RESUMEN
BACKGROUND: The SPAN trial (Stroke Preclinical Assessment Network) is the largest preclinical study testing acute stroke interventions in experimental focal cerebral ischemia using endovascular filament middle cerebral artery occlusion (MCAo). Besides testing interventions against controls, the prospective design captured numerous biological and procedural variables, highlighting the enormous heterogeneity introduced by the multicenter structure that might influence stroke outcomes. Here, we leveraged the unprecedented sample size achieved by the SPAN trial and the prospective design to identify the biological and procedural variables that affect experimental stroke outcomes in transient endovascular filament MCAo. METHODS: The study cohort included all mice enrolled and randomized in the SPAN trial (N=1789). Mice were subjected to 60-minute MCAo and followed for a month. Thirteen biological and procedural independent variables and 4 functional (weight loss and 4-point neuroscore on days 1 and 2, corner test on days 7 and 28, and mortality) and 3 tissue (day 2, magnetic resonance imaging infarct volumes and swelling; day 30, magnetic resonance imaging tissue loss) outcome variables were prospectively captured. Multivariable regression with stepwise elimination was used to identify the predictors and their effect sizes. RESULTS: Older age, active circadian stage at MCAo, and thinner and longer filament silicone tips predicted higher mortality. Older age, larger body weight, longer anesthesia duration, and longer filament tips predicted worse neuroscores, while high-fat diet and blood flow monitoring predicted milder neuroscores. Older age and a high-fat diet predicted worse corner test performance. While shorter filament tips predicted more ipsiversive turning, longer filament tips appeared to predict contraversive turning. Age, sex, and weight interacted when predicting the infarct volume. Older age was associated with smaller infarcts on day 2 magnetic resonance imaging, especially in animals with larger body weights; this association was most conspicuous in females. High-fat diet also predicted smaller infarcts. In contrast, the use of cerebral blood flow monitoring and more severe cerebral blood flow drop during MCAo, longer anesthesia, and longer filament tips all predicted larger infarcts. Bivariate analyses among the dependent variables highlighted a disconnect between tissue and functional outcomes. CONCLUSIONS: Our analyses identified variables affecting endovascular filament MCAo outcome, an experimental stroke model used worldwide. Multiple regression refuted some commonly reported predictors and revealed previously unrecognized associations. Given the multicenter prospective design that represents a sampling of real-world conditions, the degree of heterogeneity mimicking clinical trials, the large number of predictors adjusted for in the multivariable model, and the large sample size, we think this is the most definitive analysis of the predictors of preclinical stroke outcome to date. Future multicenter experimental stroke trials should standardize or at least ensure a balanced representation of the biological and procedural variables identified herein as potential confounders.
RESUMEN
The Stroke Preclinical Assessment Network (SPAN) is a multicenter preclinical trial platform using rodent models of transient focal cerebral ischemia to address translational failure in experimental stroke. In addition to centralized randomization and blinding and large samples, SPAN aimed to introduce heterogeneity to simulate the heterogeneity embodied in clinical trials for robust conclusions. Here, we report the heterogeneity introduced by allowing the 6 SPAN laboratories to vary most of the biological and experimental model variables and the impact of this heterogeneity on middle cerebral artery occlusion (MCAo) performance. We included the modified intention-to-treat population of the control mouse cohort of the first SPAN trial (n=421) and examined the biological and procedural independent variables and their covariance. We then determined their impact on the dependent variables cerebral blood flow drop during MCAo, time to achieve MCAo, and total anesthesia duration using multivariable analyses. We found heterogeneity in biological and procedural independent variables introduced mainly by the site. Consequently, all dependent variables also showed heterogeneity among the sites. Multivariable analyses with the site as a random effect variable revealed filament choice as an independent predictor of cerebral blood flow drop after MCAo. Comorbidity, sex, use of laser Doppler flow to monitor cerebral blood flow, days after trial onset, and maintaining anesthesia throughout the MCAo emerged as independent predictors of time to MCAo. Total anesthesia duration was predicted by most independent variables. We present with high granularity the heterogeneity introduced by the biological and model selections by the testing sites in the first trial of cerebroprotection in rodent transient filament MCAo by SPAN. Rather than trying to homogenize all variables across all sites, we embraced the heterogeneity to better approximate clinical trials. Awareness of the heterogeneity, its sources, and how it impacts the study performance may further improve the study design and statistical modeling for future multicenter preclinical trials.
