Báo cáo khoa học: Post-ischemic brain damage: pathophysiology and role of inflammatory mediators ppt

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

Post-ischemic brain damage: pathophysiology and role of inflammatory mediators

Diana Amantea1, Giuseppe Nappi2, Giorgio Bernardi3, Giacinto Bagetta1,4and Maria T Corasaniti5

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.

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)-1b and 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]

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-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

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 Ca2+ 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

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-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 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-1b represents 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.

Focal brain ischemia produced by either permanent or

transient MCAO in rats results in a significant

induc-tion of IL-1b mRNA [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-1b production 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-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

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-1b into mature IL-1b has 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-1b detected 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-1b in 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-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] 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]

IL-6 expression significantly increases in the acute

phase of cerebral ischemia [156,157] and remains

ele-vated in neurons and reactive microglia of the ischemic

penumbra up to 14 days after the ischemic insult

[158,159] In patients with acute brain ischemia,

plasma concentrations of IL-6 are strongly associated

with stroke severity and long-term clinical outcome

[160–162] In a double-blind clinical trial on patients

with acute stroke, intravenous administration of

human recombinant IL-1ra ameliorates clinical

out-come and reduces blood concentrations of IL-6 [163]

This is in contrast to the results from animal studies

suggesting that IL-6 may exert a neuroprotective role

during stroke In fact, intracerebroventricular injection

of recombinant IL-6 reduces ischemic brain damage

induced by permanent MCAO in rat [164] It has been

suggested that increased levels of the endogenous

cytokine prevent damaged neurons from undergoing

apoptosis via signal transduction and activator of

transcription-3 activation [165]

Among other cytokines involved in stroke

patho-physiology, IL-10 and transforming growth factor-beta

have been demonstrated to have anti-inflammatory

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 astrocytes [175] This chemokine has been suggested to promote neuroprotection by increasing bone marrow-derived cell targeting to the ischemic brain and by improving local cerebral blood flow [176,177] The cru-cial involvement of chemokines in regulating cell migration, promoting the interaction of stem cells with ischemia-damaged host tissue, might be useful for improving the clinical application of stem cell therapy Another chemokine implicated in ischemic patho-physiology is fractalkine, whose expression is increased

in neurons and in some endothelial cells after a focal ischemic insult Interestingly, expression of its receptor, CX3CR1, was observed only in microglia⁄ macrophages, suggesting that fractalkine is involved in neuron– microglia signalling [178] In fact, this chemokine participates in leukocyte migration and in the activa-tion and chemoattracactiva-tion of microglia into the

infract-ed tissue [178] Indeed, fractalkine-deficient mice exhibit a smaller infarct size and lower mortality after transient focal cerebral ischemia, further underlying the detrimental effect of this chemokine on stroke outcome [179]

Conclusions

Neuroinflammatory mechanisms activated following an ischemic insult play a complex role in the pathophysi-ology of cerebral ischemia (Fig 2) The induction of pro-inflammatory genes may occur very early after the insult and commonly aggravate tissue damage Thus, early inflammatory responses appear to contribute to ischemic injury, whereas late responses may represent 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

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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