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1.
J Neurosci Methods ; 249: 99-105, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25936850

RESUMO

BACKGROUND: Stroke is the third leading cause of death and the leading cause of long-term disability in North America. On average, someone in the US has a stroke every 45 s, and worldwide, stroke claims 15 million lives each year. Therefore, reliable stroke models are vital to the production of effective new therapies for the treatment of this devastating cerebral vascular accident. NEW METHOD: Middle cerebral artery occlusion (MCAo) is considered to be the most clinically relevant surgical model of ischemic stroke, in which a variety of methods may be employed to block the MCA (the most common being through insertion of a monofilament). In this study, we have compared two different approaches that are currently used arbitrarily in various laboratories worldwide: one involving insertion of a monofilament via the common carotid artery (Koizumi et al.) and one via the external carotid artery (Longa et al.). RESULTS AND COMPARISONS WITH EXISTING METHODS: We assessed various parameters, including: mortality rates, neurological scores, inflammation levels, cellular trafficking (using intravital microscopy) and infarct volumes in mice after using each of the two approaches. We found that the Longa method produced a greater, and robust, inflammatory response, versus the Koizumi method. CONCLUSIONS: In conclusion, we suggest that the Longa method is superior for the study of both short and long-term outcomes of ischemic stroke. These results have considerable implications on stroke model selection for researchers.


Assuntos
Infarto da Artéria Cerebral Média/cirurgia , Acidente Vascular Cerebral/cirurgia , Procedimentos Cirúrgicos Vasculares/métodos , Animais , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Acidente Vascular Cerebral/etiologia , Procedimentos Cirúrgicos Vasculares/instrumentação
2.
Am J Physiol Heart Circ Physiol ; 296(3): H689-97, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19112095

RESUMO

Hypercholesterolemia is associated with phenotypic changes in endothelial cell function that lead to a proinflammatory and prothrombogenic state in different segments of the microvasculature. CD40 ligand (CD40L) and its receptor CD40 are ubiquitously expressed and mediate inflammatory responses and platelet activation. The objective of this study was to determine whether CD40/CD40L, in particular T-cell CD40L, contributes to microvascular dysfunction induced by hypercholesterolemia. Intravital microscopy was used to quantify blood cell adhesion in cremasteric postcapillary venules, endothelium-dependent vasodilation responses in arterioles, and microvascular oxidative stress in wild-type (WT) C57BL/6, CD40-deficient ((-/-)), CD40L(-/-), or severe combined immune deficient (SCID) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 wk. WT-HC mice exhibited an exaggerated leukocyte and platelet recruitment in venules and impaired vasodilation responses in arterioles compared with ND counterparts. A deficiency of CD40, CD40L, or lymphocytes attenuated these responses to HC. The HC phenotype was rescued in CD40L(-/-) and SCID mice by a transfer of WT T cells. Bone marrow chimeras revealed roles for both vascular- and blood cell-derived CD40 and CD40L in the HC-induced vascular responses. Hypercholesterolemia induced an oxidative stress in both arterioles and venules of WT mice, which was abrogated by either CD40 or CD40L deficiency. The transfer of WT T cells into CD40L(-/-) mice restored the oxidative stress. These results implicate CD40/CD40L interactions between circulating cells and the vascular wall in both the arteriolar and venular dysfunction elicited by hypercholesterolemia and identify T-cell-associated CD40L as a key mediator of these responses.


Assuntos
Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Hipercolesterolemia/imunologia , Inflamação/imunologia , Músculo Esquelético/irrigação sanguínea , Linfócitos T/imunologia , Transferência Adotiva , Animais , Arteríolas/imunologia , Plaquetas/imunologia , Transplante de Medula Óssea , Antígenos CD40/deficiência , Antígenos CD40/genética , Ligante de CD40/deficiência , Ligante de CD40/genética , Adesão Celular , Citocinas/sangue , Modelos Animais de Doenças , Endotélio Vascular/imunologia , Hipercolesterolemia/complicações , Hipercolesterolemia/fisiopatologia , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Microcirculação , Estresse Oxidativo , Transdução de Sinais , Linfócitos T/transplante , Quimeras de Transplante/imunologia , Vasodilatação , Vênulas/imunologia
3.
Free Radic Biol Med ; 43(1): 22-30, 2007 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-17561090

