Repurposing the KCa3.1 Blocker Senicapoc for Ischemic Stroke.

Senicapoc, a small molecule inhibitor of the calcium-activated potassium channel KCa3.1, was safe and well-tolerated in clinical trials for sickle cell anemia. We previously reported proof-of-concept data suggesting that both pharmacological inhibition and genetic deletion of KCa3.1 reduces infarction and improves neurologic recovery in rodents by attenuating neuroinflammation. Here we evaluated the potential of repurposing senicapoc for ischemic stroke. In cultured microglia, senicapoc inhibited KCa3.1 currents with an IC50 of 7 nM, reduced Ca2+ signaling induced by the purinergic agonist ATP, suppressed expression of pro-inflammatory cytokines and enzymes (iNOS and COX-2), and prevented induction of the inflammasome component NLRP3. When transient middle cerebral artery occlusion (tMCAO, 60 min) was induced in male C57BL/6 J mice, twice daily administration of senicapoc at 10 and 40 mg/kg starting 12 h after reperfusion dose-dependently reduced infarct area determined by T2-weighted magnetic resonance imaging (MRI) and improved neurological deficit on day 8. Ultra-high-performance liquid chromatography/mass spectrometry analysis of total and free brain concentrations demonstrated sufficient KCa3.1 target engagement. Senicapoc treatment significantly reduced microglia/macrophage and T cell infiltration and activation and attenuated neuronal death. A different treatment paradigm with senicapoc started at 3 h and MRI on day 3 and day 8 revealed that senicapoc reduces secondary infarct growth and suppresses expression of inflammation markers, including T cell cytokines in the brain. Lastly, we demonstrated that senicapoc does not impair the proteolytic activity of tissue plasminogen activator (tPA) in vitro. We suggest that senicapoc could be repurposed as an adjunctive immunocytoprotective agent for combination with reperfusion therapy for ischemic stroke.

Microglial depletion abolishes ischemic preconditioning in white matter.

Ischemic preconditioning (IPC) is a phenomenon whereby a brief, non-injurious ischemic exposure enhances tolerance to a subsequent ischemic challenge. The mechanism of IPC has mainly been studied in rodent stroke models where gray matter (GM) constitutes about 85% of the cerebrum. In humans, white matter (WM) is 50% of cerebral volume and is a critical component of stroke damage. We developed a novel CNS WM IPC model using the mouse optic nerve (MON) and identified the involved immune signaling pathways. Here we tested the hypothesis that microglia are necessary for WM IPC. Microglia were depleted by treatment with the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622. MONs were exposed to transient ischemia in vivo, acutely isolated 72 h later, and subjected to oxygen-glucose deprivation (OGD) to simulate a severe ischemic injury (i.e., stroke). Functional and structural axonal recovery was assessed by recording compound action potentials (CAPs) and by microscopy using quantitative stereology. Microglia depletion eliminated IPC-mediated protection. In control mice, CAP recovery was improved in preconditioned MONs compared with non-preconditioned MONs, however, in PLX5622-treated mice, we observed no difference in CAP recovery between preconditioned and non-preconditioned MONs. Microgliadepletion also abolished IPC protective effects on axonal integrity and survival of mature (APC+ ) oligodendrocytes after OGD. IPC-mediated protection was independent of retinal injury suggesting it results from mechanistic processes intrinsic to ischemia-exposed WM. We conclude that preconditioned microglia are critical for IPC in WM. The "preconditioned microglia" phenotype might protect against other CNS pathologies and is a neurotherapeutic horizon worth exploring.

A Subpopulation of Microglia Generated in the Adult Mouse Brain Originates from Prominin-1-Expressing Progenitors.

