Pathophysiology, treatment, and animal and cellular models of human ischemic stroke

A significant portion of ischemia-induced neuronal damage is mediated by excessive accumulation of excitatory amino acids, leading to toxic increases in intracellular calcium [20]. Although this is an intrinsic defensive response to protect against ischemia by activating a reaction to severe cell stress, paradoxically, this increase in intracellular calcium activates multiple signaling pathways, which ultimately leads to cell death. Soon after reduction or termination of cerebral blood flow, energy-dependent cellular pumps fail due to a fall in glucose-dependent ATP generation, resulting in the flow of numerous ionic species into the cell. This results in cellular swelling through osmosis and cellular depolarization. Calcium ions (Ca²⁺) enter the cell through voltage-dependent and ligand-gated ion channels, resulting in activation of a number of proteases, kinases, lipases, and endonucleases, triggering of the intrinsic apoptotic pathway and thus ending in cell death by [18, 21]. Glutamate, which is the major excitatory neurotransmitter in the brain, accumulates in the extracellular space following ischemia, and activates its receptors [22]. Glutamate receptor activation induces alterations in the concentration of intracellular ions, most notably Ca²⁺ and sodium ions (Na⁺) (Figure 1). Elevations of intracellular Na⁺ can be detrimental to neuronal survival at earlier time points after ischemia [23]. However, experimental studies groups suggest that glutamate toxicity is primarily dependent on Ca²⁺ influx [24, 25]. Collectively, these results suggest that cellular self-harm processes exist within the brain itself, but also, that stroke-induced central nervous system (CNS) damage may be reduced by medicinal strategies to relieve the tendency of the brain to injure itself, under conditions of stroke. The inflammatory paradox of cellular self-injury is amplified by the special sensitivity of CNS neurons to sudden deprivation of oxygen and glucose; the catastrophic temporal and anatomical nature of stroke conspires with these realities to produce consequences that are difficult to treat with medicines, and thus far, this has been a challenge beyond the capacities of modern medicine.

Increasing evidence suggests that oxidative stress and apoptosis are closely linked phenomena in the pathophysiology of ischemic stroke. Neurons are normally exposed to a baseline level of oxidative stress from both exogenous and endogenous sources, as are all cells in the body. Free radicals are highly reactive molecules with one or more unpaired electrons. Free radicals can react with DNA, proteins, and lipids, causing varying degrees of damage and dysfunction [26, 27]. Numerous experimental and clinical observations have shown increased free radical formation during all forms of stroke injury [26, 28]. Free radicals, involved in stroke-induced brain injury, include superoxide anion radical, hydroxyl radical and nitric oxide (NO). The damaging effects of free radicals are normally prevented or reduced by antioxidant enzymes and free radical scavengers [29, 30]. The primary source of oxygen-derived free radicals (often referred to as 'reactive oxygen species') during ischemic-stroke injury is the mitochondria, which produce superoxide anion radicals during the electron transport process [28]. Another potentially important source of superoxide in post-ischemic neurons, is the metabolism of arachidonic acid through the cyclooxygenase and lipooxygenase pathways [31, 32]. Oxygen free radicals can also be generated by activated microglia and infiltrating peripheral leukocytes via the NADPH oxidase system following reperfusion of ischemic tissue [33]. This oxidation causes further tissue damage, and is thought to be an important trigger molecule for apoptosis after ischemic stroke.

NO is generated from L-arginine through one of several NO synthase (NOS) isoforms. The neuronal form (nNOS), which requires calcium/calmodulin for activation, is expressed by subpopulations of neurons throughout the brain [34]. Inducible NOS (iNOS) is expressed by inflammatory cells such as microglia and monocytes. These two isoforms are, for the most part, damaging to the brain under ischemic conditions. A third isoform found in endothelial cells (eNOS), has vasodilatory effects and is likely to play a beneficial role by improving local blood flow [35]. NMDA receptor activation has been shown to stimulate nitric oxide (NO) production by nNOS, and to possibly play a role in excitotoxic-mediated injury in ischemic stroke [36]. NO diffuses freely across membranes and can react with superoxide at it point of generation to produce peroxynitrite (ONOO^-), another highly reactive oxygen species [29]. Both oxygen-derived free radicals and reactive nitrogen species are involved in activating several pathways involved in cell death following stroke, such as apoptosis and inflammation [37‐39]. A reduction of oxygen supply also leads to the accumulation of lactate via anaerobic glycolysis and so to acidosis [40‐43].

