RESUMO
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.
Assuntos
Trombose Intracraniana/enzimologia , AVC Isquêmico/enzimologia , Ativação de Neutrófilo , Neutrófilos/enzimologia , Piruvato Quinase/metabolismo , Animais , Feminino , Inflamação/enzimologia , Inflamação/genética , Trombose Intracraniana/genética , AVC Isquêmico/genética , Masculino , Camundongos , Camundongos Knockout para ApoE , Piruvato Quinase/genéticaRESUMO
BACKGROUND: Elucidating mechanisms of brain damage in cerebral venous thrombosis (CVT) would be instrumental to develop targeted therapies and improve prognosis prediction. Matrix metalloproteinase-9 (MMP-9), a gelatinase that degrades major components of the basal lamina, has been associated to blood-brain barrier disruption. We aimed to assess, in patients with CVT, the temporal change in serum concentrations of MMP-9 and its association with key imaging and clinical outcomes. METHODS: Pathophysiology of Venous Infarction-PRediction of InfarctiOn and RecanalIzaTion in CVT (PRIORITy-CVT) was a multicenter prospective cohort study of patients with newly diagnosed CVT. Serial collection of peripheral blood samples performed on day 1, 3, and 8, and standardized magnetic resonance imaging on day 1, 8, and 90. MMP-9 was quantified using enzyme-linked immunosorbent assay in 59 patients and 22 healthy controls. Primary outcomes were parenchymal brain lesion, early evolution of brain lesion, early recanalization, and functional outcome on day 90. RESULTS: CVT patients with parenchymal brain lesion had higher baseline concentrations of MMP-9 compared with controls (adjusted p = 0.001). The area under receiver operating characteristic curve value for MMP-9 for predicting brain lesion was 0.71 (95% confidence interval [CI]: 0.57-0.85, p = 0.009). Patients with venous recanalization showed early decline of circulating MMP-9 and significantly lower levels on day 8 (p = 0.021). Higher MMP-9 on day 8 was associated with persistent venous occlusion (odds ratio: 1.20 [per 20 ng/mL], 95% CI: 1.02-1.43, p = 0.030). CONCLUSION: We report a novel relationship among MMP-9, parenchymal brain damage, and early venous recanalization, suggesting that circulating MMP-9 is a dynamic marker of brain tissue damage in patients with CVT.
Assuntos
Veias Cerebrais , Trombose Intracraniana/enzimologia , Metaloproteinase 9 da Matriz/sangue , Trombose Venosa/enzimologia , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Angiografia Cerebral , Veias Cerebrais/diagnóstico por imagem , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Trombose Intracraniana/sangue , Trombose Intracraniana/diagnóstico por imagem , Angiografia por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Flebografia , Portugal , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Fatores de Tempo , Trombose Venosa/sangue , Trombose Venosa/diagnóstico por imagem , Adulto JovemRESUMO
Previously, we reported that phospholipase D1 (PLD1) and PLD2 inhibition by selective PLD1 and PLD2 inhibitors could prevent platelet aggregation in humans, but not in mice. Moreover, only the PLD1 inhibitor, but not PLD2 inhibitor, could effectively prevent thrombus formation in mice, indicating that PLD might play different roles in platelet function in humans and mice. Although PLD1 and PLD2 were reported to be implicated in thrombotic events, the role of PLD in mice remains not completely clear. Here, we investigated the role of PLD1 and PLD2 in acute pulmonary thrombosis and transient middle cerebral artery occlusion-induced brain injury in mice. The data revealed that inhibition of PLD1, but not of PLD2, could partially prevent pulmonary thrombosis-induced death. Moreover, concurrent PLD1 and PLD2 inhibition could considerably increase survival rate. Likewise, inhibition of PLD1, but not PLD2, partially improved ischemic stroke and concurrent inhibition of PLD1, and PLD2 exhibited a relatively better protection against ischemic stroke, as evidenced by the infarct size, brain edema, modified neurological severity score, rotarod test, and the open field test. In conclusion, PLD1 might play a more important role than PLD2, and both PLD1 and PLD2 could act synergistically or have partially redundant functions in regulating thrombosis-relevant events.
