Astrocytes: Targets for Neuroprotection in Stroke

Abstract

In the past two decades, over 1000 clinical trials have failed to demonstrate a benefit in treating stroke, with the exception of thrombolytics. Although many targets have been pursued, including antioxidants, calcium channel blockers, glutamate receptor blockers, and neurotrophic factors, often the focus has been on neuronal mechanisms of injury. Broader attention to loss and dysfunction of non-neuronal cell types is now required to increase the chance of success. Of the several glial cell types, this review will focus on astrocytes. Astrocytes are the most abundant cell type in the higher mammalian nervous system, and they play key roles in normal CNS physiology and in central nervous system injury and pathology. In the setting of ischemia astrocytes perform multiple functions, some beneficial and some potentially detrimental, making them excellent candidates as therapeutic targets to improve outcome following stroke and in other central nervous system injuries. The older neurocentric view of the central nervous system has changed radically with the growing understanding of the many essential functions of astrocytes. These include K⁺ buffering, glutamate clearance, brain antioxidant defense, close metabolic coupling with neurons, and modulation of neuronal excitability. In this review, we will focus on those functions of astrocytes that can both protect and endanger neurons, and discuss how manipulating these functions provides a novel and important strategy to enhance neuronal survival and improve outcome following cerebral ischemia.

1. INTRODUCTION

Stroke is the third leading cause of death in the United States and results in substantial health-care expenditures; the mean lifetime cost resulting from an ischemic stroke is estimated at $140,048 per patient, and this estimation is higher for people over 45 years. Nationwide in 2010, the estimated direct and indirect costs of stroke totaled $73.7 billion [1]. Although many clinical trials have been completed in stroke patients, none of these have demonstrated protective efficacy except for thrombolysis [2, 3]. In the case of cardiac arrest and resuscitation only hypothermia has been shown to have clinical utility [4]. In some sense the two therapies that have been effective thus far clinically have broad targets, and do not only affect a single injury mechanism. In contrast, of the failed trials, many targeted neuron-specific injury mechanisms [5]. This may reflect too narrow a view of what is needed for brain preservation. A large body of work has shown that astrocytes play key roles both in normal and pathological central nervous system functioning [6]. Astrocytes are the most abundant brain cell type, and in addition to their multiple important homeostatic roles, they organize the structural architecture of the brain, help organize communication pathways, and modulate neuronal plasticity (for recent review see [7, 8]). Thus, astrocytes are now thought to be important potential targets for manipulation.

Ischemic stroke is caused by an interruption of cerebral blood flow that leads to stress, cell death, and inflammation. Neurons are more susceptible to injury than astrocytes when studied under some in vitro conditions [9, 10]. Neurons have less endogenous antioxidants and are susceptible to excito-toxicity [10]. Both normally and after ischemia, astrocytes support neurons by providing antioxidant protection [11, 12], substrates for neuronal metabolism [13], and glutamate clearance REF. Although astrocytes are sometimes more resilient than neurons, injury can result in impaired astrocyte function even when astrocytes do not die. Impaired astrocyte function can amplify neuronal death [14]. Therefore, many recent efforts have focused on the astrocyte-neuron interaction and how astrocyte function can be improved after stroke to enhance neuronal support and survival [10, 15, 16]. A growing body of data demonstrates that astrocytes play a multifaceted and complex role in the response to ischemia, with potential to both enhance and impair neuronal survival and regeneration [17]. Many recent studies focus on the astrocyte-neuron interaction and several investigate ways in which astrocyte function can be improved after stroke to enhance neuronal survival.

This review provides a brief overview of the pathophysiological events underlying ischemic brain damage, and considers how these events affect astrocyte-mediated support of neurons. In addition, we discuss some experimental approaches to enhance the neuronal supportive role of astrocytes as a novel strategy against stroke. Finally, we explore how these approaches may eventually be applied in the clinical setting to improve stroke outcome for patients.