Besides the production of different species of oxygen radicals, acidosis also interferes with intracellular protein synthesis. Lipid peroxidation appears to play a prominent role in the pathogenesis of stroke. The mechanism, whereby membrane lipid peroxidation induces neuronal apoptosis, involves generation of an aldehyde called 4-hydroxynonenal (4-HNE), which covalently modifies membrane transporters such as the Na⁺/K⁺ ATPase, glucose transporters and glutamate transporters, thereby impairing their function [44]. Whilst potentially damaging via their direct actions, Ca²⁺ and free radicals can also activate neuroprotective transcription factors, including nuclear factor-κB (NF-κB), hypoxia-inducible factor 1 and interferon regulatory factor 1 [17]. Some of these transcription factors induce the expression of inflammatory cytokines (for example, IL-1, IL-6 and TNF-α) and chemokines (for example, IL-8 and MCP-1), endothelial cell adhesion molecules (for example, selectins, ICAM-1 and VCAM-1), and other proinflammatory genes (for example, interferon-inducible protein-10) [17, 45].

There are several resident cell populations within brain tissue that are able to secrete proinflammatory mediators after an ischemic insult. These include endothelial cells, astrocytes, microglia and neurons. Activation of transcription factors results in increased protein levels for cytokines and increased expression of endothelial cell adhesion molecules (CAMs) in post-stroke brain tissue [46‐48]. A major role in brain inflammation following stroke is attributed to microglia, especially in the penumbral region of damage [49]. Activated microglia produce numerous proinflammatory cytokines, as well as toxic metabolites and enzymes [47, 50]. In addition to microglial cells, astrocytes also have an important part in stroke-induced brain inflammation. These cells can produce both proinflammatory cytokines and neuroprotective factors, such as erythropoietin, TGFβ1, and metallothionein-2 [4]. Because of the mixed nature of microglial and astrocyte products (both destructive and protective factors), the overall role of glia may differ at different time points following stroke insult, with protective or regenerative activities occurring days to weeks after the onset of ischemia [49, 51]. These factors add layers of complexity, in both adducing their pathophysiological roles in stroke, and in the goal of developing new therapeutics for stroke therapy.

The brain endothelium is quite distinctive compared with other organs, as evidenced by the blood brain barrier (BBB). However, it responds to stroke injury with increased permeability and diminished barrier function, along with degradation of the basal lamina of the vessel wall, as occurs as in other organs after ischemic injury [52]. Similarly, there is considerable evidence that acute ischemic stroke enhances the interactions of brain endothelium with extravascular CNS cells (astrocytes, microglia, neurons), as well as intravascular cells (platelets, leukocytes), and that these interactions contribute to the injury process [45]. The net result of all these responses to stroke is that the cerebral vasculature assumes the following phenotypes: 1) poor capillary perfusion of brain tissue, 2) pro-adhesive for circulating cells, 3) pro-inflammatory, 4) pro-thrombogenic, and 5) diminished endothelial barrier function. These changes in normal physiological functions cumulate with each of these inflammatory responses tilting in the same detrimental direction, such that the end-result is harmful to the host CNS cells and tissues. Indeed, this is the central problem facing rapid and effective treatment of stroke.

There is a large body of evidence that implicates leukocytes in the pathogenesis of stroke injury. The contention that leukocytes mediate reperfusion-induced tissue injury and microvascular dysfunction is supported by three major lines of evidence: 1) leukocytes accumulate in post-ischemic tissues prior to the onset of tissue injury, 2) animals rendered neutropenic exhibit a diminished injury response to ischemic stroke, and 3) prevention of leukocyte-endothelial cell adhesion with monoclonal antibodies (mAbs) directed against specific leukocyte or endothelial CAMs also affords substantial protection against stroke injury [45]. Polymorphonuclear leukocytes, of which neutrophils predominate, are therefore heavily implicated in worsening stroke outcome. Neutrophils adhere to endothelial ischemic brain vasculature in the acute phase following stroke, and infiltrate into brain parenchyma [53‐56]. Rodents, with reduced circulating neutrophils (through the use of antineutrophil serum), show reduced infarct volumes and improved neurological outcomes, also indicating a pathogenic role of neutrophil accumulation and activation following stroke [57, 58]. In contrast, the pathophysiological significance of lymphocyte recruitment into the brain after ischemic stroke remains uncertain. However, recent studies have shown important roles for T-lymphocytes in mediating reperfusion injury in post-ischemic brain tissue [8, 59]. How lymphocytes and neutrophils may interact in stroke pathophysiology is yet unknown.

It has been claimed that the focal stroke models are of greater relevance than other models to the typical human stroke situation. However, global ischemia of the brain is of clear clinical relevance to cardiac arrest and asphyxia in humans [12]. In addition, it should be noted that the physiological, biochemical and functional measurements made during recovery from a global model of reversible ischemia may be important in identifying the molecular and cellular mechanisms and pharmacological actions of potential neuroprotective agents. Global models of cerebral ischemia may thus be as useful as broader focal models, provided that care is taken in the interpretation of the data. The three most widely used animals in models of global ischemia are: 1) rat - four-vessel occlusion (4-VO) or two-vessel occlusion (2-VO) combined with hypotension; 2) gerbil - 2-VO; and 3) mouse - 2-VO. A 2-VO mouse model was developed for studies in transgenic mice and is now being routinely utilised [12, 68, 72].

