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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.
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There is a critical need for cerebro-protective interventions to improve the suboptimal outcomes of patients with ischemic stroke who have been treated with reperfusion strategies. We found that nuclear pyruvate kinase muscle 2 (PKM2), a modulator of systemic inflammation, was upregulated in neutrophils after the onset of ischemic stroke in both humans and mice. Therefore, we determined the role of PKM2 in stroke pathogenesis by using murine models with preexisting comorbidities. We generated novel myeloid cell-specific PKM2-/- mice on wild-type (PKM2fl/flLysMCre+) and hyperlipidemic background (PKM2fl/flLysMCre+Apoe-/-). Controls were littermate PKM2fl/flLysMCre- or PKM2fl/flLysMCre-Apoe-/- mice. Genetic deletion of PKM2 in myeloid cells limited inflammatory response in peripheral neutrophils and reduced neutrophil extracellular traps after cerebral ischemia and reperfusion, suggesting that PKM2 promotes neutrophil hyperactivation in the setting of stroke. In the filament and autologous clot and recombinant tissue plasminogen activator stroke models, irrespective of sex, deletion of PKM2 in myeloid cells in either wild-type or hyperlipidemic mice reduced infarcts and enhanced long-term sensorimotor recovery. Laser speckle imaging revealed improved regional cerebral blood flow in myeloid cell-specific PKM2-deficient mice that was concomitant with reduced post-ischemic cerebral thrombo-inflammation (intracerebral fibrinogen, platelet [CD41+] deposition, neutrophil infiltration, and inflammatory cytokines). Mechanistically, PKM2 regulates post-ischemic inflammation in peripheral neutrophils by promoting STAT3 phosphorylation. To enhance the translational significance, we inhibited PKM2 nuclear translocation using a small molecule and found significantly reduced neutrophil hyperactivation and improved short-term and long-term functional outcomes after stroke. Collectively, these findings identify PKM2 as a novel therapeutic target to improve brain salvage and recovery after reperfusion.
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Trombosis Intracraneal/enzimología , Accidente Cerebrovascular Isquémico/enzimología , Activación Neutrófila , Neutrófilos/enzimología , Piruvato Quinasa/metabolismo , Animales , Femenino , Inflamación/enzimología , Inflamación/genética , Trombosis Intracraneal/genética , Accidente Cerebrovascular Isquémico/genética , Masculino , Ratones , Ratones Noqueados para ApoE , Piruvato Quinasa/genéticaRESUMEN
BACKGROUND: Obesity-induced hyperglycemia is a significant risk factor for stroke. Integrin α9ß1 is expressed on neutrophils and stabilizes adhesion to the endothelium via ligands, including Fn-EDA (fibronectin containing extra domain A) and tenascin C. Although myeloid deletion of α9 reduces susceptibility to ischemic stroke, it is unclear whether this is mediated by neutrophil-derived α9. We determined the role of neutrophil-specific α9 in stroke outcomes in a mice model with obesity-induced hyperglycemia. METHODS: α9Neu-KO (α9fl/flMRP8Cre+) and littermate control α9WT (α9fl/flMRP8Cre-) mice were fed on a 60% high-fat diet for 20 weeks to induce obesity-induced hyperglycemia. Functional outcomes were evaluated up to 28 days after stroke onset in mice of both sexes using a transient (30 minutes) middle cerebral artery ischemia. Infarct volume (magnetic resonance imaging) and postreperfusion thrombo-inflammation (thrombi, fibrin, neutrophil, phospho-nuclear factor kappa B [p-NFκB], TNF [tumor necrosis factor]-α, and IL [interleukin]-1ß levels, markers of neutrophil extracellular traps) were measured post 6 or 48 hours of reperfusion. In addition, functional outcomes (modified Neurological Severity Score, rota-rod, corner, and wire-hanging test) were measured for up to 4 weeks. RESULTS: Stroke upregulated neutrophil α9 expression more in obese mice (P<0.05 versus lean mice). Irrespective of sex, deletion of neutrophil α9 improved functional outcomes up to 4 weeks, concomitant with reduced infarct, improved cerebral blood flow, decreased postreperfusion thrombo-inflammation, and neutrophil extracellular traps formation (NETosis) (P<0.05 versus α9WT obese mice). Obese α9Neu-KO mice were less susceptible to thrombosis in FeCl3 injury-induced carotid thrombosis model. Mechanistically, we found that α9/cellular fibronectin axis contributes to NETosis via ERK (extracellular signal-regulated kinase) and PAD4 (peptidyl arginine deiminase 4), and neutrophil α9 worsens stroke outcomes via cellular fibronectin-EDA but not tenascin C. Obese wild-type mice infused with anti-integrin α9 exhibited improved functional outcomes up to 4 weeks (P<0.05 versus vehicle). CONCLUSIONS: Genetic ablation of neutrophil-specific α9 or pharmacological inhibition improves long-term functional outcomes after stroke in mice with obesity-induced hyperglycemia, most likely by limiting thrombo-inflammation.
