Predicting outcomes after transient ischemic attack and stroke.

PURPOSE OF REVIEW: Predicting functional outcome and mortality after stroke, with or without thrombolysis, is a critical role of neurologists. This article reviews the predictors of outcome after ischemic stroke. RECENT FINDINGS: Several scores were recently designed to predict (1) mortality and poor functional outcome after ischemic stroke, (2) the functional outcome and risk of symptomatic intracranial hemorrhage (sICH) after thrombolysis, and (3) the risk of stroke following TIA. Validation of these prediction instruments is ongoing, and studies will be critical to determine the general applicability of these scores. SUMMARY: Although several scores were developed to predict mortality and outcome after stroke, it may be premature to employ these prediction scores to determine individual patient outcome. Similarly, prediction scores should not be used to deny patients tissue plasminogen activator (tPA), even if the scores predict that the patient has a high likelihood of sICH or poor outcome after thrombolysis.

Neurological diseases as primary gliopathies: a reassessment of neurocentrism.

Diseases of the human brain are almost universally attributed to malfunction or loss of nerve cells. However, a considerable amount of work has, during the last decade, expanded our view on the role of astrocytes in CNS (central nervous system), and this analysis suggests that astrocytes contribute to both initiation and propagation of many (if not all) neurological diseases. Astrocytes provide metabolic and trophic support to neurons and oligodendrocytes. Here, we shall endeavour a broad overviewing of the progress in the field and forward the idea that loss of homoeostatic astroglial function leads to an acute loss of neurons in the setting of acute insults such as ischaemia, whereas more subtle dysfunction of astrocytes over periods of months to years contributes to epilepsy and to progressive loss of neurons in neurodegenerative diseases. The majority of therapeutic drugs currently in clinical use target neuronal receptors, channels or transporters. Future therapeutic efforts may benefit by a stronger focus on the supportive homoeostatic functions of astrocytes.

Cocultures of neurons and astrocytes as a model for examining hypoxia-induced neuronal death.

Astrocytes perform critical functions necessary for neuronal survival. Thus, examining the influence of astrocyte function on neuronal cell death during disease, including hypoxia/ischemia, has become an important avenue of investigation. In this chapter we detail the methodology and potential pitfalls for establishing cocultures of astrocytes and cortical neurons for studying hypoxia-induced neuronal death. In brief, astrocyte cultures are first established until they reach confluence. The medium is exchanged from a medium that supports astrocyte growth to a medium that supports neuronal viability 24 h before adding neurons to the astrocyte monolayer. After the neurons mature, the cultures are exposed to severe hypoxia and neuronal death is quantified 1-2 days later.

Prophylactic neuroprotection against stroke: low-dose, prolonged treatment with deferoxamine or deferasirox establishes prolonged neuroprotection independent of HIF-1 function.

Prophylactic neuroprotection against stroke could reduce stroke burden in thousands of patients at high risk of stroke, including those with recent transient ischemic attacks (TIAs). Prolyl hydroxylase inhibitors (PHIs), such as deferoxamine (DFO), reduce stroke volume when administered at high doses in the peristroke period, which is largely mediated by the hypoxia-inducible transcription factor (HIF-1). Yet, in vitro experiments suggest that PHIs may also induce neuroprotection independent of HIF-1. In this study, we examine chronic, prophylactic, low-dose treatment with DFO, or another iron chelator deferasirox (DFR), to determine whether they are neuroprotective with this paradigm and mediate their effects through a HIF-1-dependent mechanism. In fact, prophylactic administration of low-dose DFO or DFR significantly reduces stroke volume. Surprisingly, DFO remained neuroprotective in mice haploinsufficient for HIF-1 (HIF-1+/-) and transgenic mice with conditional loss of HIF-1 function in neurons and astrocytes. Similarly, DFR was neuroprotective in HIF-1+/- mice. Neither DFO nor DFR induced expression of HIF-1 targets. Thus, low-dose chronic administration of DFO or DFR induced a prolonged neuroprotective state independent of HIF-1 function. As DFR is an orally administered and well-tolerated medication in clinical use, it has promise for prophylaxis against stroke in patients at high risk of stroke.

Targeting astrocytes for stroke therapy.

