalance the ROS devas-
tation [82]. Furthermore, ROS have profound effects on the
cerebral vasculature which ultimately influence cerebral blood
flow. Hence, the H2O2, O2, and ONOO exert their effects by
increasing vasodilation, aggregation of the platelets, increased
endothelial permeability, and focal lesions in endothelial cells
[99].
The NO molecule which is generated by the ischemia-
activated NOS combines with superoxide to produce
peroxynitrite, a potent oxidant [23]. RNS have important cel-
lular effects, such as facilitating the opening of the mitochon-
drial permeability transition pore (mPTP), inhibition of impor-
tant mitochondrial enzymes, DNA damage, and activation of
the transient receptor potential cation channel, subfamily M,
member 7 (TRPM7) channels that are permeable to the Ca2+
ions [100, 101]. Also, NO modifies protein groups through
post-translational alterations that affect cell survival by chang-
ing the functions of critical proteins such as caspases,
metalloproteases, and the glycolytic enzyme GAPDH [102,
103]. In particular, NO by covalently attaching to cysteine
residues forms S-nitrosothiol derivatives, which impact pro-
tein function, thus affecting S-nitrosylation, but this aspect is
underexplored in ischemic mechanisms [20].
Despite the deleterious effects of reactive radical overpro-
duction which are destructive to brain cells, at homeostatic
levels, these radicals are required signaling molecules contrib-
uting to normal neuronal function [104]. Therefore, it is im-
portant that novel therapies only scavenge deleterious radicals
without interfering with endogenous signaling [105]. The ef-
fects of free radical injuries are normally inhibited or lessened
Neurol Sci (2017) 38:1167–1186
by free radical scavengers and antioxidant enzymes [83, 106].
The activity of antioxidant and detoxifying enzymes maintain
redox homeostasis. These enzymes include superoxide dis-
mutase (SOD), glutathione peroxidase (GSHPx), glutathione
reductase, and glutathione-S-transferase (GST), and their ac-
tivity is studied in ischemic stroke progression [107, 108]. The
SOD enzymes including manganese SOD (MnSOD) and ex-
tracellular SOD (EcSOD) assist in brain recovery following
ischemic reperfusion damage [109, 110].
Antioxidant enzymes are encoded by specific genes that
bear an antioxidant response element (ARE) within their pro-
moters. The activation of this element is mainly regulated by
nuclear factor erythroid-2 related factor 2 (Nrf2) [111].
Postischemic oxidative stress induces antioxidant responses
that are mediated by Nrf2, and this factor is neuroprotective
against stroke injuries [112, 113]. It has been found that ex-
pression of Nrf2 is upregulated in the penumbra, indicating
the value and contribution of this factor in neuronal protection
and survival [114]. Nrf2 regulates several downstream protec-
tive proteins, including glutathione-S transferase (GST),
NAD(P)H: quinone oxidoreductase-1 (NQO1) and
hemeoxygenase-1 (HO-1). These proteins are all involved in
cytoprotection against various oxidative insults in the brain
[115, 116]. Thus, Nrf2 is located in the center of the neuro-
protective pathway [117]. Studies have demonstrated that
Nrf2 also contributes to protective effects against mitochon-
drial dysfunction, which occur during ischemic stroke [118].
Postischemic inflammation
Inflammation is a critical step in the pathophysiology of cere-
brovascular diseases, especially ischemic stroke [119]. Many
studies have indicated that postischemic neuro-inflammation
is an important factor for ischemic long-term prognoses [120,
121]. Brain inflammation is a therapeutic target for some brain
diseases such as ischemia and trauma [122]. Following brain
ischemia, inflammatory responses are initiated as a result of
several agents such as ROS formation, necrotic cells, and im-
paired tissues. These agents cause activation of inflammatory
cells [123–126]. Moreover, these activated inflammatory cells
participate in tissue remodeling after cerebral injuries [127].
The inflammatory process involves several different cell
types, inflammatory cytokines, and cellular receptors such as
toll-like receptors (TLRs) (Fig. 1).
The cellular neuro-inflammatory response
Following brain injury, the initial inflammatory response is
mediated by the activation and recruitment of microglial cells
[129–131]. Microglial cells are the resident macrophages of
the brain, and they are highly activated after brain insult [132].
Studies have demonstrated that suppression of the microglial
Neurol Sci (2017) 38:1167–1186
Fig. 1 Schematic of the effects of cerebral ischemia on the inflammatory
cytokines and immune cells and their contribution to neurotoxicity.
Cerebral ischemia causes lessening of blood supply in the penumbra
zone, and triggers a complex cascade of events that include
excitotoxicity and oxidative stress in the ischemic neurons. These
cascades lead to microglial activation and release of pro-inflammatory
cytokines such as TNF-α, IL-1β, and IL-6. These cytokines can potenti-
ate inflammation by recruitment and infiltration of the neutrophils, mono-
cytes, and T cells into the brain lesion. After core zone ischemia, neurons
undergo irreversible neuronal damage due to necrotic cell death. DAMPs
released by necrotic cells are detected by receptors such as TLRs, which
are expressed by immune cells such as NK cells, which are used as an
example of lymphocytes. In the early stages of stroke, fractalkine released
1171
from ischemic neurons recruits NK cells into the ischemic zones [128].
