MINIREVIEW
Post-ischemic brain damage: pathophysiology and role of
inflammatory mediators
Diana Amantea
1
, Giuseppe Nappi
2
, Giorgio Bernardi
3
, Giacinto Bagetta
1,4
and Maria T. Corasaniti
5
1 Department of Pharmacobiology, University of Calabria, Rende (CS), Italy
2 IRCCS ‘‘C. Mondino Institute of Neurology’’ Foundation, Pavia, Italy and Department of Clinical Neurology and Otorhinolaryngology,
‘La Sapienza’ University, Rome, Italy
3 IRCCS-Santa Lucia Foundation, Centre of Excellence in Brain Research and Department of Neuroscience, ‘‘Tor Vergata’’ University, Rome,
Italy
4 University Centre for Adaptive Disorders and Headache, Section of Neuropharmacology of Normal and Pathological Neuronal Plasticity,
University of Calabria, Rende (CS), Italy
5 Department of Pharmacobiological Sciences, ‘‘Magna Graecia’’ University, Catanzaro, Italy and Experimental Neuropharmacology Center
‘‘Mondino-Tor Vergata’’, IRCCS-C. Mondino Foundation, Rome, Italy
Stroke is a major cause of death and long-term disabil-
ity worldwide and is associated with significant clinical
and socioeconomical implications, emphasizing the
need for effective therapies. In fact, current therapeutic
approaches, including antiplatelet and thrombolytic
drugs, only partially ameliorate the clinical outcome of
stroke patients because such drugs are aimed at
preserving or restoring cerebral blood flow rather than
at preventing the actual mechanisms associated with
neuronal cell death [1,2].
Keywords
brain ischemia; cytokines; matrix
metalloproteinases; microglia;
neuroinflammation
Correspondence
D. Amantea, Department of
Pharmacobiology, University of Calabria, via
P. Bucci, Ed. Polifunzionale, 87036
Arcavacata di Rende (CS), Italy
Fax: +39 0984 493271
Tel: +39 0984 493270
E-mail: diana.amantea@unical.it
(Received 28 June 2008, revised 7 October
2008, accepted 21 October 2008)
doi:10.1111/j.1742-4658.2008.06766.x
Neuroinflammatory mediators play a crucial role in the pathophysiology of
brain ischemia, exerting either deleterious effects on the progression of tis-
sue damage or beneficial roles during recovery and repair. Within hours
after the ischemic insult, increased levels of cytokines and chemokines
enhance the expression of adhesion molecules on cerebral endothelial cells,
facilitating the adhesion and transendothelial migration of circulating
neutrophils and monocytes. These cells may accumulate in the capillaries,
further impairing cerebral blood flow, or extravasate into the brain paren-
chyma. Infiltrating leukocytes, as well as resident brain cells, including
neurons and glia, may release pro-inflammatory mediators, such as cyto-
kines, chemokines and oxygen ⁄nitrogen free radicals that contribute to the
evolution of tissue damage. Moreover, recent studies have highlighted the
involvement of matrix metalloproteinases in the propagation and regula-
tion of neuroinflammatory responses to ischemic brain injury. These
enzymes cleave protein components of the extracellular matrix such as
collagen, proteoglycan and laminin, but also process a number of cell-sur-
face and soluble proteins, including receptors and cytokines such as inter-
leukin-1b. The present work reviewed the role of neuroinflammatory
mediators in the pathophysiology of ischemic brain damage and their poten-
tial exploitation as drug targets for the treatment of cerebral ischemia.
Abbreviations
BBB, blood–brain barrier; COX-2, cyclooxygenase-2; ICAM-1, intercellular adhesion molecule 1; ICE, interleukin-1b-converting enzyme; IL,
interleukin; IL-1ra, interleukin-1 receptor antagonist; iNOS, inducible nitric oxide synthase; MCAO, middle cerebral artery occlusion; MCP-1,
monocyte chemotactic protein-1; MMP, matrix metalloproteinase; NO, nitric oxide; TNF, tumor necrosis factor.
FEBS Journal 276 (2009) 13–26 ª2008 The Authors Journal compilation ª2008 FEBS 13
The development of tissue damage after an ischemic
insult occurs over time, evolving within hours or sev-
eral days and is dependent on both the intensity and
the duration of the flow reduction, but also on flow-
independent mechanisms, especially in the peri-infarct
brain regions [3].