Stroke remains a major health problem and is a leading cause of death and disability. Past research and neurotherapeutic clinical trials have targeted the molecular mechanisms of neuronal cell death during stroke, but this approach has uniformly failed to reduce stroke-induced damage or to improve functional recovery. Beyond the intrinsic molecular mechanisms inducing neuronal death during ischemia, survival and function of astrocytes is absolutely required for neuronal survival and for functional recovery after stroke. Many functions of astrocytes likely improve neuronal viability during stroke. For example, uptake of glutamate and release of neurotrophins enhances neuronal viability during ischemia. Under certain conditions, however, astrocyte function may compromise neuronal viability. For example, astrocytes may produce inflammatory cytokines or toxic mediators, or may release glutamate. The only clinical neurotherapeutic trial for stroke that specifically targeted astrocyte function focused on reducing release of S-100ß from astrocytes, which becomes a neurotoxin when present at high levels. Recent work also suggests that astrocytes, beyond their influence on cell survival, also contribute to angiogenesis, neuronal plasticity, and functional recovery in the several days to weeks after stroke. If these delayed functions of astrocytes could be targeted for enhancing stroke recovery, it could contribute importantly to improving stroke recovery. This review focuses on both the positive and the negative influences of astrocytes during stroke, especially as they may be targeted for translation to human trials.

During hypoxia, HUMMR joins the mitochondrial dance.

Mitochondrial distribution is integrally related to cellular function. Highly polarized cells, such as neurons, likely depend on mitochondrial transport to maintain proper synaptic function and neurite plasticity. In some cases, mitochondrial transport is also required for cellular migration and proper calcium signaling in non-neuronal cells. Over the past few years, much progress has been made in identifying proteins that control mitochondrial transport and distribution. Miro and Milton, which are two outer mitochondrial membrane proteins, tether mitochondria to kinesin motor proteins. Our recent work identified a novel protein, HUMMR, which interacts with Miro. While present in normoxia, HUMMR protein abundance is markedly induced by hypoxia through a HIF-1 dependent mechanism. Knock down of HUMMR function diminishes the number of mitochondria in the axon, an effect that was more prominent in neurons exposed to hypoxia. Interestingly, in hypoxic neurons, knock down of HUMMR also diminished the number of anterograde moving mitochondria, but increased the number of retrograde moving mitochondria. Thus, HUMMR is a protein which biases mitochondrial movement in the anterograde direction in response to hypoxia. The implication for this biased transport of mitochondria during hypoxia on neuronal function and viability is yet to be discerned. Regardless, since hypoxia is prominent during ischemia and in solid tumors, HUMMR likely contributes to mitochondrial distribution under these conditions. As such, HUMMR may influence cellular function that is dependent upon the correct mitochondrial localization.

HUMMR, a hypoxia- and HIF-1alpha-inducible protein, alters mitochondrial distribution and transport.

Mitochondrial transport is critical for maintenance of normal neuronal function. Here, we identify a novel mitochondria protein, hypoxia up-regulated mitochondrial movement regulator (HUMMR), which is expressed in neurons and is markedly induced by hypoxia-inducible factor 1 alpha (HIF-1alpha). Interestingly, HUMMR interacts with Miro-1 and Miro-2, mitochondrial proteins that are critical for mediating mitochondrial transport. Interestingly, knockdown of HUMMR or HIF-1 function in neurons exposed to hypoxia markedly reduces mitochondrial content in axons. Because mitochondrial transport and distribution are inextricably linked, the impact of reduced HUMMR function on the direction of mitochondrial transport was also explored. Loss of HUMMR function in hypoxia diminished the percentage of motile mitochondria moving in the anterograde direction and enhanced the percentage moving in the retrograde direction. Thus, HUMMR, a novel mitochondrial protein induced by HIF-1 and hypoxia, biases mitochondria transport in the anterograde direction. These findings have broad implications for maintenance of neuronal viability and function during physiological and pathological states.

The Janus-faced effects of hypoxia on astrocyte function.

Astrocytes are increasingly recognized for their impact on neuronal function and viability in health and disease. Hypoxia has Janus-faced influences on astrocytes and their ability to support neuronal viability. For example, hypoxia induces astrocyte-dependent protection of neurons following hypoxia preconditioning. Yet, hypoxia induces processes in astrocytes that augment neuronal death in other situations, such as the coincidence of hypoxia with inflammatory signaling. A complex array of gene expression is induced by hypoxia within astrocytes and neurons through multiple transcription factors and intracellular molecular pathways. The hypoxia inducible factors (HIFs) are transcription factors that are likely instrumental in orchestrating adaptive and pathological functions of astrocytes. As such, the HIFs are postulated to mediate both adaptive and pathological functions during hypoxia/ ischemia. Identifying the conditions under which hypoxia induces signaling in astrocytes that alters autonomous or neuronal survival will undoubtedly have important implications regarding the development of new strategies for stroke treatment.

Loss of c/EBP-beta activity promotes the adaptive to apoptotic switch in hypoxic cortical neurons.