These NK cells affect ischemic neurons by releasing cytokines, mainly
TNF, that increase glutamate release and result in neuronal hyperactivity
and excitotoxicity [128]. NK cells also release cytokines, such as IFN-γ
and GM-CSF, that activate microglia and macrophages and condition
astrocytes, which in turn secrete inflammatory mediators such as IL-1β,
IL-6, and NO [128]. Thus, excitotoxicity and inflammation induced by
microglia and NK cells contribute to neurotoxicity and apoptotic cell
death. Abbreviations: DAMPs damage-associated molecular patterns,
TLRs toll-like receptors, NK cells natural killer cells, TNF tumor necrosis
factor, GM-CSF granulocyte macrophage colony-stimulating factor, NO
nitric oxide
1172
cells reduced postischemic damage, and these cells may be-
come part of an attractive therapeutic strategy for ischemic
stroke [133, 134]. Ischemia activates microglia and transform
them into the phagocytic cells that release a variety of cyto-
toxic and/or cytoprotective substances. The neuroprotective
effects of microglia are exerted by generating neurotrophic
factors such as insulin-like growth factor I (IGF-I) and brain-
derived neurotrophic factor (BDNF). In response to the ische-
mia, activated microglial cells distribute pro-inflammatory cy-
tokines including tumor necrosis factor α (TNF-α), interleu-
kin-1β(IL-1β), interleukin-6 (IL-6), as well as other potential
cytotoxic molecules such as prostanoids, ROS, and NO
[135–137]. While the initial purpose of microglial activation
is the brain’s effort to protect neurons, the over-activation of
microglia results in deleterious inflammation, increasing the
probability of neuronal death [138–140].
Previously, it was accepted that blood brain barrier (BBB)
prevented blood inflammatory cells (monocytes and neutro-
phils) from entering the brain and the predominant opinion
was that only microglial cells were involved in inflammation
of the brain. However, infiltration of neutrophils and mono-
cytes into spinal cord injury sites has been described and their
contribution to the inflammation has been demonstrated
[141]. Also, an increase in neutrophil and monocyte counts
has been reported in transient ischemia and in stroke. [142].
Leukocyte transmigration through BBB appears to be regulat-
ed by CAMs (ICAM-1, VCAM-1) and chemokine signaling
processes [143]. Moreover, astrocytes and even neurons con-
tribute to brain inflammation, and these cells produce several
anti-inflammatory factors and chemokines that recruit mono-
cytes [144–149]. Inflammation is regulated both negatively
and positively by neuronal cells [150–152], and the number
of circulating monocytes is increased following stroke.
Neutrophilia and lymphocytopenia have also been observed
in cerebral ischemic patients [142]. The distinct aspects of
ischemic stroke are characterized by neuronal necrosis and
excess infiltration of immune cells [20, 153]. Hence, brain
inflammation is a complex process in which several immune
cells play a role.
Astrocytes, similar to the microglial cells, could deliver
inflammatory factors such as chemokines, cytokines, and
NO [154]. Furthermore, there are some cytokines delivered
from several peripheral cells such as T lymphocytes, mono-
nuclear phagocytes, natural killer (NK) cells, and poly morph
nuclear leukocytes, and they are involved in the ischemic in-
flammation [155]. NK cells are innate lymphocytes that can
be rapidly mobilized in the primary phases of immune re-
sponses. During stroke, NK cells infiltrate ischemic lesions
of the brain. Following cerebral ischemia, fractalkine is de-
rived from the ischemic neurons, which subsequently recruit
NK cells, and determine the size of brain lesions in a T and B
cell-independent manner. Also, NK cells exacerbate the brain
infarction by increasing local inflammation and neuronal
Neurol Sci (2017) 38:1167–1186
hyperactivity, and finally catalyze neuronal death [128, 156].
Data from experimental models showed that lymphocytes in-
filtrate the brain immediately after ischemic stroke, with neu-
trophils migrating into the brain parenchyma immediately af-
ter the initial injury, followed by macrophages and natural
killer cells [128, 157–159]. Subsequently, T and B lympho-
cytes enter the lesion [157].
In ischemic brain tissue, ROS induce the expression of the
pro-inflammatory genes such as nuclear factor kappa B (NF-
kB), interferon regulator factor 1 (IRF 1), hypoxia inducible
factor 1 (HIF 1), and STAT3. Thus, these inflammatory factors
increase the expression of cytokines and also of adhesion
molecules. The adhesion molecules are upregulated in the first
few days following ischemic stroke, and they are responsible
for the leukocyte migration through the brain endothelium
[134, 160]. Neutrophils also infiltrate the brain facilitated by
disruption of the BBB disruption, and it has been mediated by
cell adhesion molecules including selectins, integrins, and im-
munoglobulins [161]. When neutrophils infiltrate the ische-
mic brain, they release oxygen-free radicals and proteolytic
enzymes that damage to the tissue. However, in animals, de-
ficient in protein kinase C infarct volumes are reduced, be-
cause protein kinase C has significant roles in neutrophil ad-
hesion, degranulation, and superoxide generation [161, 162].
