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


  • Diana Amantea,

    1.  Department of Pharmacobiology, University of Calabria, Rende (CS), Italy
    Search for more papers by this author
  • Giuseppe Nappi,

    1.  IRCCS “C. Mondino Institute of Neurology” Foundation, Pavia, Italy and Department of Clinical Neurology and Otorhinolaryngology, ‘La Sapienza’ University, Rome, Italy
    Search for more papers by this author
  • Giorgio Bernardi,

    1.  IRCCS-Santa Lucia Foundation, Centre of Excellence in Brain Research and Department of Neuroscience, “Tor Vergata” University, Rome, Italy
    Search for more papers by this author
  • Giacinto Bagetta,

    1.  Department of Pharmacobiology, University of Calabria, Rende (CS), Italy
    2.  University Centre for Adaptive Disorders and Headache, Section of Neuropharmacology of Normal and Pathological Neuronal Plasticity, University of Calabria, Rende (CS), Italy
    Search for more papers by this author
  • Maria T. Corasaniti

    1.  Department of Pharmacobiological Sciences, “Magna Graecia” University, Catanzaro, Italy and Experimental Neuropharmacology Center “Mondino-Tor Vergata”, IRCCS-C. Mondino Foundation, Rome, Italy
    Search for more papers by this author

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


Neuroinflammatory mediators play a crucial role in the pathophysiology of brain ischemia, exerting either deleterious effects on the progression of tissue 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 parenchyma. Infiltrating leukocytes, as well as resident brain cells, including neurons and glia, may release pro-inflammatory mediators, such as cytokines, 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 regulation 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-surface and soluble proteins, including receptors and cytokines such as interleukin-1β. The present work reviewed the role of neuroinflammatory mediators in the pathophysiology of ischemic brain damage and their potential exploitation as drug targets for the treatment of cerebral ischemia.


blood–brain barrier




intercellular adhesion molecule 1


interleukin-1β-converting enzyme




interleukin-1 receptor antagonist


inducible nitric oxide synthase


middle cerebral artery occlusion


monocyte chemotactic protein-1


matrix metalloproteinase


nitric oxide


tumor necrosis factor

Stroke is a major cause of death and long-term disability 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].

The development of tissue damage after an ischemic insult occurs over time, evolving within hours or several 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 consequence of the acute energy failure and loss of ionic gradients associated with permanent and anoxic depolarization [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 compartments. At this stage, cellular damage is mainly triggered 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 secondary 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 infiltration 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 accumulate 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 oxygen/nitrogen free radicals [16]. Four to six hours after ischemia, astrocytes become hypertrophic, followed by activation of microglial cells that evolve into an ameboid type with an enlarged cell body and shortened cellular processes. Twenty-four hours after focal ischemia, 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 deleterious role, macrophages and microglial cells also contribute 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 contribution 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 cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 [22,23], and chemokines, such as monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1 alpha [24–26]. These mediators induce the expression of the adhesion molecules 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 molecules 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 damage [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 receptors 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 transcription-3, and early growth response-1 have been demonstrated 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 expression 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-1β and TNF-α, contributing to post-ischemic brain damage [64–66].


Both in human stroke and in animal models, neutrophils, vascular cells and, most notably, neurons, show increased expression of COX-2, an enzyme implicated in post-ischemic inflammation through the production of toxic prostanoids and superoxide [59,67–69]. COX-2-deficient mice develop less inflammation 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 damage 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 vasodilatation, 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 pathophysiology [73,75–77], by using chimeric iNOS-deficient 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]. Moreover, 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 extracellular 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 expression 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 demonstrated 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 compartment and in non-neuronal, presumably glial, cells at later reperfusion times [95].

Treatment with MMP inhibitors or MMP neutralizing antibodies has been reported to decrease infarct volume and to prevent BBB disruption after permanent or transient MCAO in rodents [84,87,96]. We have previously demonstrated that systemic administration 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 damage 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 endogenous inhibitors (tissue inhibitor of matrix metalloproteinases) 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 production 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-1β, into its biologically active form both in vitro [106] and under ischemic conditions in vivo [97]. Indeed, we have demonstrated that systemic administration of a neuroprotective dose of GM6001 prevents the early increase of IL-1β 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 damage under various neuropathological conditions, including brain ischemia.