Asunto(s)
Ataque Isquémico Transitorio , Accidente Cerebrovascular , Ratones , Animales , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media , Proyectos de Investigación , Circulación Cerebrovascular/fisiología , Estudios Multicéntricos como AsuntoRESUMEN
Thrombolytic stroke therapy with tissue plasminogen activator (tPA) is limited by risks of hemorrhagic transformation (HT). We have reported that a new 12/15-lipoxygenase (12/15-LOX) inhibitor ML351 reduced tPA related HT in mice subjected to experimental stroke under anticoagulation. In this study, we asked whether ML351 can ameliorate tPA induced HT in an embolic stroke model. Rats were subjected to embolic middle cerebral artery occlusion with 2 or 3 hr ischemia and tPA infusion, with or without ML351. Regional cerebral blood flow was monitored 2 hr after ischemia and continuously monitored for 1 hr after treatment for determining reperfusion. Hemoglobin was determined in brain homogenates and infarct volume was quantified at 24 hr after stroke.12/15-LOX, cluster of differentiation 68(CD68), immunoglobulin G (IgG), and tight junction proteins expression was detected by immunohistochemistry. ML351 significantly reduced tPA related hemorrhage after stroke without affecting its thrombolytic efficacy. ML351 also reduced blood-brain barrier disruption and improved preservation of junction proteins. ML351 and tPA combination improved neurological deficit of rats even though ML351 did not further reduce the infarct volume compared to tPA alone treated animals. Pro-inflammatory cytokines were suppressed by ML351 both in vivo and in vitro experiments. We further showed that ML351 suppressed the expression of c-Jun-N-terminal kinase (JNK) in brains and microglia cultures, whereas exogenous 12-HETE attenuated this effect in vitro. In conclusion, ML351 and tPA combination therapy is beneficial in ameliorating HT after ischemic stroke. This protective effect is probably because of 12/15-LOX inhibition and suppression of JNK-mediated microglia/macrophage activation.
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Embolia Intracraneal/tratamiento farmacológico , Accidente Cerebrovascular Isquémico/tratamiento farmacológico , Isoxazoles/uso terapéutico , Inhibidores de la Lipooxigenasa/uso terapéutico , Naftalenos/uso terapéutico , Activador de Tejido Plasminógeno/uso terapéutico , Animales , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Infarto Encefálico/patología , Circulación Cerebrovascular , Citocinas/antagonistas & inhibidores , Quimioterapia Combinada , Glucosa/deficiencia , Hipoxia Encefálica/metabolismo , Embolia Intracraneal/complicaciones , Accidente Cerebrovascular Isquémico/etiología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Microglía/efectos de los fármacos , Microglía/metabolismo , Ratas , Ratas Sprague-Dawley , ReperfusiónRESUMEN
Investigators acknowledge the limitations of rodent or non-human primate stroke models, hundreds of putative neuroprotectants have been evaluated in preclinical models, but not one has entered the clinical realm. Initial studies focused on the neuron, but in recent years the focus has widened to also include other neural cells including astrocytes, pericytes and endothelial cells, which together form the neurovascular unit. Some new developments raise renewed hope for neuroprotection: the appearance of new compounds with multiple mechanisms of action, or the promulgation of new standards for a rigorous preclinical testing. At the bedside in the last 5 years, uric acid and nerinetide are the only compounds tested for clinical efficacy in randomised controlled trials (RCTs), where all patients had to receive reperfusion therapies, either intravenous thrombolysis and/or mechanical thrombectomy. In addition, otaplimastat, 3K3A-activated protein C (APC), intra-arterial verapamil and intra-arterial hypothermia were also assessed in combination with reperfusion therapy, but in RCTs that only included feasibility or safety outcomes. Some of these compounds yielded promising results which are discussed in this review. Altogether, a deeper knowledge of the mechanisms involved in the ischaemic death process at the neurovascular unit, an improved preselection and evaluation of drugs at the preclinical stage and the testing of putative neuroprotectants in enriched clinical studies of patients receiving reperfusion therapies, might prove more effective than in the past to reverse a dismal situation that has lasted already too long.