2. ASTROCYTE VIABILITY AFTER ISCHEMIA

2.1. In Vitro Studies

In vitro studies have provided substantial insight into the mechanisms governing the survival of astrocytes following simulated ischemia. These investigations have shown that astrocytes are generally more resistant than neurons to oxygen-glucose deprivation (OGD) performed in media at physiologically normal pH, an in vitro model of ischemia [10, 18]. Most neurons in astrocyte-neuronal co-cultures will die after 60–90 min of OGD, while astrocyte cultures only suffer a similar extent of injury after 4–6 hours [9, 18, 19]. Different astrocyte populations exist and astrocytes isolated from different brain regions such as cortex, striatum, and hippocampus differ in their sensitivity to OGD [15, 20, 21]. Furthermore, Lukaszevicz and colleagues [22] reported that protoplasmic astrocytes lose their integrity faster than fibrous astrocytes, which may explain the regional differences in susceptibility to ischemia between white matter astrocytes which are fibrous and grey matter astrocytes that are protoplasmic. Although less susceptible to OGD-induced damaged in vitro studies have highlighted certain elements that are highly toxic to astrocytes. For example, acidosis has been found to be very effective in killing astrocytes [23–26], in contrast to neurons, which are protected in acidic conditions [24, 26].

2.2. Focal Cerebral Ischemia

Much of the information about the recovery of astrocytes in vivo has been provided by studies using immunohistological markers for astrocyte specific proteins, such as glial fibrillary acidic protein (GFAP) and glutamine synthetase GS; Fig. 1. Using these markers as tools, several investigations suggest that astrocytes are better preserved than neurons in animal models of stroke outside the core where all cells die [27–29]. Though neuronal markers are decreased as soon as 1 hour after MCAO, GFAP expression is preserved over the first 3 hours of reperfusion after 2 hour MCAO [29] and GS is increased 3 hours following a 3 hour MCAO [28]. At later reperfusion periods, GFAP increases in the peri-infarct area that later develops into the glial scar [29–32]. In contrast, Liu and colleagues [33] reported the deterioration of some astrocyte markers prior to that of neuronal markers. Discrepancy in findings may be due to differences in detection (i.e., protein vs. mRNA) and injury paradigms.

Fig. (1).

Expression of different astrocytic proteins following stroke. Increased expression of GFAP is a hallmark of astrocytes activation, as is induction/re-expression of vimentin. Astrocytes normally express glutamine synthetase (GS) and S100β, genes that are widely expressed in both reactive and non-reactive astrocytes. The GFAP and S100β labelling are for the same cell, while the Vim and GS staining labels are for of other cells. Scale bar, 50µm.

2.3. Forebrain Ischemia

Excitotoxic neuronal injury is a common mechanism in both acute and chronic neurodegenerative diseases. It has long been appreciated that inhibition of astrocyte glutamate uptake [34, 35], and more recently inhibition of astrocyte mitochondrial function [36], impairs neuronal survival from excitotoxic injury. Brief forebrain ischemia is a model of the delayed hippocampal neuronal loss seen in patients following cardiac arrest and resuscitation, and in part involves excitotoxicity. Increased generation of reactive oxygen species (ROS) and mitochondrial dysfunction in CA1 astrocytes contributes to ischemia-induced loss of GLT-1 and ultimately to delayed death of CA1 neurons [15]. Our studies and those of other laboratories have demonstrated that selective dysfunction of hippocampal CA1 subregion astrocytes, with loss of glutamate transport activity and immunoreactivity for glutamate transporter 1 (GLT-1), occurs at early reperfusion times, hours to days before the death of CA1 neurons [15, 37, 38].

The heterogeneous degeneration of astrocytic processes and mitochondria was tightly associated with the appearance of disseminated selective neuronal necrosis and its maturation after temporary ischemia [39]. By electronmicroscopy the same investigators [40] found that focal infarction is exacerbated by temporary microvascular obstruction due to compression by swollen astrocytic end-feet. However, hypoxia has multiple effects on astrocytes and their ability to support neuronal viability [41]. For example, hypoxia induces astrocyte-dependent protection of neurons following hypoxic preconditioning. Yet, hypoxia induces processes in astrocytes that augment neuronal death in other situations, such as the coincidence of hypoxia with inflammatory signaling.