This is induced by temporarily ligating the carotid arteries, with no reduction in blood pressure. Because there are no posterior communicating arteries in gerbils, this produces profound forebrain ischemia. The changes in regional CNS blood flow are similar to those in the rat models; blood flow in the cortex is ~1%, and in the hippocampus ~4% of control values [77].

Mouse models of global stroke are similar to rat 2-VO models using bilateral occlusion of the common carotid arteries. Several studies have demonstrated mouse global ischemia models with quantitatively uniform injury to hippocampal CA1 neurons, using techniques including 2-VO and 2-VO with hypotension [78]. However, because of the variability of collateral flow via the posterior communicating artery, it has been difficult to obtain uniform injury in the CA1 region, whilst still retaining high survival and experimental success rates. One model combines basilar artery occlusion with bilateral common carotid artery occlusion (three-vessel occlusion; [79]). However, in this model the animal survival rate is low, and CA1 neuronal injury is inconsistent. Because the mouse basilar artery runs through a narrow groove in the brain stem, and is attached to the pia mater and arachnoid membrane with arachnoid trabeculae, it is technically difficult to isolate and occlude.

Complete global ischemia is generally achieved by neck-cuff, cardiac arrest, or by ligating or compressing all arteries from the heart [80]. Blood flow to the whole brain is zero or <1% in these models. Due to a very high mortality associated with this model, it is not widely used.

Focal ischemic stroke models, whether in larger mammals such as cats, dogs or non-human primates, or in rodents, usually involve occlusion of one MCA [18, 81‐83]. Focal ischemia is differentiated from global ischemia in two ways. First, even at the core of the lesion, the blood flow is almost always higher than during global ischemia, so that longer insults are required to cause damage than for global ischemia. Secondly, there is a significant gradation of ischemia from the core of the lesion to its outermost boundary, and hence there are different metabolic conditions within the affected site. Because of its duration and heterogeneity, the insult is much more complex than in global ischemia, but it is an invaluable and realistic model for human stroke, and is thus widely studied.

There are two models of focal ischemic stroke, 1) transient focal ischemia and 2) permanent focal ischemia. In transient focal ischemia models, vessels are blocked for periods of up to 3 hours, followed by prolonged reperfusion; whereas, in permanent focal ischemia, the arterial blockage is maintained throughout an experiment, usually for one or more days.

Another in vitro approach of studying neurons in the CNS are organotypic brain slice culture techniques. These have many advantages as the neuronal morphology, cellular and anatomical relations and network connections are maintained in these types of cultures [97‐99]. Organotypic slice cultures of the brain have been used increasingly to examine neuronal cell death, mechanisms of cell migration, myelination, electrophysiological activities and synapse plasticity. They have also been used to investigate basic cellular mechanisms and treatment strategies for ischemic stroke [100‐103]. The principle of this approach is based on ex vivo cultures derived from different anatomical regions of the CNS [104], and the donor sources most commonly used are rats and mice.

Most organotypic brain slice cultures are obtained from neonatal (P0-P10) animals. The two main culturing methods for slice cultures include: the roller drum technique introduced by Gähwiler [105], and the interface cultures developed by Stoppini [106]. Slices maintained in stationary culture with the interface method are ideally suited for three-dimensional structure studies, whereas those cultured in roller tubes are often employed for imaging experiments. In OGD studies using these cultures, the cell death and apoptotic changes in neurons can be studied by looking at cellular uptake of propidium iodide and Fluro-jade staining, TUNEL staining and immunofluorescent staining for cleaved caspase-3, respectively. With growing demands for working experimental models that can replace or reduce animal experiments, such cultures certainly seem to offer a lot of promise in studying ischemic stroke in vitro.