Assuntos
Trombose Intracraniana/enzimologia , AVC Isquêmico/enzimologia , Fosfolipase D/metabolismo , Animais , Trombose Intracraniana/patologia , AVC Isquêmico/patologia , Masculino , Camundongos , Camundongos Endogâmicos ICRRESUMO
Epigenetic processes play important roles in brain responses to ischemic injury. We studied effects of photothrombotic stroke (PTS, a model of ischemic stroke) on the intracellular level and cellular localization of histone deacetylases HDAC3, HDAC4 and HDAC6 in the rat brain cortex, and tested the potential neuroprotector ability of their inhibitors. The background level of HDAC3, HDAC4 and HDAC6 in the rat cerebral cortex was relatively low. HDAC3 localized in the nuclei of some neurons and few astrocytes. HDAC4 was found in the neuronal cytoplasm. After PTS, their levels in penumbra did not change, but HDAC4 appeared in the nuclei of some cells. Its level in the cytoplasmic, but not nuclear fraction of penumbra decreased at 24, but not 4 h after PTS. HDAC6 was upregulated in neurons and astrocytes in the PTS-induced penumbra, especially in the nuclear fraction. Unlike HDAC3 and HDAC4, HDAC6 co-localized with TUNEL-positive apoptotic cells. Inhibitory analysis confirmed the involvement of HDAC6, but not HDAC3 and HDAC4 in neurodegeneration. HDAC6 inhibitor HPOB, HDAC2/8 inhibitor α-phenyl tropolone, and non-specific histone deacetylase inhibitor sodium valproate, but not HDAC3 inhibitor BRD3308, or HDAC4 inhibitor LMK235, decreased PTS-induced infarction volume in the mouse brain, reduced apoptosis, and recovered the motor behavior. HPOB also restored PTS-impaired acetylation of α-tubulin. α-phenyl tropolone restored acetylation of histone H4 in penumbra cells. These results suggest that histone deacetylases HDAC6 and HDAC2 are the possible molecular targets for anti-ischemic therapy, and their inhibitors α-phenyl tropolone, HBOP and sodium valproate can be considered as promising neuroprotectors.
Assuntos
Córtex Cerebral/enzimologia , Desacetilase 6 de Histona/biossíntese , Inibidores de Histona Desacetilases/farmacologia , Trombose Intracraniana/enzimologia , Acidente Vascular Cerebral/enzimologia , Tropolona/farmacologia , Ácido Valproico/farmacologia , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Expressão Gênica , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/biossíntese , Trombose Intracraniana/patologia , Trombose Intracraniana/prevenção & controle , Lasers Semicondutores/efeitos adversos , Masculino , Camundongos , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Ratos , Acidente Vascular Cerebral/patologia , Acidente Vascular Cerebral/prevenção & controle , Tropolona/uso terapêutico , Ácido Valproico/uso terapêuticoRESUMO
(3ß,5α,16α,20S)-4,4,14-trimethyl-3,20-bis(methylamino)-9,19-cyclopregnan-16-ol-dihydrochloride (JLX001), a derivative of cyclovirobuxine D (CVB-D), is a novel compound from synthesis. This study aims to confirm the therapeutic effect of JLX001 on cerebral ischemia and researchits antiplatelet and antithrombosis activities via thromboxane (TXA2)/phospholipase C-ß-3(PLCß3)/protein kinase C (PKC) pathway suppression. The therapeutic effects of JLX001 was evaluated by infarct sizes, brain edema and neurological scores in Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO). Brain TXA2 and prostacyclin (PGI2) were measured by enzyme-linked immunosorbentassay (ELISA). P-PLCß3and activated PKC were detected by immunohistochemical method. Adenosine diphosphate (ADP) or 9, 11-dieoxy-11α, 9α-epoxymethanoeprostaglandin F2α (U46619) was used as platelet agonist in the in vivo and in vitro platelet aggregation experiments. Clotting time and bleeding time were determined. Besides, two whole-animal experiments including arteriovenous shunt thrombosis and pulmonary thromboembolism model were conducted. Results showed that JLX001 treatment markedly alleviated cerebral infarcts, edema, and neurological scores in permanent middle cerebral artery occlusion (pMCAO) rats. Brain TXA2 level, p-PLCß3and activated PKC were decreased, while PGI2level had no significant change. Besides, JLX001 inhibited platelet aggregation induced by ADP or U46619 and exhibited anti-coagulation effects with a minor bleeding risk. In the two whole-animal experiments, JLX001 inhibited thrombus formation. In summary, JLX001 attenuates cerebral ischemia injury and the underlying mechanisms relate to inhibiting platelet activation and thrombus formation via TXA2/PLCß3/PKC pathway suppression.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Infarto da Artéria Cerebral Média/prevenção & controle , Trombose Intracraniana/prevenção & controle , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Triterpenos/farmacologia , Animais , Aspirina/farmacologia , Comportamento Animal/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Edema Encefálico/sangue , Edema Encefálico/patologia , Edema Encefálico/prevenção & controle , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/farmacologia , Epoprostenol/metabolismo , Feminino , Infarto da Artéria Cerebral Média/sangue , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/patologia , Trombose Intracraniana/sangue , Trombose Intracraniana/enzimologia , Trombose Intracraniana/patologia , Masculino , Camundongos Endogâmicos ICR , Fosfolipase C beta/metabolismo , Inibidores da Agregação Plaquetária/uso terapêutico , Proteína Quinase C/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Tromboxano A2/metabolismo , Triterpenos/uso terapêuticoRESUMO
BACKGROUND: The presence of Janus Kinase 2 (JAK2) V617F mutation represents a major diagnostic criterion for detecting myeloproliferative neoplasms (MPN) and even in the absence of overt MPN, JAK2 V617F mutation is associated with splanchnic vein thrombosis. However, the actual prevalence and diagnostic value of the JAK2 V617F mutation in patients with cerebral venous thrombosis (CVT) are not known. The aims of this study were to assess the prevalence of JAK2 V617F mutation in a large group of consecutive CVT patients, to detect clinical, biological, and radiological features associated with the mutation, and to determine the long-term venous thrombosis recurrence rate in CVT patients with JAK2 mutation but without overt MPN in order to recommend the best preventive treatment. METHODS: This was a prospective study conducted on consecutive patients with a first-ever radiologically confirmed CVT. JAK2 V617F mutation analysis was assessed in all the study subjects. JAK2 V617F-positive patients were followed up to detect new venous thrombotic events. RESULTS: Of the 125 included subjects, 7 were found to have JAK2 V617F mutation (5.6%; 95% CI 2.3-11.2). Older age (p = 0.039) and higher platelet count (p = 0.004) were independently associated with JAK2 V617F positivity in patients without overt MPN. During a mean follow-up period of 59 (SD 46) months, 2 JAK2 V617F-positive patients presented with 4 new venous thromboembolic events. CONCLUSIONS: Screening for the JAK2 V617F mutation in CVT patients seems to be useful even in the absence of overt MPN and/or in the presence of other risk factors for CVT because of its relatively high prevalence and the risk of thrombosis recurrence.