RESUMO

Elevated cholesterol levels promote proinflammatory and prothrombogenic responses in venules and impaired endothelium-dependent arteriolar dilation. Although NAD(P)H oxidase-derived superoxide has been implicated in the altered vascular responses to hypercholesterolemia, it remains unclear whether this oxidative pathway mediates the associated arteriolar dysfunction and platelet adhesion in venules. Platelet and leukocyte adhesion in cremasteric postcapillary venules and arteriolar dilation responses to acetylcholine were monitored in wild-type (WT), Cu,Zn-superoxide dismutase transgenic (SOD-TgN), and NAD(P)H oxidase-knockout (gp91(phox-/-)) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 weeks. HC elicited increased platelet and leukocyte adhesion in WT mice versus ND. Cytosolic subunits of NAD(P)H oxidase (p47phox and p67phox) were expressed in platelets. This was not altered by hypercholesterolemia; however, platelets and leukocytes from HC mice exhibited elevated generation of reactive oxygen species compared to ND mice. Hypercholesterolemia-induced leukocyte recruitment was attenuated in SOD-TgN-HC and gp91(phox-/-)-HC mice. Recruitment of platelets derived from WT-HC mice in venules of SOD-TgN-HC or gp91(phox-/-)-HC recipients was comparable to ND levels. Adhesion of SOD-TgN-HC platelets paralleled the leukocyte response and was attenuated in SOD-TgN-HC recipients, but not in WT-HC recipients. However, gp91(phox-/-)-HC platelets exhibited low levels of adhesion comparable to those of WT-ND in both hypercholesterolemic gp91(phox-/-) and WT recipients. Arteriolar dysfunction was evident in WT-HC mice, compared to WT-ND. Overexpression of SOD or, to a lesser extent, gp91(phox) deficiency restored arteriolar vasorelaxation responses toward WT-ND levels. These findings reveal a novel role for platelet-associated NAD(P)H oxidase in producing the thrombogenic phenotype in hypercholesterolemia and demonstrate that NAD(P)H oxidase-derived superoxide mediates the HC-induced arteriolar dysfunction.


Assuntos
Plaquetas/enzimologia , Hipercolesterolemia/complicações , Glicoproteínas de Membrana/fisiologia , NADPH Oxidases/fisiologia , Trombose/enzimologia , Trombose/etiologia , Animais , Artérias/fisiopatologia , Adesão Celular , Colesterol/sangue , Leucócitos/fisiologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos , NADPH Oxidase 2 , NADPH Oxidases/genética , Fenótipo , Adesividade Plaquetária , Subunidades Proteicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Vasodilatação , Vênulas/fisiopatologia
4.
Microcirculation ; 13(5): 377-88, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16815823

RESUMO

OBJECTIVE: To define the contribution of platelets, specifically platelet-associated P-selectin, to the altered venular and arteriolar responses induced by hypercholesterolemia. METHODS: Leukocyte and platelet recruitment in cremasteric venules, and endothelium-dependent relaxation (EDR) in arterioles were determined using intravital videomicroscopy. Wild-type (WT) mice were placed on a normal or high cholesterol diet. Hypercholesterolemic mice were treated with blocking antibodies against either P-selectin or PSGL-1, or were depleted of neutrophils (ANS) or platelets (APS). Bone marrow chimeras (P-selectin deficiency in platelets, but not in endothelial cells) were produced by transplanting bone marrow from P-selectin-/- into WT mice (P-sel-/---> WT). RESULTS: Hypercholesterolemia (HC) elicited the recruitment of adherent platelets and leukocytes in venules and an impaired EDR in arterioles. The exaggerated cell adhesion responses were absent in hypercholesterolemic mice treated with ANS, anti-P-selectin or anti-PSGL-1 antibodies and in P-sel-/---> WT chimeras. The hypercholesterolemia-induced impairment of arteriolar EDR was significantly blunted in mice rendered either neutropenic or thrombocytopenic, and in P-sel-/---> WT chimeras. CONCLUSIONS: The findings indicate that platelet-associated P-selectin contributes to the recruitment of leukocytes and platelets in venules of hypercholesterolemic mice and that the P-selectin-mediated adhesive interactions also contribute to the impaired arteriolar function induced by hypercholesterolemia.