Microglia maintain brain health and play important roles in disease and injury. Despite the known ability of microglia to proliferate, the precise nature of the population or populations capable of generating new microglia in the adult brain remains controversial. We identified Prominin-1 (Prom1; also known as CD133) as a putative cell surface marker of committed brain myeloid progenitor cells. We demonstrate that Prom1-expressing cells isolated from mixed cortical cultures will generate new microglia in vitro To determine whether Prom1-expressing cells generate new microglia in vivo, we used tamoxifen inducible fate mapping in male and female mice. Induction of Cre recombinase activity at 10 weeks in Prom1-expressing cells leads to the expression of TdTomato in all Prom1-expressing progenitors and newly generated daughter cells. We observed a population of new TdTomato-expressing microglia at 6 months of age that increased in size at 9 months. When microglia proliferation was induced using a transient ischemia/reperfusion paradigm, little proliferation from the Prom1-expressing progenitors was observed with the majority of new microglia derived from Prom1-negative cells. Together, these findings reveal that Prom1-expressing myeloid progenitor cells contribute to the generation of new microglia both in vitro and in vivo Furthermore, these findings demonstrate the existence of an undifferentiated myeloid progenitor population in the adult mouse brain that expresses Prom1. We conclude that Prom1-expressing myeloid progenitors contribute to new microglia genesis in the uninjured brain but not in response to ischemia/reperfusion.SIGNIFICANCE STATEMENT Microglia, the innate immune cells of the CNS, can divide to slowly generate new microglia throughout life. Newly generated microglia may influence inflammatory responses to injury or neurodegeneration. However, the origins of the new microglia in the brain have been controversial. Our research demonstrates that some newly born microglia in a healthy brain are derived from cells that express the stem cell marker Prominin-1. This is the first time Prominin-1 cells are shown to generate microglia.

The role of microglia in ischemic preconditioning.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a brief ischemic stimulus confers protection against a subsequent prolonged ischemic event. Initially thought to be due to mechanistic changes in neurons, our understanding of IPC has evolved to encompass a global reprogramming of the Central Nervous System (CNS) after transient ischemia/reperfusion that requires innate immune signaling pathways including Toll-like receptors (TLRs) and Type I interferons. Microglia are the CNS resident neuroimmune cells that express these key innate immune receptors. Studies suggest that microglia are required for IPC-mediated neuronal and axonal protection. Multiple paradigms targeting TLRs have converged on a distinctive Type I interferon response in microglia that is critical for preconditioning-mediated protection against ischemia. These pathways can be targeted through administration of TLR agonists, cytokines including interferon-ß, and pharmaceutical agents that induce preconditioning through cross-tolerance mechanisms. Transcriptomic analyses and single cell RNA studies point to specific gene expression signatures in microglia that functionally shift these mutable cells to an immunomodulatory or protective phenotype. Although there are technological challenges and gaps in knowledge to overcome, the targeting of specific molecular signaling pathways in microglia is a promising direction for development of novel and effective pharmacotherapies for stroke. Studies on preconditioning in animal models, including nonhuman primates, show promise as prophylactic preconditioning treatments for selected at risk patient populations. In addition, our growing understanding of the mechanisms of IPC-mediated protection is identifying novel cellular and molecular targets for therapeutic interventions that could apply broadly to both acute stroke and chronic vascular cognitive impairment patients.

Ischemic preconditioning induces cortical microglial proliferation and a transcriptomic program of robust cell cycle activation.

Ischemic preconditioning (IPC) is an experimental phenomenon in which a subthreshold ischemic insult applied to the brain reduces damage caused by a subsequent more severe ischemic episode. Identifying key molecular and cellular mediators of IPC will provide critical information needed to develop novel therapies for stroke. Here we report that the transcriptomic response of acutely isolated preconditioned cortical microglia is dominated by marked upregulation of genes involved in cell cycle activation and cellular proliferation. Notably, this transcriptional response occurs in the absence of cortical infarction. We employed ex vivo flow cytometry, immunofluorescent microscopy, and quantitative stereology methods on brain tissue to evaluate microglia proliferation following IPC. Using cellular colocalization of microglial (Iba1) and proliferation (Ki67 and BrdU) markers, we observed a localized increase in the number of microglia and proliferating microglia within the preconditioned hemicortex at 72, but not 24, hours post-IPC. Our quantification demonstrated that the IPC-induced increase in total microglia was due entirely to proliferation. Furthermore, microglia in the preconditioned hemisphere had altered morphology and increased soma volumes, indicative of an activated phenotype. Using transgenic mouse models with either fractalkine receptor (CX3CR1)-haploinsufficiency or systemic type I interferon signaling loss, we determined that microglial proliferation after IPC is dependent on fractalkine signaling but independent of type I interferon signaling. These findings suggest there are multiple distinct targetable signaling pathways in microglia, including CX3CR1-dependent proliferation that may be involved in IPC-mediated protection.

Correction to: Neuroimmune Response in Ischemic Preconditioning.