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Neuroprotección , Accidente Cerebrovascular/terapia , Humanos , Fármacos Neuroprotectores/uso terapéutico , Reperfusión/métodos , Accidente Cerebrovascular/complicaciones , Investigación Biomédica TraslacionalRESUMEN
Background and Purpose- Subarachnoid hemorrhage (SAH) is a devastating form of stroke. Oxidative stress contributes to brain injury, but the mechanisms have been poorly studied. Here, we evaluated the role of 12/15-lipoxygenase (12/15-LOX), an enzyme known to cause cell death in ischemic stroke, on brain injury in a mouse model of SAH. Methods- C57Bl6 wild-type mice and Alox15 knockout mice were subjected to SAH using a direct blood injection technique. In SAH wild-type mice, half received the 12/15-LOX inhibitor ML351 and half received vehicle. Immunohistochemistry, brain edema, blood-brain barrier leakage and functional outcomes were assessed 1 and 3 days after SAH induction. Results- SAH led to increased 12/15-LOX in macrophages of the brain parenchyma, adjacent to the subarachnoid blood. Neuronal cell death after SAH was reduced by ML351 and in Alox15 knockout mice. Similarly, SAH induced brain edema, which was 12/15-LOX dependent. Finally, Alox15 gene knockout and inhibitor treatment in wild-type mice with SAH led to an improved behavioral outcome. Conclusions- 12/15-LOX is overexpressed in macrophages after SAH in mice, and inhibition of the 12/15-LOX pathway decreases brain injury and improves neurological outcome. This study suggests 12/15-LOX as a novel therapeutic target to limit brain injury after SAH.
Asunto(s)
Araquidonato 12-Lipooxigenasa , Araquidonato 15-Lipooxigenasa , Lesiones Encefálicas , Isoxazoles/farmacología , Inhibidores de la Lipooxigenasa/farmacología , Macrófagos , Naftalenos/farmacología , Estrés Oxidativo , Hemorragia Subaracnoidea , Animales , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/metabolismo , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/enzimología , Lesiones Encefálicas/genética , Lesiones Encefálicas/patología , Modelos Animales de Enfermedad , Macrófagos/enzimología , Macrófagos/patología , Ratones , Ratones Noqueados , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Hemorragia Subaracnoidea/tratamiento farmacológico , Hemorragia Subaracnoidea/enzimología , Hemorragia Subaracnoidea/genética , Hemorragia Subaracnoidea/patologíaRESUMEN
Background and Purpose- Dual antiplatelet treatment poses a risk for increased hemorrhagic transformation (HT) following intravenous thrombolysis and mechanical thrombectomy. The aim of this study was to implement a model of experimental stroke with tissue-type plasminogen activator (tPA)-associated HT in mice on dual antiplatelet treatment to enable mechanistic studies and also to allow for an initial assessment of therapeutic approaches to limit HT. Methods- Male C57BL6 mice were fed with Aspirin and Clopidogrel via drinking water for 3 days. Subsequently, mice were subjected to 2-hour transient middle cerebral artery occlusion, and tPA was infused when indicated. HT was quantified by measuring hemorrhaged areas in brain sections with ImageJ. TTC staining was used to determine infarct size. Platelet function was tested in vitro using flow cytometry and in vivo with standard tail bleeding tests. Results- Both flow cytometry and tail bleeding volumes indicated significantly reduced platelet function following Aspirin and Clopidogrel treatment. While tPA administered 2 hours after onset of middle cerebral artery occlusion did not cause bleeding in control mice (0.51±0.13 mm2), HT significantly increased by 18.9±5.4 mm2 (P=0.0045) in Aspirin and Clopidogrel mice treated with tPA. HT in aspirin and clopidogrel mice not treated with tPA was nonsignificantly elevated by 8.0±4.6 mm2 (P=0.3784) compared with controls. Infarct sizes did not differ between groups. The HT persisted when the tPA dosage was reduced. Conclusions- We successfully established a translational stroke model of tPA treatment under dual antiplatelet treatment. The impaired platelet function led to an increased risk for HT in tPA-treated mice. Reducing the dosage of tPA did not prevent this hemorrhagic complication.
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Aspirina/uso terapéutico , Clopidogrel/uso terapéutico , Fibrinolíticos/uso terapéutico , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Hemorragias Intracraneales/epidemiología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Activador de Tejido Plasminógeno/uso terapéutico , Animales , Trombosis Coronaria/tratamiento farmacológico , Modelos Animales de Enfermedad , Quimioterapia Combinada , Masculino , Ratones , Pruebas de Función Plaquetaria , Prevención Secundaria , Accidente Cerebrovascular/tratamiento farmacológico , Terapia Trombolítica/métodosRESUMEN
Ischemic strokes are caused by one or more blood clots that typically obstruct one of the major arteries in the brain, but frequently also result in leakage of the blood-brain barrier and subsequent hemorrhage. While it has long been known that the enzyme 12/15-lipoxygenase (12/15-LOX) is up-regulated following ischemic strokes and contributes to neuronal cell death, recent research has shown an additional major role for 12/15-LOX in causing this hemorrhagic transformation. These findings have important implications for the use of 12/15-LOX inhibitors in the treatment of stroke.