While the initial infiltration of immune cells into the ischemic
brain exacerbates tissue damage and worsening of neurologi-
cal deficits, there is a subsequent beneficial effect, including
the release of cytokines that promote glial scar formation ac-
companied by phagocytosis of cellular debris that is essential
for effective wound healing [23].
Cytokines and the neuro-inflammatory response
Cytokines are key players in the inflammatory mechanism and
contribute stroke-related brain injury [163]. During ischemia,
pro-inflammatory cytokines and chemokines appear to exac-
erbate stroke-related brain injury. The major pro-
inflammatory cytokines are TNF-α, IL-1β, IL-6, and
chemokines. However, anti-inflammatory cytokines such as
IL-10 and TGF-β may have neuroprotective effects [161,
164, 165]. Pro-inflammatory and anti-inflammatory cytokines
are secreted from different cells in the brain, including microg-
lia, astrocytes, endothelial cells, and neurons [155]. In animal
models, it has been demonstrated that there is an increase in
the production of pro-inflammatory cytokines and a lessening
in production of anti-inflammatory cytokines associated with
increasing infarct size [166].
The use different ischemia models has resulted in the ob-
servation that production of IL-1 is increased after permanent
or transient cerebral ischemia in microglia, astrocytes, and
neurons [161]. Calcium entry increases due to the actions of
IL-1 on the NMDARs, resulting in neuronal cell death [167].
Furthermore, IL-1 can potentiate inflammation by recruitment
Neurol Sci (2017) 38:1167–1186
and adhesion of neutrophils. IL-1 has been shown to increases
leukocyte infiltration by increasing the expression of adhesion
molecules such as E-selectin, ICAM-1, ICAM-2, and VCAM-
1 on the endothelial cells and causing breakdown of the blood
brain barrier [161, 168–170]. Adhesion molecules such as
ICAM-1 and VCAM-1 are the major ligands for leukocyte
integrins to attach to endothelial cells. Under normal condi-
tions, ICAM-1 is detected on a small number of CNS
microvessels and massively upregulated by inflammatory
stimuli [171].
It has also been shown that administration of recombinant
IL-1 receptor antagonists decreases tissue necrosis and infarct
volume [172], and mouse models with IL1-αβ knock-out
have smaller infarct volumes than controls [173].
Furthermore, intra-cerebroventricular administration of IL-
1β increased infiltration of neutrophils into the brain, as well
as increasing cerebral edema and infarct size [161].
In rats subjected to experimental ischemia, IL-6 is elevated
within 3–3.5 h of stroke, with peak expression at 12 h and
continuing for at least 24 h [174]. IL-6 is a pro-inflammatory
cytokine whose serum levels correlate with larger infarct size
and poorer clinical outcome [161, 175]. However, following
stroke, IL-6 signaling is important for tissue remodeling and
recovery via activation of transcription factor signal transduc-
er and activator of transcription (STAT) [176–178]. In addi-
tion, IL-6 demonstrates anti-inflammatory properties by in-
ducing IL-1 receptor antagonist synthesis, inhibiting TNF-α,
and inducing apoptosis in neutrophils [23, 179]. However, IL-
6 also increases brain damage by elevating body temperature
after stroke [180]. Thus, IL-6 has both beneficial and detri-
mental effects in the context of stroke.
Several studies demonstrate a positive correlation between
TNF-α and the degree of ischemic damage [181]. Hence, in
rat ischemic models, administration of anti-TNF antibody and
TNF-α binding protein decreases infarct volume [182, 183].
TNF-α promotes neutrophil infiltration into the brain by in-
ducing expression of adhesion molecules in cerebral endothe-
lial cells. Additionally, TNF-α disrupts the BBB and stimu-
lates the induction of other inflammatory mediators [184,
185].
IL-10 is an anti-inflammatory cytokine that can inhibit pro-
inflammatory cytokine production and chemokine secretion
and prevents antigen delivery by macrophages and microglia
[186]. Moreover, IL-10 can neutralize the deleterious effects
of TNF-α by preventing activation of signaling pathways trig-
gered by TNF-α [187]. Also, TGF-β is a neuroprotective
factor against neuro-inflammation [188] and it has neuropro-
tective effects against ischemic insult [161, 189]. Moreover,
administration of TGF-β reduces the number of circulating
neutrophils, which improves postischemic injury [190].
After ischemic stroke, chemokines attract neutrophils to ische-
mic tissue, and blocking these chemokines decreases cerebral
edema and infarction [161].
1173
NF-kB is a heteromeric transcription factor widely known
to be associated with inflammatory responses following ische-
mia [191–193]. It is involved in activation of several pro-
inflammatory genes including TNF-α, ICAM-1, IL-6, iNOS,
and cyclooxygenase 2 (COX2) [194, 195]. Several studies
have revealed that inhibition of the NF-kB pathway prevents
production of pro-inflammatory cytokines [196–198].
Matrix metalloproteinases (MMPs) are a family of proteo-
lytic enzymes that are responsible for remodeling the extracel-
lular matrix and that can degrade all its components.
Expression of MMPs is increased in the brain in response to
injury [199]. Following injury, expression of MMPs has been
shown in some cells such as neurons, astrocytes, microglia,
and endothelial cells. Experimental studies have shown that
MMPs such as MMP-9 and MMP-2 are responsible for dis-
ruption of BBB and hemorrhagic transformation following
ischemic stroke [200–202]. MMP-9 is associated with
neuro-inflammation, and its inhibition could have a beneficial
effect on the outcome of stroke [203].