Assuntos
Análise Mutacional de DNA , Testes Genéticos/métodos , Trombose Intracraniana/genética , Janus Quinase 2/genética , Mutação , Trombose Venosa/genética , Adulto , Idoso , Anticoagulantes/uso terapêutico , Feminino , Predisposição Genética para Doença , Humanos , Trombose Intracraniana/diagnóstico por imagem , Trombose Intracraniana/tratamento farmacológico , Trombose Intracraniana/enzimologia , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Fenótipo , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Recidiva , Fatores de Risco , Fatores de Tempo , Trombose Venosa/diagnóstico por imagem , Trombose Venosa/tratamento farmacológico , Trombose Venosa/enzimologiaRESUMO
BACKGROUND: Blood-brain barrier (BBB) disruption mediated by proteases plays a pivotal role in neural tissue damage after acute ischemic stroke. In an animal stroke model, the activation of matrix metalloproteinases (MMPs), especially MMP-9, was significantly increased and it showed potential association with blood-brain barrier (BBB) disruption and cerebral edema. Theoretically, it is expected that early blockade of expression and activation of MMP-9 after ischemic stroke provides neuroprotective effects from secondary neural tissue damage. This study was aimed to determine the ability of rutin to influence MMP-9 expression, activity and BBB disruption using a photothrombotic focal ischemic model in rats. METHODS: Adult male Sprague-Dawley rats, weighing between 250 and 300 g (aged 8 weeks) received focal cerebral ischemia by photothrombosis using Rose Bengal (RB) and cold light. Injured animals were divided into two groups; one group received 50mg/kg of rutin intraperitoneally, starting 1h after injury and at 12h intervals for 3 days, while animals in the control group received weight-adjusted doses of saline vehicle over the same period. In each group, the expressions and activities of MMP-9 were assessed by Western blot and gelatin zymography at 6, 24, 48, and 72 h after photothrombotic insult. The effects of rutin on BBB disruption and functional outcomes were also determined. RESULTS: Western blot and zymographic analysis showed up-regulated MMP-9 expression and activity in the ischemic cortex. The expression and activity of MMP-9 were significantly elevated at 6h after photothrombotic insult, which remained up-regulated for at least until 72 h after injury. In the rutin-treated group, MMP-9 expression and activity were significantly attenuated at 6, 24, and 48 h compared to the control group. Relative to the control group, BBB permeability was significantly reduced in the rutin-treated group. The results of the rotarod test revealed that rutin treatment significantly improved functional outcomes. CONCLUSIONS: Rutin treatment starting 1h after injury attenuated BBB disruption during photothrombotic focal ischemia, which was partly, at least, achieved through inhibitory effects on MMP-9 expression and activity. The results of this study suggest that rutin might be useful in clinical trials aimed to improve the outcome of patients suffering from acute ischemic stroke.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Modelos Animais de Doenças , Trombose Intracraniana/tratamento farmacológico , Metaloproteinase 9 da Matriz/biossíntese , Estimulação Luminosa/efeitos adversos , Rutina/uso terapêutico , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/enzimologia , Isquemia Encefálica/enzimologia , Isquemia Encefálica/etiologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Trombose Intracraniana/enzimologia , Trombose Intracraniana/etiologia , Masculino , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/fisiologia , Rosa Bengala/toxicidade , Rutina/farmacologia , Resultado do TratamentoRESUMO
BACKGROUND: The study aimed at evaluating the contribution of genetic variations in the drug metabolizing enzyme, CYP2C9, and the influence of co-medication with the antiepileptic drug, phenytoin, to variability in acenocoumarol response, in patients with cerebral venous thrombosis (CVT). METHODS: 476 acenocoumarol-treated CVT patients (153 males and 323 females) were genotyped for CYP2C9*2 and CYP2C9*3 polymorphisms by PCR-RFLP method. Mean acenocoumarol dose required for achieving and maintaining a stable international normalized ratio (INR) was calculated for different genotypes. The effect of co-administration with phenytoin was determined. RESULTS: Genotype distributions of CYP2C9 were as follows: 83%CYP2C9*1/*1, 8.6%CYP2C9*1/*3, 5.9%CYP2C9*1/*2, 1.9%CYP2C9*3/*3, 0.4%CYP2C9*2/*3 and 0.2%CYP2C9*2/*2. During the initiation phase of anticoagulation the CYP2C9*2 allele was independently associated with low acenocoumarol dose requirement (Adjusted OR 5.38; 