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon in which a brief period of cerebral ischemia confers transient tolerance to subsequent ischemic challenge. Research on IPC has implicated cellular, molecular, and systemic elements of the immune response in this phenomenon. Potent molecular mediators of IPC include innate immune signaling pathways such as Toll-like receptors and type 1 interferons. Brain ischemia results in release of pro- and anti-inflammatory cytokines and chemokines that orchestrate the neuroinflammatory response, resolution of inflammation, and transition to neurological recovery and regeneration. Cellular mediators of IPC include microglia, the resident central nervous system immune cells, astrocytes, and neurons. All of these cell types engage in cross-talk with each other using a multitude of signaling pathways that modulate activation/suppression of each of the other cell types in response to ischemia. As the postischemic neuroimmune response evolves over time there is a shift in function toward provision of trophic support and neuroprotection. Peripheral immune cells infiltrate the central nervous system en masse after stroke and are largely detrimental, with a few subtypes having beneficial, protective effects, though the role of these immune cells in IPC is largely unknown. The role of neural progenitor cells in IPC-mediated neuroprotection is another active area of investigation as is the role of microglial proliferation in this setting. A mechanistic understanding of these molecular and cellular mediators of IPC may not only facilitate more effective direct application of IPC to specific clinical scenarios, but also, more broadly, reveal novel targets for therapeutic intervention in stroke.

Ischemia/Reperfusion Induces Interferon-Stimulated Gene Expression in Microglia.

Innate immune signaling is important in the pathophysiology of ischemia/reperfusion (stroke)-induced injury and recovery. Several lines of evidence support a central role for microglia in these processes. Recent work has identified Toll-like receptors (TLRs) and type I interferon (IFN) signaling in both ischemia/reperfusion-induced brain injury and ischemic preconditioning-mediated neuroprotection. To determine the effects of "ischemia/reperfusion-like" conditions on microglia, we performed genomic analyses on wild-type (WT) and TLR4-/- cultured microglia after sequential exposure to hypoxia/hypoglycemia and normoxia/normoglycemia (H/H-N/N). We observed increased expression of type 1 IFN-stimulated genes (ISGs) as the predominant transcriptomal feature of H/H-N/N-exposed WT, but not TLR4-/-, microglia. Microarray analysis on ex vivo sorted microglia from ipsilateral male mouse cortex after a transient in vivo ischemic pulse also demonstrated robust expression of ISGs. Type 1 IFNs, including the IFN-αs and IFN-ß, activate the interferon-α/ß receptor (IFNAR) complex. We confirmed both in vitro H/H-N/N- and in vivo ischemia/reperfusion-induced microglial ISG responses by quantitative real-time PCR and demonstrated that both were dependent on IFNAR1. We characterized the effects of hypoxia/hypoglycemia on phosphorylation of signal transducer and activator of transcription 1 (STAT1), release of type 1 IFNs, and surface expression of IFNAR1 in microglia. We demonstrated that IFN-ß induces dose-dependent secretion of ISG chemokines in cultured microglia and robust ISG expression in microglia both in vitro and in vivo Finally, we demonstrated that the microglial ISG chemokine responses to TLR4 agonists were dependent on TLR4 and IFNAR1. Together, these data suggest novel ischemia/reperfusion-induced pathways for both TLR4-dependent and -independent, IFNAR1-dependent, type 1 IFN signaling in microglia.SIGNIFICANCE STATEMENT Stroke is the fifth leading cause of death in the United States and is a leading cause of serious long-term disability worldwide. Innate immune responses are critical in stroke pathophysiology, and microglia are key cellular effectors in the CNS response to ischemia/reperfusion. Using a transcriptional analysis approach, we identified a robust interferon (IFN)-stimulated gene response within microglia exposed to ischemia/reperfusion in both in vitro and in vivo experimental paradigms. Using a number of complementary techniques, we have demonstrated that these responses are dependent on innate immune signaling components including Toll-like receptor-4 and type I IFNs. We have also elucidated several novel ischemia/reperfusion-induced microglial signaling mechanisms.

Stroke: Basic and Clinical.