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Accidente Cerebrovascular , Trombosis , Masculino , Femenino , Ratones , Animales , Neutrófilos/patología , Fibronectinas , Ratones Obesos , Ratones Noqueados , Accidente Cerebrovascular/patología , Trombosis/patología , Inflamación/patología , FN-kappa B , Infarto , Obesidad/complicaciones , Obesidad/metabolismo , Ratones Endogámicos C57BLRESUMEN
Cerebral ischemia and reperfusion initiate cellular events in brain that lead to neurological disability. Investigating these cellular events provides ample targets for developing new treatments. Despite considerable work, no such therapy has translated into successful stroke treatment. Among other issues-such as incomplete mechanistic knowledge and faulty clinical trial design-a key contributor to prior translational failures may be insufficient scientific rigor during preclinical assessment: nonblinded outcome assessment; missing randomization; inappropriate sample sizes; and preclinical assessments in young male animals that ignore relevant biological variables, such as age, sex, and relevant comorbid diseases. Promising results are rarely replicated in multiple laboratories. We sought to address some of these issues with rigorous assessment of candidate treatments across 6 independent research laboratories. The Stroke Preclinical Assessment Network (SPAN) implements state-of-the-art experimental design to test the hypothesis that rigorous preclinical assessment can successfully reduce or eliminate common sources of bias in choosing treatments for evaluation in clinical studies. SPAN is a randomized, placebo-controlled, blinded, multilaboratory trial using a multi-arm multi-stage protocol to select one or more putative stroke treatments with an implied high likelihood of success in human clinical stroke trials. The first stage of SPAN implemented procedural standardization and experimental rigor. All participating research laboratories performed middle cerebral artery occlusion surgery adhering to a common protocol and rapidly enrolled 913 mice in the first of 4 planned stages with excellent protocol adherence, remarkable data completion and low rates of subject loss. SPAN stage 1 successfully implemented treatment masking, randomization, prerandomization inclusion/exclusion criteria, and blinded assessment to exclude bias. Our data suggest that a large, multilaboratory, preclinical assessment effort to reduce known sources of bias is feasible and practical. Subsequent SPAN stages will evaluate candidate treatments for potential success in future stroke clinical trials using aged animals and animals with comorbid conditions.
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Isquemia Encefálica , Accidente Cerebrovascular , Anciano , Animales , Encéfalo , Isquemia Encefálica/terapia , Estudios de Factibilidad , Humanos , Infarto de la Arteria Cerebral Media/terapia , Masculino , Ratones , Accidente Cerebrovascular/terapiaRESUMEN
ABSTRACT: Neutrophils contribute to deep vein thrombosis (DVT) by releasing prothrombotic neutrophil extracellular traps (NETs). NET formation (known as NETosis) is an energy-intensive process that requires an increased rate of aerobic glycolysis. The metabolic enzymes pyruvate dehydrogenase kinases (PDKs) inhibit the pyruvate dehydrogenase complex to divert the pyruvate flux from oxidative phosphorylation toward aerobic glycolysis. Herein, we identified that the combined deletion of PDK2 and PDK4 (PDK2/4-/-) renders mice less susceptible to DVT (measured by thrombus incidence, weight, and length) in the inferior vena cava-stenosis model at day 2 after surgery. Compared with wild-type (WT) mice, the venous thrombus obtained from PDK2/4-/- mice exhibited reduced citrullinated histone content, a known marker of NETs. In line with in vivo observations, phorbol 12-myristate 13-acetate (PMA)-stimulated PDK2/4-/- neutrophils displayed reduced NETosis and secretion of cathepsin G and elastase compared with PMA-stimulated WT neutrophils. The formation of platelet aggregates mediated by PMA-stimulated PDK2/4-/- neutrophils were significantly reduced compared with PMA-stimulated WT neutrophils. Finally, PDK2/4-/- neutrophils exhibited reduced levels of intracellular Ca2+ concentration, extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation, and glycolytic proton efflux rate (a measure of aerobic glycolysis), known to facilitate NETosis. Together, these findings elucidate, to our knowledge, for the first time, the fundamental role of PDK2/4 in regulating NETosis and acute DVT.
