Cerebral Stoke Molecular Mechanismsdoc

=== Cerebral stoke – molecular mechanisms ===

CEREBRAL STROKE: MOLECULAR MECHANISMS

Background.

The one of the first definition of stroke has been made by M.Goldstein and H.Barnett in 1989 year, who defined it as an "acute neurologic dysfunction of vascular origin with sudden (within seconds) or at least rapid (within hours) occurrence of symptoms and signs corresponding to the involvement of focal areas in the brain" [1]. Update, stroke or cerebral vascular accident is recognize as a neurologic event due to impaired cerebral circulation, being the third leading cause of death in many countries, including the United States [2]. Or, stroke is an abnormal condition of the brain characterized by occlusion by an embolus, thrombus, or cerebrovascular hemorrhage or vasospasm, resulting in ischemia of the brain tissues normally perfused by the damaged vessels. The sequalae of a CVA depend on the location and extent of ischemia. It is the most common and lethal neurological disorder, and the number one cause of disability in adults. The primary cause of stroke is cerebrovascular disease caused by one of several pathologic processes involving the blood vessels of the brain and often occurs in well-appearing adults.

Risk factors for stroke include family history, age >55 years old, hypertension, high cholesterol, cigarette smoking, diabetes, obesity, cardiovascular disease, previous history of stroke or TIA, high levels of homocysteine, and use of birth control or other hormone therapies. The subtypes of stroke are ischemia, infarction, and hemorrhage. Ischemia and infarction are the result of atherosclerotic development of thrombi and emboli. Decreased and/or absent cerebral circulation causes neuronal cellular injury and death. Intra-cerebral hemorrhage occurs from rupture of cerebral vessels often as the result of hypertension. Patient assessment and diagnosis include the use of computed tomography scans, magnetic resonance imaging, and the National Institute of Health Stroke Scale, and treatment depends on the etiology of the stroke. Thrombolytic therapy is the mainstay of treatment for thrombotic and embolic events. Current recommendations for future stroke care include the development of designated stroke centers. Directions for research in stroke treatment includes examining neuroprotective therapies.

Historical glance:

In 1994 the Stroke Council of the American Heart Association (AHA) published the first guidelines for the management of acute ischemic stroke. In 1996, the U.S. Food and Drug Administration (FDA) approved intravenous (IV) tissue plasminogen activator (tPA) as the first medication to treat acute ischemic stroke. The AHA subsequently published a supplement to the 1994 guidelines that addressed tPA. The advent of new therapies for acute ischemic stroke has brought about higher expectations for improved recovery and good outcome. The AHA published guidelines for the early management of ischemic stroke in 2003 and updated them in 2005 and in 2007.

Pathophysiological entity:

A stroke occurs when there is interruption of the blood supply to a particular area of the brain, ultimately leading to cell injury, ATO depletion, boosting of inflammatory response, oxidative stress activation and finally cell death (Fig.1).

Figure 1. Synoptic scheme of cerebral blood flow decline consequences

The early clinical manifestations of stroke include: sudden weakness or numbness of the face, arm, or leg; sudden dimness or loss of vision, particularly in one eye; sudden difficulty speaking or understanding speech; sudden severe headache with no known cause; unexplained dizziness, unsteadiness, or sudden falls.

Strokes can be classified according to American Heart Association [3] as either:

1) Ischemic   or   2) Hemorrhagic

1) Ischemic Strokes

Ischemic strokes are the most common, accounting for up to 80% of strokes, and occur when there is an occlusion of a blood vessel impairing the flow of blood to the brain.

In regard to N.Beauchamp et al. (1998) related evidences average blood flow in the brain is about 58-60 ml/100g brain weight per minute. At level of 30 ml/100 g, neuronal dysfunction begins to occur and at 12 ml/100 mg cell death begins to occur [4].

Ischemic strokes are divided into:

[A] thrombotic      [B] embolic      [C] systemic hypoperfusion

Thrombotic stroke

In thrombotic stroke, a thrombus or clot gradually builds up in the artery, eventually occluding it and disturbing blood flow to tissue downstream from the thrombus. Since blockage of the artery is gradual, onset of thrombotic strokes is generally slower than that of embolic strokes. Three primary influences predispose to the formation of thrombi, the so-called Virchow triad: 

The first factor of Virchow triad is endothelial injury which could be in pathogenic relation to following disorders:

– atherosclerosis;

– vasculitis;

– endocardial injury following MI;

– hypertension;

– bacterial endotoxins.

The second factor of Virchow triad is blood stasis or turbulent blood flow whose traits follow as:

– turbulence over atherosclerotic plaques;

– stasis in aneurysms;

– stasis in noncontractile endocardium following MI;

– stasis due to atrial fibrillation;

– stasis due to vascular occlusion from sickle-cell anemia.

The third factor of Virchow triad is hypercoagulability. Blood is proven to clotting especially in cases of:

– genetic hypercoagulable states (excess of factor V Leiden, proteins C and S deficiency, antithrombin III deficiency, etc.);

– acquired hypercoagulability (prolonged immobilization, cancer, prosthetic cardiac valves, heparin induced thrombocytopenia).

Between cerebral vascular accident incidence and rate of inherited or acquired thrombophilia is found a strong correlation. Also, a touch relation is emphasized between stroke platelet number elevation as well as between stroke subtypes and genetic variants of fibrinolytic system [5]. Inhibitor of plasminogen activator decrease is a strong factor leading to thrombotic stroke. Interestingly, in young patients with stroke, lupus anticoagulant is accompanied with an imbalance of the fibrinolytic system as a result of higher levels of plasminogen activator inhibitor

Embolic stroke

In an embolic stroke, a piece of material (or embolus) travels from a distant location and lodges in the blood vessel, occluding it. The most common type of embolus is a blood clot. Because the blockage arrives from another location, the onset of embolic strokes is usually quicker than that of thrombotic strokes. As well, because of this, treatment of the stroke must also include determining the source of the embolus so as to prevent further emboli. Because a blood clot is the most common type of embolus, all of the risk factors listed above for thrombotic stroke (Virchow Triad) also apply to embolic strokes.
A most often occurred cause of embolic stroke is those induced by fat thrombus (e.g. from bone marrow in a broken bone).

Other emboli are due to air (from accidental injection), cancer cells metastasizing from a distant site, and bacteria (i.e. in bacterial endocarditis).

Embolism to the brain could be arterial or cardiac by origin. Commonly recognized cardiac pathologic situations for embolism include atrial fibrillation, sinoatrial disorder, recent acute myocardial infarction, subacute bacterial endocarditis, cardiac tumors, and even valvular disorders, both native and artificial.

Stroke is a main and important sequela in patients with acute myocardial infarction (AMI), occurring in 1% to 3% of all infarctions and in 2% to 6% of patients with anterior wall infarctions. T. Podolecki et al. (2012) consider that highest risk of stroke refers to the first month after AMI beginning [6].