3. REACTIVE ASTROGLIA: GOOD OR BAD AFTER STROKE?

The astrocyte response to ischemia has traditionally been viewed as detrimental to recovery, as the astrocyte-rich glial scar has both physical and chemical inhibitory properties [42, 43]. As components of the glial scar, astrocytes exhibit hypertrophied, interdigitated processes that form a physical barrier. Astrocytes produce inhibitory molecules including chondroitin sulfate proteoglycans (CSPGs) that contribute to chemical inhibition [44, 45]. In the acute setting, astrocytic gap junctions may remain open following ischemia [46], allowing substances such as proapoptotic factors to spread through the syncytium, thereby expanding the size of the infarct [47]. As discussed below, astrocytes can also produce a variety of pro-inflammatory cytokines.

Many studies have shown that decreased astrogliosis often correlates with decreased infarct size. Nonspecific inhibition of cell proliferation following ischemia using a cyclin kinase inhibitor decreases astrocyte proliferation and results in improved functional recovery [48]. In addition, treatment with alpha-melanocyte stimulating hormone [49], cysteinyl leukotriene receptor antagonist [50], cliostazol [51], and caffeic acid [52] result in reduced infarct size accompanied by a decrease in astrogliosis. Treadmill exercise [28] and acupuncture [53] are similarly associated with improved outcome and reduced astrogliosis. Thus, results from several studies suggest that treatments that decrease infarct size are often accompanied by attenuated astrocyte response. Despite the frequent association of decreased astrogliosis with improved outcome, it is difficult to determine cause and effect, since the extent of astrogliosis likely reflects the severity of the injury, as well as influencing it.

In addition to their role in glial scar formation, astrocytes also respond to ischemia with functions important for neuroprotection and repair. These include protecting spared tissue from further damage [14], taking up excess glutamate as discussed above, rebuilding the blood brain barrier [54, 55], and producing neurotrophic factors [10]. GFAP knockout mice exhibit larger lesions than their wild-type littermates following focal ischemia [56], and mice lacking both GFAP and vimentin have impaired astrocyte activation, decreased glutamate uptake abilities, and attenuated PAI-1 expression after ischemia [57]. Application of astrocyte-conditioned media after transient MCAO results in decreased infarct volume and regained blood-brain barrier function [58], suggesting that factors released by astrocytes following ischemia are important for neuroprotection.

Although few studies other than the use of animals lacking vimentin and GFAP have specifically targeted astrocyte activation after ischemia, there is correlational evidence suggesting that astrogliosis may be beneficial. Environmental enrichment, which results in reduced infarct size and improved recovery following ischemia, also leads to increased astrocyte proliferation [59, 60]. After focal ischemia, aged rats exhibit increased tissue damage and increased astrocyte hypertrophy, but have decreased astrocyte proliferation compared to young rats [61]. Systemic infusion of bone marrow stromal cells following MCAO increases gliogenesis and decreases lesion size [62, 63]. In addition, administration of transforming growth factor α (TGFα), a known mitogen for astrocytes [64], following MCAO leads to reduced infarct size and improved functional recovery [65]. Furthermore, ischemic preconditioning that produces a neuroprotective state leads to prolonged astrocyte expression of Hsp27 [66]. Finally, mice lacking connexin 43, the gap junction connecting astrocyte networks that is needed for proper neurotransmitter and potassium regulation, have increased infarcts following MCAO [67]. Thus, astrocytes have the potential to be both detrimental and beneficial following ischemic insult, making them promising targets for manipulation to improve outcome.