A recent study has also demonstrated that the extracellular MMP inducer is strongly upregulated in endothelial cells and astrocytes of peri-focal regions 2–7 days after permanent MCAO in mice. The expression 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]. Accordingly, 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].


After an ischemic insult, several cytokines are upregulated in cells of the immune system, but also in resident brain cells, including neurons and glia [110]. While some cytokines, such as IL-1, appear to exacerbate cerebral injury, others (e.g. IL-6, IL-10 and transforming growth factor-beta) seem to provide neuroprotection [111].

The pro-inflammatory cytokine IL-1β represents a crucial mediator of neurodegeneration induced by excitatory or traumatic brain injury and, most notably, by experimental cerebral ischemia in rodents [112] (Fig. 1). Focal brain ischemia produced by either permanent or transient MCAO in rats results in a significant induction of IL-1β mRNA [23,113,114]. Accordingly, IL-1β 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 cerebral ischemia are endothelial cells, microglia and macrophages, 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-1β production by astrocytes and microglia during ischemic injury in rats [121–123]. Moreover, there is evidence suggesting that activation of the Toll-like receptor-4 may be responsible for (pro-)IL-1β production following cerebral ischemia [124].

Figure 1.

 Putative mechanisms implicated in IL-1β-induced neuroinflammation after stroke injury. CNS, central nervous system.

Intracerebral injection of IL-1β neutralizing antibody to rats reduces ischemic brain damage [125], and both intracerebroventricular and systemic administration of IL-1 receptor antagonist (IL-1ra) markedly reduces brain damage induced by focal stroke, further implicating IL-1β in ischemic pathophysiology [126–129]. IL-1β expression is closely associated with an upregulation of ICAM and endothelial leucocyte adhesion 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-1β-dependent injury is mediated by the activation of ICAM-1 [41].

IL-1β is synthesized as a precursor molecule, pro-IL-1β, which is cleaved and converted into the mature, biologically active form of the cytokine by caspase-1, formerly referred to as interleukin-1β-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 caspase-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-1β 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-1β, 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-1β into mature IL-1β has been demonstrated to occur after co-incubation with recombinant 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-1β 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 ischemia-reperfusion injury appears to be dependent on MMP activity because systemic administration of the MMP inhibitor, GM6001, prevents the early increase of mature IL-1β in the ischemic cortex [97] (Fig. 1). As cytokines, such as IL-1β, regulate the expression and the activation of MMPs, a complex cross-regulation does occur between these neuroinflammatory mediators, and further studies are needed to understand their spatio-temporal occurrence during stroke injury.

Despite being structurally and functionally correlated 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-1β, brain injury induced by focal ischemia is characterized by a significant and rapid upregulation of TNF-α, as demonstrated both in animal models and in stroke patients. Increased expression of TNF-α 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-α receptor, p75, in resident microglia and infiltrating macrophages of the injured hemisphere [145,148].

Administration of neutralizing antibodies raised against TNF-α or soluble TNF receptor 1 results in reduced infarct size in rats subjected to permanent MCAO, suggesting that the cytokine exacerbates ischemic injury [28,149–151]. However, to date, the role of TNF-α 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 following transient focal ischemia [153]. Moreover, ischemic preconditioning by TNF-α 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 elevated 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 outcome 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 pathophysiology, IL-10 and transforming growth factor-beta have been demonstrated to have anti-inflammatory effects, providing significant protection against ischemic brain damage [166].


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 tissue 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 deficient in the gene for the MCP-1 receptor, CCR2, transient focal ischemia results in reduced infarct size, edema, leukocyte infiltration and expression of inflammatory mediators [173]. Moreover, MCP-1, as well as stromal cell-derived factor-1α, have been shown to trigger migration of newly formed neuroblasts from neurogenic regions to ischemic damaged areas [169,174].

Stromal cell-derived factor-1α 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 crucial 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 pathophysiology 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 activation and chemoattraction of microglia into the infracted 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].


Neuroinflammatory mechanisms activated following an ischemic insult play a complex role in the pathophysiology 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.

Figure 2.

 Main pathways implicated in the neuroinflammatory response to ischemic injury. CBF, cerebral blood flow; MIP-1α, macrophage inflammatory protein-1 alpha; ROS, reactive oxygen species.

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 randomized 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 intravenously 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 concentrations 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.


Financial support from the Italian Ministry of University and Research (PRIN prot. 2006059200_002) is gratefully acknowledged.