Tissue plasminogen activator (tPA) was first approved in the USA 25 years ago for those who had experienced a recent occlusion (<3 h) of a cerebral vessel. Now, advances in clot retrieval (stentriever), in concert with tPA, heralds new optimism for ischemic stroke victims, but adds more pressure to identify therapies that will minimize hypoxic damage, protect compromised cells, and promote rehabilitation. In the past preclinical investigations have been poor at predicting potential clinical therapy, but they have contributed enormously to understanding post-stroke pathology. Current clinical trials ( www.strokecenter.org/trials ) anticipate a broad range of approaches: from hypothermia, to cell therapy, to neuroprotection.

Microglial Interferon Signaling and White Matter.

Microglia, the resident immune cells of the CNS, are primary regulators of the neuroimmune response to injury. Type I interferons (IFNs), including the IFNαs and IFNß, are key cytokines in the innate immune system. Their activity is implicated in the regulation of microglial function both during development and in response to neuroinflammation, ischemia, and neurodegeneration. Data from numerous studies in multiple sclerosis (MS) and stroke suggest that type I IFNs can modulate the microglial phenotype, influence the overall neuroimmune milieu, regulate phagocytosis, and affect blood-brain barrier integrity. All of these IFN-induced effects result in numerous downstream consequences on white matter pathology and microglial reactivity. Dysregulation of IFN signaling in mouse models with genetic deficiency in ubiquitin specific protease 18 (USP18) leads to a severe neurological phenotype and neuropathological changes that include white matter microgliosis and pro-inflammatory gene expression in dystrophic microglia. A class of genetic disorders in humans, referred to as pseudo-TORCH syndrome (PTS) for the clinical resemblance to infection-induced TORCH syndrome, also show dysregulation of IFN signaling, which leads to severe neurological developmental disease. In these disorders, the excessive activation of IFN signaling during CNS development results in a destructive interferonopathy with similar induction of microglial dysfunction as seen in USP18 deficient mice. Other recent studies implicate "microgliopathies" more broadly in neurological disorders including Alzheimer's disease (AD) and MS, suggesting that microglia are a potential therapeutic target for disease prevention and/or treatment, with interferon signaling playing a key role in regulating the microglial phenotype.

Senicapoc: Repurposing a Drug to Target Microglia KCa3.1 in Stroke.

Stroke is the leading cause of serious long-term disability and the fifth leading cause of death in the United States. Treatment options for stroke are few in number and limited in efficacy. Neuroinflammation mediated by microglia and infiltrating peripheral immune cells is a major component of stroke pathophysiology. Interfering with the inflammation cascade after stroke holds the promise to modulate stroke outcome. The calcium activated potassium channel KCa3.1 is expressed selectively in the injured CNS by microglia. KCa3.1 function has been implicated in pro-inflammatory activation of microglia and there is recent literature suggesting that this channel is important in the pathophysiology of ischemia/reperfusion (stroke) related brain injury. Here we describe the potential of repurposing Senicapoc, a KCa3.1 inhibitor, to intervene in the inflammation cascade that follows ischemia/reperfusion.

Neuroimmune Response in Ischemic Preconditioning.

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon in which a brief period of cerebral ischemia confers transient tolerance to subsequent ischemic challenge. Research on IPC has implicated cellular, molecular, and systemic elements of the immune response in this phenomenon. Potent molecular mediators of IPC include innate immune signaling pathways such as Toll-like receptors and type 1 interferons. Brain ischemia results in release of pro- and anti-inflammatory cytokines and chemokines that orchestrate the neuroinflammtory response, resolution of inflammation, and transition to neurological recovery and regeneration. Cellular mediators of IPC include microglia, the resident central nervous system immune cells, astrocytes, and neurons. All of these cell types engage in cross-talk with each other using a multitude of signaling pathways that modulate activation/suppression of each of the other cell types in response to ischemia. As the postischemic neuroimmune response evolves over time there is a shift in function toward provision of trophic support and neuroprotection. Peripheral immune cells infiltrate the central nervous system en masse after stroke and are largely detrimental, with a few subtypes having beneficial, protective effects, though the role of these immune cells in IPC is largely unknown. The role of neural progenitor cells in IPC-mediated neuroprotection is another active area of investigation as is the role of microglial proliferation in this setting. A mechanistic understanding of these molecular and cellular mediators of IPC may not only facilitate more effective direct application of IPC to specific clinical scenarios, but also, more broadly, reveal novel targets for therapeutic intervention in stroke.

Ischemic Preconditioning in White Matter: Magnitude and Mechanism.