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Araquidonato 15-Lipooxigenasa , Isquemia Encefálica , Hemorragia , Accidente Cerebrovascular , Barrera Hematoencefálica/patología , Encéfalo/patología , Isquemia Encefálica/complicaciones , Isquemia Encefálica/fisiopatología , Hemorragia/etiología , Hemorragia/fisiopatología , Humanos , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/fisiopatologíaRESUMEN
BACKGROUND AND PURPOSE: For stroke prevention, patients with atrial fibrillation typically receive oral anticoagulation. The commonly used anticoagulant warfarin increases the risk of hemorrhagic transformation (HT) when a stroke occurs; tissue-type plasminogen activator treatment is therefore restricted in these patients. This study was designed to test the hypothesis that 12/15-lipoxygenase (12/15-LOX) inhibition would reduce HT in warfarin-treated mice subjected to experimental stroke. METHODS: Warfarin was dosed orally in drinking water, and international normalized ratio values were determined using a Coaguchek device. C57BL6J mice or 12/15-LOX knockout mice were subjected to transient middle cerebral artery occlusion with 3 hours severe ischemia (model A) or 2 hours ischemia and tissue-type plasminogen activator infusion (model B), with or without the 12/15-LOX inhibitor ML351. Hemoglobin was determined in brain homogenates, and hemorrhage areas on the brain surface and in brain sections were measured. 12/15-LOX expression was detected by immunohistochemistry. RESULTS: Warfarin treatment resulted in reproducible increased international normalized ratio values and significant HT in both models. 12/15-LOX knockout mice suffered less HT after severe ischemia, and ML351 reduced HT in wild-type mice. When normalized to infarct size, ML351 still independently reduced hemorrhage. HT after tissue-type plasminogen activator was similarly reduced by ML351. CONCLUSIONS: In addition to its benefits in infarct size reduction, 12/15-LOX inhibition also may independently reduce HT in warfarin-treated mice. ML351 should be further evaluated as stroke treatment in anticoagulated patients suffering a stroke, either alone or in conjunction with tissue-type plasminogen activator.
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Anticoagulantes/toxicidad , Araquidonato 12-Lipooxigenasa/deficiencia , Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/deficiencia , Araquidonato 15-Lipooxigenasa/metabolismo , Hemorragia Cerebral/enzimología , Accidente Cerebrovascular/enzimología , Warfarina/toxicidad , Animales , Hemorragia Cerebral/inducido químicamente , Hemorragia Cerebral/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Accidente Cerebrovascular/tratamiento farmacológicoRESUMEN
Lipoxygenases (LOXs) form a heterogeneous class of lipid peroxidizing enzymes, which have been implicated not only in cell proliferation and differentiation but also in the pathogenesis of various diseases with major public health relevance. As other fatty acid dioxygenases LOXs oxidize polyunsaturated fatty acids to their corresponding hydroperoxy derivatives, which are further transformed to bioactive lipid mediators (eicosanoids and related substances). On the other hand, lipoxygenases are key players in the regulation of the cellular redox homeostasis, which is an important element in gene expression regulation. Although the first mammalian lipoxygenases were discovered 40 years ago and although the enzymes have been well characterized with respect to their structural and functional properties the biological roles of the different lipoxygenase isoforms are not completely understood. This review is aimed at summarizing the current knowledge on the physiological roles of different mammalian LOX-isoforms and their patho-physiological function in inflammatory, metabolic, hyperproliferative, neurodegenerative and infectious disorders. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".
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Ácidos Grasos Insaturados/metabolismo , Lipooxigenasas/metabolismo , Animales , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/enzimología , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Enfermedades del Sistema Nervioso Central/enzimología , Homeostasis , Humanos , Inflamación/metabolismo , Isoenzimas , Inhibidores de la Lipooxigenasa/uso terapéutico , Lipooxigenasas/química , Lipooxigenasas/clasificación , Lipooxigenasas/genética , Enfermedades Metabólicas/tratamiento farmacológico , Enfermedades Metabólicas/enzimología , Modelos Moleculares , Oxidación-Reducción , Conformación Proteica , Transducción de SeñalRESUMEN
Human reticulocyte 12/15-lipoxygenase (h12/15-LOX) is a lipid-oxidizing enzyme that can directly oxidize lipid membranes in the absence of a phospholipase, leading to a direct attack on organelles, such as the mitochondria. This cytotoxic activity of h12/15-LOX is up-regulated in neurons and endothelial cells after a stroke and thought to contribute to both neuronal cell death and blood-brain barrier leakage. The discovery of inhibitors that selectively target recombinant h12/15-LOX in vitro, as well as possessing activity against the murine ortholog ex vivo, could potentially support a novel therapeutic strategy for the treatment of stroke. Herein, we report a new family of inhibitors discovered in a High Throughput Screen (HTS) that are selective and potent against recombinant h12/15-LOX and cellular mouse 12/15-LOX (m12/15-LOX). MLS000099089 (compound 99089), the parent molecule, exhibits an IC50 potency of 3.4±0.5 µM against h12/15-LOX in vitro and an ex vivo IC50 potency of approximately 10 µM in a mouse neuronal cell line, HT-22. Compound 99089 displays greater than 30-fold selectivity versus h5-LOX and COX-2, 15-fold versus h15-LOX-2 and 10-fold versus h12-LOX, when tested at 20 µM inhibitor concentration. Steady-state inhibition kinetics reveals that the mode of inhibition of 99089 against h12/15-LOX is that of a mixed inhibitor with a Kic of 1.0±0.08 µM and a Kiu of 6.0±3.3 µM. These data indicate that 99089 and related derivatives may serve as a starting point for the development of anti-stroke therapeutics due to their ability to selectively target h12/15-LOX in vitro and m12/15-LOX ex vivo.