4. ASTROCYTE-MEDIATED INFLAMMATION AFTER STROKE: A DOUBLE-EDGED SWORD

Inflammation plays both detrimental and beneficial roles in brain ischemia, depending upon the timing and severity of the inflammation. Within minutes after injury, injured neurons in the core and penumbra of the lesion and glial cells in the core produce pro-inflammatory mediators, cytokines, and reactive oxygen species, which activate both astrocytes and microglia [68]. Activated astrocytes can produce the proinflammatory cytokines IL-6, TNFα, IL-1α and β, interferon γ, and others [68–70]. High levels of these cytokines can be detrimental to ischemic recovery [71–75] by directly inducing apoptosis of neuronal cells and/or increasing toxic nitric oxide levels [76] and inhibiting neurogenesis [77]. Indeed, inactivation of astrocyte NfκB signaling, shown to induce astrocyte production of pro-inflammatory cytokines [78], decreases cytokine production and protects neurons after ischemic injury [79]. Hsp72 overexpression is associated with lower NfκB activation and lower TNFα [80]. In addition to cytokines, reactive astrocytes also produce chemokines following ischemia [81]. Chemokines upregulate adhesion molecules in vascular endothelial cells, resulting in attraction of immune cells, which may worsen ischemia-induced damage [82]. Overall, some aspects of the local inflammatory response contribute to secondary injury to potentially viable tissue and lead to apoptotic and necrotic neuronal cell death hours to days after injury [83], while other aspects are beneficial.

Although the potential benefits of inflammation after stroke have received relatively little attention so far, indirect evidence suggests that some specific inflammatory reactions are neuroprotective and neuroregenerative [84–91]. In addition to providing defense against the invasion of pathogens, inflammation is also involved in clearing damaged tissue, and in angiogenesis, tissue remodeling, and regeneration [89]. This is probably best studied in wound healing, which is severely compromised if inflammation is inhibited [89, 91]. There is also evidence suggesting that specific inflammatory factors can be protective in some circumstances. IL-6, produced by astrocytes acutely after MCAO [69], is likely neuroprotective early after ischemia [84]. Interestingly, ischemic preconditioning resulting in protection appears to be dependent on TLR-4 signaling, and is accompanied by increased TNFα, NFκB, and COX-2 expression [90]. Indeed, in vitro work has shown that administration of TNFα in combination with Hsp70 results in decreased expression of pro-apoptotic proteins following hypoxia [88]. Thus, it is important to consider these factors, along with timing, when trying to determine the best strategy to reduce damage and improve recovery and regeneration.

5. ASTROCYTE SUPPORT OF NEURONS AFTER STROKE

5.1. Antioxidant Production

One hallmark of the cellular response to ischemia is a rapid, dramatic increase in damaging free radicals, including nitric oxide (NO), superoxide, and peroxynitrite [92]. Nitric oxide synthetase levels increase as soon as 10 minutes after induction of MCAO [93], followed by NO production that persists for at least one week after MCAO [94]. Nitric oxide can cause cell death by inducing the release of cytochrome-c from mitochondria, leading to apoptosis [95]. Nitric oxide can also induce necrotic death [96]. Furthermore, the production of nitric oxide and other free radicals can modify oxidative metabolism and impair ATP production [13, 19]. Changes in mitochondrial properties can further limit oxidative metabolism [97]. Not surprisingly, several studies have shown that antioxidant treatment enhances neuroprotection and recovery after stroke [98–101].

The release of glutathione and SOD by astrocytes has been reported and is suggested to play an important role in maintaining and enhancing neuronal survival, yet they are able to reduce ascorbate for further neuronal antioxidant defense Fig. (2) [10, 102–106]. Interestingly, neurons cocultured with astrocytes exhibit higher levels of glutathione compared with neurons cultured alone [107]. Although astrocytes upregulate SOD after cerebral ischemia [108], they do not appear to increase levels of glutathione in ischemic conditions [109]. It is unknown whether ischemia alters astrocytic ascorbate levels, but osmotic swelling from ischemia results in increased astrocyte release of ascorbate in vitro [110], suggesting that similar mechanisms may occur in vivo.

Fig. (2).