95%CI 1.65-17.49; p=0.005). Similarly, the adjusted odds ratio for requiring a low dose during the induction phase in patients bearing the CYP2C9*3 allele was 12.79 (95%CI 4.74-34.57; p<0.0001). During the maintenance phase, CYP2C9*2 and CYP2C9*3 alleles were associated with 19-fold (Adjusted OR 19.67; 95%CI 2.46-157.19; p=0.005) and 11.9-fold odds (Adjusted OR 11.98; 95%CI 2.61-55.08; p=0.001) of requiring a low dose. Clinical covariates such as age, alcohol consumption, postpartum state and oral contraceptive intake also influenced acenocoumarol dosage. Co-medication with phenytoin was associated with lower dose requirement across genotypes during the initiation phase. However, during the maintenance phase, phenytoin-treated patients of all genotypes required higher doses of acenocoumarol. CONCLUSION: This study emphasizes the fact that polymorphisms in CYP2C9 gene and co-medication with phenytoin alter the anticoagulant effect of acenocoumarol.
Assuntos
Acenocumarol/uso terapêutico , Citocromo P-450 CYP2C9/genética , Trombose Intracraniana/tratamento farmacológico , Fenitoína/uso terapêutico , Trombose Venosa/tratamento farmacológico , Adolescente , Adulto , Idoso , Citocromo P-450 CYP2C9/metabolismo , Interações Medicamentosas , Feminino , Genótipo , Humanos , Trombose Intracraniana/enzimologia , Trombose Intracraniana/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Prevalência , Trombose Venosa/enzimologia , Trombose Venosa/genética , Adulto JovemRESUMO
Delayed complications of subarachnoid hemorrhage (SAH) such as angiographic vasospasm, cortical spreading ischemia, microcirculatory dysfunction, and microthrombosis are reported in both patients and animal models of SAH. We demonstrated previously that SAH is associated with increased oxidative stress in the brain parenchyma, and that this correlates with dysfunction of endothelial nitric oxide synthase (eNOS) (homodimeric uncoupling). Uncoupling of eNOS exacerbated oxidative stress and enhanced nitric oxide (NO) depletion, and was associated with multiple secondary complications such as microthrombosis, neuronal apoptosis, and release of reactive oxygen species. Thus, we hypothesized that genetic abbrogation of eNOS would confer a beneficial effect on the brain after SAH. Using a prechiasmatic injection model of SAH, we show here that eNOS knockout (KO) significantly alleviates vasospasm of the middle cerebral artery and reduces superoxide production. Endothelial nitric oxide synthase KO also affected other nitric oxide synthase isoforms. It significantly increases neuron nitric oxide synthase expression but has no effect on inducible nitric oxide synthase. Endothelial nitric oxide synthase KO decreases Zn(2+) release after SAH, reduces microthrombi formation, and prevent neuronal degeneration. This work is consistent with our findings where, after SAH, increased oxidative stress can uncouple eNOS via Zn(2+) thiolate oxidation, or theoretically by depletion or oxidation of tetrahydrobiopterin, resulting in a paradoxical release of superoxide anion radical, further exacerbating oxidative stress and microvascular damage.
Assuntos
Trombose Intracraniana/prevenção & controle , Óxido Nítrico Sintase Tipo III/genética , Hemorragia Subaracnóidea/complicações , Vasoespasmo Intracraniano/prevenção & controle , Animais , Western Blotting , Circulação Cerebrovascular/fisiologia , Modelos Animais de Doenças , Fibrinogênio/metabolismo , Imuno-Histoquímica , Trombose Intracraniana/enzimologia , Trombose Intracraniana/etiologia , Trombose Intracraniana/patologia , Camundongos , Camundongos Knockout , Neurônios/enzimologia , Neurônios/patologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/fisiologia , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Hemorragia Subaracnóidea/enzimologia , Superóxidos/metabolismo , Vasoespasmo Intracraniano/enzimologia , Vasoespasmo Intracraniano/etiologia , Vasoespasmo Intracraniano/patologia , Zinco/metabolismoRESUMO
The association between the methylenetetrahydrofolate reductase (MTHFR) gene and cerebral venous thrombosis (CVT) remains controversial. This study principally investigated the potential role of the MTHFR A1298C variant and CVT. The genotyping of the A1298C variant of the MTHFR gene was performed in 35 CVT patients and 200 healthy controls. The frequency of A1298C genotype among CVT patients was significantly higher compared with controls (Pâ<â10(-3)), suggesting an association between this polymorphism and CVT. To our knowledge, there are no previous reports assessing the correlation between the MTHFR A1298C variant and CVT. Large study populations would be required to understand the contribution of this marker in the risk of CVT.