U. Kajemoto et al. (2014) have demonstrated that an adequate treatment of AMI, especially by use of platelet receptor P2Y12 inhibitor lead to significant decrease of stroke risk within 1-2 months period of post-infarct evolution [7]. Statins also are quit useful medicines concerning reducing of stroke risk due to improvement of endothelial function and respectively of hemostasis (anti-thrombotic effect among other pleiotropic effects).

The majority of strokes after AMI are thought to be embolic, originated from left ventricular wall mural thrombi. However, a lot of thrombi may be atherothrombotic or, in the acute phase, secondary to hemodynamic compromise. Although majority of strokes happen in the first 3-6 weeks after the infarction, real risk for stroke remains for an indefinite time.

Echocardiographic studies have demonstrated that left ventricular mural thrombosis occurs in up to 40% of patients with anterior wall MI, particularly in association with wall motion abnormalities. Mural thrombosis is uncommon with inferior wall MI. Risk factors for left ventricular mural thrombosis are large infarctions, left ventricular dilation, or congestive heart failure.

Atrial fibrillation may occur after AMI as an independent risk factor. Atrial fibrillation (AF) describes the rapid, irregular beating of the left atrium. These rapid contractions of the heart are weaker than normal contractions, resulting in slow flow of blood in the atrium. The blood pools and becomes sluggish and can result in the formation of blood clots. If a clot leaves the heart and travels to the brain, it can cause a stroke by blocking the flow of blood through cerebral arteries. Some people with AF have no symptoms, but others may experience a fluttering feeling in the area of the chest above the heart, chest pain, lightheadness or fainting, shortness of breath, and fatigue. AF is diagnosed by an electrocardiogram, and other tests are often performed to rule out contributing causes, such as high blood pressure, an overactive thyroid gland, heart failure, faulty heart valves, lung disease, and stimulant or alcohol abuse. Some people will have no identifiable cause for their AF.

The original studies comparing anticoagulant therapy with heparin and warfarin in the treatment of AMI showed, in addition to reduction in mortality and recurrent infarction, a large reduction in risk of stroke.

More recent long-term studies with oral anticoagulants in patients who survived AMI showed a reduction in risk of stroke of 40% to 50% over a 3-year period. The reduction in risk of stroke with oral anticoagulant therapy is greater than that demonstrated with aspirin, the most widely used antithrombotic treatment following AMI. Bleeding events, including intracerebral bleeding, are an uncommon but serious occurrence in patients treated with long-term oral anticoagulants. The rate for intracranial bleeding in long-term studies is approximately 1% per year. Patients identified as being at high risk for systemic embolism are similar to those at risk for left ventricular mural thrombosis. The presence of left ventricular mural thrombosis increases the risk of stroke.

Systemic hypoperofusion

In systemic hypoperfusion, blood flow is decreased to all vascular parts of the body. This most commonly occurs due to decline of the heart to pump blood or because of loss of blood during blood letting. Because the reduction in blood flow is entirely, all zones of the brain are aletred, particularly watershed areas. They are those areas of the brain that lie between the territories and fields of the major arteries. These areas are supplied by the smallest vessels and therefore are most susceptible to damage if case of blood circulation failure.

Stroke due to hypoperfusion occurs in severe stenosis of the carotid and basilar artery, and when there is microstenosis of the small deep arteries. The effects of perfusion failure fall on the most distal territories before the most proximal territories, a process termed “border zone” or “watershed infarction”, and is a repercussion of hemodynamic mismatch.

Hemodynamic stroke is considered as a type of ischemic stroke that is caused by hypoperfusion rather than by embolism or local vasculopathy. It can be caused by systemic diseases such as heart failure or hypotension, but also by severe obstruction of the carotid or vertebral arteries [8].

Patients with haemodynamic stroke or transient ischaemic attack might show specific clinical features that distinguish them from patients with embolism or local small-vessel disease. Ancillary assay of cerebral perfusion can show whether blood flow to the brain is compromised and provide important prognostic value. Management of patients who have hypoperfusion as the major cause of ischaemic stroke or as a contributing factor needs probably specific patterns. Improvement means increasing of cerebral blood flow and further researches are necessary for final definition of criteria for the diagnosis of haemodynamic pattern of stroke.

Large artery atherosclerotic plaque and contribution to stroke.

Many studies having pathogenetic goal show that atherosclerotic lesions are not randomly distributed along the cerebral arterial tree. The carotid artery system is mostly affected at the common carotid artery bifurcation, and the internal segment related to middle cerebral artery. Concerning the vertebrobasilar circulation, the first and fourth segments of the vertebral artery as well as the first segment of the basilar artery are frequently affected. Factors that support these lesions to become symptomatic are still not understood, but a stenosis more than 70% is linearly associated with increased risk of distal brain infarct.

Cooperative studies have shown that in symptomatic patients with >70% carotid stenosis, carotid endarterectomy is effective in reducing the risk of subsequent ipsilateral stroke. The accuracy of cerebral angiography in determining the severity of stenosis of extracranial and intracranial lesions has been questioned. There is no consensus regarding a method to measure the degree of stenosis from radiographic films. It is frequently impossible to differentiate between recanalized emboli, thrombi, and actual atherosclerotic plaque at the site of artery occlusion. Angiography is considered the gold standard for assessment of the cerebral vasculature. Magnetic resonance angiography, color duplex, and transcranial Doppler are acceptable noninvasive techniques to screen patients with suspected lesions.

Artery-to-artery embolism is thought to be the most common cause of cerebral infarction associated with plaques of the large cerebral arteries. Watershed infarcts secondary to the hemodynamic compromise may be less common. In situ thrombosis may occur. Embolic infarcts associated with these plaques usually involve the middle and posterior artery territories and vary in size. These lesions tend to involve the cerebral cortex and frequently are wedge-shaped on neuroimaging studies.

Vasculitis

Inflammatory conditions can involve the cerebral vasculature. Some, like granulomatous angiitis, are primarily limited to intracranial arteries and arterioles. Others usually—but not always—present with systemic manifestations by the time the effects of cerebral involvement become clinically evident. Giant cell arteritis, systemic lupus erythematosus, and polyarteritis nodosa are examples of this group. These diseases are etiologically and pathologically heterogeneous. Their causes are poorly understood, and the bedside diagnosis is problematic for lack of an accurate noninvasive test and the relatively nonspecific nature of clinical manifestations. Because of studies reporting favorable results with immunosuppressive therapy, meningeal or cerebral biopsy is indicated in selected patients. The mechanism of stroke varies: necrotizing vasculitis, hypercoagulable state, artery-to-artery or cardiac embolism. An inflammatory infiltration of the arterial wall can be seen in patients with bacterial or tuberculous meningitis, cerebral cysticercosis, fungal infection, and herpes zoster arteritis. Diagnosis and treatment are specific for each instance.