Timed pregnant female mice or rats at days fifteen to seventeen of gestation (E15-17) are normally used as a source of embryos for primary cortical neuronal collection [107]. The pregnant mice are anaesthetized and fetuses are collectively removed. The mouse fetuses are decapitated and the heads separated and collected into ice-cold HEPES-buffered (10 mM) Hanks balanced saline solution (HBBS) lacking Ca²⁺ and Mg²⁺. The brains are then removed and placed in HBSS in petri dishes, where the meninges and blood vessels are stripped off the surface. Under sterile conditions, the cerebral hemispheres are dissected from the brains with the aid of a dissecting microscope and a trans-illumination light source. The isolated cerebral tissues are then pooled into another petri dish containing HBSS and minced into smaller pieces for enzymatic dissociation. The tissues are then transferred to sterile 15 ml tubes containing 3-5 ml of HBSS containing 0.2% trypsin. Following a 15-20 min incubation in the trypsin solution, the tissue pieces are rinsed with fresh HBSS and incubated for 5 min in 0.1% soybean trypsin inhibitor to halt the protease reaction. Following another wash in HBBS, cells are further dissociated by triturating the tissue, using a narrowed bore sterile pasteur pipette, until most of the visible-sized tissue pieces have been disrupted. Aliquots of cell suspension are added to growth substrate-coated cell culture dishes containing Neurobasal medium with B-27 supplement (Invitrogen, USA). The culture plate-coating substrates that are known to work well for primary neuronal culture are poly-L-lysine, poly-D-lysine and polyethylene imine (PEI) [107, 108]. The choice of substrate depends on the nature of experiment [107]. After an overnight coating of the plates at room temperature, the dishes are washed 2-3 times with phosphate buffered saline (PBS) and allowed to dry. The dishes are sterilized by exposure to UV light for 10 min and culture medium is added to the dishes. Cells are allowed to attach to the substrate-coated dishes for 3-6 hours and maintained in a humidified incubator (37°C with air and 6% CO₂). The cell culture medium is then replaced with fresh medium a few hours later. Generally the neurons are allowed to mature over 5-7 days before experimentation.

Astrocytes account for about 50% of the cell population in adult mammalian brains. In addition to providing structural, metabolic and trophic support to neurons, their role in protecting neurons from injurious stimuli is slowly being unraveled [109‐111]. Consequently, studying them under in vitro conditions is cardinal to understanding their role in stroke pathology. Astrocyte cell cultures are normally obtained from one to three day postnatal (P1-P3) mouse/rat pups. The heads are removed from pups and placed in a Petri dish with ice-cold HBSS containing 2% sucrose. The meninges are then stripped off and the mice cerebra are dissected aseptically from the residual parts of the brain. The cerebra are then cut carefully into small pieces. The tissue pieces are trypsinised following a 10 min incubation with 0.25% trypsin. Trypsin inactivation is achieved by adding Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and an antibiotics mix of penicillin (100 IU/ml), streptomycin (100 mg/ml) and amphotericin (0.25 mg/ml). The cerebra are dissociated by passing them through a fire-polished Pasteur pipette and the resultant cell suspension is allowed to settle for about 5 min, to allow for decantation of the large, uncleaved cell clusters. The supernatant is then centrifuged for about 5 min at 1500-2000 rpm at room temperature. The cell pellet is then dispersed in the new volume of the medium and plated on either T-75 or T-25 culture flasks at a density of about 5 million cells/ml. Following the initial seeding of cells, the medium is replaced once every 3 days. The resulting culture obtained is a mixed culture of astrocytes, microglia and oligodendrocytes. Microglia are microscopically visible on days 6-7 of the culture, remaining suspended in media as well as adhered to the bed layer of astrocytes. The remaining microglia and oligodendrocytes are separated from the bed layer of astrocytes by shaking the culture flasks on a rotary shaker placed in the incubator for at least 10 hours [112] and the media from this flask is then harvested to obtain pure microglial cultures. The remaining undetached cells are relatively pure astrocytes, and are detached following trypsin incubation for about 10 min. After addition of an equal amount of the new growth medium, the cells are centrifuged and the cell pellet is re-dispersed by gentle pipetting and seeded onto new culture flasks. Following a 3 hour incubation, this medium is aspirated out and new volume (about 10 mL) of growth medium is added. This applied procedure allows for a high purity of astrocytes to be obtained with routine culturing [113].

For GD, cultured neurons are incubated in glucose-free Locke's medium, pH 7.2, supplemented with gentamycin (5 mg/L) for 6, 12 or 24 hours. In order to mimic in vitro, transient focal ischemic strokes that occur in vivo, the neuronal cultures are exposed to OGD. The original glucose-containing neurobasal medium is replaced with a glucose-free Locke's buffer containing no serum. All media changes are followed by washes with sterile PBS (pH 7.4). Following this, the cultures dishes are exposed to hypoxia (PO₂ <50 mm Hg) by placing them in a small, 3 L, airtight experimental hypoxia chamber (Billups-Rothenberg, San Diego, CA) with inflow and outflow connectors and circulating a hypoxic gas mixture of 95% N₂/5% CO₂ mixture for 15-20 min [84]. Cultures are exposed to conditions of OGD for 1, 3, 6, 12 or 24 hrs.