Assuntos
Trombose Intracraniana/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Trombose Venosa/genética , Adolescente , Adulto , Idoso , Feminino , Predisposição Genética para Doença , Humanos , Trombose Intracraniana/enzimologia , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Pessoa de Meia-Idade , Polimorfismo Genético , Fatores de Risco , Trombose Venosa/enzimologia , Adulto JovemRESUMO
BACKGROUND: The accumulation of toxic free radicals plays a pivotal role in the early molecular cascades of blood-brain barrier (BBB) disruption mediated by matrix metalloproteinases (MMPs) activation in ischemic stroke. Theoretically, it is expected that early blockade of activation of MMPs may provide protective effects from secondary neural tissue damage. The present study was designed to determine the ability of melatonin to influence MMP-9 activity and BBB disruption, in a focal ischemia rat model induced by photothrombosis. METHODS: Adult, male, 8-week Sprague-Dawley rats weighing 230-300 g received focal cerebral ischemia by photothrombosis using Rose Bengal (RB). The injured animals were divided into two groups. One group received 50mg/kg of melatonin intraperitoneally, starting 1h after injury and at 12h intervals for 3 days. The control group received weight-adjusted doses of saline vehicle. In each group, MMP-9 expression and activity were assessed by Western blot and gelatin zymography, respectively, at various times. The effects of melatonin on BBB disruption and brain edema were also determined. RESULTS: MMP-9 activity and expression were significantly elevated at 24h in the ischemic cortex, which remained up-regulated at least until 72 h after injury. Melatonin treatment significantly attenuated MMP-9 activity and expression at 24, 48, and 72 h after ischemic injury. Relative to control group, BBB permeability was significantly reduced in the melatonin-treated group. The water content was decreased by melatonin treatment, although there was no statistically significant difference. CONCLUSIONS: Melatonin treatment starting 1h after injury attenuated BBB disruption during focal ischemia, which is at least partly due to inhibition of MMP-9 activity. Melatonin might have a potential role in clinical trials aimed to improve the outcome of patients suffering cerebral ischemia.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Isquemia Encefálica/prevenção & controle , Trombose Intracraniana/tratamento farmacológico , Metaloproteinase 9 da Matriz/efeitos dos fármacos , Melatonina/uso terapêutico , Animais , Água Corporal , Edema Encefálico/etiologia , Edema Encefálico/prevenção & controle , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/enzimologia , Modelos Animais de Doenças , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Injeções Intraperitoneais , Trombose Intracraniana/induzido quimicamente , Trombose Intracraniana/enzimologia , Masculino , Melatonina/farmacologia , Fotoquímica , Prosencéfalo/enzimologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Rosa Bengala/efeitos da radiação , Rosa Bengala/toxicidadeRESUMO
BACKGROUND: It is currently unclear whether or not cerebral venous thrombosis, such as splanchnic venous thrombosis, can be the first manifestation of an underlying myeloproliferative neoplasm. OBJECTIVE: To determine the prevalence of the JAK2 V617F mutation in patients with a first episode of cerebral venous thrombosis. PATIENTS: In this retrospective cohort study, patients with cerebral venous thrombosis were tested for the JAK2 V617F mutation and were followed until the development of a myeloproliferative neoplasm or censored at the end of follow-up. RESULTS: Ten of 152 patients (6.6%) carried the JAK2 V617F mutation. Three of them had known acquired risk factors for thrombosis, and five had thrombophilia. Six patients met the diagnostic criteria for myeloproliferative neoplasm at the time of cerebral venous thrombosis, and three additional patients developed the disease during the follow-up (median duration 7.8 years, range 6 months to 21.3 years), giving an annual incidence of 0.26% patient-years (95% confidence interval 0.05-0.64). The last patient has no evidence of disease after 3 years of follow-up. Patients without the JAK2 V617F mutation at the time of cerebral venous thrombosis were retested at the end of the follow-up and remained negative, with normal blood counts (log-rank test χ(2) : 159 [P<0.0001]). CONCLUSIONS: Cerebral venous thrombosis can be the first symptom of a myeloproliferative neoplasm. Patients with cerebral venous thrombosis can carry the JAK2 V617F mutation, irrespective of blood count.
