RESUMO
BACKGROUND AND PURPOSE: Oxidative stress has been reported to be a main cause of neuronal cell death in ischemia reperfusion injury (IRI). Nuclear factor-erythroid 2-related factor 2 (Nrf2) is an important factor involved in anti-oxidative responses. We previously reported that bardoxolone methyl (BARD), an Nrf2 activator, prevented damage induced by IRI. In this study, we investigated the effect of BARD on hemorrhagic transformation in the context of blood brain barrier (BBB) protection. METHODS: Mice received pre-treatment with warfarin (4.0 mg/kg, p.o.). IRI was subsequently induced 18 h after the warfarin administration by transient middle cerebral artery occlusion (MCAO) for 6 h. BARD (0.06, 0.2, 0.6 or 2.0 mg/kg) or saline was injected intravenously immediately after reperfusion. The infarct volume, neurological score, intracranial hemorrhage volume, and BBB permeability were evaluated 24 h after MCAO. The survival rate and behavioral functional recovery were evaluated for 7 days following IRI. Furthermore, the effects of BARD on BBB components were investigated by western blotting and immunostaining analysis. RESULTS: BARD suppressed warfarin-mediated increases in the intracranial hemorrhage volume without affecting the infarct volume. BBB permeability was also suppressed by administration of BARD. Western blotting showed that BARD increased expression of BBB components such as endothelial cells, pericytes, and tight junction proteins. Furthermore, immunostaining showed that BARD induced localization of Nrf2 to endothelial cells and pericytes. CONCLUSIONS: BARD suppressed the exacerbation hemorrhage caused by warfarin pretreatment and ameliorated BBB disruption by protecting endothelial cells, pericytes, and tight junction protein expressions. These results indicate that Nrf2 activators may be an effective therapy against hemorrhagic transformation caused by anticoagulant drugs.
Assuntos
Anticoagulantes/administração & dosagem , Barreira Hematoencefálica/metabolismo , Isquemia Encefálica/metabolismo , Hemorragias Intracranianas/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ácido Oleanólico/análogos & derivados , Varfarina/administração & dosagem , Animais , Antígenos CD/metabolismo , Comportamento Animal/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Isquemia Encefálica/complicações , Isquemia Encefálica/patologia , Caderinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Hemorragias Intracranianas/etiologia , Hemorragias Intracranianas/patologia , Masculino , Camundongos , Fator 2 Relacionado a NF-E2/agonistas , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ácido Oleanólico/administração & dosagem , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Análise de Sobrevida , Proteínas de Junções Íntimas/metabolismoRESUMO
Recurrent pregnancy loss (RPL), which mostly is of unknown etiology (unexplained RPL, uRPL), is defined as three or more consecutive spontaneous abortions. Some women with uRPL display a higher fraction and cytotoxicity of natural killer (NK) cells in the periphery and endometrium. Therefore, some uRPL cases have been explained by autoimmune abnormalities. The efficacy of intravenous immunoglobulin (IVIg) for uRPL has been confirmed in several clinical trials; however, its mechanism remains unknown, mainly because the abortion mechanism remains to be elucidated. In the present study, we analyzed the mechanisms of both abortion and IVIg action using a uRPL mouse model in which abortion was induced by lipopolysaccharide injection. IVIg attenuated the abortion rate in the uRPL model mice. The suppressive effect of IVIg was maximized by high dose administration early after lipopolysaccharide injection. Specifically, we discovered the presence of two distinct uterine NK (uNK) subsets: CD44(bright) and CD44(mid) In uRPL model mice, we observed an increase in the number of CD44(bright) uNK cells, while the CD44(mid) uNK subset remained unchanged. Furthermore, when abortion was reduced by IVIg administration, the cell number of the CD44(bright) uNK subset did not increase, which might allow differentiating pathological from normal uNK cells based on CD44 expression. Based on these results, we propose not only an effective administration protocol of IVIg to the uRPL model mice, but also a novel mechanism of abortion related to the increase in the CD44(bright) subset and of IVIg, which suppresses the increase of the CD44(bright) subset.