2) Hemorrhagic Strokes

Despite less common (~15% of strokes), hemorrhagic strokes due to bleeding can be much more serious. This is because in addition to the interruption of blood supply to the target tissue, the hemorrhage (bleed) can also cause increasing intracranial pressure which can physically impinge on brain tissue, further impairing perfusion of the cerebrum.
There are many causes of cerebral hemorrhage, the most common of which follow as below indicsted:

1. chronic arterial hypertension;

2. aneurysm;

3. vascular malformation (a-v malformation, cavernoma, telangectasias, venous angioma);

4. abnormally fragile vessels (amyloid angiopathy, vasculitis, sickle-cell anemia);

5. bleeding diatheses (anticoagulants, fibrinolytics, thromocytopenia, leukemia, hemophilia);

6. drug abuse;

7. head trauma (primary hemorrhagic contusion, shear injury, vascular avulsion);

8. hemorrhage into preexisting lesions (primary brain tumors, metastases, granulomas);

9. secondary hemorrhage into pre-existing infarct.

Pathogenesis and mechanisms. 

An ischemic stroke results when cerebral blood flow to an area of the brain is interrupted. Ischemia produces impaired energy metabolism and depolarization of cells that leads to an accumulation of calcium ions in the intracellular space, elevated lactate levels, acidosis, and production of free radicals. If the disruption is severe enough, cell death occurs. Activation of the N-methyl-D-aspartate receptor by an increase in glutamate leads to a cascade of chemical reactions that ultimately leads to cell death (“theory of excitotoxicity”). Modulators of this receptor include polyamines, glycine, magnesium, zinc, and phencyclidine. Magnesium delivered during ambulance transfer is under active investigation as a neuroprotective agent. However, magnesium delivered in the first hour after stroke fails to improve outcome. Normal adult brain cerebral blood flow is 50 to 60 mL/100g/minute. When cerebral blood flow falls below 18 mL/100g/minute in baboons, sensory evoked potentials disappear. In the same experiment, when cerebral blood flow fell below 12 mL/100g/minute, infarction occurred. Therefore, cerebral blood flow between 10 and 20 mL/100g/minute is considered consistent with ischemic penumbra. Cerebral blood flow below 10 mL/100g/minute is considered compatible with infarction. These delineations are not absolute because time is also a factor in the fate of tissue. Cerebral blood flows of 5 mL/100g/minute result in infarction within 30 minutes, whereas those between 5 and 15 mL/100g/minute result in infarction after 1 to 3 hours.

The pathological characteristics of ischemic stroke are dependent on the mechanism of the stroke, the size of the obstructed artery, and the availability of collateral blood flow. There may be advanced changes of atherosclerosis visible within arteries. The surface of the brain in the area of infarction appears pale. With ischemia due to hypotension or hemodynamic changes, the arterial border zones may be involved. A wedge-shaped area of infarction in the center of an arterial territory may result if there is occlusion of a main artery in the presence of collateral blood flow. In the absence of collateral blood flow, the entire territory supplied by an artery may be infarcted. With occlusion of a major artery, such as the internal carotid artery, there may be a multilobar infarction with surrounding edema. There may be flattening of the gyri and obliteration of the sulci with cerebral edema. A lacunar infarction in subcortical regions or the brainstem may be barely visible, with a size of 1.5 cm or less. Emboli to the brain tend to lodge at the junction between the cerebral cortex and the white matter. There may be early reperfusion of the infarct when the clot lyses, leading to hemorrhagic transformation. Over time the necrotic tissue is absorbed (leaving a cystic cavity) and is surrounded by a glial scar.

The initiation, progression, and activation of atherosclerosis are predominantly inflammatory conditions produced by a “response to injury” mechanism after exposure to certain injurious vascular risk factors. Individual genetic profiles can affect pathophysiological mediators of plaque development, symptomatic manifestations, and recovery from strokes associated with cerebrovascular atherosclerosis. A number of stimuli can initiate endothelial injury, which, in turn, leads to a cascade of events that result in lipid deposition and inflammatory cell migration. This inflammatory process includes the increased expression of adhesion molecules, cytokines, chemokines, metalloproteinases, and antigen-mediated activation of macrophages and T-lymphocytes. As the plaque matures, platelet aggregation and clot formation with or without plaque rupture may ensue. The clinical result is an atherothrombotic ischemic stroke.

Microscopic pathologic events. Microscopic changes after infarction depend on the age of the infarction and may be delayed up to 6 hours after infarction. Initially there is neuronal swelling, followed by shrinkage, hyperchromasia, and pyknosis. Chromatolysis appears and the nuclei become eccentric. There is swelling and fragmentation of the astrocytes and endothelial swelling. Neutrophils infiltrate as early as 4 hours after the ischemia and become abundant by 36 hours. Within 48 hours, the microglia proliferate and ingest the products of myelin breakdown and form macrophages. There is neovascularity with proliferation of capillaries and increased prominence of the existing capillaries. The elements in the area of necrosis are gradually reabsorbed and a cavity, consisting of glial and fibrovascular elements, forms. In a large infarction, there are 3 distinct zones: an inner area of coagulative necrosis; a central zone of vacuolated neuropil, leukocytic infiltrates, swollen axons, and thickened capillaries; and an outer marginal zone of hyperplastic astrocytes and variable changes in nuclear staining.

Genetics. As with most diseases, stroke is a result of the interaction between genetics and environmental exposure. There are a number of genetic causes of stroke. These disorders often result in an early age of stroke onset (i.e. younger than 40 years of age). Some inherited diseases predispose to accelerated atherosclerosis, such as the hereditary dyslipoproteinemias. A number of inherited diseases are associated with nonatherosclerotic vasculopathies, including cerebral autosomal dominant arteriopathy with subcortical infarcts and. Inherited cardiac disorders that predispose to stroke include familial atrial myxomas, hereditary cardiomyopathies, and hereditary cardiac conduction disorders. Inherited hematologic abnormalities that are associated with venous stroke include deficiencies of protein C, S, and antithrombin III. Other hematologic abnormalities including mutation of factor V Leiden, polymorphism of thermolabile methylenetetrahydrofolate reductase, and G20210A mutation of the prothrombin gene have been associated with venous and arterial stroke. Sickle cell disease is a well-known cause of stroke and frequently leads to strokes during childhood. Finally, rare inherited metabolic disorders that can cause stroke include mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes and homocystinuria [9,10].

Other potential genetic factors, particularly for carotid atherosclerosis, include PDE4D, interleukin-1 receptor antagonist (IL-1ra), toll-like receptor-4 (TL4), 5-lipoxygenase (5-LO), interleukin-6 (IL-6), hepatic lipases, cyclooxygenase 2 (COX-2), and matrix metalloproteinase polymorphisms (MMP). More recently, the potential importance of certain microRNAs (miRNAs) has been demonstrated in the walls of unstable plaques [11,12].