Mechanisms of astrocyte support of neurons important in stroke. Antioxidant defense includes release of glutathione and ascorbate. Regulation of extracellular levels of ions and neuro-transmitters, especially K⁺ and glutamate, strongly influences neuronal excitability. Elevated extracellular K⁺ triggers astrocyte glycolysis and enhances lactate and pyruvate release which support neuronal metabolism. Sodium dependent glutamate uptake by astrocytes activates the Na⁺/K⁺ ATPase, stimulating glycolytic activity and production of lactate. Astrocytes and neurons are also coupled by the glutamate-glutamine cycle. Astrocytes take up glutamate, convert it to glutamine, and release glutamine to the extracellular space where it is taken up by neurons and used to synthesize glutamate to replenish the neurotransmitter pool.

Several treatments that attenuate ischemic injury result in increased glutathione levels [111, 112]. SOD converts superoxide into oxygen and hydrogen peroxide. Similar to glutathione, many treatments that ameliorate stroke damage are accompanied by an increase in SOD [113, 114]. Furthermore, rodents overexpressing SOD1 have significantly smaller injuries after both focal and global ischemia [115, 116], while mice with decreased SOD1 have larger infarcts [117]. Finally, ascorbate can also reduce oxidative stress [118]. Treatment with dehydroascorbic acid, a blood-brain-barrier-permeable precursor to ascorbic acid, is protective after MCAO [119]. Dehydroascorbic acid is taken up by astrocytes and released as ascorbic acid [12], a process increased by propofol [120], a treatment that can be protective after stroke [121]. In summary, astrocytes are important producers of antioxidants in the normal CNS, and astrocyte production of these molecules after stroke may enhance neuronal survival and protect astrocyte function.

5.2. Glutamate Regulation

Astrocytes are key players in the regulation of neuro-transmitters in the CNS. Astrocytes make glutamine, the precursor for the neurotransmitters glutamate and GABA [122] Fig. (2). Astrocyte production of neurotransmitter precursors is impaired after MCAO, and alterations in neuro-transmitter levels occur throughout the brain following stroke, possibly contributing to neuronal death [123, 124].

Astrocytes are primarily responsible for glutamate uptake in the normal brain using the astrocyte specific glutamate transporters GLAST and GLT-1 (Fig. 2) [125–127], as excess glutamate leads to cell death via excitotoxicity [128]. Glutamate transporter levels in astrocytes decrease acutely following global ischemia [38, 129] and neonatal hypoxia-ischemia [130], most likely exacerbating neuronal death as a result of glutamate-induced excitoxicity. Despite the therapeutic potential of increasing astrocyte glutamate transport after stroke, few groups have explored this possibility. Carnosine, shown to be protective after focal ischemia, may partially be effective because it prevents loss of GLT-1 on astrocytes, resulting in attenuated excitotoxicity [131]. In a more direct assessment of how post-ischemic astrocyte glutamate transporters contribute to neuronal survival, our laboratory has shown that upregulation of GLT-1 on astrocytes using ceftriaxone protects CA1 neurons after global ischemia [129], similar to its effects in focal cerebral ischemia [132].

5.3. Potassium Uptake and Energy Metabolism

Astrocytes also regulate neuronal activation by extracellular potassium uptake [133] Fig. (2). Neurons release potassium after activation, and increased extracellular potassium leads to neuronal hyperexcitability [133], a phenomenon that occurs in ischemic conditions [134]. In addition to regulating neuronal activation, proper maintenance of ion gradients, such as potassium, is important in regulating cell volume in both normal and ischemic conditions [135, 136]. Astrocytes increase potassium transporter activity in response to transient in vitro ischemia [137]. Due to its effects on both neuronal activity and cell volume, increasing astrocytic potassium uptake may be a possible therapeutic target for stroke.