Assuntos
Aborto Habitual/prevenção & controle , Receptores de Hialuronatos/metabolismo , Imunoglobulinas Intravenosas/uso terapêutico , Células Matadoras Naturais/metabolismo , Aborto Habitual/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Progesterona/sangue , Baço/metabolismo , Útero/metabolismoRESUMO
BACKGROUND AND PURPOSE: Although long-term treatment with the oral anticoagulant warfarin is widely used to prevent cardioembolic ischemic stroke, it has been reported that warfarin can exacerbate hemorrhagic transformation (HT) after cerebral ischemia. We investigated whether cilostazol, a phosphodiesterase-III inhibitor, suppressed the warfarin-induced HT after cerebral ischemia in mice. METHODS: Male ddY mice were treated with oral warfarin before 3-hour middle cerebral artery occlusion followed by 21-hour reperfusion to induce HT. The duration of warfarin pretreatment was determined by measurement of prothrombin time-international normalized ratio value. Cilostazol or vehicle was administered by intraperitoneal injection immediately after reperfusion. The infarct volume, brain swelling, and brain hemoglobin content were evaluated at 24 hours after middle cerebral artery occlusion. We also evaluated the survival rate of each treated group for 7 days after surgery. To investigate the mechanism underlying cilostazol's effects, the proteins involved in vascular endothelial integrity were investigated using Western blotting. RESULTS: HT volume was exacerbated by warfarin treatment, and cilostazol (3 mg/kg, i.p.) suppressed this exacerbation (sham, mean±SD, 29.2±13.4 mg/dL; vehicle, 33.3±11.9 mg/dL; warfarin, 379.4±428.9 mg/dL; warfarin+cilostazol 1 mg/kg, 167.5±114.2 mg/dL; warfarin+cilostazol 3 mg/kg, 116.9±152.3 mg/dL). Furthermore, cilostazol improved survival rate and upregulated the expression of tight junction proteins and vascular endothelial cadherin. CONCLUSIONS: Cilostazol reduced the warfarin-related risk of HT after ischemia by protecting the vascular endothelial cells. This result suggested that cilostazol administration in patients with acute ischemic stroke might reduce HT.
Assuntos
Anticoagulantes/efeitos adversos , Isquemia Encefálica/tratamento farmacológico , Hemorragia Cerebral/induzido quimicamente , Hemorragia Cerebral/prevenção & controle , Inibidores da Fosfodiesterase 3/farmacologia , Tetrazóis/farmacologia , Varfarina/efeitos adversos , Animais , Anticoagulantes/farmacologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/mortalidade , Cilostazol , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Masculino , Camundongos , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Fatores de Tempo , Varfarina/farmacologiaRESUMO
Oxidative stress plays a critical role in mediating tissue injury and neuron death during ischemia-reperfusion injury (IRI). The Keap1-Nrf2 defense pathway serves as a master regulator of endogenous antioxidant defense, and Nrf2 has been attracting attention as a target for the treatment of IRI. In this study, we evaluated Nrf2 expression in IRI using OKD (Keap1-dependent oxidative stress detector) mice and investigated the neuroprotective ability of an Nrf2 activator. We demonstrated temporal changes in Nrf2 expression in the same mice with luciferase assays and an Nrf2 activity time course using Western blotting. We also visualized Nrf2 expression in the ischemic penumbra and investigated Nrf2 expression in mice and humans using immunohistochemistry. Endogenous Nrf2 upregulation was not detected early in IRI, but expression peaked 24h after ischemia. Nrf2 expression was mainly detected in the penumbra, and it was found in neurons and astrocytes in both mice and humans. Intravenous administration of the Nrf2 activator bardoxolone methyl (BARD) resulted in earlier upregulation of Nrf2 and heme oxygenase-1. Furthermore, BARD decreased infarction volume and improved neurological symptoms after IRI. These findings indicate that earlier Nrf2 activation protects neurons, possibly via effects on astrocytes.
Assuntos
Isquemia Encefálica/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Western Blotting , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Imunoprecipitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Traumatismo por Reperfusão/patologiaRESUMO
Diabetes is a crucial risk factor for stroke and is associated with increased frequency and poor prognosis. Although endothelial dysfunction is a known contributor of stroke, the underlying mechanisms have not been elucidated. The aim of this study was to elucidate the mechanism by which chronic hyperglycemia may contribute to the worsened prognosis following stroke, especially focusing on mitochondrial alterations. We examined the effect of hyperglycemia on hemorrhagic transformation at 24 hours after middle cerebral artery occlusion (MCAO) in streptozotocin (STZ) -induced diabetic mice. We also examined the effects of high-glucose exposure for 6 days on cell death, mitochondrial functions and morphology in human brain microvascular endothelial cells (HBMVECs) or human endothelial cells derived from induced pluripotent stem cells (iCell endothelial cells). Hyperglycemia aggravated hemorrhagic transformation, but not infarction following stroke. High-glucose exposure increased apoptosis, capase-3 activity, and release of apoptosis inducing factor (AIF) and cytochrome c in HBMVECs as well as affected mitochondrial functions (decreased cell proliferation, ATP contents, mitochondrial membrane potential, and increased matrix metalloproteinase (MMP)-9 activity, but not reactive oxygen species production). Furthermore, morphological aberration of mitochondria was observed in diabetic cells (a great deal of fragmentation, vacuolation, and cristae disruption). A similar phenomena were seen also in iCell endothelial cells. In conclusion, chronic hyperglycemia aggravated hemorrhagic transformation after stroke through mitochondrial dysfunction and morphological alteration, partially via MMP-9 activation, leading to caspase-dependent apoptosis of endothelial cells of diabetic mice. Mitochondria-targeting therapy may be a clinically innovative therapeutic strategy for diabetic complications in the future.