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon whereby brief ischemic exposure confers tolerance to a subsequent ischemic challenge. IPC has not been studied selectively in CNS white matter (WM), although stroke frequently involves WM. We determined whether IPC is present in WM and, if so, its mechanism. We delivered a brief in vivo preconditioning ischemic insult (unilateral common carotid artery ligation) to 12- to 14-week-old mice and determined WM ischemic vulnerability [oxygen-glucose deprivation (OGD)] 72 h later, using acutely isolated optic nerves (CNS WM tracts) from the preconditioned (ipsilateral) and control (contralateral) hemispheres. Functional and structural recovery was assessed by quantitative measurement of compound action potentials (CAPs) and immunofluorescent microscopy. Preconditioned mouse optic nerves (MONs) showed better functional recovery after OGD than the non-preconditioned MONs (31 ± 3 vs 17 ± 3% normalized CAP area, p < 0.01). Preconditioned MONs also showed improved axon integrity and reduced oligodendrocyte injury compared with non-preconditioned MONs. Toll-like receptor-4 (TLR4) and type 1 interferon receptor (IFNAR1), key receptors in innate immune response, are implicated in gray matter preconditioning. Strikingly, IPC-mediated WM protection was abolished in both TLR4(-/-) and IFNAR1(-/-) mice. In addition, IPC-mediated protection in WM was also abolished in IFNAR1(fl/fl) LysM(cre), but not in IFNAR1(fl/fl) control, mice. These findings demonstrated for the first time that IPC was robust in WM, the phenomenon being intrinsic to WM itself. Furthermore, WM IPC was dependent on innate immune cell signaling pathways. Finally, these data demonstrated that microglial-specific expression of IFNAR1 plays an indispensable role in WM IPC. SIGNIFICANCE STATEMENT: Ischemic preconditioning (IPC) has been studied predominantly in gray matter, but stroke in humans frequently involves white matter (WM) as well. Here we describe a novel, combined in vivo/ex vivo mouse model to determine whether IPC occurs in WM. It does. Using genetically altered mice, we identified two innate immune cell receptors, Toll-like receptor 4 and type 1 interferon receptor (IFNAR1), that are required for IPC-mediated protection in WM. Furthermore, using microglia-targeted IFNAR1 knockdown, we demonstrate that interferon signaling specifically in microglia is essential for this protection. The discovery of IPC as an intrinsic capability of WM is novel and important. This is also the first in vivo demonstration that cell-type-specific expression of an individual gene plays an indispensable role in IPC-mediated protection.

IgM-Dependent Phagocytosis in Microglia Is Mediated by Complement Receptor 3, Not Fcα/µ Receptor.

Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with Igs or complement. Microglia recognize these opsonized pathogens by Fc or complement receptors triggering phagocytosis. In this study, we investigated the role of Fcα/µR, the less-studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fcα/µR. We also showed that anti-Staphylococcus aureus IgM markedly increased the rate of microglial S. aureus phagocytosis. To unequivocally test the role of Fcα/µR in IgM-mediated phagocytosis, we performed experiments in microglia from Fcα/µR(-/-) mice. Surprisingly, we found that IgM-dependent phagocytosis of S. aureus was similar in microglia derived from wild-type or Fcα/µR(-/-) mice. We hypothesized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this, we used immunologic and genetic inactivation of complement receptor 3 components (CD11b and CD18) as well as C3. IgM-, but not IgG-mediated phagocytosis of S. aureus was reduced in wild-type microglia and macrophages following preincubation with an anti-CD11b blocking Ab. IgM-dependent phagocytosis of S. aureus was also reduced in microglia derived from CD18(-/-) and C3(-/-) mice. Taken together, our findings implicate complement receptor 3 and C3, but not Fcα/µR, in IgM-mediated phagocytosis of S. aureus by microglia.

Functional polymorphisms in toll-like receptor 4 are associated with worse outcome in acute ischemic stroke patients.

Toll-like receptor-4 (TLR4) is important in neuroinflammation. Single nucleotide polymorphisms (SNPs) in TLR4, including 1063 A/G [Asp299Gly] and 1363 C/T [Thr399Ile], are associated with altered immune responses but their effect on acute ischemic stroke (AIS) outcome is unknown. We collected demographic, clinical, laboratory, radiologic, and genotype data on 113 AIS patients and performed multivariate analyses to assess associations between TLR4 SNP haplotype and either neurological outcome, infection, or inflammatory markers. In adjusted analyses, TLR4 SNPs were associated with worse outcome as well as increases in circulating leukocytes, C-reactive protein, and interleukin-1 receptor antagonist. In AIS, variations in TLR4 may influence neurological outcome (for video abstract, please see Supplemental digital content 1 file, http://links.lww.com/WNR/A274).