Inadequate blood flow in a single brain artery can often be compensated for by an efficient collateral system, particularly between the carotid and vertebral arteries via anastomoses at the circle of Willis and, to a lesser extent, between major arteries supplying the cerebral hemispheres. However, normal variations in the circle of Willis and in the caliber of various collateral vessels, atherosclerosis, and other acquired arterial lesions can interfere with collateral flow, increasing the chance that blockage of one artery will cause brain ischemia. Some neurons die when perfusion is < 5% of normal for > 5 min; however, the extent of damage depends on the severity of ischemia. If it is mild, damage proceeds slowly; thus, even if perfusion is 40% of normal, 3 to 6 h may elapse before brain tissue is completely lost. However, if severe ischemia (ie, decrease in perfusion) persists > 15 to 30 min, all of the affected tissue dies (infarction). Damage occurs more rapidly during hyperthermia and more slowly during hypothermia. If tissues are ischemic but not yet irreversibly damaged, promptly restoring blood flow may reduce or reverse injury. For example, intervention may be able to salvage the moderately ischemic areas (penumbras) that often surround areas of severe ischemia (these areas exist because of collateral flow). Mechanisms of ischemic injury include edema, microvascular thrombosis, programmed cell death (apoptosis), and infarction with cell necrosis. Inflammatory mediators (eg, IL-1B, tumor necrosis factor-α) contribute to edema and microvascular thrombosis. Edema, if severe or extensive, can increase intracranial pressure. Many factors may contribute to necrotic cell death; they include loss of ATP stores, loss of ionic homeostasis (including intracellular Ca accumulation), lipid peroxidative damage to cell membranes by free radicals (an iron-mediated process), excitatory neurotoxins (eg, glutamate), and intracellular acidosis due to accumulation of lactate.

The effects of ischemia are fairly rapid because the brain does not store glucose, the chief energy substrate and is incapable of anaerobic metabolism.1 Non-traumatic intracerebral hemorrhage represents approximately 10% to 15% of all strokes. Intracerebral hemorrhage originates from deep penetrating vessels and causes injury to brain tissue by disrupting connecting pathways and causing localized pressure injury. In either case, destructive biochemical substances released from a variety of sources play an important role in tissue destruction. Focal Ischemic Injury A thrombus or an embolus can occlude a cerebral artery and cause ischemia in the affected vascular territory. It is often not possible to distinguish between a lesion caused by a thrombus and one caused by an embolus. Thrombosis of a vessel can result in artery-to-artery embolism. Mechanisms of neuronal injury at the cellular level are governed by hypoxia or anoxia from any cause that is reviewed below. At a gross tissue level, the vascular compromise leading to acute stroke is a dynamic process that evolves over time. The progression and the extent of ischemic injury is influenced by many factors. Rate of onset and duration: the brain better tolerates an ischemic event of short duration or one with slow onset. Collateral circulation: the impact of ischemic injury is greatly influenced by the state of collateral circulation in the affected area of the brain. A good collateral circulation is associated with a better outcome. Health of systemic circulation: Constant cerebral perfusion pressure depends on adequate systemic blood pressure. Systemic hypotension from any reason can result in global cerebral ischemia. Hematological factors: a hypercoagulable state increases the progression and extent of microscopic thrombi, exacerbating vascular occlusion. Temperature: elevated body temperature is associated with greater cerebral ischemic injury. Glucose metabolism: hyper- hypoglycemia can adversely influence the size of an infarct. In response to ischemia, the cerebral autoregulatory mechanisms compensate for a reduction in CBF by local vasodilatation, opening the collaterals, and increasing the extraction of oxygen and glucose from the blood. However, when the CBF is reduced to below 20 ml/100g/min, an electrical silence ensues and synaptic activity is greatly diminished in an attempt to preserve energy stores. CBF of less than 10ml/100g/min results in irreversible neuronal injury. Mechanisms of neuronal injury Formation of microscopic thrombi responsible for impairment of microcirculation in the cerebral arterioles and capillaries is a complex phenomenon. Formation of a micro thrombus is triggered by ischemia-induced activation of destructive vasoactive enzymes that are released by endothelium, leucocytes, platelets and other neuronal cells. Mechanical “plugging” by leucocytes, erythrocytes, platlets and fibrin ensues. At a molecular level, the development of hypoxic- ischemic neuronal injury is greatly influenced by “overreaction” of certain neurotransmitters, primarily glutamate and aspartate. This process called “excitotoxicity” is triggered by depletion of cellular energy stores. Glutamate, which is normally stored inside the synaptic terminals, is cleared from the extracellular space by an energy dependent process. The greatly increased concentration of glutamate (and aspartate) in the extracellular space in a depleted energy state results in the opening of calcium channels associated with N-methy1-D-asapartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxanole propionate (AMPA) receptors. Persistent membrane depolarization causes influx of calcium, sodium, and chloride ions and efflux of potassium ions. Intracellular calcium is responsible for activation of a series of destructive enzymes such as proteases, lipases, and endonucleases that allow release of cytokines and other mediators, resulting in the loss of cellular integrity. Inflammatory response to tissue injury is initiated by the rapid production of many different inflammatory mediators, tumor necrosis factor being one of the key agents. Leukocyte recruitment to the ischemic areas occurs as early as thirty minutes after ischemia and reperfusion. In addition to contributing to mechanical obstruction of microcirculation, the leucocytes also activate vasoactive substances such as oxygen free radicals, arachidonic acid metabolites (cytokines), and nitric acid. The cellular effects of these mediators include vasodilatation, vasoconstriction, increased permeability, increased platelets aggregation, increased leukocyte adherence to the endothelial wall, and immunoregulation. Endothelial cells are one of the first cell types to respond to hypoxia. This response occurs at morphological, biochemical and immunological levels, causing a variety of physiological and pharmacological effects. This results in a reduction in the luminal patency of the capillary vessel. Mechanical plugging by erythrocytes, leukocytes, and platelets ensues. At a biochemical level, endothelial cells mediate the effects of vasoactive agents such as endothelin peptides, eicosanoids, and smooth muscle relaxant (probably nitric acid), which in part modulate the vascular tone of the microcirculation. Activation of endothelial adhesion molecules promotes leukocyte adherence to the endothelial wall, a key process in the initiation of the inflammatory process.

Ischemic penumbra. Within an hour of hypoxic-ischemic insult, there is a core of infarction surrounded by an oligemic zone called the ischemic penumbra (IP) where autoregulation is ineffective. The critical time period during which this volume of brain tissue is at risk is referred to as the “window of opportunity” since the neurological deficits created by ischemia can be partly or completely reversed by reperfusing the ischemic yet viable brain tissue within a critical time period (2 to 4 hours?). Ischemic penumbra is characterized by some preservation of energy metabolism because the cerebral blood flow in this area is 25% to 50% of normal. Cellular integrity and function are preserved in this area of limited ischemia for variable periods of time. The pathophysiology of IP is closely linked to generation of spontaneous waves of depolarization (SWD). SWD can originate from multiple foci; some from the ischemic core and others form ischemic foci within the periinfarct zone (penumbra). Sustained increases of synaptic glutamate and extracellular potassium ions are closely associated with the development of SWD. Glutamate receptor antagonists that block transmembrane calcium flux and prevent intracellular calcium accumulation are known to suppress SWD. Hypoxic or rapid depolarizations eventually supervene just before irreversible neuronal death.