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Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Inhibidores de la Lipooxigenasa/farmacología , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Ensayos Analíticos de Alto Rendimiento , Humanos , Inhibidores de la Lipooxigenasa/química , Ratones , Modelos Moleculares , Estructura Molecular , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
12/15-lipoxygenase (12/15-LOX) is involved in organelle homeostasis by degrading mitochondria in maturing red blood cells and by eliminating excess peroxisomes in liver. Furthermore, 12/15-LOX contributes to diseases by exacerbating oxidative stress-related injury, notably in stroke. Nonetheless, it is unclear what the consequences are of abolishing 12/15-LOX activity. Mice in which the alox15 gene has been ablated do not show an obvious phenotype, and LOX enzyme inhibition is not overtly detrimental. We show here that liver histology is also unremarkable. However, electron microscopy demonstrated that 12/15-LOX knockout surprisingly leads to increased macroautophagy in the liver. Not only macroautophagy but also mitophagy and pexophagy were increased in hepatocytes, which otherwise showed unaltered fine structure and organelle morphology. These findings were substantiated by immunofluorescence showing significantly increased number of LC3 puncta and by Western blotting demonstrating a significant increase for LC3-II protein in both liver and brain homogenates of 12/15-LOX knockout mice. Inhibition of 12/15-LOX activity by treatment with four structurally different inhibitors had similar effects in cultured HepG2 hepatoma cells and SH-SY5Y neuroblastoma cells with significantly increased autophagy discernable already after 2 hours. Hence, our study reveals a link between ablation or inhibition of 12/15-LOX and stimulation of macroautophagy. The enhanced macroautophagy may be related to the known tissue-protective effects of LOX ablation or inhibition under various diseased conditions caused by oxidative stress and ischemia. This could provide an important cleaning mechanism of cells and tissues to prevent accumulation of damaged mitochondria and other cellular components.
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Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/genética , Autofagia/efectos de los fármacos , Autofagia/genética , Eliminación de Gen , Inhibidores de la Lipooxigenasa/farmacología , Animales , Células Hep G2 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Tiempo , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND AND PURPOSE: We explored the hypothesis that injured neurons release lipocalin-2 as a help me signal. METHODS: In vivo lipocalin-2 responses were assessed in rat focal cerebral ischemia and human stroke brain samples using a combination of ELISA and immunostaining. In vitro, microglia and astrocytes were exposed to lipocalin-2, and various markers and assays of glial activation were quantified. Functional relevance of neuron-to-glia lipocalin-2 signaling was examined by transferring conditioned media from lipocalin-2-activated microglia and astrocytes onto neurons to see whether activated glia could protect neurons against oxygen-glucose deprivation and promote neuroplasticity. RESULTS: In human stroke samples and rat cerebral ischemia, neuronal expression of lipocalin-2 was significantly increased. In primary cell cultures, exposing microglia and astrocytes to lipocalin-2 resulted in glial activation. In microglia, lipocalin-2 converted resting ramified shapes into a long-rod morphology with reduced branching, increased interleukin-10 release, and enhanced phagocytosis. In astrocytes, lipocalin-2 upregulated glial fibrillary acid protein, brain-derived neurotropic factor, and thrombospondin-1. Conditioned media from lipocalin-2-treated astrocytes upregulated synaptotagmin, and conditioned media from lipocalin-2-treated microglia upregulated synaptophysin and post-synaptic density 95 (PSD95) and protected neurons against oxygen-glucose deprivation. CONCLUSIONS: These findings provide proof of concept that lipocalin-2 is released by injured neurons as a help me distress signal that activates microglia and astrocytes into potentially prorecovery phenotypes.