Astrocytes are also integral to normal maintenance of neuronal metabolism. When astrocytes take up extracellular glutamate as a result of neuronal activity, the Na⁺/ K⁺-ATPase, along with AMPA signaling, triggers astrocyte uptake of glucose from the blood, as astrocytic endfeet contact capillaries [138, 139]. This glucose is then made into lactate, a substrate for neuronal energy, to further “fuel” active neurons [140] Fig. (2). As mentioned above, astrocytes produce glutathione. In addition to its antioxidant properties, glutathione is needed for the conversion of methylglyoxal, a toxic by-product of metabolism, into D-Lactate by glyoxalase 1 [141]. Although the role of astrocyte metabolism is relatively well-established in normal tissue, the post-ischemic role of astrocyte metabolism maintenance is less clear [142]. After ischemia, astrocytes upregulate glucose transporters in order to provide energy to stressed/dying neuronal cells [143, 144]. Ethyl pyruvate, a derivative of the energy substrate pyruvate, is neuroprotective after stroke only when astrocytes are viable, suggesting that astrocytes are necessary for improvement in post-ischemic energy metabolism [122].

6. NOVEL STRATEGIES TO IMPROVE THE NEURONAL SUPPORTIVE ROLE OF ASTROCYTES

Although few studies have specifically targeted astrocytes for repair after stroke, there is some evidence that this can be a successful strategy. Recent results indicate that induction of BDNF in astrocytes by galectin-1 reduces neuronal apoptosis in ischemic boundary zone and improves functional recovery [145]. In addition, protection by pyruvate against glutamate neurotoxicity is mediated by astrocytes through a glutathione-dependent mechanism [146]. Our recent study demonstrated that enhancing astrocyte resistance to ischemic stress by overexpressing protective proteins or antioxidant enzyme results in improved survival of CA1 neurons following forebrain ischemia Fig. (3) [16]. Two well-studied protective proteins, heat shock protein 72 (Hsp72) and mitochondrial SOD, were genetically targeted for expression in astrocytes using the astrocyte-specific human GFAP promoter. In both cases protection was accompanied by preservation of the astrocytic glutamate transporter GLT-1, and reduced evidence of oxidative stress in the CA1 region [16]. Similarly, selective overexpression of excitatory amino acid transporter 2 (EAAT2) in astrocytes enhances neuroprotection from moderate hypoxia-ischemia [147].

Fig. (3).

Targeted over-expression of Hsp72 in astrocytes reduces the vulnerability of CA1 neurons to forebrain ischemia. Selective overexpression of Hsp72 in astrocytes by expressing it from the astrocyte specific GFAP promoter was achieved by unilateral stereotaxic injection of the expression plasmid just above the CA1 region of the hippocampus (black arrows for microinjection tracks) 2 days before rats were subjected to 15 min forebrain ischemia. Selective loss of CA1 hippocampal neurons (between white arrows in middle panel) was observed at 7 days reperfusion by cresyl violet staining. The loss of CA1 hippocampal neurons was significantly reduced with astrocytic Hsp72 overex-pression (right panel) compared to the neuronal loss seen with injection of control DNA (middle panel). Modified from [16].

7. TRANSLATING INSIGHTS INTO PROTECTION INTO CLINICAL APPLICATIONS

Many factors have been identified that likely contribute to the failure in translation seen so far with stroke therapies. Currently, the only approved stroke therapy is thrombolysis induced by intravenous administration of recombinant tissue plasminogen activator [148]; however, because of a short therapeutic time window, only a small fraction of patients benefit from this treatment. Hypothermia is the only accepted acute treatment to reduce brain injury following cardiac arrest and resuscitation [4]. Thus far many clinical trials have focused on treatments that would likely be beneficial to neurons, with fewer studies focused on mechanisms that might benefit all cell types or specifically targeting other cell types, such as astrocytes. Often the consequence of these treatments on the astrocyte response is not considered. Several examples of past and ongoing clinical trials are discussed below, with specific attention to how these treatments may alter astrocyte response or viability.