Neuronal death. The two processes by which injured neurons are known to die are coagulation necrosis and apoptosis. Coagulation necrosis (CN) refers to a process in which individual cells die among living neighbor cells without eliciting an inflammatory response. This type of cell death is attributed to the effects of physical, chemical, or osmotic damage to the plasma membrane. This is in contrast to liquefaction necrosis, which occurs when cells die, leaving behind a space filled by “inflammatory response” or pus. In CN, the cell initially swells then shrinks and undergoes pyknosis – a term used to describe marked nuclear chromatin condensation. This process evolves over 6 to 12 hours. By 24 hours extensive chromatolysis occurs resulting in pan-necrosis. Astrocytes swell and fragment, myelin sheaths degenerate. Irreversible cellular injury as demonstrated by eosinophilic cytoplasm and shrunken nuclei after arterial occlusion. The morphology of dying cells in coagulation necrosis is different than that of cell death due to apoptosis. The term apoptosis is derived from the study of plant life wherein deciduous trees shed their leaves in the fall. This is also called “programmed cell death”, because the leaves are programmed to die in response to seasonal conditions. Similarly, cerebral neurons are “programmed” to die under certain conditions, such as ischemia. During apoptosis, nuclear damage occurs first. The integrity of the plasma and the mitochondrial membrane is maintained until late in the process. Ischemia activates latent “suicide” proteins in the nuclei, which starts an autolytic process resulting in cell death. This autolytic process is mediated by DNA cleavage. Apoptotic mechanisms begin within 1 hour after ischemic injury whereas CN begins by 6 hours after arterial occlusion. This observation has an important bearing on future directions of research. The manner by which apoptosis evolves is a focus of much research, because, hypothetically, neuronal death can be prevented by modifying the process of DNA cleavage that seems to be responsible for apoptosis. However, strokes caused by vasospasm (migraine, following SAH, hypertensive encephalopathy) and some form of “arteritis” stand out among the more infrequent causes of stroke. Thrombosis Atherosclerosis is the most common pathological feature of vascular obstruction resulting in thrombotic stroke. Atherosclerotic plaques can undergo pathological changes such as ulcerations, thrombosis, calcifications, and intra-plaque hemorrhage. The susceptibility of the plaque to disrupt, fracture or disrupt or ulcerate depends on the structure of the plaque, and its composition and consistency. Disruption of endothelium that can occur in the setting of any of these pathological changes initiates a complicated process that activates many destructive vasoactive enzymes. Platelet adherence and aggregation to the vascular wall follow, forming small nidi of platelets and fibrin. Leucocytes that are present at the site within 1 hour of the ictus mediate an inflammatory response.38-43 In addition to atherosclerosis, other pathological conditions that cause thrombotic occlusion of a vessel include clot formation due to hypercoagulable state, fibromuscular dysplasia, arteritis (Giant cell and Takayasu), and dissection of a vessel wall. In contrast to the occlusion of large atherosclerotic vessels, lacunar infarcts occur as a result of occlusion of deep penetrating arteries that are 100 to 400 mm in diameter and originate for the cerebral arteries. The putamen and pallidum, followed by pons, caudate nucleus, and internal capsule are the most frequently affected sites. The size of a lacunar infarct is only about 20 mm in diameter. The incidence of lacunar infarcts is 10% to 30% of all strokes depending on race and preexisting hypertension and diabetes mellitus. The small arteriole, most frequently as a result of chronic hypertension lengthens, becomes tortuous and develops subintimal dissections and micro-aneurysms rendering the arteriole susceptible to occlusion from micro-thrombi. Fibrin deposition resulting in lipohyalinosis is considered to be the underlying pathological mechanism. The three main factors associated with “red infarcts” or hemorrhagic infarctions include the size of the infarct, richness of collateral circulation, and the use of anticoagulants and interventional therapy with thrombolytic agents. Large cerebral infarctions are associated with a higher incidence of hemorrhagic transformation. Hypertension is not considered to be an independent risk factor for hemorrhagic transformation of an ischemic infarct.

Some neurons are more susceptible to ischemia than others. These include the pyramidal cell layer of the hippocampus and the Purkinje cell layer of the cerebellar cortex. Cerebral gray matter is also particularly vulnerable. Abundance of glutamate in these neurons renders them more susceptible to global ischemia. Global ischemia causes the greatest damage to areas between the territories of the major cerebral and cerebellar arteries known as the “boundary zone” or “watershed area.” The parietal-temporal-occipital triangle at the junction of the anterior, middle, and posterior cerebral arteries is most commonly affected. Watershed infarction in this area causes a clinical syndrome consisting of paralysis and sensory loss predominantly involving the arm; the face is not affected and speech is spared. Watershed infarcts make up approximately 10% of all ischemic strokes and almost 40% of these occur in patients with carotid stenosis or occlusion.

Old age is associated with an enhanced susceptibility to stroke and poor recovery from brain injury, but the cellular processes underlying these phenomena are uncertain. Therefore studying the basic mechanism underlying functional recovery after brain ischemia in aged subjected it is of considerable clinical interest. Potential mechanisms include neuroinflammation, changes in brain plasticity-promoting factors, unregulated expression of neurotoxic factors, or differences in the generation of scar tissue that impedes the formation of new axons and blood vessels in the infarcted region. Available data indicate that behaviorally, aged rats were more severely impaired by ischemia than were young rats, and they also showed diminished functional recovery. Further, as compared to young rats, aged rats develop a larger infarct area, as well as a necrotic zone characterized by a higher rate of cellular degeneration, and a larger number of apoptotic cells. In both old and young rats, the early intense proliferative activity following stroke leads to a precipitous formation of growth-inhibiting scar tissue, a phenomenon amplified by the persistent expression of neurotoxic factors. Reduced transcriptional activity in the healthy, contralateral hemisphere in conjunction with an early upregulation of DNA damage related genes and the early induction of proapoptotic genes in the periinfarct area of aged rats are likely to account for poor neurorehabilitation after stroke in aged rats. Finally, the regenerative potential of the rat brain is largely preserved up to 20 months of age but gene expression is temporally displaced, has lower amplitude, and is sometimes of relatively short duration. Most interestingly, it has recently been shown that the human brain can respond to stroke with increased progenitor proliferation in aged patients opening the possibilities to utilize this intrinsic attempt for neuroregeneration of the human brain as a potential therapy for stroke. Given the heterogeneity of stroke, a universal anti-inflammatory solution may be a distant prospect, but probably neuroprotective drug cocktails targeting inflammatory pathways in combination with thrombolysis may be a possibility for acute stroke treatment in the future.