The TLRs and the neuro-inflammatory response
Activation of toll-like receptors (TLRs) leads to cytokine and
chemokine production, which initiates the inflammatory re-
sponse. In the central nervous system, TLRs are expressed
on endothelial cells, microglia, astrocytes, oligodendrocytes,
and neurons [204–207]. Thirteen TLRs have been identified,
and TLR4 signaling contributes to postischemic inflammatory
injuries [208, 209]. In response to hypoxia, TLR4 expression
is upregulated on the surface of microglial cells [210].
After cerebral ischemia in mice, TLR4 mRNA expression
increased between 1 and 22 h post-stroke [211]. Furthermore,
TLR2 mRNA expression also increased following brain is-
chemia [212]. Endogenous ligands such as Hsp70 are upreg-
ulated in the brain tissue following ischemia and can cause a
detrimental response to cerebral ischemia by TLR activation.
In animals missing either TLR2 or TLR4, there are smaller
infarct volumes than in wild-type animals following experi-
mental ischemia [213]. In TLR4 and TLR2 knockout models,
lesion size is reduced between 1 and 3 days after stroke [211,
214]. TLRs can activate NF-κB which induces the expression
of pro-inflammatory genes, cytokines, and adhesion mole-
cules [215].
Innate and adaptive immune responses following
stroke
In response to injuries, the first line of defense is the innate
immune system in which macrophages, neutrophils, dendritic
cells, natural killer cells, and γδT cells are involved in the
innate immune response to cerebral damage. Following ische-
mic brain injury, a variety of danger signals are released from
1174
intracellular compartments or generated by the action of lytic
enzymes escaping from dead cells on matrix proteins, which
are usually called danger-associated molecular pattern mole-
cules (DAMPs) [216]. These signals include purines (ATP,
UTP, and their catabolites), cytokines, and chemokines,
among other molecules. DAMPs activate their receptors, in-
cluding TLRs and scavenger receptors, on microglia, astro-
cytes, perivascular macrophages, and brain endothelial cells
[217–221]. These signals activate microglia that then release
inflammatory cytokines, such as IL-1β and TNF-α. Also,
activated microglia induce release of chemokines, which in-
clude CCL2 and CCL3, to recruit leukocyte infiltration [222].
Following cerebral ischemia, the CCL2 and receptor CC che-
mokine receptor 2 (CCR2) axis is responsible for the recruit-
ment of macrophages [223]. Ischemia also activates macro-
phages that are present in the perivascular space, and which
release pro-inflammatory cytokines, chemokines, ROS, and
NO [224, 225]. Cellular adhesion molecules, including
ICAM-1, P-selectin, and E-selectin, are expressed in the in-
flamed endothelial cells of ischemic brain. Adhesion mole-
cules appear to be involved in the trans-endothelial migration
of neutrophils into the brain. Following acute stroke, neutro-
phil infiltration and infarct volumes were reduced in ICAM-1-
deficient or P-selectin-deficient mice compared with wild-
type mice [226–228]. Many studies have highlighted that fol-
lowing cerebral ischemia, activated neutrophils greatly con-
tribute to elevation of MMP-9 [229], which, in turn, causes
BBB breakdown, stimulates peripheral leukocyte infiltration,
and ultimately results in neuronal damage [230]. Therefore,
inhibition of neutrophils could be a reasonable peripheral tar-
get for stroke treatment.
Adaptive immunity that requires a relatively long time to
become protective is an antigen-specific defense mechanism,
which includes the T and B lymphocytes that are related to the
post-injury inflammatory response. Antibodies against brain-
derived antigens develop following ischemic stroke, leading
to the sensitization of circulating T cells against brain antigens.
These antigens can be processed by antigen-presenting cells
(APC), such as dendritic cells, with MHC molecules on the
cell surface. T cell receptors can recognize the MHC and
brain–antigen complex and activate the adaptive immune sys-
tem. In rodent and human stroke, APC numbers are increased
in the ischemic brain and reduced in the periphery [158,
231–233]. Following stroke, both CD4+ T-helper cells and
larger numbers of CD8+ cytotoxic T cells are recruited into
the ischemic brain [158, 234]. The CD4+ helper T cells help to
activate and direct other immune cells, including CD8+ cyto-
toxic T cells, which are capable of directly inducing cell death
[235]. The regulatory T cells (Treg) exert their neuroprotective
function by secretion of inhibitory cytokines, such as TGF-β,
IL-10 [187], and IL-35 that is a novel anti-inflammatory cy-
tokine [236, 237]. Several studies show that T helper cell
subpopulations such as pro-inflammatory Th1, Th17, and γδ
Neurol Sci (2017) 38:1167–1186
T cells have differential effects on stroke outcome, which have
been associated with increased inflammatory damage and
even worse outcomes [158, 187, 238, 239]. Studies showed
that following transient cerebral ischemia, B cell deficiency
caused further deterioration of histological damage and func-
tional outcomes, while adoptive transfer of B cells into these
mice improved neurological function and reduced ischemic
infarct size. The authors also showed that IL-10 was respon-
sible for the B cell-mediated neuroprotection [240]. Therefore,
in cerebral ischemia/reperfusion injury, the role of B lympho-
cytes might be protective rather than pathogenic.