Assuntos
Coagulação Sanguínea/genética , Trombose Intracraniana/genética , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/epidemiologia , Trombose Venosa/genética , Adolescente , Adulto , Idoso , Distribuição de Qui-Quadrado , Intervalo Livre de Doença , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Incidência , Trombose Intracraniana/enzimologia , Trombose Intracraniana/mortalidade , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/sangue , Transtornos Mieloproliferativos/mortalidade , Fenótipo , Prevalência , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Trombose Venosa/enzimologia , Trombose Venosa/mortalidade , Adulto JovemRESUMO
Matrix metalloproteinases (MMP) have been thought to be involved in stroke pathogenesis. MMP-9 contributes to tissue destruction. Our aim was to analyze the MMP-9 levels in blood within 24 hours of acute ischemic stroke onset to observe the role of MMP-9 in the pathogenesis of atherothrombotic stroke. In this study we investigated prospectively MMP-9 levels in serum from 106 patients (42 men and 64 women, mean age 71.52 +/- 6.32 years) with acute ischemic stroke in the middle cerebral artery area in the first 24 hours from the onset (mean duration 7.8 +/- 4.5 hours) as compared to 112 controls (48 men and 64 women, mean age 70.36 +/- 6.8 years). Serum samples were collected under sterile conditions and stored in aliquots at -70 degrees C until assay. Serum MMP-9 levels were determined by enzyme-linked immunosorbent assay (ELISA) in blood samples obtained on admission. Statistical analysis was performed by Mann-Whitney and Log-Likeliwood Ratio tests. All values reported are expressed as mean (x) +/- SD. Mean serum MMP-9 concentrations were higher in group with ischemic stroke 172 +/- 32.4 ng/mL, range 139.6-204.4 ng/mL vs. controls 57 +/- 9.6 ng/mL, range 47.4-66.6 ng/mL (95% CI, 3.17 to 14.18; p < 0.014). In conclusion, MMP-9 activity is associated with early acute ischemic stroke. The high levels of MMP-9 in acute ischemic stroke document the involvement of this enzyme in the regulation of inflammation in stroke.
Assuntos
Isquemia Encefálica/enzimologia , Metaloproteinase 9 da Matriz/sangue , Acidente Vascular Cerebral/enzimologia , Idoso , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Arteriosclerose Intracraniana/enzimologia , Trombose Intracraniana/enzimologia , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
We aimed to study patients with splanchnic vein thrombosis (SVT) and cerebral vein thrombosis (CVT) searching for JAK2 mutations. We evaluated 14 patients (median age: 41.5 years) with portal vein thrombosis (PVT) = 7; mesenteric vein thrombosis (MVT) = 3; and CVT = 4. JAK2 V617F was assessed by allele specific PCR of peripheral blood DNA. In addition, DNA was sequenced for other JAK2 mutations. Other inherited and acquired thrombophilia risk factors were evaluated. JAK2 V617F was positive in four out of seven patients with PVT and in one CVT patient. These five patients had a diagnosis of myelo-proliferative disorder (MPD) at the moment of the occurrence of thrombosis (n = 2) or later (n = 2). Patients with MVT and CVT were negative for JAK2 V617F, except one patient with CVT and a diagnosis of essential thrombocythemia. No other JAK2 mutations were found in this cohort. Besides MPD, other thrombophilia risk factors were identified in five patients. One patient had MPD as well as thrombophilia risk factor. In this group, 4 out of 7 of the patients with PVT carried the JAK2 V617F mutation with or without overt MPD. However, the investigation of other JAK2 mutations may not be necessary in patients with thrombosis at unusual sites.
Assuntos
Trombose Intracraniana/genética , Janus Quinase 2/genética , Veias Mesentéricas , Mutação/genética , Veia Porta , Trombose Venosa/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Humanos , Trombose Intracraniana/enzimologia , Masculino , Pessoa de Meia-Idade , Prevalência , Estudos Retrospectivos , Fatores de Risco , Trombose Venosa/enzimologiaRESUMO
We aimed to study patients with splanchnic vein thrombosis (SVT) and cerebral vein thrombosis (CVT) searching for JAK2 mutations. We evaluated 14 patients (median age: 41.5 years) with portal vein thrombosis (PVT) = 7; mesenteric vein thrombosis (MVT) = 3; and CVT = 4. JAK2 V617F was assessed by allele specific PCR of peripheral blood DNA. In addition, DNA was sequenced for other JAK2 mutations. Other inherited and acquired thrombophilia risk factors were evaluated. JAK2 V617F was positive in four out of seven patients with PVT and in one CVT patient. These five patients had a diagnosis of myeloproliferative disorder (MPD) at the moment of the occurrence of thrombosis (n = 2) or later (n = 2). Patients with MVT and CVT were negative for JAK2 V617F, except one patient with CVT and a diagnosis of essential thrombocythemia. No other JAK2 mutations were found in this cohort. Besides MPD, other thrombophilia risk factors were identified in five patients. One patient had MPD as well as thrombophilia risk factor. In this group, 4 out of 7 of the patients with PVT carried the JAK2 V617F mutation with or without overt MPD. However, the investigation of other JAK2 mutations may not be necessary in patients with thrombosis at unusual sites.