Regional microvascular tissue perfusion (cerebral blood flow) is normally monitored before, during, and after focal ischemia, using laser Doppler flowmetry. The values during ischemia are calculated as a percentage of the pre-occlusion level. The region of measurement is set at 1 mm rostral and 1 mm dorsal to the cross-over point of the left MCA and rhinal fissure, which is in the ischemic penumbra of the ischemic lesion [114]. Blood pressure, rectal temperature, and blood gases are measured during the operation in rats and mice. Normally, under controlled conditions using heating pads, there are no significant differences in temperature or blood pressure monitored at pre-, intra-, and post-ischemic time points in most studies. However, measuring and maintaining these parameters is important for proper interpretation of the technical procedure causing stroke, outcomes among animals, as well as for evaluating adhering leukocytes, which is accomplished by intravital video microscopy [115]. An estimate of shear rate in venules is obtained by fluorescence microscopy based on image analysis determinations of the maximal velocity of fluorescently-labeled red blood cells or platelets within the venules under study [116, 117]. Such estimates of pseudo-shear rate in venules are obtained using measurements of venular diameter (Dv) and the maximal velocity of flowing platelets (Vplt) according to the formula: pseudo-shear rate = (Vplt/1.6)/Dv × 8 [116].

The size of the brain infarct in focal cerebral ischemia increases during the period of reperfusion (Figure 3). This has been shown in animal models of stroke and in human stroke patients [118]. The infarct volume is normally analyzed after 12-24 hours in transient and permanent focal ischemia models. The brain is removed and coronal sections are cut (2 mm-thick slices in rats or 1-2 mm thick slices in mice) through the entire rostro-caudal extent of the cerebral cortex. The slices are immersed in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC). An edema index is calculated by dividing the total volume of the hemisphere ipsilateral to the MCA occlusion by the total volume of the contralateral hemisphere. An infarction index, the actual infarcted lesion size adjusted for edema, is then calculated for each animal [119].

The functional consequences of focal ischemic stroke injury are evaluated using a 5-point scale neurological deficit score (0, no deficit; 1, failure to extend right paw; 2, circling to the right; 3, falling to the right; and 4, unable to walk spontaneously) [120]. More recently a 14 point neurological scoring system was developed [121]. This new scoring method includes the results of motor, reflex and balance tests; a single point is awarded for the inability to perform the test or for the lack of a tested reflex.

Albumin leakage, a quantitative index of endothelial barrier dysfunction, can be applied to the brain for determination of BBB integrity. Albumin extravasation in the brain can be quantified either by fluorescence imaging of the leakage of fluorescein isothiocyanate-labeled albumin, or sulforhodamine (Texas Red)-labelled albumin from cerebral venules, or from the clearance of the same fluorochromes from blood to the artificial CSF perfusing the brain surface [122, 123]. With this imaging approach, fluorescently tagged albumin is administered intravenously to the animals 15 minutes before the baseline observation period and fluorescence intensity is detected using a silicon-intensified target camera. The fluorescence intensities within a specified segment of cerebral venules within the cranial window and in a contiguous area of perivenular interstitium are measured at various times after administration of fluorescent-albumin using a computer assisted digital imaging processor (NIH Image 1.61 on a Macintosh computer). Vascular albumin leakage is determined from the difference in fluorescence intensity between the outside and inside of the venular segment.

The brains are immediately removed and divided into contralateral and ipsilateral hemispheres. The tissue samples are weighed on an electronic analytical balance to the nearest 0.1 mg to obtain the wet weight. The tissue is then dried at 90-100°C for 24 hours to determine the dry weight. Brain water content (%) is calculated as {(wet weight-dry weight)/wet weight} × 100.

Adhesion of leukocytes and platelets following ischemia and reperfusion can be monitored in vivo using intravital video microscopy (Figure 4). The head of the animal is immobilized and a hole drilled through the skull using a high-speed micro drill (1 mm posterior from bregma and 4 mm lateral from the midline). The dura mater is not cut because the fluorescently labeled blood cells are easily observed and intracranial pressure is well-maintained with the dura mater intact. Artificial cerebrospinal fluid is placed on the exposed brain tissue. For focal ischemia models, the observation area includes the infarcted region following MCA occlusion [124]. For global ischemia models, the observation area includes the entire cerebral cortex. Platelets are isolated from a donor animal using a series of centrifugation steps and labeled ex vivo with carboxyfluorescein diacetate succinimidyl ester. Once the platelet data are collected, endogenous leukocytes are labeled in vivo by infusing rhodamine-6G (100 μl; 0.02%) over 5 min and allowed to circulate an additional 5 min before observation. An upright Nikon microscope equipped with a SIT camera (C2400-08; Hamamatsu Photonics) and a mercury lamp is used to observe the cerebral microcirculation. The images are received by a CCD video camera and recorded on a video recorder equipped with a time-date generator (WJ-810; Panasonic).