The gut microbiota and ischemic stroke
Recent studies demonstrated that antibiotic-induced alter-
ations in the intestinal flora reduce ischemic brain injury in
mice. Intestinal dysbiosis causes an increase in regulatory T
cells and a reduction in IL-17+ γδ T cells [241]. Hence, large
stroke lesions cause gut microbiota dysbiosis via stress-
mediated intestinal paralysis, which in turn affects stroke out-
come via changes in T cell homeostasis, induction of a pro-
inflammatory response, and deterioration of stroke outcome.
Therapeutic transplantation of fecal microbiota to normalize
poststroke dysbiosis normalizes the brain lesion and improves
stroke outcome. Therefore, a target of stroke-induced systemic
alterations is the gut microbiome, which is an effector with
substantial impact on stroke outcome [242]. Also, several
studies describe changes in intestinal microbiota composition
in stroke patients [243, 244]. Recent studies show that consid-
erable numbers of T cells migrate from the intestine to the
poststroke brain [242]. The gut microbiota is a key regulator
of T cell homeostasis [245]. After the first few days of stroke
insult, T cells are recruited to the injured brain [246].
Post-ischemic apoptosis
Apoptosis is involved in the pathogenesis of ischemic stroke
and is known as programmed cell death. It is activated during
development and physiological cellular turnover and in path-
ological conditions such as stroke [247–249]. The apoptotic
intrinsic stimuli are activated by the mitochondrial signaling
pathways, while extrinsic stimuli trigger cell surface death
receptors such as TNF-α, Fas (CD95/APO1), and TRAIL
(TNF related apoptosis inducing ligand) receptors [250, 251].
During ischemia, activation of NMDARs leads to accumu-
lation of the intracellular Ca2+, causing cleavage of Bcl-2
interacting domain (BID) to truncated Bid (tBid) [252]. At
the mitochondrial membrane, tBid interacts by pro-apoptotic
proteins such as Bad-Box and opens the mitochondrial transi-
tion pores (MTP). MTP opening promotes release of mito-
chondrial cytochrome c (Cytc) or apoptosis-inducing factor
(AIF) [253]. Cytc in the presence of ATP/dATP binds to the
Neurol Sci (2017) 38:1167–1186
Apaf-1 (apoptotic protease activating factor 1) and
procaspase-9 to form an apoptosome. Cytc release activates
downstream caspases of the intrinsic pathway through forma-
tion of the apoptosome that activates caspase-9 and conse-
quently caspase-3. Activated caspase-3 cleaves nuclear DNA
(nDNA) repair enzymes, causing nDNA damage and apopto-
tic cell death. Moreover, AIF is translocated to the nucleus and
causes DNA fragmentation and cell death in a caspase-
independent pathway [254]. It has been reported that caspase
proteins are activated in the penumbra zone, and their inhibi-
tion could protect against focal ischemia injuries [255].
In response to ischemia, release of the Cytc from outer
mitochondrial membrane plays a pivotal role in apoptosis ini-
tiation. As mentioned, release of Cytc caused MTP formation,
in which proteins of the Bcl-2 family could either promote
(Bax, Bak, Bad, Bim, Bid) or prevent (Bcl-2, Bcl-XL, Bcl-
w) MTP formation. In the outer membrane of the mitochon-
dria, oligomerization of Bax and/or Bak causes MTP forma-
tion, while anti-apoptotic proteins such as Bcl-2 and Bcl-XL
prevent pore formation via heterodimers forming the Bax pro-
teins [256–259]. Bax and Bak directly mediate release of the
Cytc by forming a pore in the mitochondrial membrane.
Meanwhile, Bid, Bad, and Bim appear to function as sensors
of cell stress and promote apoptosis by antagonizing the func-
tion of Bcl-2/Bcl-XL, as anti-apoptotic proteins, and/or acti-
vating the function of apoptotic function of the Bax and Bak
[257, 258, 260].
The extrinsic pathway is initiated via ligand-receptor inter-
actions, and it can induce caspase activation independent of
the release of Cytc. The TNF family of ligands such as FASL,
TNF, LT-alpha, LT-beta, CD40L, LIGHT, RANKL, and
TRAIL bind to the TNF-receptor, Fas receptor (FasR), and
TRAIL receptor that leads to the activation of death receptors
[261, 262]. These bindings result in activation of caspase-8
and caspase-10, which in turn can activate effector caspase 3
[263, 264]. Activation of caspase-3 results in the mitochon-
drial membrane permeabilization, chromatin condensation,
DNA fragmentation, and eventually cell death [265].
Increasing the Fas expression by the extrinsic caspase-
dependent pathway has been involved in the ischemic stroke.
Further studies have revealed that expression of caspase-1,
caspase-3, caspase-8, and caspase-9 and cleaved caspase-8
are increased in the ischemic penumbra zone [266]. Fas/
FasL belong to the tumor necrosis factor receptor/ligand su-
perfamily of costimulatory molecules and play an important
role in induction of apoptosis [267]. Caspase-1 and caspase-3
activation occurred only 2 h after ischemia and reperfusion
[268].