Thrombin induces release of proinflammatory chemokines interleukin-8 and interferon-γ-induced protein-10 from cultured human fetal astrocytes.

Thrombin is a multifunctional serine proteinase that induces a variety of responses from neural cells by cleavage of proteinase-activated receptors (PARs) including PAR1 and PAR4. Thrombin/PAR signaling has been implicated in the neuroinflammatory response that occurs in the brain following stroke and other central nervous system pathologies. The neuroinflammatory response involves astrocytes and results in induction of proinflammatory chemokines including interleukin-8 (IL-8 or CXCL8) and interferon-γ-induced protein-10 (IP-10 or CXCL10) in these cells. Astroctyes are known to express PARs, however the effect of thrombin on astrocytic chemokine secretion is unknown. Here we characterize the ability of thrombin to induce proliferation/metabolic activity and chemokine secretion in primary human fetal astrocytes. Thrombin induces dose-dependent astrocyte proliferation as well as release of both IL-8 and IP-10, but not IL-6 or the chemokine regulated and normal T cell expressed and secreted (RANTES). The chemokine responses were mimicked by PAR1, but not PAR4, activating peptides. Our data indicate that astrocytic chemokine release is part of the neuroinflammatory response triggered by the exposure of the central nervous system to thrombin.

Do-not-attempt-resuscitation orders and prognostic models for intraparenchymal hemorrhage.

OBJECTIVES: Statistical models predicting outcome after intraparenchymal hemorrhage include patients irrespective of do-not-attempt-resuscitation orders. We built a model to explore how the inclusion of patients with do-not-attempt-resuscitation orders affects intraparenchymal hemorrhage prognostic models. DESIGN: Retrospective, observational cohort study from May 2001 until September 2003. SETTING: University-affiliated tertiary referral hospital in Seattle, WA. PATIENTS: Four hundred twenty-four consecutive patients with spontaneous intraparenchymal hemorrhage. MEASUREMENTS AND MAIN RESULTS: We retrospectively abstracted information from medical records of intraparenchymal hemorrhage patients admitted to a single hospital. Using multivariate logistic regression of presenting clinical characteristics, but not do-not-attempt-resuscitation status, we generated a prognostic score for favorable outcome (defined as moderate disability or better at discharge). We compared observed probability of favorable outcome with that predicted, stratified by do-not-attempt-resuscitation status. We then generated a modified prognostic score using only non-do-not-attempt-resuscitation patients. Records of 424 patients were reviewed: 44% had favorable outcome, 43% had a do-not-attempt-resuscitation order, and 38% died in hospital. The observed and predicted probability of favorable outcome agreed well with all patients taken together. The observed probability of favorable outcome was significantly higher than predicted in non-do-not-attempt-resuscitation patients and significantly lower in do-not-attempt-resuscitation patients. Results were similar when applying a previously published and validated prognostic score. Our modified prognostic score was no longer pessimistic in non-do-not-attempt-resuscitation patients but remained overly optimistic in do-not-attempt-resuscitation patients. CONCLUSIONS: Although our prognostic model was well-calibrated when assessing all intraparenchymal hemorrhage patients, predictions were significantly pessimistic in patients without and optimistic in those with do-not-attempt-resuscitation orders. Such pessimism may drive decisions not to attempt resuscitation in patients in whom a favorable outcome may have been possible, thereby creating a self-fulfilling prophecy. To be most useful in clinical decision making, intraparenchymal hemorrhage prognostic models should be calibrated to large intraparenchymal hemorrhage cohorts in whom do-not-attempt-resuscitation orders were not used.

Microglia in ischemic brain injury.