The dual radiolabelled monoclonal antibody (mAb) technique is used to quantify the expression of different endothelial cell adhesion molecules, including P-selectin, ICAM-1 and VCAM-1, in the microvasculature of the brain. This method determines the relative accumulation, in any regional vascular bed, of a binding mAb to a specific endothelial surface epitope (eg, P-selectin) and an isotype-matched non-binding mAb, the latter of which is used to compensate for non-specific accumulation of the binding mAb. Different endothelial cellular adhesion molecules in the brain can be analyzed following ischemic stroke. However, adhesion molecules specifically expressed by immune cells such as LFA-1 are analyzed at least 24 hours after reperfusion. Activation of microglial cells following ischemia or reperfusion is normally analyzed using CD11b (Mac-1/CR3) antibodies.

With different MRI techniques, in vivo diagnostic and prognostic information can be obtained on edema formation, hemodynamics, tissue structure, neuronal activation, cell migration, gene expression and more. In addition, MRI can be combined with other imaging modalities such as positron emission tomography or optical imaging to obtain complementary or supplementary information. Advances in magnetic resonance technology, such as magnets with higher field strength, more powerful gradient systems and increasing availability of targeted magnetic resonance contrast agents, allow MRI research in animal models of ischemic and hemorrhagic stroke with higher sensitivity, faster acquisition, and improved specificity (see [125] for review).

Identification and quantification of specific proteins and mRNAs in situ and in brain tissue samples can provide valuable information to elucidate the signaling pathways and molecular mechanisms involved in ischemic brain damage and recovery from stroke. Proteins can be identified en masse by proteomic methods and individual proteins can be quantified by immunoblot or enzyme-linked immunosorbent assays [126, 127]. Specific mRNAs are detected and quantified by polymerase chain reaction (PCR)-based gene array methods and by real-time PCR (see [128] for review).

This test is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, whereas dead cells do not possess this ability. Hence, following OGD/GD conditions the dead cells would have altered membrane permeability, thereby facilitating the entry of this dye into the cell and staining the cytoplasm blue, and the live cells would have a clear cytoplasm (Figure 5). This test is performed by adding trypan blue into control normoxic neuronal plates containing NB medium or OGD/GD- exposed neuron plates containing glucose deprived-Locke's Buffer. Following 3-5 min incubation with trypan blue, the cells in the culture plates are fixed with 4% buffered formaldehyde and counted under a normal light microscope. In each field, the dead and total number of cells is counted and their ratio provides an estimate of percentage cell death [129].

Cell viability can also be assessed colorimetrically using the 3-(4, 5-dimethylthiazol-2-yl)-2,5 -diphenyl tetrazolium bromide (MTT) assay [130, 131]. MTT is added to the cultures and incubated at 37°C for 40 min. Following this incubation, the media is aspirated and DMSO is used to solubilise the blue formazan product. The cell plates are then assessed using a spectrophotometer at 570 nm and the live cell percentage can be compared between control normoxic and OGD-exposed cell plates.

Cells undergoing apoptosis or necrosis following OGD conditions in vitro can be visualised using fluorescent stains like propidium podide (PI) and Hoechst 33258. Similar to trypan blue, PI is taken up only by dead cells. Hoechst 33258, on the other hand, is a nuclear stain and stains all nuclei of both viable and dead cells. This method is carried out by adding PI (10 μM) to the neuronal cultures 6-12 hours after OGD. The cells are then fixed in ice-cold 4% buffered formaldehyde. Hoechst staining is carried out post-fixation by incubating the cultures with Hoechst 33258 (1 μg/mL) for 10 min at room temperature. The cells are then analyzed under a nonconfocal fluorescent microscope. The results are interpreted as follows: the nuclei of viable cells are blue- intact Hoechst positive and PI-negative, the nuclei of apoptotic cells are red- PI-positive and fragmented (or condensed), and the nuclei of necrotic cells are red- PI-positive and round [132]. Both the apoptotic and necrotic cells also stain blue Hoechst-positive.

Cell death cascades in ischemic stroke are mediated, in part, by excessive calcium influx resulting from activation of glutamate receptors and voltage-dependent calcium channels (VDCC). In addition, the function of Ca²⁺-ATPases is compromised, resulting in prolonged elevation of the intracellular calcium concentration. Drugs that block glutamate receptors (eg, MK-801) or VDCC (eg, nimodipine and flunarazine) have proven effective in rodent models of stroke [138]. At least 14 clinical trials of nimodipine in ischemic stroke were conducted beginning in the mid 1980s. Nine trials found no effect, one trial found short-term worsened outcome with treatment, and four trials found positive outcomes [139]. Clinical trials with flunarizine found no statistically significant improvement in outcome [140]. Despite this discouraging analysis, dantrolene, which blocks ryanodine receptors, has been discussed for clinical trials as the result of beneficial effects in rodent stroke models [141].