Several studies have revealed that inhibition of apoptosis
decreases ischemic injury [269]. For example, inhibition of
BCL2L11 led to reduction of apoptosis, restoration of neuro-
logical scores, and reduction of behavioral abnormalities fol-
lowing stroke [270]. BCL2L11 induce apoptosis by
1175
inactivating anti-apoptotic protein BCL2 and activating apo-
ptotic protein BAX-BAK1 [271, 272]. Following ischemia,
reducing Bcl-2 and Bcl-w increased ischemic neuronal cell
death [273]. Decreasing of the Bcl-2 proteins also resulted in
astrocyte dysfunction [274]. Moreover, following oxygen and
glucose deprivation, ischemic injuries were alleviated by en-
hancement of Bcl-2 expression in the neurons [275]. Several
studies have shown that activation of transcription factors
such as CREB and NFkB are related to cell survival and phos-
phorylate Bax and Bad thus inhibiting the release of Cytc. In
addition, inhibition of caspase 3, gene deletions of Bid, and
the use of viral vector-mediated gene transfer of Bcl-2 and
Bcl-XL are approaches that are identified to be neuroprotec-
tive [276–280].
A summary of the most important genes involved in neu-
ronal degradation and neuronal protection following ischemic
stroke is provided in Tables 1 and 2.
Intracranial atherosclerosis and ischemic stroke
Several lines of evidence show that intracranial atherosclerosis
is a common cause of ischemic stroke [281–283]. Progression
of intracranial atherosclerosis is accelerated in association
with several risk factors such as hypertension and diabetes
mellitus [282, 283]. Advanced atherosclerosis can affect
downstream or collateral perfusion because of stenosis of
blood vessels [284, 285]. Advanced atherosclerosis leads to
vasoconstriction, platelet activation, and thrombosis and intra-
cranial atherosclerosis, which potentially alters hemodynam-
ics and may cause thrombosis that has been linked to
Alzheimer’s disease (AD) [286, 287]. Atherosclerosis of ce-
rebral vessels may enhance the production of amyloid-β pep-
tide (Aβ), a peptide central to AD pathology, through induc-
tion of hypoperfusion and hypoxia. In turn, Aβ may promote
formation of atherosclerotic lesions through vascular oxida-
tive stress and endothelial dysfunction. Furthermore, athero-
sclerosis could contribute to AD pathophysiology by
impairing Aβ clearance and elevating brain levels of Aβ
[288]. Antioxidants are known to affect the progression of
experimental atherosclerosis [289], which may be why of in-
tracranial arteries are less susceptible to hypercholesterolemia
and ox-LDL [290, 291]. Furthermore, the rate of progression
of atherosclerosis can be affected by the gut microbiota [292,
293] and gut microbes can modify important factors in hemo-
stasis and thrombosis, including platelet responsiveness and
clot formation [294].
Future therapies
Despite progress in understanding the pathophysiologic
mechanisms of ischemic stroke over the last few decades,
1176 Neurol Sci (2017) 38:1167–1186

Table 1 Specific genes involved in neurodegeneration following ischemic stroke

Pathogenic category Genes Expression Function Ref.

Excitotoxicity NR2B Increased Calcium influx, stimulates the signaling components of NDC [37, 67]
AMPAR Increased Calcium influx [37]
NCX Decreased Dis-regulation of intracellular calcium and sodium homeostasis [38, 40]
mNCX Increased Cytoplasmic calcium loading [45, 46]
Calpains Increased Turns the calcium influx via the NMDAR into neuronal damage [62, 63, 76, 77]
Oxidative and NADPH oxidase Increased Produces the majority of superoxide anions [94, 95]
nitrative stress xanthine oxidase Increased ROS formation [97]
NOS Increased RNS formation [23]
Inflammation IL-1 Increased Increases leukocyte infiltration, breakdown of BBB [161, 168–170]
IL-6 Increased Elevates body temperature [180]
TNF-α Increased Neutrophil infiltration, expression of adhesion molecules, BBB [184, 185]
disruption
CAM Increased Leukocyte transmigration [143]
NF-kB Increased Activation of pro-inflammatory genes and adhesion molecules [194, 195]
MMP Increased Disruption of BBB and hemorrhagic transformation [200–202]
TLR Increased Activation of NF-κB, Cytokine and chemokine production [204–207, 215]
Apoptosis Bax, Bak Increased Promotes MTP formation, [257, 258, 260].
Bad, Bim, Bid Increased Sensors of cell stress [257, 258, 260].
Caspase-1, caspase-3, Increased DNA fragmentation and cell death [265, 266]
caspase-8, caspase-9
Fas Increased Induces caspase activation [266]
BCL2L11 Increased Inactivates BCL2 and activating BAX-BAK1 [271, 272]

NR2B N-methyl-D-aspartate receptor (NMDAR) subunit, NDC neuronal death-signaling complex, AMPAR a-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid receptor, NCX sodium–calcium exchanger, mNCX mitochondrial NCX, calpains a family of calcium-activated cysteine prote-
ases, ROS reactive oxygen species, NOS nitric oxide synthase, RNS reactive nitrative species, TNF-α tumor necrosis factor α, IL-1β interleukin-1β, IL-6
interleukin-6, BBB blood brain barrier, CAM cell adhesion molecule, NF-kB nuclear factor kappa B, IRF 1 interferon regulator factor 1, HIF 1 hypoxia
inducible factor 1, MMP matrix metalloproteinases, TLR toll-like receptor, MTP mitochondrial transition pores, Fas tumor necrosis factor receptor, Bcl-2
B cell lymphoma 2

thrombolytic therapy with tPA and endovascular stroke. Due to a narrow time window and a hemorrhagic risk,
recannulation are only FDA-approved treatments for acute this treatment is only suitable for a minority (4%) of stroke

Table 2 Specific genes involved in neuroprotection following ischemic stroke

Pathogenic category Genes Expression Function Ref.

