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 and in stroke patients. 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. 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. 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. In addition to their deleterious role, macrophages and microglial cells also contribute to tissue recovery by scavenging necrotic debris and by facilitating plasticity. Indeed, selective ablation of proliferating microglial cells exacerbates brain injury produced by transient middle cerebral artery occlusion (MCAO) in mice. 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, and chemokines, such as monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1 alpha. These mediators induce the expression of the adhesion molecules ICAM-1, P-selectin and E-selectin and integrins on endothelial cells and leukocytes, which promote the adhesion and transendothelial migration of leukocytes. By this mechanism, activated neutrophils and platelets accumulate in cerebral capillaries and further impair blood perfusion of the injured tissue. ICAM-1-deficient or P-selectin-deficient mice show smaller infarct volumes and less neutrophil infiltration following acute stroke compared with wild-type mice. However, although there was initial enthusiasm concerning the neuroprotective effect of antibodies raised against adhesion molecules in preclinical studies, administration an antibody against ICAM-1 in humans failed to improve stroke outcome.

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. 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. 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. 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. 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. 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.

Enzymes

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. COX-2-deficient mice develop less inflammation after stroke, 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. 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.

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 and in stroke patients. 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. Despite substantial evidence underlying the deleterious role of iNOS-derived NO in ischemic pathophysiology, 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, 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. Moreover, NO has been suggested to promote ischemic cell death via S-nitrosylation and, thereby, activation of matrix metalloproteinase (MMP)-9.

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. Thus, in addition to their physiological roles, such as extracellular matrix remodelling, MMPs contribute to the propagation and regulation of neuroinflammatory responses to injury. 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 and in stroke patients. Previous studies have described increased expression and activity of gelatinases in the brain following transient focal ischemia. 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.

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. 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.

MMP-9, but not MMP-2, gene knockout is associated with reduced infarct size and less BBB damage in mouse models of ischemic stroke. 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. 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, anoikis, calpain activity, death receptor activation, neurotrophic factor bioavailability and production of neurotoxic products. 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 and under ischemic conditions in vivo. 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. 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. This was associated with reduced vascular endothelial growth factor signalling resulting from MMP inhibition. 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.