Several compounds that interfere with glutamate receptor activation have been developed and tested against experimental animal models of stroke as well as against human clinical trials. The noncompetitive NMDA antagonist MK-801 (dizocilpine) improved outcome in models of focal ischemia producing up to 75% reductions in infarct volume [142, 143]. Both MK-801 and dextromorphan, another noncompetitive NMDA receptor antagonist, exhibited protective effects in experimental studies, but clinical trials were terminated early because of phencyclidine-like psychotic side effects and lack of efficacy against stroke injury [144]. Some other noncompetitive (aptiganel, ceresine,) or competitive (selfotel, eliprodil) NMDA receptor antagonists were shown to be very effective in animal stroke models, but with no significant effects in clinical trials [139]. Non-NMDA antagonists have also been developed and studied against stroke conditions. Zonampanel (YM-872) is an AMPA antagonist tested in human Phase 2 clinical trials [145]. In addition, another AMPA antagonist, SPD-502, as well as metabotropic glutamate receptor modulators, are being developed and tested against stroke injury in animals and humans [9, 139]. However, the development of anti-excitotoxic agents against stroke has thus far been disappointing.

Free radical production is enhanced in both the ischemic core and penumbra following stroke injury, and this is believed to cause much of the damage seen in these regions. There are many agents that either block free radical production or inhibit its activation that have been shown to be very effective in experimental models. Uric acid is a well-known natural antioxidant present in fluids and tissues. Administration of uric acid resulted in a large and significant reduction in ischemic damage and improved behavioral outcome [146]. Edaravone, tetramethylpyrazine, alpha-phenyl-N-tert-butyl-nitrone, FR210575 and NXY-59 are some of other free radical inhibitors that have been shown to be effective against experimental stroke injury [147, 148]. Completed clinical trials with free radical scavengers, however, have had limited success after acute ischemic stroke. The free-radical-trapping agent, NXY-59, was initially reported to be efficacious in acute ischemic stroke [149], however a follow-up trial in a larger cohort of patients failed to demonstrate efficacy [150]. EGb-761 (Tanakan^®), being developed by Ipsen, is a free radical scavenger derived from a concentrated extract of Ginkgo [151], that has recently completed a Phase 3 clinical trial with results still pending [152].

Accumulating evidence strongly suggests that apoptosis contributes to neuronal cell death in stroke injury. Caspases, a family of cysteine-aspartate proteases that include at least 14 members divided into three groups (I, II, and III), are essential players in apoptotic neuronal cell death [153]. Many groups have studied the effects of caspase inhibition on cerebral ischemia-induced neurodegeneration by using the broad spectrum caspase inhibitor z-VAD, either in the fluoromethylketone (fmk) or dichlorobenzoyloxopentanoic acid (dcb) form and z-DVED-fmk. Both inhibitors were neuroprotective in mouse models of transient cerebral ischemia and z-VAD was neuroprotective also in transient and permanent models in the rat [154]. Ac-YVAD-cmk (Ac-Tyr-Val-Ala-Asp-cmk), a caspase group I (caspase-1-like) inhibitor, also was shown to be neuroprotective in a mouse transient model of cerebral ischemia [154]. In addition, peptide-based caspase inhibitors have been shown to prevent neuronal loss in animal models of stroke [155]. To date however, the efficacy of anti-apoptotic agents in human stroke patients has not yet been tested.

Inflammation in stroke is characterized by the accumulation of leukocytes and activation of resident microglial cells. Inflammatory cells can contribute to stroke pathology through two basic mechanisms. They form aggregates in the venules after reperfusion, or, enter infarcted tissue and exacerbate cell death through production of free radicals and cytokines [8, 53]. Cell adhesion molecules such as selectins, integrins, and ICAMs permit endothelial-inflammatory cell interactions. Treatment with anti-selectin antibodies successfully decreased infarct volume by up to 70% after transient focal ischemia in mice [156]. An anti-ICAM-1 antibody has also been shown to decrease infarct size after transient, but not permanent, focal ischemia [157]. However, a recent clinical trial using the murine anti-ICAM-1 antibody enlimomab, worsened neurologic score and mortality in patients and a follow-up study using the murine anti-rat ICAM-1 antibody in rats also found an increase in infarct volume and no efficacy [158, 159]. It is believed that immune activation in response to the foreign mouse protein probably accounted for the failed clinical and follow-up experimental results [159]. Recently, a Phase 2 trial using anti-CD11b/CD18 agent UK-279276, has been completed, and demonstrated that this compound is safe and well-tolerated [160]. Other targets include the mitogen activated protein kinases (MAPK), which have been linked to inflammatory cytokine production and cell death in ischemic stroke injury. SB-239063 is a MAPK inhibitor that reduced infarct size and improved neurological outcome following focal stroke in rodents, which may be an alternative target to limit inflammation in human stroke patients [161].