Excitotoxicity NR2A Decreased Stimulates the signaling components of NSC [59, 67]
mGluR (group I) Decreased Activation of the PI3K-Akt signaling pathway [49–51]
mGluR (group II and Decreased Inhibition of presynaptic glutamate release [52]
group III)
GLT-1 Decreased Inhibits glutamate accumulation [54]
Oxidative and nitrative SOD enzymes Increased Maintains redox homeostasis [109, 110]
stress Nrf2 Increased Antioxidant responses [112, 113]
Inflammation IGF-I Increased Neurogenesis [135–137]
BDNF Increased Neurogenesis [135–137]
IL-10 Increased Inhibition of pro-inflammatory cytokine [186]
production and chemokine secretion
TGF-β Increased Reduces the number of circulating neutrophils [190]
Apoptosis Bcl-2, Bcl-w Decreased Prevents MTP formation [273]

NR2A NMDAR subunit, mGluR metabotropic glutamate receptors, GLT-1 glutamate transporter-1, NSC neuronal survival signaling complex, SOD
superoxide dismutase, Nrf2 nuclear factor erythroid-2 related factor 2, IGF-I insulin-like growth factor I, BDNF brain-derived neurotrophic factor, TGF-
β transforming growth factor beta, MTP mitochondrial transition pores, Bcl-2 B cell lymphoma 2
Neurol Sci (2017) 38:1167–1186
patients [295]. Therefore, therapies that can benefit more
stroke patients are urgently needed.
Normobaric oxygenation therapy
Currently, stroke researchers are competing to improve neu-
roprotective strategies that guard the brain from ischemia-
induced injury. As yet, neuroprotective agents that have
shown protective potential in animal models have failed in
clinical trials. Since the underlying pathophysiology of stroke
is highly complicated and exploits numerous deleterious path-
ways, a combination of neuroprotective agents may offer sub-
stantially better results than a single agent alone, by interven-
ing in multiple mechanisms. After stroke onset, normobaric
oxygenation (NBO) therapy increases tissue oxygenation,
thereby decreasing the extent of hypoxic injury. Studies have
also shown that low to moderate levels of ethanol not only
reduce the risk of stroke but also decrease its postischemic
consequences [296]. In rats following ischemic stroke, NBO
administration salvages ischemic brain tissue by diminishing
apoptotic cell death. Further, combination therapy with NBO
and ethanol greatly improves both the degree of neuroprotec-
tion and associated apoptosis [297, 298]. Other studies dem-
onstrate that acute ethanol administration reduces lactic acido-
sis after ischemic stroke. This beneficial effect of ethanol was
shown to be potentiated by NBO therapy in permanent ische-
mia. Because both NBO and ethanol are available, inexpen-
sive, and easy to administer, their combination could be ap-
plied in clinics shortly after ischemic stroke [299].
Hypothermia
Studies have shown that therapeutic hypothermia (TH) is a
promising treatment for stroke. TH therapy protects brain tis-
sue via inhibition of several pathways including oxidative
stress, inflammatory responses, metabolic disruption, and cell
death signals [300–302]. In animal models and human pa-
tients of stroke, TH therapy improves functional and behav-
ioral outcomes [300, 303, 304]. TH therapies also affect in-
flammatory responses after a hypoxic/ischemic insult in cul-
tured cells as well as in a stroke model of adult mice and can
inhibit the expression of inflammatory factors, reduce both
microglial activation and migration as well as promoting func-
tional recovery following stroke [305].
Additional studies demonstrate that the effect of a single
drug can be improved or the therapeutic time window avail-
able for administration extended if applied in combination
with hypothermia. For example, the synergistic effects of S-
emopamil, which reduces brain edema, and nimopidine with
hypothermia. The benefit of hypothermia is to extend the time
window available for Bcl-2 gene therapy [306–309].
1177
Combination therapy
MK-801, an NMDA receptor antagonist, confers neuroprotec-
tion by attenuating the NMDARs and excitotoxicity [310],
failed in clinical use due to the short therapeutic window.
Moreover, maslinic acid treatment promoted the expression
of the astrocytic glutamate transporter GLT-1 post-ischemia.
In ischemic rats, the combination of maslinic acid and MK-
801 clearly induced neuroprotection. Furthermore, the syner-
gistic effect provided by maslinic acid on the therapeutic time
window and dosage range for MK-801 is possibly attributed
to the modulation of glutamate excitotoxicity by increasing
astrocytic glutamate transporter GLT-1 levels and stimulating
astrocyte activation. Therefore, a combination of drugs
targeting astrocytic glutamate transporters, and also NMDA
receptor antagonists may be a potential treatment for ischemic
stroke [311].