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Proteínas de Fase Aguda/metabolismo , Astrocitos/metabolismo , Isquemia Encefálica/metabolismo , Corteza Cerebral/metabolismo , Lipocalinas/metabolismo , Microglía/metabolismo , Neuronas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Accidente Cerebrovascular/metabolismo , Anciano , Animales , Biomarcadores/metabolismo , Corteza Cerebral/patología , Femenino , Humanos , Lipocalina 2 , Masculino , Plasticidad Neuronal/fisiología , Fenotipo , Ratas , Ratas Wistar , Método Simple CiegoRESUMEN
Targeting newly identified damage pathways in the ischemic brain can help to circumvent the currently severe limitations of acute stroke therapy. Here we show that the activity of 12/15-lipoxygenase was increased in the ischemic mouse brain, and 12/15-lipoxygenase colocalized with a marker for oxidized lipids, MDA2. This colocalization was also detected in the brain of 2 human stroke patients, where it also coincided with increased apoptosis-inducing factor. A novel inhibitor of 12/15-lipoxygenase, LOXBlock-1, protected neuronal HT22 cells against oxidative stress. In a mouse model of transient focal ischemia, the inhibitor reduced infarct sizes both 24 hours and 14 days poststroke, with improved behavioral parameters. Even when treatment was delayed until at least 4 hours after onset of ischemia, LOXBlock-1 was protective. Furthermore, it reduced tissue plasminogen activator-associated hemorrhage in a clot model of ischemia/reperfusion. This study establishes inhibition of 12/15-lipoxygenase as a viable strategy for first-line stroke treatment.
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Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Inhibidores de la Lipooxigenasa/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/enzimología , Anciano , Animales , Femenino , Humanos , Inhibidores de la Lipooxigenasa/administración & dosificación , Masculino , Ratones , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Recovery from stroke is limited, in part, by an inhibitory environment in the postischemic brain, but factors preventing successful remodeling are not well known. Using cultured cortical neurons from mice, brain endothelial cells, and a mouse model of ischemic stroke, we show that signaling from the axon guidance molecule Sema3A via eicosanoid second messengers can contribute to this inhibitory environment. Either 90 nM recombinant Sema3A, or the 12/15-lipoxygenase (12/15-LOX) metabolites 12-HETE and 12-HPETE at 300 nM, block axon extension in neurons compared to solvent controls, and decrease tube formation in endothelial cells. The Sema3A effect is reversed by inhibiting 12/15-LOX, and neurons derived from 12/15-LOX-knockout mice are insensitive to Sema3A. Following middle cerebral artery occlusion to induce stroke in mice, immunohistochemistry shows both Sema3A and 12/15-LOX are increased in the cortex up to 2 wk. To determine whether a Sema3A-dependent damage pathway is activated following ischemia, we injected recombinant Sema3A into the striatum. Sema3A alone did not cause injury in normal brains. But when injected into postischemic brains, Sema3A increased cortical damage by 79%, and again, this effect was reversed by 12/15-LOX inhibition. Our findings suggest that blocking the semaphorin pathway should be investigated as a therapeutic strategy to improve stroke recovery.
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Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Encéfalo/metabolismo , Semaforina-3A/metabolismo , Accidente Cerebrovascular/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Animales , Araquidonato 12-Lipooxigenasa/deficiencia , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 15-Lipooxigenasa/deficiencia , Araquidonato 15-Lipooxigenasa/genética , Encéfalo/irrigación sanguínea , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/citología , Células Endoteliales/metabolismo , Inmunohistoquímica , Leucotrienos/metabolismo , Masculino , Ratones , Ratones Noqueados , Neovascularización Fisiológica , Neuronas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas de Mensajero Secundario , Semaforina-3A/antagonistas & inhibidores , Semaforina-3A/genética , Transducción de Señal , Accidente Cerebrovascular/patologíaRESUMEN
Physiological levels of H(2)S exert neuroprotective effects, whereas high concentrations of H(2)S may cause neurotoxicity in part via activation of NMDAR. To characterize the neuroprotective effects of combination of exogenous H(2)S and NMDAR antagonism, we synthesized a novel H(2)S-releasing NMDAR antagonist N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzamide (S-memantine) and examined its effects in vitro and in vivo. S-memantine was synthesized by chemically combining a slow releasing H(2)S donor 4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid (ACS48) with a NMDAR antagonist memantine. S-memantine increased intracellular sulfide levels in human neuroblastoma cells (SH-SY5Y) 10-fold as high as that was achieved by ACS48. Incubation with S-memantine after reoxygenation following oxygen and glucose deprivation (OGD) protected SH-SY5Y cells and murine primary cortical neurons more markedly than did ACS48 or memantine. Glutamate-induced intracellular calcium accumulation in primary cortical neurons were aggravated by sodium sulfide (Na(2)S) or ACS48, but suppressed by memantine and S-memantine. S-memantine prevented glutamate-induced glutathione depletion in SH-SY5Y cells more markedly than did Na(2)S or ACS48. Administration of S-memantine after global cerebral ischemia and reperfusion more robustly decreased cerebral infarct volume and improved survival and neurological function of mice than did ACS48 or memantine. These results suggest that an H(2)S-releasing NMDAR antagonist derivative S-memantine prevents ischemic neuronal death, providing a novel therapeutic strategy for ischemic brain injury.