Matrix metalloproteinases (MMPs) are enzymes that break down components of the extracellular matrix and enhance BBB breakdown after stroke, promote hemorrhage, and increase inflammation. MMP inhibitors such as BB-94 and KB-R7785 show decreased infarct volume in treated mice after permanent focal ischemia [162]. MMP inhibitors have been evaluated in patients for their anti-angiogenic properties and are well-tolerated [163]. Although chemokines can have pro- or anti-inflammatory actions, the overall effect of chemokine up-regulation in ischemia-reperfusion injury is detrimental. NR58-3.14.3, a novel broad-spectrum inhibitor of chemokine function significantly reduced lesion volume in rats by up to 50%, and this was associated with a marked functional improvement [164]. Several other anti-inflammatory cytokine approaches were tested in experimental stroke models, including various antibodies that target inflammatory proteins. However, there have been no successful clinical trials of such anti-inflammatory agents reported so far. As mentioned earlier in this review, microglial activation following stroke insult plays a role in promoting inflammatory processes, but therapeutic approaches that specifically target microglia are currently lacking.

Brain tissue injury following ischemic stroke results from the complex interplay of excitotoxicity, oxidative stress, inflammation and apoptosis. As mentioned above, two decades of basic research targeting single stroke injury mechanisms, in single-cell types, or in single-injury mechanisms in multiple cell types, have failed completely when applied in clinical trials of human strokes. On the basis of the complexity of events in cerebral ischemia and the disappointing results from human clinical stroke trials using single agent, it is unrealistic to anticipate that a single neuroprotective drug will demonstrate benefits in human stroke. Given this consistent finding, we believe that a new pleiotropic approach in stroke treatment is required, and that targeting more diverse pathogenic events in multiple cell types may prove a superior approach over the classical, single-target one.

We have recently identified Gamma-secretase inhibitors (GSIs) as a novel and potent stroke therapy [63]. Specifically, we reported that ischemic stroke can transiently activate gamma-Secretase (γ-secretase), and a single treatment with GSIs reduced ischemic brain damage and improved recovery by targeting diverse pathogenic mechanisms in multiple cell types [63]. The concept raised by our findings, that GSIs are potentially neuroprotective after stroke, was broadened by another study which showed that GSIs are beneficial for the treatment of traumatic brain injury [165].

Intravenous immunoglobulin (IVIG) is a therapeutic modality approved for the treatment of various conditions, and is increasingly used for autoimmune disorders to suppress immune-mediated tissue damage, particularly in neuro-autoimmune diseases [166]. We have recently shown that administration of IVIG to mice subjected to experimental stroke almost entirely eliminated mortality and reduced the size of brain infarction by 50-60% [84]. Moreover, not only was the infarcted area reduced, but also, within this ischemic region, neurons were spared and only occasional cell loss was observed. In additional studies we provided evidence that IVIG can directly protect neurons against ischemia-like conditions [84]. The efficacy of IVIG against stroke-induced brain injury in our study was due, in part, to its ability to selectively neutralize complement components, and by reducing cell adhesion molecule production and subsequent infiltration of inflammatory cells, and thus reducing inflammation in the infarcted region [84].

In further studies, our group provided evidence that IVIG can directly protect neurons against ischemia-like conditions. We found that OGD in cultured neurons caused an increase in levels of cleaved (enzymatically active) caspase-3 (a marker of apoptosis) and a progressive decrease in neuronal viability. Treatment with IVIG suppressed the OGD-induced increases in activated caspase-3 levels, suggesting the IVIG protects against neuronal cell death directly, by as yet unknown mechanisms [84]. In light of the extensive clinical experience with IVIG for other indications [85, 166, 167], our results may support consideration of the development of clinical trials to evaluate the use of IVIG in human stroke patients.

Similar to IVIG and GSIs, statins may have potential pleiotropic effects against ischemic stroke-induced brain injury [168]. Statins reduce cholesterol levels, which have been related to a reduction in vascular event risk, but they also have pleiotropic effects such as regulation of NO and glutamate metabolism, modulation of inflammatory repsonses, reduction in platelet aggregation, immune dampening activity, and anti-apoptotic effects, as well as potentially promoting angiogenesis [169‐171]. Recent clinical studies suggest a neuroprotective effect of statins during the acute phase of stroke. It has been found that patients under treatment with statins prior to suffering stroke, showed higher probability of favourable outcome at three months, compared with those without previous treatment with statins, despite similar stroke severity at admission [172]. This beneficial effect is also observed in those patients who received thrombolytic therapy with rtPA [173]. Furthermore, recent prospective data indicated that the cessation of statin medication in acute ischemic stroke patients confers a significantly higher likelihood of early neurological deterioration and poorer outcomes [168]. The promising pleiotropic action of statins could also be extended to the field of neurorepair after ischemic stroke.