Several clinical trials have targeted manipulation of the inflammatory response to ischemia, as stroke patients with systemic inflammation exhibit poorer outcomes [149]. Although anti-inflammatory therapy decreases infarct size and improves neurological sequelae in experimental animal models of stroke [150], human trials with anti-neutrophil therapy have not shown a clear benefit [151, 152]. In addition, recent clinical trials in which anti-CD11/18 antibodies were administered to human subjects were unsuccessful [153]. Likewise, a double-blinded, placebo-controlled clinical trial in which anti–ICAM-1 antibody was administered within 6 hours of stroke symptoms showed disappointing results [151]. In understanding these results it is important to recall that while experimental stroke is relatively homogeneous concerning size, territory, and etiology, with more consistent inflammatory response, human stroke is extremely heterogeneous [154], with different vascular territories and extents of injury. In addition, these mediators are known to affect many organ systems beyond the central nervous system. Systemic administration of anti-inflammatory agents may have exacerbated the relative state of immunocompromise seen in stroke patients, thereby confounding the outcome. Furthermore, inflammation and astrocyte response are likely closely connected. Although there is little evidence for a direct relationship between neutrophils and astrocytes, it has been shown that mice with a blunted inflammatory response exhibit increased loss of GFAP-positive astrocytes after cortical stab injury [155]. Because astrocytic glial scar formation is important in protection of spared tissue from further damage [156], it is possible that treatments that drastically attenuate inflammation lead to a stunted astrocyte response that is deleterious to recovery.

Another drug that has advanced to clinical study is DP-b99, currently in phase III studies for acute stroke. DP-b99 is a membrane active chelator derivative of the known calcium chelator, BAPTA spell out [157]. A lipophilic chelator of calcium, zinc and copper ions, DP-b99 sequesters metal ions only within and in to cell membranes. This clinical trial is especially attractive because sequestration of calcium, zinc, and copper are potentially beneficial not only to neurons, but also to astrocytes. It has been shown in Alzheimer’s disease that beta amyloid increases astrocyte calcium influx, which causes decreased glutathione levels [158]. Zinc chloride is toxic to astrocytes as well as neurons in vitro [159]. Similarly, astrocytes exposed to neocuprine exhibit increased copper influx and undergo apoptotic cell death [160]. Approaches that benefit multiple cell types, including astrocytes, are more likely to be successful.

Other current ongoing clinical trials focus on neuroprotective agents for the purpose of aiding neurological recovery after stroke. Minocycline (Phase I), edavarone (Phase IV), propanolol (a β-blocker; phase II and III), and more recently arundic acid have been previously shown to be protective and enhance neuronal survival in stroke [161–165], though by targeting different mechanisms. Some additional completed and ongoing trials are summarized in Table 1. Preclinical research needs to consider these clinical results, and assess effects on astrocytes as well as neurons.

Table 1.

Overview of Some Completed and Ongoing Clinical Trials for Stroke

Mechanism and Compound
Anticoagulants
Ancrod® (Viprinex)
Aspirin added to IV TPA
Angiotensin II Receptor Antagonist
Diovan® (valsartan)
Losartan and amlodipine
Calcium Channel Blockers
Cilnidipine
Amlodipine
Inhibiton of Myeloperoxidases
Dapsone
Antiplatelet Therapy
Eptifibatide and rt-PA
Glutamate Antagonists
YM872
Granulocyte Colony Stimulating Factor
AX200 (G-CSF)
Free Radical Scavenging
NXY-059

Sources: http://clinicaltrials.gov/; http://strokecenter.stanford.edu/trials/

Although anti-inflammatory strategies to diminish ischemic brain injury have failed thus far, continued elucidation of the complex interactions involved in modulating the inflammatory response may still enable novel therapeutic approaches that translate successfully into clinical efficacy.

CONCLUSIONS

Traditionally, stroke research has focused on neurons and often ignored effects on glial cells. It is increasingly evident that glia are vital to both normal CNS functioning and also play important roles in neuropathological conditions. Although astrocytes form an inhibitory glial scar following ischemia, they also perform functions necessary for neuronal survival and well-being, such as maintaining low extracellular glutamate levels and providing antioxidant protection. Because they have a great many functions, astrocytes are attractive candidates as therapeutic targets. By striving to shift astrocytes towards a pro-reparative, neuronal-supportive phenotype following stroke, future clinical therapies may well be more successful in protecting neurons from ischemic damage and promoting repair.