A few minutes after the onset of ischemia, tissue
damage occurs in the centre of ischemic injury, where
cerebral blood flow is reduced by more than 80%. In
this core region, cell death rapidly develops as a conse-
quence of the acute energy failure and loss of ionic
gradients associated with permanent and anoxic depo-
larization [4,5]. A few hours later, the infarct expands
into the penumbra, an area of partially preserved
energy metabolism, as a result of peri-infarct spreading
depression and molecular injury pathways that are
activated in the cellular and extracellular compart-
ments. At this stage, cellular damage is mainly trig-
gered by excitotoxicity, mitochondrial disturbances,
reactive oxygen species production and programmed
cell death [6]. The evolution of tissue damage further
perpetuates for days or even weeks as a result of sec-
ondary phenomena such as vasogenic edema and
delayed inflammatory processes [3].
There is increasing evidence demonstrating that
neuroinflammatory processes play a pivotal role in the
pathophysiology of brain ischemia. The inflammatory
cascade is characterized by an immediate phase, which
is initiated a few hours after stroke and may last for
days and weeks as a delayed tissue reaction to injury
[5,7]. In addition to their deleterious contribution to
ischemic tissue damage, inflammatory mediators may
also exert beneficial effects on stroke recovery [8–10].
Mechanisms of post-ischemic
inflammation
Cellular response to injury
Post-ischemic inflammation is characterized by a rapid
activation of resident microglial cells and by infiltra-
tion of neutrophils and macrophages in the injured
parenchyma, as demonstrated both in animal models
[11,12] and in stroke patients [13–15]. Within hours
after the ischemic insult, increased levels of cytokines
and chemokines enhance the expression of adhesion
molecules, such as intercellular adhesion molecule 1
(ICAM-1), on cerebral endothelial cells, facilitating the
adhesion and transendothelial migration of circulating
neutrophils and monocytes. These cells may accumu-
late in the capillaries, further impairing cerebral blood
flow, or may extravasate into the brain parenchyma
where they release neurotoxic substances, including
pro-inflammatory cytokines, chemokines and oxy-
gen ⁄nitrogen free radicals [16]. Four to six hours after
ischemia, astrocytes become hypertrophic, followed by
activation of microglial cells that evolve into an ame-
boid type with an enlarged cell body and shortened
cellular processes. Twenty-four hours after focal ische-
mia, an intense microglial reaction develops in the
ischemic tissue, particularly in the penumbra, and
within days most microglial cells transform into
phagocytes [7,17,18]. Activation of microglial cells
enhances the inflammatory process and contributes to
tissue injury, as demonstrated by the evidence that
minocycline or other immunosuppressant drugs reduce
infarct damage by preventing microglial activation
induced by stroke [19,20]. In addition to their deleteri-
ous role, macrophages and microglial cells also con-
tribute to tissue recovery by scavenging necrotic debris
and by facilitating plasticity [16]. Indeed, selective
ablation of proliferating microglial cells exacerbates
brain injury produced by transient middle cerebral
artery occlusion (MCAO) in mice [21]. Therefore,
depending on the pathophysiologic context, the contri-
bution of inflammatory cells to tissue damage may be
different.
Adhesion molecules
The recruitment and infiltration of leukocytes into the
brain is promoted by the expression of receptors and
adhesion molecules induced by neuroinflammatory
mediators that are rapidly released from injured tissue
following ischemic insult. Indeed, focal ischemia is
associated with significantly elevated levels of cyto-
kines, such as tumor necrosis factor (TNF)-a, inter-
leukin (IL)-1band IL-6 [22,23], and chemokines, such
as monocyte chemotactic protein-1 (MCP-1) and mac-
rophage inflammatory protein-1 alpha [24–26]. These
mediators induce the expression of the adhesion mole-
cules ICAM-1 [27–30], P-selectin and E-selectin [31,32]
and integrins [33,34] on endothelial cells and leukocytes,
which promote the adhesion and transendothelial
migration of leukocytes [13,35,36]. By this mechanism,
activated neutrophils and platelets accumulate in
cerebral capillaries and further impair blood perfusion
of the injured tissue [37,38]. ICAM-1-deficient or
P-selectin-deficient mice show smaller infarct volumes
and less neutrophil infiltration following acute stroke
compared with wild-type mice [39–41]. However,
although there was initial enthusiasm concerning the
neuroprotective effect of antibodies raised against
adhesion molecules in preclinical studies [32,40,42],
administration an antibody against ICAM-1 in
humans failed to improve stroke outcome [43,44].
Neuroinflammatory mediators in brain ischemia D. Amantea et al.