Cerebral collateral flow
Cerebral collateral flow is a powerful determinant of ischemic
stroke outcome, which is recruited after arterial occlusion.
Approaches to support collateral flow in stroke models may
promote the translational development of therapeutic strate-
gies for enhancing collateral flow, and also may have a role
in the hyper-acute phase of ischemic stroke, prior to recanali-
zation therapies [312]. Exploring the modulatory mechanisms
of cerebral collateral flow may improve the outcome of ische-
mic stroke by rescuing cerebral perfusion in the penumbra
region, a potentially viable region of the ischemic area.
These protective mechanisms may have been impaired by
vascular risk factors such as hypertension, diabetes, and acute
ischemia. These risk factors result in insufficient collateral
flow arising from adjacent normally perfused territories, and
increase susceptibility to focal ischemic injury. The fate of the
ischemic penumbra is mainly dependent on the efficiency of
collateral flow following vascular occlusion [313]. In stroke
patients, the degree of collateralization has been revealed to be
a predictor of damage [314] and functional outcome [315].
Research has shown that intravenous and intra-arterial reper-
fusion therapy with the presence of good collaterals leads to
better clinical outcomes. Important advances in the imaging of
collateral blood vessels and collateral blood flow by multi-
modal CT and MRI techniques seem to be the most hopeful
methods for the routine assessment and quantification of col-
lateral blood flow in patients with acute ischemic stroke [316].
Pre- and post-conditioning
Conditioning consists of premeditated application of a non-
detrimental physiologic or pharmacologic stimulus or stimu-
lus train pre- and post-stroke (pre- and post-conditioning, re-
spectively) with the intention that the brain becomes
1178
transiently more resistant to ischemia [317–319]. It has been
shown that pre- and post-conditioning with an ischemic stim-
ulus are both able to produce neuroprotection in stroke models
[320]. Ischemic pre- and post-conditioning, as promising en-
dogenous mechanisms of neuroprotection [321], have the po-
tential to affect several protective pathways simultaneously
[320]. Several studies showed that patients with a history of
transient ischemic attack and who have undergone precondi-
tioning may have reduced morbidity following stroke [322].
Studies demonstrate that postischemic inflammatory ele-
ments are reduced or abolished in the stroke-tolerant brain.
In particular, preconditioning attenuates the numbers of acti-
vated neutrophils that are induced by ischemia, and also de-
creases plasma levels of monocytes and T lymphocytes [323].
In addition, expression of ICAM-1 and VCAM-1 in cerebral
endothelial cells are reduced by conditioning, [324, 325].
Hence, leukocyte movements along inflamed endothelia and
leukocyte diapedesis into the brain parenchyma were quanti-
tatively reduced in preconditioned animals [326, 327].
Following stroke, the extent of peripheral lymphopenia and
Fig. 2 Important pathways in ischemic stroke. Following ischemia, the deprivation of oxygen and glucose to the brain can trigger a cascade of events
that leads to neuronal cell death
Neurol Sci (2017) 38:1167–1186
monocyte activation were lessened in postconditioned ani-
mals [328]. Furthermore, angiogenic responses were in-
creased in the conditioned brain following stroke. Indeed, in
the neonatal rat brain, angiogenesis-related genes were upreg-
ulated in response to preconditioning, so postischemic lessen-
ing in capillary density was ameliorated in these animals
[329]. Post-conditioning could thus be seen as a strategy for
neuroprotection in the reperfused brain following stroke
[330].
Conclusion
Ischemic stroke-induced brain injury results from the interac-
tion of complex pathophysiological processes such as
excitotoxicity, oxidative stress, inflammation, and apoptosis
(Fig. 2). Current treatments for ischemic stroke injuries have
proved inadequate, owing to the complexity of events in ce-
rebral ischemia and the disappointing results from single agent
trials. Therefore, it may be unrealistic to hope that a single
Neurol Sci (2017) 38:1167–1186
neuroprotective drug will be beneficial in treatment of stroke.
Despite several agents that can prevent the cascade of events
leading to ischemic injuries, there is no obvious neuroprotec-
tive agent yet available to improve stroke outcome in humans.
Given the complexity of ischemic stroke injuries, an effec-
tive stroke treatment will probably need a combined approach.
Fortunately, several experimental discoveries of combination
drug therapy for stroke have revealed drugs that target cellular
and molecular mechanisms involved in ischemic stroke injury.
These drugs can act together either synergistically or additive-
ly. Combination therapy for cerebral ischemia can be more
effective and has fewer adverse effects than single therapies.
Further studies have shown that single-drug effects can be
improved or their time window for administration prolonged
in combination with hypothermia, a potent therapy that targets
multiple mechanisms of injury. Furthermore, cerebral collat-
eral flow and pre- and post-conditioning are a valuable new
strategies for neuroprotection following ischemic stroke.
Therefore, due to the extensive overlap of the detrimental
mechanisms triggered by brain ischemia, combination thera-
py, cerebral collateral flow, and pre- and post-conditioning
may represent a promising therapeutic approaches for the
treatment of stroke.
Acknowledgements This work was supported by Ahvaz Jundishapur
University Grant.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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