Nuestro objetivo fue estudiar pacientes con trombosis de las venas esplácnicas (TVE) o trombosis de las venas cerebrales (TVC) en búsqueda de mutaciones del gen quinasa Janus 2 (JAK2). Se estudiaron 14 pacientes (media de edad: 41.5 años) con trombosis de la vena porta (TVP n = 7), trombosis de la vena mesentérica (TVM, n = 3) y TVC (n = 4). La mutación V617F del gen JAK2 fue evaluada por reacción en cadena de la polimerasa (PCR) alelo-específica en muestras de sangre periférica. Además, se realizó secuenciación de ADN en búsqueda de otras mutaciones del gen JAK2 distintas de V617F. También se investigaron factores genéticos y adquiridos para trombofilia. JAK2 V617F fue positiva en 4 de 7 pacientes con TVP y en un paciente con TVC. Estos 5 pacientes con la mutación tuvieron diagnóstico de síndrome mieloproliferativo (SMP) en el momento de la detección de la trombosis (n = 2) o después (n = 3). Un paciente con TVP sufrió el episodio trombótico 18 años después del diagnóstico del SMP y la mutación JAK2 V617F fue negativa. No se encontraron otras mutaciones del gen JAK2 en este grupo d e pacientes. Además del diagnóstico de SMP, se identificaron otros factores de riesgo para trombofilia en 4 pacientes. Un paciente tuvo un factor de riesgo para trombofilia además del diagnóstico de SMP. La mutación JAK2 V617F se presentó en 4/7 de los pacientes con TVP con o sin un diagnóstico obvio de SMP. La investigación de otras mutaciones podría no ser necesaria en pacientes con trombosis en sitios poco frecuentes.
Assuntos
Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Trombose Intracraniana/genética , /genética , Veias Mesentéricas , Mutação/genética , Veia Porta , Trombose Venosa/genética , Trombose Intracraniana/enzimologia , Prevalência , Estudos Retrospectivos , Fatores de Risco , Trombose Venosa/enzimologiaRESUMO
We studied whether endothelial nitric oxide synthase (eNOS) is upregulated and uncoupled in large cerebral arteries after subarachnoid hemorrhage (SAH) and also whether this causes cerebral vasospasm in a mouse model of anterior circulation SAH. Control animals underwent injection of saline instead of blood (n=16 SAH and n=16 controls). There was significant vasospasm of the middle cerebral artery 2 days after SAH (lumen radius/wall thickness ratio 4.3 ± 1.3 for SAH, 23.2 ± 2.1 for saline, P<0.001). Subarachnoid hemorrhage was associated with terminal deoxynucleotidyl transferase dUTP nick-end labeling, cleaved caspase-3, and Fluoro-Jade-positive neurons in the cortex and with CA1 and dentate regions in the hippocampus. There were multiple fibrinogen-positive microthromboemboli in the cortex and hippocampus after SAH. Transgenic mice expressing lacZ under control of the eNOS promoter had increased X-gal staining in large arteries after SAH, and this was confirmed by the increased eNOS protein on western blotting. Evidence that eNOS was uncoupled was found in that nitric oxide availability was decreased, and superoxide and peroxynitrite concentrations were increased in the brains of mice with SAH. This study suggests that artery constriction by SAH upregulates eNOS but that it is uncoupled and produces peroxynitrite that may generate microemboli that travel distally and contribute to brain injury.
Assuntos
Óxido Nítrico Sintase Tipo III/metabolismo , Hemorragia Subaracnóidea/enzimologia , Animais , Arteríolas/enzimologia , Arteríolas/patologia , Western Blotting , Caspase 3/metabolismo , Artérias Cerebrais/enzimologia , Artérias Cerebrais/patologia , Fibrinogênio/metabolismo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Trombose Intracraniana/enzimologia , Trombose Intracraniana/patologia , Óperon Lac/genética , Camundongos , Camundongos Transgênicos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Ácido Peroxinitroso/metabolismo , Hemorragia Subaracnóidea/genética , Superóxidos/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo , Vasoespasmo Intracraniano/enzimologiaRESUMO
The sole FDA-approved treatment for acute stroke is recombinant tissue-type plasminogen activator (rtPA). However, rtPA aggravates the impairment of cerebrovasodilation induced by global hypoxia/ischemia; this impairment is attenuated by the preinjury treatment with the plasminogen activator inhibitor derivative EEIIMD. MAPK (a family of kinases, p38, and JNK) is upregulated after cerebral ischemia. In this study, we determined whether the novel plasminogen activator inhibitor-derived peptide, Ac-RMAPEEIIMDRPFLYVVR-amide, (PAI-1-DP) given 30 min before or 2 h after, focal central nervous system injury induced by photothrombosis would preserve responses to cerebrovasodilators and the role of p38 and JNK MAPK in such effects. Cerebrospinal fluid JNK and p38 levels were elevated by photothrombotic injury, an effect potentiated by rtPA. Cerebrovasodilation was blunted by photothrombosis and reversed to vasoconstriction by rtPA but restored to dilation by PAI-1-DP pre- and posttreatment. PAI-1-DP blocked JNK, but preserved p38 MAPK upregulation after photothrombosis. The JNK MAPK antagonist SP600125 prevented, and the p38 antagonist SB203580 potentiated, impaired cerebrovasodilation after photothrombosis. These data indicate that rtPA impairs cerebrovasodilation after injury by activating JNK, while p38 MAPK is protective, and that the novel peptide PAI-1-DP protects by inhibiting activation of JNK by rtPA. JNK MAPK inhibitors, including PAI-1-DP, may offer a novel approach to increase the benefit-to-risk ratio of thrombolytic therapy and enable its use in central nervous system ischemic disorders.