14 FEBS Journal 276 (2009) 13–26 ª2008 The Authors Journal compilation ª2008 FEBS
Transcription factors
In rodent models of transient MCAO, inflammatory
genes (including cytokines, chemokines, adhesion mol-
ecules and pro-inflammatory enzymes) are upregulated
a few hours after the insult and remain elevated for
days [45–49]. The expression of these pro-inflammatory
genes is regulated by transcription factors that are
strongly stimulated by the ischemic insult and may
exert opposing effects on the evolution of tissue dam-
age [50]. Some transcription factors, such as cyclic
AMP response element-binding protein, hypoxia
inducible factor-1, nuclear factor-E2-like factor 2,
c-fos, p53 and peroxisome proliferator-activated recep-
tors alpha and gamma, are known to prevent ischemic
brain damage [51–57]. By contrast, nuclear factor-
kappaB, activating transcription factor-3, CCAAT-
enhancer binding protein-beta, interferon regulatory
factor-1, signal transduction and activator of transcrip-
tion-3, and early growth response-1 have been demon-
strated to mediate post-ischemic neuronal damage
[49,58–63]. Many transcription factors, including
nuclear factor-kappaB, interferon regulatory factor-1,
early growth response-1 and CCAAT-enhancer binding
protein-beta promote pro-inflammatory gene expres-
sion that, in turn, contributes to secondary neuronal
death [50,63]. Recent evidence suggests that the high-
mobility-group box 1 protein prompts the induction of
pro-inflammatory mediators, including the inducible
form of nitric oxide synthase (iNOS), cyclooxygenase-2
(COX-2), IL-1band TNF-a, contributing to post-
ischemic brain damage [64–66].
Enzymes
Both in human stroke and in animal models, neu-
trophils, vascular cells and, most notably, neurons,
show increased expression of COX-2, an enzyme impli-
cated in post-ischemic inflammation through the
production of toxic prostanoids and superoxide
[59,67–69]. COX-2-deficient mice develop less inflam-
mation after stroke [69], and post-ischemic treatment
with COX-2 inhibitors reduces blood–brain barrier
(BBB) damage and leukocyte infiltration induced by
transient focal cerebral ischemia in rat [70]. Moreover,
it has been recently suggested that COX-2-derived
prostaglandin E2 may contribute to ischemic cell dam-
age by disrupting Ca
2+
homeostasis in neurons via
activation of prostaglandin E2 EP1 receptors [71].
Infiltrating neutrophils, microglia ⁄macrophages and
endothelial cells may release toxic amounts of nitric
oxide (NO) via the iNOS isoform, which is strongly
induced following the ischemic insult both in animal
models [72,73] and in stroke patients [74]. Immediately
after brain ischemia, NO produced by endothelial
NOS exerts beneficial effects by promoting vasodilata-
tion, whereas NO produced during later stages of
injury by overactivation of neuronal NOS and de novo
expression of iNOS contributes to ischemic tissue
injury [75]. Despite substantial evidence underlying the
deleterious role of iNOS-derived NO in ischemic path-
ophysiology [73,75–77], by using chimeric iNOS-defi-
cient mice, a recent study has suggested that this
enzyme may not be implicated in the development
of brain damage induced by transient focal ischemia
[78], but further evidence is needed to confirm this
hypothesis.
Excessive production of NO by iNOS is responsible
for cytotoxicity by inhibiting ATP-producing enzymes,
by producing peroxynitrite and by stimulating other
pro-inflammatory enzymes such as COX-2 [79]. More-
over, NO has been suggested to promote ischemic cell
death via S-nitrosylation and, thereby, activation of
matrix metalloproteinase (MMP)-9 [80].
Recent studies have highlighted the involvement of
MMPs in ischemic pathophysiology. MMPs cleave
protein components of the extracellular matrix, such as
collagen, proteoglycan and laminin, but also process a
number of cell-surface and soluble proteins, including
receptors, cytokines and chemokines [81]. Thus, in
addition to their physiological roles, such as extra-
cellular matrix remodelling, MMPs contribute to the
propagation and regulation of neuroinflammatory
responses to injury [82,83]. Two members of this class
of proteases, the gelatinases MMP-2 and MMP-9, have
been strongly implicated in ischemic pathophysiology
because they contribute to the disruption of the BBB
and hemorrhagic transformation following injury both
in animal models [84–87] and in stroke patients [88–
90]. Previous studies have described increased expres-
sion and activity of gelatinases in the brain following
transient focal ischemia [85,91–94]. Moreover, in a rat
model of transient MCAO, we have recently demon-
strated that gelatinolytic activity increases very early
after the start of reperfusion in the regions supplied by
the middle cerebral artery. Enzyme activity was mainly
detected in neuronal nuclei during the early stages
after the insult, but also appeared in the cytosolic com-
partment and in non-neuronal, presumably glial, cells
at later reperfusion times [95].