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Neutrófilos , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Trombosis de la Vena , Animales , Trombosis de la Vena/etiología , Trombosis de la Vena/metabolismo , Ratones , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Neutrófilos/metabolismo , Ratones Noqueados , Modelos Animales de Enfermedad , Trampas Extracelulares/metabolismo , Susceptibilidad a Enfermedades , Eliminación de GenRESUMEN
Resting platelets rely on oxidative phosphorylation (OXPHOS) and aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) for their energy requirements. In contrast, platelet activation exhibits an increased rate of aerobic glycolysis relative to OXPHOS. Mitochondrial enzymes pyruvate dehydrogenase kinases (PDKs) phosphorylate the pyruvate dehydrogenase (PDH) complex to inhibit its activity, thereby diverting the pyruvate flux from OXPHOS to aerobic glycolysis upon platelet activation. Of 4 PDK isoforms, PDK2 and PDK4 (PDK2/4) are predominantly associated with metabolic diseases. Herein, we report that the combined deletion of PDK2/4 inhibits agonist-induced platelet functions, including aggregation, integrin αIIbß3 activation, degranulation, spreading, and clot retraction. In addition, collagen-mediated PLCγ2 phosphorylation and calcium mobilization were significantly reduced in PDK2/4-/- platelets, suggesting impaired GPVI signaling. The PDK2/4-/- mice were less susceptible to FeCl3-induced carotid and laser-induced mesenteric artery thrombosis without any effect on hemostasis. In adoptive transfer experiments, thrombocytopenic hIL-4Rα/GPIbα-transgenic mice transfused with PDK2/4-/- platelets exhibited less susceptibility to FeCl3 injury-induced carotid thrombosis compared with hIL-4Rα/GPIbα-Tg mice transfused with WT platelets, suggesting a platelet-specific role of PDK2/4 in thrombosis. Mechanistically, the inhibitory effects of PDK2/4 deletion on platelet function were associated with reduced PDH phosphorylation and glycoPER in activated platelets, suggesting that PDK2/4 regulates aerobic glycolysis. Finally, using PDK2 or PDK4 single KO mice, we identified that PDK4 plays a more prominent role in regulating platelet secretion and thrombosis compared with PDK2. This study identifies the fundamental role of PDK2/4 in regulating platelet functions and identifies the PDK/PDH axis as a potentially novel antithrombotic target.
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Proteínas Serina-Treonina Quinasas , Trombosis , Ratones , Animales , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Ratones Noqueados , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Hemostasis , Trombosis/etiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Piruvatos , Glucólisis , OxidorreductasasRESUMEN
BACKGROUND: Easy bruising is included as a major or minor criterion for the classification of multiple types of Ehlers-Danlos syndrome (EDS). Despite a longstanding recognition of the association between EDS and bleeding, we still lack a definitive understanding of the frequency, severity, and types of bleeding complications in patients with EDS. OBJECTIVES: To evaluate hemorrhagic symptoms using the International Society of Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT) in a cohort of patients with defined types of EDS. METHODS: We utilized the ISTH-BAT to characterize hemorrhagic symptoms and their severity in a cohort of 52 patients with classical, classical-like, hypermobile, or vascular EDS and a matched group of 52 healthy control subjects. RESULTS: The mean ISTH-BAT score was 0.1 for healthy subjects and 9.1 for patients with EDS (p < .0001). An abnormal ISTH-BAT score was observed in 32 of 52 (62%) patients with EDS and 0 of 52 healthy controls (p < .0001). The most frequent bleeding symptoms were bruising, muscle hematomas, menorrhagia, epistaxis, bleeding from the oral cavity, and bleeding after tooth extraction. Menorrhagia that was life-threatening or required surgery was reported in 7 of 52 (14%) patients with EDS. CONCLUSION: Patients with multiple types of EDS exhibit a wide range of bleeding symptoms ranging from mild to life-threatening episodes.