The mechanisms leading to cellular damage from ischemia-reperfusion (I/R) injury are complex and multi-factorial. Accumulating evidence suggests an important role for oxidative stress in the regulation of neuro-inflammation following stroke. Gene expression studies have revealed that the increase in oxygen radicals post-ischemia triggers the expression of a number of pro-inflammatory genes. These genes are regulated by the transcription factor, nuclear factor-kappa-B (NF-kappaB) which is redox-sensitive. It is hypothesised that changes in the oxidative state may modulate alterations in the neuro-inflammatory response following an I/R injury. Furthermore, NF-kappaB is involved in the transcriptional regulation of adhesion molecules, which play an important role in leukocyte-endothelium interactions. Recent studies have demonstrated that adhesion molecule-mediated leukocyte recruitment is associated with increased tissue damage in stroke, while mice lacking key adhesion molecules conferred neuro-protection. Nevertheless, the involvement of oxidative stress in leukocyte recruitment and the subsequent regulated cell injury is yet to be elucidated. While leukocyte infiltration into the ischemic brain is detrimental, leukocyte accumulation in the microvasculature was shown to be one of the many factors implicated in reduced reperfusion. Although this "no-reflow" phenomenon was confirmed in a variety of animal models of cerebral ischemia, the exact mechanism is still uncertain. This review aims to highlight the impact that oxidative stress has in the regulation of post-ischemic neuro-inflammation and the implication for the cerebral microvasculature after injury.

Cerebral ischemia induces a complex series of molecular pathways involving signaling mechanisms, gene transcription, and protein formation. The proteases and free radicals involved are important, both individually and in concert, at each of the steps in the injury cascade. Matrix metalloproteinases (MMPs) and serine proteases are essential in the breakdown of the extracellular matrix around cerebral blood vessels and neurons, and their action leads to opening of the blood-brain barrier, brain edema, hemorrhage, and cell death. Reactive oxygen and nitrogen species affect the signaling pathways that induce the enzymes, the stability of the mRNA, and their activation processes.

Mice that either lack MMP genes or overexpress free radical-removing genes exhibit diminished cerebral damage after stroke. Drugs that block MMP activity, or are free radical scavengers, significantly reduce ischemic damage. Understanding the relationship between proteases and free radicals in cerebral ischemia is critical for the design of therapeutic agents aimed at controlling cell death in ischemic tissues.

Range of pathological events.

The most upstream consequence of cerebral ischemia fundamentally is composed of an energetic problem. In the area of reduced blood supply, adenosine triphosphate (ATP) consumption continues despite insufficient synthesis, causing total ATP levels to drop and lactate acidosis to develop with concomitant loss of ionic homeostasis in neurons. Once this initial step has taken place, an ischemic cascade follows involving a multimodal and multicell series of downstream mechanisms. The reader is referred to several excellent reviews for an in-depth discussion of the molecular details. Here, we will summarize the key steps and focus on mechanisms of acute neuronal injury. Severe cerebral ischemia leads to a loss of energy stores resulting in ionic imbalance and neurotransmitter release and inhibition of reuptake. It is especially the case for glutamate, the main excitotoxic neurotransmitter. Glutamate binds to ionotropic N-Methyl-D-aspartate (NMDA) and a-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (iGluRs), promoting a major influx of calcium. This calcium overload triggers phospholipases and proteases.