Treatment with MMP inhibitors or MMP neutraliz-
ing antibodies has been reported to decrease infarct
volume and to prevent BBB disruption after perma-
nent or transient MCAO in rodents [84,87,96]. We
have previously demonstrated that systemic adminis-
tration of the MMP inhibitor, GM6001, at a dose that
D. Amantea et al. Neuroinflammatory mediators in brain ischemia
FEBS Journal 276 (2009) 13–26 ª2008 The Authors Journal compilation ª2008 FEBS 15
significantly prevents the increase of MMP-2 and
MMP-9 in the ischemic hemisphere, results in reduced
infarct volume in rats subjected to transient MCAO
[97].
MMP-9, but not MMP-2 [98], gene knockout is
associated with reduced infarct size and less BBB dam-
age in mouse models of ischemic stroke [87,99,100].
The mechanisms of brain damage involve gelatinase-
mediated disruption of the BBB integrity, of edema
and hemorrhagic transformation, as well as of white
matter myelin degradation [83,99]. Recent work has
also emphasized the role of MMPs and their endoge-
nous inhibitors (tissue inhibitor of matrix metallopro-
teinases) in the regulation of neuronal cell death
through the modulation of excitotoxicity [101], anoikis
[80], calpain activity [102], death receptor activation
[103], neurotrophic factor bioavailability [104] and pro-
duction of neurotoxic products [80,105]. Moreover,
these proteases may regulate inflammatory processes
because they have been involved in the processing of
pro-inflammatory cytokines, such as IL-1b, into its
biologically active form both in vitro [106] and under
ischemic conditions in vivo [97]. Indeed, we have dem-
onstrated that systemic administration of a neuropro-
tective dose of GM6001 prevents the early increase of
IL-1bin the cortex of rats subjected to transient
MCAO. This suggests that, in addition to extracellular
matrix degradation, MMPs might elicit some direct,
pathogenic effects that contribute to brain tissue dam-
age under various neuropathological conditions,
including brain ischemia.
A recent study has also demonstrated that the extra-
cellular MMP inducer is strongly upregulated in endo-
thelial cells and astrocytes of peri-focal regions
2–7 days after permanent MCAO in mice. The expres-
sion of the extracellular MMP inducer has been
spatially and temporally associated with the delayed
increase of MMP-9, suggesting its involvement in
neurovascular remodelling after stroke [107]. Accord-
ingly, inhibition of MMP-9 between 7 and 14 days after
stroke results in a substantial reduction in the number
of neurons and new vessels implicated in neurovascular
remodelling [108]. This was associated with reduced
vascular endothelial growth factor signalling resulting
from MMP inhibition [108]. These findings underscore
the complexity of MMP activity during tissue injury,
ranging from detrimental effects during the early phases
after stroke to beneficial roles at later stages [109].
Cytokines
After an ischemic insult, several cytokines are upregu-
lated in cells of the immune system, but also in resi-
dent brain cells, including neurons and glia [110].
While some cytokines, such as IL-1, appear to exacer-
bate cerebral injury, others (e.g. IL-6, IL-10 and trans-
forming growth factor-beta) seem to provide
neuroprotection [111].
The pro-inflammatory cytokine IL-1brepresents a
crucial mediator of neurodegeneration induced by excit-
atory or traumatic brain injury and, most notably, by
experimental cerebral ischemia in rodents [112] (Fig. 1).
Neurons, other cells
Endothelium
Fig. 1. Putative mechanisms implicated in
IL-1b-induced neuroinflammation after
stroke injury. CNS, central nervous system.
Neuroinflammatory mediators in brain ischemia D. Amantea et al.
16 FEBS Journal 276 (2009) 13–26 ª2008 The Authors Journal compilation ª2008 FEBS
Focal brain ischemia produced by either permanent or
transient MCAO in rats results in a significant induc-
tion of IL-1bmRNA [23,113,114]. Accordingly, IL-1b
protein levels increase very early following permanent
MCAO [115,116] and peak within hours of reperfusion
in transient focal ischemic models in rodents
[97,117,118]. The main source of the cytokine after cere-
bral ischemia are endothelial cells, microglia and macro-
phages, although it may also be expressed by neurons
and astrocytes [119,120]. Activation of p38 mitogen-
activated protein kinase has been suggested to underlie
IL-1bproduction by astrocytes and microglia during
ischemic injury in rats [121–123]. Moreover, there is evi-
dence suggesting that activation of the Toll-like recep-
tor-4 may be responsible for (pro-)IL-1bproduction
following cerebral ischemia [124].