Assuntos
Circulação Cerebrovascular/efeitos dos fármacos , Trombose Intracraniana/tratamento farmacológico , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Peptídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Antracenos/farmacologia , Modelos Animais de Doenças , Eritrosina , Feminino , Fibrinolíticos/toxicidade , Imidazóis/farmacologia , Trombose Intracraniana/enzimologia , Trombose Intracraniana/etiologia , Trombose Intracraniana/fisiopatologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lasers de Estado Sólido , Masculino , Piridinas/farmacologia , Proteínas Recombinantes/toxicidade , Suínos , Fatores de Tempo , Ativador de Plasminogênio Tecidual/toxicidade , Vasoconstrição/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: Intravenous administration of recombinant tissue plasminogen activator (rtPA) is known as the only approved treatment for acute ischemic stroke. However, it is still controversial whether acute ischemic stroke patients with atrial fibrillation should receive rtPA therapy. METHODS: We studied 99 patients altogether who belonged to three different groups based on the patient characteristics: (1) atrial fibrillation rtPA-treated group consisting of 22 ischemic stroke patients with atrial fibrillation treated with rtPA within 4.5 hours after the onset of stroke; (2) atrial fibrillation non-rtPA-treated group consisting of 44 acute ischemic stroke patients with atrial fibrillation matching in age and baseline National Institutes of Health Stroke Scale (NIHSS); (3) the non-atrial fibrillation rtPA-treated group consisting of 33 patients without atrial fibrillation treated with rtPA. RESULTS: The median time for the administration of rtPA was 199.6 +/- 50.0 minutes. More patients had favorable outcomes (90 day modified Rankin Scale 0-1) in the atrial fibrillation rtPA-treated group than the atrial fibrillation non-rtPA-treated group (36.4 versus 13.6%; odds ratio=2.667; 95% confidence interval: 1.056-6.735; p=0.033). The mortality at day 90 was lower in the rtPA-treated group than the non-rtPA-treated group (18.2 versus 20.5%; p=0.827), although the incidence of symptomatic intracranial hemorrhage was higher (18.2 versus 6.8%; p=0.184). Patients in the atrial fibrillation rtPA-treated group had fewer favorable outcomes than non-atrial fibrillation rtPA-treated group (36.4 versus 51.6%; p=0.076), but their baseline NIHSS was higher (12.0 +/- 7.1 versus 9.1 +/- 7.3; p=0.161). CONCLUSION: As compared with non-rtPA-treated patients, rtPA treated within 4.5 hours after the onset of stroke significantly improved clinical outcomes in atrial fibrillation patients. Thrombolytic treatment increases intracranial hemorrhage rate but does not increase mortality.
Assuntos
Fibrilação Atrial/complicações , Hipóxia-Isquemia Encefálica/sangue , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/tratamento farmacológico , Terapia Trombolítica/métodos , Ativador de Plasminogênio Tecidual/administração & dosagem , Doença Aguda , Idoso , Feminino , Fibrinolíticos/administração & dosagem , Fibrinolíticos/efeitos adversos , Humanos , Hipóxia-Isquemia Encefálica/enzimologia , Trombose Intracraniana/sangue , Trombose Intracraniana/tratamento farmacológico , Trombose Intracraniana/enzimologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Acidente Vascular Cerebral/enzimologia , Terapia Trombolítica/normas , Terapia Trombolítica/estatística & dados numéricos , Fatores de Tempo , Ativador de Plasminogênio Tecidual/efeitos adversos , Resultado do TratamentoAssuntos
Trombose Intracraniana/genética , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/genética , Trombose Venosa/genética , Idoso , Feminino , Predisposição Genética para Doença , Humanos , Trombose Intracraniana/diagnóstico , Trombose Intracraniana/enzimologia , Trombose Intracraniana/terapia , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/complicações , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/terapia , Fenótipo , Fatores de Risco , Resultado do Tratamento , Trombose Venosa/diagnóstico , Trombose Venosa/enzimologia , Trombose Venosa/terapiaRESUMO
The amyloid beta-protein precursor (AbetaPP) is best recognized as the precursor to the Abeta peptide that accumulates in the brains of patients with Alzheimer's disease, but less is known about its physiological functions. Isoforms of AbetaPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AbetaPP regulates cerebral thrombosis in vivo (Xu et al., 2005, 2007). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AbetaPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here, we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AbetaPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model, both AbetaPP(-/-) and APLP2(-/-) mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a prothrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage, both AbetaPP(-/-) and APLP2(-/-) mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AbetaPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury.