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

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 ª 2008 The Authors Journal compilation ª 2008 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,

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

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-1b and 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

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

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-1b in 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

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-1b production
following cerebral ischemia [124].
Intracerebral injection of IL-1b neutralizing 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-1b in ischemic pathophysiology [126–
129]. IL-1b expression 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-1b is 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

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-a has 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-a receptor, p75, in
resident microglia and infiltrating macrophages of the
injured hemisphere [145,148].
Administration of neutralizing antibodies raised
against TNF-a or 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-a has 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-a has been suggested to
occur via p55 receptor upregulation in neurons [154].

effects, providing significant protection against ische-
mic brain damage [166].
Chemokines
Chemokines are regulatory polypeptides that mediate
cellular communication and leukocyte recruitment in
inflammatory and immune responses. Increased
mRNA expression for MCP-1 and macrophage
inflammatory protein-1 alpha has been described in
the rat brain after focal cerebral ischemia, and both
chemokines have been suggested to contribute to tis-
sue damage via recruitment of inflammatory cells
[25,167,168]. Expression of MCP-1 has been described
in neurons 12 h after focal brain ischemia, but also in
astrocytes and microglia at later stages following the
insult [26,169]. The MCP-1 levels are also increased
in the cerebrospinal fluid of stroke patients [170].
MCP-1 is a major factor driving leukocyte infiltration
in the brain parenchyma [171]. Mice deficient in
MCP-1 develop less infarct volume as a consequence
of focal brain ischemia [172]. Similarly, in mice defi-
cient in the gene for the MCP-1 receptor, CCR2,
transient focal ischemia results in reduced infarct size,
edema, leukocyte infiltration and expression of inflam-
matory mediators [173]. Moreover, MCP-1, as well as
stromal cell-derived factor-1a, have been shown to
trigger migration of newly formed neuroblasts from
neurogenic regions to ischemic damaged areas
[169,174].
Stromal cell-derived factor-1a expression is increased
in the ischemic penumbra, particularly in perivascular

endogenous mechanisms of recovery and repair. The
switch from detrimental to beneficial effects seems to
depend on the strength and the duration of the insult
and is crucial for determining the time-window for an
effective pharmacotherapy.
Given its pivotal role in stroke pathophysiology, the
IL-1 system represents an attractive therapeutic target
(Fig. 1). Indeed, IL-1ra reduces brain injury in animal
models of cerebral ischemia and, in a recent random-
ized clinical trial, intravenous administration of
recombinant human IL-1ra in patients with acute
stroke provided evidence for safety and for effective
reduction of peripheral inflammatory markers [163].
Recombinant human IL-1ra administered intrave-
nously has also been shown to penetrate the human
brain at experimentally therapeutic concentrations
[180], although its slow penetration into cerebrospinal
fluid [181] will probably result in subtherapeutic con-
centrations during the crucial early hours of an acute
Neuroinflammatory mediators in brain ischemia D. Amantea et al.
18 FEBS Journal 276 (2009) 13–26 ª 2008 The Authors Journal compilation ª 2008 FEBS
stroke. Further work is necessary to identify a suitable
therapeutic regime prior to phase II ⁄ III clinical trials.
Acknowledgements
Financial support from the Italian Ministry of Univer-
sity and Research (PRIN prot. 2006059200_002) is
gratefully acknowledged.
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