Intracerebral injection of IL-1bneutralizing anti-
body to rats reduces ischemic brain damage [125], and
both intracerebroventricular and systemic administra-
tion of IL-1 receptor antagonist (IL-1ra) markedly
reduces brain damage induced by focal stroke, further
implicating IL-1bin ischemic pathophysiology [126–
129]. IL-1bexpression is closely associated with an
upregulation of ICAM and endothelial leucocyte adhe-
sion molecule, which reach a peak between 6 and 12 h
after the onset of ischemia [130]. ICAM-1-deficient
mice suffer smaller infarcts after transient MCAO,
suggesting that part of the IL-1b-dependent injury is
mediated by the activation of ICAM-1 [41].
IL-1bis synthesized as a precursor molecule, pro-
IL-1b, which is cleaved and converted into the mature,
biologically active form of the cytokine by caspase-1,
formerly referred to as interleukin-1b-converting
enzyme (ICE) [131–133]. Inhibition of caspase-1 by
Ac-YVAD.cmk affords neuroprotection in rodent
models of permanent [134] or transient [117] MCAO,
and evidence from knockout mice indicates that cas-
pase-1 is important in the development of cerebral
ischemic damage [135,136]. However, to date, it is not
clear whether neuroprotection yielded by caspase-1-
preferring inhibitors is mediated by reduced IL-1b
production or by interference with the cell-death
process [137]. Although most studies have clearly
established the role of ICE in the maturation of IL-1b,
evidence from ICE-deficient mice and from in vitro
studies suggests that cytokine activation might also
involve other mechanisms [138–140]. Interestingly,
in vitro studies have described the involvement of
MMPs in cytokine processing. The conversion of
recombinant pro-IL-1binto mature IL-1bhas been
demonstrated to occur after co-incubation with recom-
binant MMP-2 or MMP-9, the latter operating a more
effective and rapid cleavage [106].
We have recently demonstrated that the early
increase of IL-1bdetected in the ischemic cortex of
rats subjected to transient MCAO is not associated
with increased activity of caspase-1 [97]. By contrast,
as discussed above, cytokine production during ische-
mia-reperfusion injury appears to be dependent on
MMP activity because systemic administration of the
MMP inhibitor, GM6001, prevents the early increase
of mature IL-1bin the ischemic cortex [97] (Fig. 1). As
cytokines, such as IL-1b, regulate the expression and
the activation of MMPs, a complex cross-regulation
does occur between these neuroinflammatory media-
tors, and further studies are needed to understand their
spatio-temporal occurrence during stroke injury.
Despite being structurally and functionally corre-
lated with IL-1, results from animal studies suggest
that IL-18 is not involved in stroke pathophysiology
[141]. However, blood levels of the cytokine increase in
acute stroke patients and appear to be predictive of
unfavourable clinical outcome [142,143].
In addition to IL-1b, brain injury induced by focal
ischemia is characterized by a significant and rapid
upregulation of TNF-a, as demonstrated both in ani-
mal models and in stroke patients. Increased expres-
sion of TNF-ahas been described in neurones,
especially during the first hours after the ischemic
insult, and at later stages in microglia ⁄macrophages
and in cells of the peripheral immune system [22,144–
147]. A focal ischemic insult has also been shown to
upregulate expression of the TNF-areceptor, p75, in
resident microglia and infiltrating macrophages of the
injured hemisphere [145,148].
Administration of neutralizing antibodies raised
against TNF-aor soluble TNF receptor 1 results in
reduced infarct size in rats subjected to permanent
MCAO, suggesting that the cytokine exacerbates ische-
mic injury [28,149–151]. However, to date, the role of
TNF-ahas not been fully clarified because neuronal
damage caused by focal brain ischemia is exacerbated
in mice genetically deficient in p55 TNF receptors
[152]. The pleiotropic activities of TNF are mediated
by two structurally related, but functionally distinct,
receptors, namely p55 and p75. Selective deletion of
the p55 gene results in increased brain damage, as
compared with wild-type and p75-deficient mice fol-
lowing transient focal ischemia [153]. Moreover, ische-
mic preconditioning by TNF-ahas been suggested to
occur via p55 receptor upregulation in neurons [154].
Thus, the roles of p55 and p75 in modulating cell
death ⁄survival remain unclear, as both receptors may
activate intracellular mechanisms contributing either to
the induction of cell-death mechanisms or to anti-
inflammatory and anti-apoptotic functions [155].
D. Amantea et al. Neuroinflammatory mediators in brain ischemia
FEBS Journal 276 (2009) 13–26 ª2008 The Authors Journal compilation ª2008 FEBS 17