Asunto(s)
Lesiones Encefálicas/prevención & control , Isquemia Encefálica/tratamiento farmacológico , Sulfuro de Hidrógeno/química , Neuronas/patología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Isquemia Encefálica/patología , Muerte Celular , Línea Celular Tumoral , Células Cultivadas , Diseño de Fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Glucosa/metabolismo , Glutatión/química , Humanos , Isquemia , Masculino , Memantina/farmacología , Ratones , Ratones Endogámicos C57BL , Modelos Químicos , Oxígeno/química , Daño por ReperfusiónRESUMEN
Oxidative stress involving premyelinating oligodendrocytes (OLs) is a major factor in the pathogenesis of preterm white matter injury. In animal and cell culture studies, activation of the lipid-oxidizing enzyme 12/15-lipoxygenase (12/15-LOX) plays a central role as an inflammatory mediator in the pathology of oxidative stress and OL cell death, as well as ischemia and neuronal death. The role of 12/15-LOX, however, is unclear in the developing human brain. The mechanism of 12/15-LOX involves the production of reactive oxygen species through the metabolism of arachidonic acid, as well as direct detrimental effects on organelle membranes. Here we tested the hypothesis that the density of 12/15-LOX-expressing cells is increased in periventricular leukomalacia (PVL). Using immunocytochemistry (ICC) in human paraffin-embedded tissue, 12/15-LOX expression was seen in macrophages of the focally necrotic lesions in the periventricular white matter, as well as in glial cells throughout the surrounding white matter with reactive gliosis. Interestingly, no significant 12/15-LOX expression was detected in neurons in the cerebral cortex overlying the damaged white matter. Using a scoring system from 0 to 3, we assessed the density of 12/15-LOX-expressing cells in diffusely gliotic white matter from 20 to 43 postconceptional (PC) weeks in 19 PVL cases (median = 36 PC weeks) and 10 control (non-PVL) cases (median = 34 PC weeks). The density of 12/15-LOX-positive cells was significantly increased in the diffuse component of PVL (score = 1.17 ± 0.15) compared to controls (score = 0.48 ± 0.21; p = 0.014). Using double-label ICC, 12/15-LOX was observed in PVL in OLs of the O4 and O1 premyelinating stages, as well as in mature OLs as determined with the mature OL marker adenomatous polyposis coli (APC). In addition, 12/15-LOX expression was present in a population of CD68-positive activated microglia. There was no 12/15-LOX expression in reactive astrocytes. Finally we observed terminal deoxynucleotide transferase dUTP nick end-labeling-positive cells within the white matter of PVL that expressed 12/15-LOX and/or within close proximity of 12/15-LOX-positive cells. Our data support a role for 12/15-LOX activation as an inflammatory mediator of injury in PVL, with a contribution of 12/15-LOX to PVL-induced damage to or cell death of OLs, including those at the O1 and O4 stages.