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Síndrome de Ehlers-Danlos , Trastornos Hemorrágicos , Menorragia , Femenino , Humanos , Hemostasis , Síndrome de Ehlers-Danlos/complicaciones , Síndrome de Ehlers-Danlos/diagnóstico , EpistaxisRESUMEN
BACKGROUND: Mitochondrial calcium uniporter b (MCUb) is a negative regulator of the mitochondrial calcium uniporter (MCU) and is known to limit mitochondrial calcium ion (Ca2+) uptake. The role of MCUb in platelet function remains unclear. OBJECTIVES: Utilizing MCUb-/- mice, we examined the role of MCUb in regulating platelet function and thrombosis. METHODS: Platelet activation was evaluated in agonist-induced standardized in vitro assays. Susceptibility to arterial thrombosis was evaluated in FeCl3 injury-induced carotid artery and laser injury-induced mesenteric artery thrombosis models. The glycolytic proton efflux rate and oxygen consumption rate were measured to evaluate aerobic glycolysis. RESULTS: Upon stimulation, MCUb-/- platelets exhibited reduced cytoplasmic Ca2+ responses concomitant with increased mitochondrial Ca2+ uptake. MCUb-/- platelets displayed reduced agonist-induced platelet aggregation and spreading on fibrinogen and decreased α and dense-granule secretion and clot retraction. MCUb-/- mice were less susceptible to arterial thrombosis in FeCl3 injury-induced carotid and laser injury-induced mesenteric thrombosis models with unaltered tail bleeding time. In adoptive transfer experiments, thrombocytopenic hIL-4Rα/GPIbα-transgenic mice transfused with MCUb-/- platelets were less susceptible to FeCl3 injury-induced carotid thrombosis compared with hIL-4Rα/GPIbα-Tg mice transfused with wild type platelets, suggesting a platelet-specific role of MCUb in thrombosis. MCUb-/- stimulated platelets exhibited reduced glucose uptake, decreased glycolytic rate, and lowered pyruvate dehydrogenase phosphorylation, suggesting that mitochondrial Ca2+ mediates bioenergetic changes in platelets. CONCLUSION: Our findings suggest that mitochondrial Ca2+ signaling and glucose oxidation are functionally linked in activated platelets and reveal a novel role of MCUb in platelet activation and arterial thrombosis.
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Hemostasis , Trombosis , Ratones , Animales , Agregación Plaquetaria , Plaquetas , Activación Plaquetaria , Ratones Transgénicos , Ratones Noqueados , CalcioRESUMEN
Human diseases may be modeled in animals to allow preclinical assessment of putative new clinical interventions. Recent, highly publicized failures of large clinical trials called into question the rigor, design, and value of preclinical assessment. We established the Stroke Preclinical Assessment Network (SPAN) to design and implement a randomized, controlled, blinded, multi-laboratory trial for the rigorous assessment of candidate stroke treatments combined with intravascular thrombectomy. Efficacy and futility boundaries in a multi-arm multi-stage statistical design aimed to exclude from further study highly effective or futile interventions after each of four sequential stages. Six independent research laboratories performed a standard focal cerebral ischemic insult in five animal models that included equal numbers of males and females: young mice, young rats, aging mice, mice with diet-induced obesity, and spontaneously hypertensive rats. The laboratories adhered to a common protocol and efficiently enrolled 2615 animals with full data completion and comprehensive animal tracking. SPAN successfully implemented treatment masking, randomization, prerandomization inclusion and exclusion criteria, and blinded assessment of outcomes. The SPAN design and infrastructure provide an effective approach that could be used in similar preclinical, multi-laboratory studies in other disease areas and should help improve reproducibility in translational science.