Understanding the mechanisms governing the switch between hypoxia-induced adaptive and pathological transcription may reveal novel therapeutic targets for stroke. Using an in vitro hypoxia model that temporally separates these divergent responses, we found apoptotic signaling was preceded by a decline in c/EBP-beta activity and was associated with markers of ER-stress including transient eIF2alpha phosphorylation, and the delayed induction of the bZIP proteins ATF4 and CHOP-10. Pretreatment with the eIF2alpha phosphatase inhibitor salubrinal blocked the activation of caspase-3, indicating that ER-related stress responses are integral to this transition. Delivery of either full-length, or a transcriptionally inactive form of c/EBP-beta protected cultures from hypoxic challenge, in part by inducing levels of the anti-apoptotic protein Bcl-2. These data indicate that the pathologic response in cortical neurons induced by hypoxia involves both the loss of c/EBP-beta-mediated survival signals and activation of pro-death pathways originating from the endoplasmic reticulum.

The good, the bad, and the cell type-specific roles of hypoxia inducible factor-1 alpha in neurons and astrocytes.

Hypoxia inducible factor-1alpha (HIF-1alpha) is a key regulator of oxygen homeostasis, because it is responsible for the regulation of genes involved in glycolysis, erythropoiesis, angiogenesis, and apoptosis. In the CNS, HIF-1alpha is stabilized by insults associated with hypoxia and ischemia. Because its many target genes mediate both adaptive and pathological processes, the role of HIF-1alpha in neuronal survival is debated. Although neuronal HIF-1alpha function has been the topic of several studies, the role of HIF-1alpha function in astrocytes has received much less attention. To characterize the role of HIF-1alpha in neurons and astrocytes, we induced loss of HIF-1alpha function specifically in neurons, astrocytes, or both cell types in neuron/astrocyte cocultures exposed to hypoxia. Although loss of HIF-1alpha function in neurons reduced neuronal viability during hypoxia, selective loss of HIF-1 function in astrocytes markedly protected neurons from hypoxic-induced neuronal death. Although the pathological processes induced by HIF-1alpha in astrocytes remain to be defined, induction of inducible nitric oxide synthase likely contributes to the pathological process. This study delineates, for the first time, a cell type-specific action for HIF-1alpha within astrocytes and neurons.

In cultured astrocytes, p53 and MDM2 do not alter hypoxia-inducible factor-1alpha function regardless of the presence of DNA damage.

A principal molecular mechanism by which cells respond to hypoxia is by activation of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha). Several studies describe a binding of p53 to HIF-1alpha in a protein complex, leading to attenuated function, half-life, and abundance of HIF-1alpha. However, these reports almost exclusively utilized transformed cell lines, and many employed transfection of p53 or HIF-1alpha plasmid constructs and/or p53 and HIF-1alpha reporter constructs as surrogates for endogenous protein activity and target expression, respectively. Thus, it remains an open and important question as to whether p53 inhibits HIF-1alpha-mediated transactivation of endogenous HIF-1alpha targets in nontransformed cells. After determining in primary astrocyte cultures the HIF-1alpha targets that were most dependent on HIF-1alpha function, we examined the effect of the loss of p53 function either alone or in combination with MDM2 on expression of these targets. Although p53 null astrocyte cultures resulted in markedly increased HIF-1alpha-dependent target expression compared with controls, this altered expression was determined to be the result of increased cell density of p53 null cultures and the accompanying acidosis, not loss of p53 protein. Although activation of p53 by DNA damage induced p53 target expression in astrocytes, it did not alter hypoxia-induced HIF-1alpha target expression. Finally, a combined loss of MDM2 and p53 did not alter HIF-1alpha target expression compared with loss of p53 alone. These data strongly suggest that p53 and MDM2 do not influence the hypoxia-induced transactivation of HIF-1alpha targets, regardless of p53 activation, in primary astrocytes.

Blockade of gap junctions in vivo provides neuroprotection after perinatal global ischemia.

BACKGROUND AND PURPOSE: We investigated the contribution of gap junctions to brain damage and delayed neuronal death produced by oxygen-glucose deprivation (OGD). METHODS: Histopathology, molecular biology, and electrophysiological and fluorescence cell death assays in slice cultures after OGD and in developing rats after intrauterine hypoxia-ischemia (HI). RESULTS: OGD persistently increased gap junction coupling and strongly activated the apoptosis marker caspase-3 in slice cultures. The gap junction blocker carbenoxolone applied to hippocampal slice cultures before, during, or 60 minutes after OGD markedly reduced delayed neuronal death. Administration of carbenoxolone to ischemic pups immediately after intrauterine HI prevented caspase-3 activation and dramatically reduced long-term neuronal damage. CONCLUSIONS: Gap junction blockade may be a useful therapeutic tool to minimize brain damage produced by perinatal and early postnatal HI.