In addition, the glutamate receptors promote an excessive sodium and water influx with concomitant cell swelling, edema and shrinking of extracellular space. Massive calcium influx activates a catabolic process mediated by proteases, lipases, and nucleases. In experimental models, blockade of GluRs can reduce infarction volume via three ways: first, reducing calcium influx and its related proteases activations; second, attenuating cortical spreading depressions (CSDs); third, triggering in neurons the subfamily of metabotropic mGluRs toward prosurvival or prodeath signaling. This glutamate excitotoxicity concept raised major hope for stroke new therapies. Despite validation of the concept in many experimental models, all the trials targeting NMDA and AMPA receptors failed to improve outcome of the patients, likely for reasons beyond methodological shortcomings in study designs. High calcium, sodium, and adenosine diphosphate (ADP) levels in ischemic cells stimulate excessive mitochondrial oxygen radical production within other sources of free radicals production such as prostaglandin synthesis and degradation of hypoxanthine. These reactive oxygen species (ROS) directly damage lipids, proteins, nucleic acid, and carbohydrates. They are especially toxic for the cells because baseline levels and any corresponding upregulation of antioxidant enzymes [superoxyde dismutase (SOD), catalase, glutathione] and scavenging mechanisms (a-tocopherol, vitamin C) are too slow to offset ROS production. Downstream of free radicals, other neuronal death mechanisms will also be induced involving, e.g. mitochondrial transition pore formation, the lipoxygenase cascade, the activation of poly ADPribose polymerase (PARP) and amplified ionic imbalance via secondary recruitment of calcium-permeable transient receptor potential ion (TRPM) channels. Furthermore, ROS and reactive nitrogen species also has the potential of modifying endogenous functions of proteins, which may be neuroprotective. Ultimately, these multimodal cascades will result in a complex mix of neuronal death comprising, necrosis, apoptosis, and autophagy. In parallel with the molecular events briefly outlined here, a physiologic process called cortical spreading depression (CSD) has also been recently identified as a candidate target for stroke. CSD is an intense depolarization of neuronal and glial membranes slowly propagating by way of gray matter contiguity at a speed of two- to six-millimeters per minute. It is characterized by near complete breakdown of ion gradients, near complete sustained depolarization, extreme shunt of neuronal membrane resistance, loss of electrical activity, and neuronal swelling and distortion of dendritic spines, associated with a shrinkage of extracellular volume. CSD occurs when extracellular K+ concentrations exceed a critical threshold. Glutamate, inhibitors of the sodium pump, hypoxia, hypoglycemia, ischemia, electrical stimulation, and status epilepticus are other well-described triggers. Gene mutations affecting either ions channels such as calcium channel or the astrocytic sodium pump decrease the level for CSD. Interestingly, the Neurogenic locus notch homolog protein 3 (NOTCH3) gene mutation causing cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy syndrome (CADASIL) is also associated with susceptibility to CSD linking CSD with the neurovascular unit. Moreover, CSD is illustrating another aspect of the normal neurovascular coupling of regional cerebral blood flow (rCBF) and physiological neuronal activity. Under physiological conditions, CSD is associated with a major CBF rise in an attempt to match to the increase in metabolic demand. This sharp hyperemia is followed by a long lasting oligemia associated with abnormal vascular responses. Under pathological conditions such as ischemia, an inverse hemodynamic response characterized by a CBF reduction spreading ischemia can be observed. In ischemia, these waves of CSD originating in the peri infarct area can invade repeatedly peri-ischemic tissue, adding a major metabolic demand on penumbra, and at term, the spreading depolarization expands the volume of infarction. After decades of clinical doubt, CSD has now been demonstrated in patients suffering from brain trauma, sub-arachnoid hemorrhage, spontaneous intracerebral hematoma, and ischemic strokes. It is tempting to target CSD as they increase the volume of infarction. NMDA antagonists can effectively suppress CSDs. But this class of drugs has failed before in many clinical stroke trials. Some differences between animal and human ischemic brains such as presence of gyri or astrocytes– neurons ratio may explain differences in occurrence and consequences of CSD. Nontraumatic methods to study CSD in humans and better methods to block CSD and improve stroke outcomes will have to be developed. Ultimately, blocking integrated mechanisms of neuronal death in the ischemic cascade may be difficult. But this is not because these targets are invalid. Indeed, the fundamental neurobiology of these molecular mechanisms is sound. From a clinical perspective, blocking these early targets is difficult because the therapeutic time windows may be extremely narrow. Furthermore, the majority of these pathways do not necessarily occur during the period of arterial occlusion but when the tissue is reperfused or some significant blood flow still persists. Hence, the clinical rationale herein might not be driven by a stand-alone neuroprotective treatment but instead aimed at a combination therapy to be given along with thrombolytic or mechanical reperfusion. Indeed, a recent powerful study provides proof of concept. Tymianski and colleagues recently showed that a compound that blocks the postsynaptic density protein post-synaptic density protein-95 (PSD-95) was able to reduce infarction in a nonhuman primate model of transient focal cerebral ischemia. The majority of our mechanisms and targets in experimental models remain mostly focused on neurons in gray matter. In human stroke, however, a signifi- cant portion of white matter injury is surely involved. Nonneuronal cells and white matter are extremely vulnerable to ischemic stress. White matter is primarily composed of axonal bundles ensheathed with myelin. The cells forming these sheaths are the oligodendrocytes, which tend to be arranged in rows parallel to axonal tracts. Just before and after birth, oligodendrocyte precursor cells (OPCs) multiply rapidly, mature into OLGs, and develop processes for myelin formation. White matter blood flow is lower than in gray matter, and there is little collateral blood supply in the deep white matter. Hence, white matter ischemia will be typically severe with rapid cell swelling and tissue edema. White matter ischemia activates several kinds of proteases, which weaken the structural integrity of axons and myelin sheath. Demyelinated axons may be more susceptible to ischemic injury due to their heightened metabolic requirements caused by loss of energy-efficient salutatory conduction and leaky sodium channels. Moreover, mechanisms responsible for ischemic cell death may be different between gray matter and white matter. While an increase in intracellular Ca2+ is involved in both white and gray matter ischemia, the routes of Ca2+ entry might differ. In white matter, pathological Ca2+ entry occurs in part due to increased intracellular Na+ and membrane depolarization. In contrast, voltage-dependent Ca2+ channels and glutamate-activated ionic receptors in gray matter have been traditionally viewed as the primary routes of pathological Ca2+ entry. For a more detailed analysis of mechanistic and cell-based models of white matter injury, please refer to a previous review on this topic. Although there are no validated drugs to prevent white matter injury in stroke, promising leads may perhaps be found in other fields. A noncompetitive NMDA receptor antagonist memantine is now licensed for moderate-to-severe Alzheimer’s disease in the United States and the European Union. At least two studies suggest that memantine may be protective to white matter. Furthermore, antioxidant drugs would be also potent therapeutic candidates for white matter stroke. Although the radical spin trap disufenton sodium (NXY)-059 failed in a final phase 3 clinical trial; it is now suspected that drug penetration through the blood–brain barrier might have been low for this specific compound. ROS are known to be deeply involved in white matter pathophysiology. Axons contain abundant mitochondria, which is an organelle for a source of ROS. Free radical scavengers signifi- cantly reduce white matter injury in rodent stroke models. Whether we can develop a radical scavenger that effectively penetrates white matter in large human brains remains to be seen. Another potential example may be found in the antiplatelet drug cilostazol, a type III phosphodiesterase (PDE3) inhibitor. Cilostazol is approved for the treatment of intermittent claudication and improves pain-free walking distance in patients with peripheral arterial disease. But recent report proposed that cilostazol may also be used for white matter protection. Future studies are warranted to dissect the mechanisms how PDE3 inhibition contributes to defend white matter from ischemic stress. No matter how compelling our molecular targets might be, it may still be difficult to reach all stroke patients in time for acute therapy. Hence, we will also need to consider how to efficiently promote rehabilitation and repair after stroke. This may be especially relevant for white matter where ‘repairing or readapting the wiring’ in neuronal networks is essential for functional recovery. In the chronic phase after stroke, several endogenous responses should be induced for repairing white matter damage. Although the population of OPCs is relatively low in the adult white matter, OPCs may play substantial roles for remyelination after injury. During development, OPCs migrate from the ventricular zone to their final destination and then differentiate to from myelin sheaths. But, after brain injury, they are guided to the site for contributing to myelin repair. It remains to be fully elucidated how oligodendrogenesis occurs. But it is likely that multiple cell– cell trophic interactions may be involved. Recent findings suggest that there might exist an oligovascular niche in white matter wherein cell–cell trophic coupling between cerebral endothelium and oligodendrocyte helps sustain ongoing oligodendrogenesis and angiogenesis. In addition, astrocytes may secrete trophic factors to support OPC survival. Hence, cell–cell trophic coupling in white matter may provide an essential mechanism for repairing damaged white matter in the chronic stroke phase. In this regard, cell-based treatment would be promising as a restorative therapy in white matter stroke. Some experimental studies suggest that injected stem cells (and their derivatives) could survive, differentiate into not only neurons/astrocytes but also oligodendrocytes. Furthermore, cellular therapies may be additionally beneficial because these cells produce a rich broth of trophic factors that stimulate endogenous repair of host tissue. Inflammation and microglia Inflammation is an important part of stroke pathophysiology, especially in the context of reperfusion. Restoring CBF is an obvious and primary goal. But as discussed earlier, ischemia– reperfusion itself can also set off numerous cascades of secondary injury. Reactive radicals will be generated, blood–brain barrier integrity may be compromised, and multimodal neuronal death processes composed of programmed necrosis, apoptosis, and autophagy may still continue unabated. Along with these central neuronal responses, an activation of peripheral immune responses is now known to occur as well.