Asunto(s)
Araquidonato 12-Lipooxigenasa/biosíntesis , Araquidonato 15-Lipooxigenasa/biosíntesis , Leucomalacia Periventricular/enzimología , Microglía/enzimología , Oligodendroglía/enzimología , Araquidonato 12-Lipooxigenasa/análisis , Araquidonato 15-Lipooxigenasa/análisis , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Recién Nacido , Leucomalacia Periventricular/patologíaRESUMEN
Introduction: Lipoxygenases (LOXs) have essential roles in stroke, atherosclerosis, diabetes, and hypertension. 12/15-LOX inhibition was shown to reduce infarct size and brain edema in the acute phase of experimental stroke. However, the significance of 12/15-LOX on neuroinflammation, which has an essential role in the pathophysiology of stroke, has not been clarified yet. Methods: In this study, ischemia/recanalization (I/R) was performed by occluding the proximal middle cerebral artery (pMCAo) in mice. Either the 12/15-LOX inhibitor (ML351, 50 mg/kg) or its solvent (DMSO) was injected i.p. at recanalization after 1 h of occlusion. Mice were sacrificed at 6, 24, and 72-h after ischemia induction. Infarct volumes were calculated on Nissl-stained sections. Neurological deficit scoring was used for functional analysis. Lipid peroxidation was determined by the MDA assay, and the inflammatory cytokines IL-6, TNF-alpha, IL-1beta, IL-10, and TGF-beta were quantified by ELISA. The inflammasome proteins NLRP1 and NLRP3, 12/15-LOX, and caspase-1 were detected with immunofluorescence staining. Results: Infarct volumes, neurological deficit scores, and lipid peroxidation were significantly attenuated in ML351-treated groups at 6, 24, and 72-h. ELISA results revealed that the pro-inflammatory cytokines IL-1beta, IL-6, and TNF-alpha were significantly decreased at 6-h and/or 24-h of I/R, while the anti-inflammatory cytokines IL-10 and TNF-alpha were increased at 24-h or 72-h of ML351 treatment. NLRP1 and NLRP3 immunosignaling were enhanced at three time points after I/R, which were significantly diminished by the ML351 application. Interestingly, NLRP3 immunoreactivity was more pronounced than NLRP1. Hence, we proceeded to study the co-localization of NLRP3 immunoreactivity with 12/15-LOX and caspase-1, which indicated that NLRP3 was co-localized with 12/15-LOX and caspase-1 signaling. Additionally, NLRP3 was found in neurons at all time points but in non-neuronal cells 72 h after I/R. Discussion: These results suggest that 12/15-LOX inhibition suppresses ischemia-induced inflammation in the acute and subacute phases of stroke via suppressing inflammasome activation. Understanding the mechanisms underlying lipid peroxidation and its associated pathways, like inflammasome activation, may have broader implications for the treatment of stroke and other neurological diseases characterized by neuroinflammation.
RESUMEN
The concept of the neurovascular unit emphasizes the importance of cell-cell signaling between neural, glial, and vascular compartments. In neurogenesis, for example, brain endothelial cells play a key role by supplying trophic support to neural progenitors. Here, we describe a surprising phenomenon where brain endothelial cells may release trans-differentiation signals that convert astrocytes into neural progenitor cells in male mice after stroke. After oxygen-glucose deprivation, brain endothelial cells release microvesicles containing pro-neural factor Ascl1 that enter into astrocytes to induce their trans-differentiation into neural progenitors. In mouse models of focal cerebral ischemia, Ascl1 is upregulated in endothelium prior to astrocytic conversion into neural progenitor cells. Injecting brain endothelial-derived microvesicles amplifies the process of astrocyte trans-differentiation. Endothelial-specific overexpression of Ascl1 increases the local conversion of astrocytes into neural progenitors and improves behavioral recovery. Our findings describe an unexpected vascular-regulated mechanism of neuroplasticity that may open up therapeutic opportunities for improving outcomes after stroke.
Asunto(s)
Células-Madre Neurales , Accidente Cerebrovascular , Masculino , Ratones , Animales , Astrocitos , Células Endoteliales , Células Cultivadas , Transdiferenciación CelularRESUMEN
Oxidative stress is a pathway of injury that is common to almost all neurological conditions. Hence, methods to scavenge radicals have been extensively tested for neuroprotection. However, saving neurons alone may not be sufficient in treating CNS disease. In this study, we tested the cytoprotective actions of the glutathione precursor gamma-glutamylcysteine ethyl ester (GCEE) in brain endothelium. First, oxidative stress was induced in a human brain microvascular endothelial cell line by exposure to H(2)O(2). Addition of GCEE significantly reduced formation of reactive oxygen species, restored glutathione levels which were reduced in the presence of H(2)O(2), and decreased cell death during H(2)O(2)-mediated injury. Next, we asked whether GCEE can also protect brain endothelial cells against oxygen-glucose deprivation (OGD). As expected, OGD disrupted mitochondrial membrane potentials. GCEE was able to ameliorate these mitochondrial effects. Concomitantly, GCEE significantly decreased endothelial cell death after OGD. Lastly, our in vivo experiments using a mouse model of brain trauma show that post-trauma (10 min after controlled cortical impact) administration of GCEE by intraperitoneal injection results in a decrease in acute blood-brain barrier permeability. These data suggest that the beneficial effects of GCEE on brain endothelial cells and microvessels may contribute to its potential efficacy as a neuroprotective agent in traumatic brain injury.