Microglia are resident CNS immune cells that are active sensors in healthy brain and versatile effectors under pathological conditions. Cerebral ischemia induces a robust neuroinflammatory response that includes marked changes in the gene-expression profile and phenotype of a variety of endogenous CNS cell types (astrocytes, neurons and microglia), as well as an influx of leukocytic cells (neutrophils, macrophages and T-cells) from the periphery. Many molecules and conditions can trigger a transformation of surveying microglia to microglia of an alerted or reactive state. Here we review recent developments in the literature that relate to microglial activation in the experimental setting of in vitro and in vivo ischemia. We also present new data from our own laboratory demonstrating the direct effects of in vitro ischemic conditions on the microglial phenotype and genomic profile. In particular, we focus on the role of specific molecular signaling systems, such as hypoxia inducible factor-1 and Toll-like receptor-4, in regulating the microglial response in this setting. We then review histological and novel radiological data that confirm a key role for microglial activation in the setting of ischemic stroke in humans. We also discuss recent progress in the pharmacologic and molecular targeting of microglia in acute ischemic stroke. Finally, we explore how recent studies on ischemic preconditioning have increased interest in pre-emptively targeting microglial activation in order to reduce stroke severity.

Regulation of Fcgamma receptors and immunoglobulin G-mediated phagocytosis in mouse microglia.

As resident macrophages in the CNS, microglia can transform from a surveillance state to an activated phenotype in response to brain injury. During this transition microglia become highly capable phagocytic cells. Invading pathogens undergo opsonization with immunoglobulins and microglia recognize these opsonized pathogens through interaction with their cognate F(c) receptors. In mice, both FcgammaRI and FcgammaRIIb receptors are involved in IgG-mediated phagocytosis of opsonzied pathogens. At sites of inflammation, microglial activity is regulated by T-cell derived cytokines. Here we first investigated the effects of IFN-gamma, IL-4, IL-13 and GM-CSF on expression of FcgammaRI and FcgammaRIIb mRNA levels in both primary microglia and microglial cell line N9. Using quantitative real-time PCR we show that IFN-gamma induced a 4-fold increase in the mRNA level of FcgammaRI but did not induce changes in FcgammaRIIb expression. IL-4 and IL-13 induced approximately 2-fold increases in expression of FcgammaRIIb mRNA, but had no effect on FcgammaRI expression. GM-CSF increased both FcgammaRI and FcgammaRIIb mRNA expression. We then characterized the ability of these same cytokines to regulate phagocytosis of immune complexes composed of IgG and the bacteria Staphylococcus aureus. IFN-gamma and GM-CSF both induced approximately 2-fold increases in IgG-mediated phagocytosis whereas IL-4 and IL-13 both decreased IgG-mediated phagocytosis by about one-third. None of the cytokines influenced basal levels of phagocytosis. These findings demonstrate a highly selective cytokine-induced regulation of both phagocytosis-related Fcgamma receptor subtypes and IgG-mediated phagocytosis itself in microglia. This selective regulation has implications for our understanding of the pathophysiology of CNS infection and autoimmune disease.

Prior antiplatelet therapy, platelet infusion therapy, and outcome after intracerebral hemorrhage.

BACKGROUND: Recent studies examining the effect of prior antiplatelet therapy (APT) on outcome in patients with spontaneous intracerebral hemorrhage (ICH) have shown conflicting results. The effect of platelet infusion therapy (PIT) on outcome in patients with ICH taking APT is unknown. METHODS: We reviewed records of patients with ICH admitted to a single hospital, excluding those with international normalized ratio greater than or equal to 1.5. Baseline characteristics were compared by APT status in all patients and by PIT status in the subgroup of patients on APT. We used multivariate analyses to generate propensity and prognostic scores for exposures (APT and PIT) and outcomes (favorable outcome and hospital death), respectively. We examined the associations between exposures and outcomes and adjusted these for propensity and/or prognostic scores. We then validated our findings with a sensitivity analysis. RESULTS: Of 368 patients identified, 121 (31.3%) were taking APT, mostly aspirin. Patients on APT were older and more likely to have vascular comorbidities than those not. The APT group also had a higher initial Glasgow Coma Scale score at presentation. In analyses adjusted for both propensity and prognostic scores, APT was associated with a higher likelihood of hospital death (odds ratio [OR] 2.4; 95% confidence interval [CI] 1.1-5.6); PIT did not prevent hospital death (OR 1.2; 95% CI 0.3-5.5) or increase favorable outcome (OR 1.4; 95% CI 0.4-5.4). CONCLUSIONS: In patients with ICH, APT is associated with an increased risk of hospital death. In the subgroup of patients on APT, PIT did not prevent death or improve outcome.