Proof of concept has been repeatedly obtained – blocking various steps in the inflammatory cascade prevents central accumulation of deleterious immune cells such as neutrophils and T cells, and improve outcomes, at least in experimental models, Many recent excellent reviews on this topic have been published and readers are referred to these for more in-depth discussions. In this section, we will focus on microglia, to ask whether these central responders in fact may comprise promising targets. Microglia are resident immune cells of the central nervous system (CNS) and serve as sensors and effectors in the normal and pathologic brain. Microglia are involved in most CNS pathologies and constantly monitor the microenvironment and respond to any kind of pathologic change, thus exerting typical macrophagic functions, such as phagocytosis, secretion of proinflammatory cytokines, and antigen presentation. Microglia have important roles even in normal brain. In fact, it has been suggested that the term ‘resting microglia’ should be changed to ‘surveying microglia’ to describe how microglia continuously monitor the healthy CNS – these are not functionally silent cells (71,73). Microglial processes are highly dynamic in intact cortex. A recent imaging study demonstrated that the dynamic motility of ‘resting’ microglial processes in vivo may be because microglia constantly monitor and respond to the functional status of synapses. In the adult brain, microglia contribute to synapse remodeling and neurogenesis. Furthermore, microglia may also interact with axons, be involved in blood vessel formation, and act as phagocytes for the removal of dying cells during the process of programmed cell death. After an ischemic lesion, resident microglia are activated within minutes of ischemia onset and accumulate at the lesion site and in the penumbra. Postischemic microglial proliferation peaks at 48–72 h after focal cerebral ischemia and may last for several weeks after initial injury. Upon activation, microglia undergo proliferating, morphological change from a ramified to few and thicker processes or amoeboid appearance, producing cytokines, chemokines, and growth factors, generating reactive oxygen and nitrogen species, increasing expression of immunomodulatory surface markers, and having the ability of presenting antigen. Traditionally, activated microglia would be viewed as deleterious actors in stroke. Overactivated microglia can be neurotoxic by releasing ROS via NADPH oxidase, proinflammatory cytokines [e.g. tumor necrosis factor (TNF)-a, interleukin (IL)-1b], and neurovascular proteases such as matrix metalloproteinase (MMP-9). But it is now increasingly recognized that microglial activation may not always be bad after stroke. Microglial activation is not a univalent state, and activated microglia can exhibit phenotypic and functional diversity depending on the nature, strength, and duration of the stimulus. At least two activated phenotypes, ‘classically activated’ (also called M1) or an ‘alternatively activated’ (also called M2), have been identified. Inflammatory microglia (M1) release a number of proinflammatory cytokines such as TNF-a and IL-1b, and ROS and nitrous oxide (NO). While anti-inflammatory microglia (M2) are considered less inflammatory than M1 cells, they are characterized by reduced NO production and increased production of anti-inflammatory cytokines and neurotrophic factors such as GDNF, BDNF, bFGF, insulin-like growth factor (IGF)-1, TGF-b, and VEGF. Indeed, beyond stroke per se, whether microglial activation is neurotoxic is a long-standing debate in neurobiology. Some degree of microglial activation reflects its important physiological function to protect neurons and the integrity of CNS, while overactivation or loss of microglial functions exacerbates a preexisting neuropathology or this function gets lost in neurodegenerative diseases. There is clear evidence shows that activated microglia can maintain and support neuronal survival by release of neurotrophic and antiinflammatory molecules, the clearance of toxic products or invading pathogens, as well as the guidance of stem cells to inflammatory lesion sites to promote neurogenesis. Hence, microglia can be potentially beneficial in some circumstances. Selective ablation of proliferating resident microglia results in a significant increase in the size of the infarction associated with an increase in apoptotic neurons and a decrease in the levels of IGF-1 in transient focal cerebral ischemia. Moreover, these results reveal a marked neuroprotective potential of proliferating microglia serving as an endogenous pool of neurotrophic molecules such as IGF-1. Exogenous microglia isolated from brain cultures injected into the subclavian artery of Mongolian gerbils results in increased numbers of surviving neurons in ischemic hippocampus. Administration of exogenous microglia increases the expression of BDNF and GDNF in the ischemic hippocampus, which may explain the positive effect of microglia on neuronal survival. Similarly, exogenous microglia microinjected into the ventricles of rats can migrate into brain parenchyma and significantly decrease neuronal loss induced by focal ischemia and reperfusion. The most likely reason why the activation of microglia has contradictory influence on stroke outcome lies in the timing and the degree of the expression of the cytokines. TNF-a is a prime instance. Overexpression of TNF-a is detrimental to stroke outcome. However, some studies have shown that microglia-derived TNF-a can be neuroprotective in cerebral ischemia in mice and that TNF-a-p55 receptor knockout mice have an increased infarction volume. It is important to understand the different states of microglia in diseases, and consequently, modulating the duration and the magnitude the expression of cytokines and growth factors will critically affect the results. From a clinical perspective, targeting inflammation may not be easy. Previous clinical trials attempting to block intercellular adhesion molecule (ICAM)-1 on cerebral endothelium did not work and treated patients actually got worse. Differences in systemic blood and immune responses in animal models vs. human patients may make it difficult to safely and effectively broadly block inflammatory pathways. In this regard, searching for ‘druggable’ mechanisms in central compartment microglia may represent an alternative approach. However, careful attention will have to be paid to the notion that microglial responses after stroke might be biphasic as well, i.e. a mix of deleterious and beneficial effects that evolve over time. Conclusions The search for effective acute stroke treatments has encountered many failures in clinical trials. In contrast, stroke prevention has gained major successes in recent years. For example, lowering blood pressure is associated with a significant reduction of stroke risk. Why? In part, this is because stroke prevention strategies address efficient targets that share an underlying linear and monotonic model. Taking hypertension as a paradigm, shear stress increase related to high blood pressure increases production of free radicals via NADPH activation and uncoupling of nitric oxide synthase, key steps in hypertension-related vascular oxidative stress. Once these proximal triggers are initiated, parallel downstream actions are induced. Loss of vasoregulatory nitric oxide leads to vasoconstriction, microvascular flow impairment, leukocytes adhesion, and smooth muscle cell proliferation. This integrated response then promotes cytokine and matrix metalloproteasemediated inflammation in systemic and cerebral blood vessels. Thus, a ‘single’ vascular risk factor is able to trigger oxidative stress and inflammation to deleteriously affect the entire neurovascular unit (neuronal, glial, and vascular compartments). From the molecular level to cellular injuries and vascular and brain consequences, each step of severity in risk factors amplifies the consequences, and can be integrated to the higher level. These effects accumulate over time – the longer a subject is exposed to a risk factor, the more severe are the consequences. The effects of ‘risk factors’ are inherently complex. But these effects can be easily detected even in simplified animal models. For example, the free radical scavenger NXY-059 was much less effective in spontaneously hypertensive rats compared with normotensive rats. Hence, any translation of therapies from models to humans should require rigorous attention to how these targets are influenced by the entire range of risk factors inherent in stroke patients. If such conceptual models work for stroke prevention, can they also work for our ongoing search for acute stroke treatments? The concept of the neurovascular unit has been useful in this regard. For perhaps far too long, we were focused only on the ‘neurobiology’ of stroke. But brain function (and dysfunction) is not only based on neurons but on cell–cell signaling between all cell types. Thus, stroke therapeutics should seek not only to prevent neuron death but to rescue functional crosstalk between all cells in the neurovascular unit. Embedded into this conceptual framework are the spatial and temporal aspects of stroke pathophysiology. Cell–cell interactions evolve depending on baseline risk factors and inflammation. And ultimately, the model is not monotonic – the same mediators can be deleterious or beneficial depending on the state of tissue injury and progression.