Assuntos
Arteríolas/fisiopatologia , Plaquetas/fisiologia , Hipercolesterolemia/sangue , Hipercolesterolemia/fisiopatologia , Leucócitos/fisiologia , Animais , Adesão Celular , Movimento Celular , Quimera , Endotélio Vascular/fisiopatologia , Contagem de Leucócitos , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/fisiologia , Selectina-P/genética , Selectina-P/fisiologia , Contagem de Plaquetas
5.
Microcirculation ; 12(2): 183-94, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15828130

RESUMO

OBJECTIVE: Obese patients with sepsis have higher morbidity and mortality than lean counterparts, but the mechanisms involved are unknown. The authors examined the inflammatory and thrombogenic responses of the cerebral microvasculature to sepsis induced by cecal ligation and perforation in obese and lean wild-type mice. METHODS: Leukocyte and platelet adhesion in cerebral microvasculature and behavioral responses were measured in wild-type and obese mice 4 h postperforation. P-selectin expression in different vascular beds was assessed 6 h postperforation. The effects of immunoblockade of P-selectin, ICAM-1, and CD18 on leukocyte and platelet recruitment were evaluated in obese septic animals. RESULTS: Cerebral venules of obese and wild-type mice assumed a proinflammatory and prothrombogenic phenotype 4 h post-perforation, with greatly exaggerated responses in obese mice compared to the lean counterparts. These enhanced responses were attenuated by blocking P-selectin, CD18, or ICAM-1. Obese mice also exhibited a more profound behavioral deficit after sepsis, which appears to be unrelated to the recruitment of leukocytes and platelets. Cecal ligation and perforation-induced P-selectin expression was greater in obese mice compared with lean counterparts. CONCLUSIONS: These findings suggest that the increased morbidity to sepsis in obesity may result from exaggerated microvascular inflammatory and thrombogenic responses that include the activation of endothelial cells with subsequent expression of adhesion molecules, such as P-selectin.


Assuntos
Encéfalo/irrigação sanguínea , Inflamação/etiologia , Microcirculação/fisiopatologia , Obesidade/complicações , Sepse/complicações , Animais , Encéfalo/fisiopatologia , Adesão Celular , Moléculas de Adesão Celular/análise , Endotélio Vascular/patologia , Leucócitos/fisiologia , Camundongos , Camundongos Obesos , Atividade Motora , Selectina-P/análise , Adesividade Plaquetária
6.
Hypertension ; 45(2): 209-15, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15655122

RESUMO

Hypercholesterolemia elicits an inflammatory response in the microvasculature that is accompanied by an increased expression of angiotensin II type-1 receptors (AT1-R) on platelets, leukocytes, and endothelial cells. AT1-R blockade attenuates inflammatory responses to angiotensin II (eg, adhesion molecule expression and reactive oxygen species production). We investigated whether AT1-R antagonism attenuates the platelet and leukocyte recruitment induced by acute hypercholesterolemia in postcapillary venules. Leukocyte and platelet adhesion and oxidative stress were quantified by intravital microscopy in cremaster muscle, and P-selectin and AT1-R expression was determined in mice placed on a normal diet (ND) or high-cholesterol diet (HCD) for 2 weeks. Platelet and leukocyte adhesion was significantly elevated by hypercholesterolemia. In HCD mice receiving losartan (HCD-Los) in drinking water, platelet and leukocyte recruitment was reduced to ND levels. Increased platelet adhesion was observed in HCD mice receiving platelets from HCD-Los mice, consistent with a direct beneficial action of losartan on the vessel wall. Hypercholesterolemia elicited an oxidative stress in venules and an increased expression of P-selectin and AT1-R. The oxidative stress and AT1-R upregulation were reduced by losartan, but the P-selectin response was not. We propose that AT1-R engagement contributes to the prothrombogenic and proinflammatory state induced in venules by hypercholesterolemia.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Hipercolesterolemia/complicações , Losartan/farmacologia , Trombose/prevenção & controle , Vasculite/prevenção & controle , Vênulas/efeitos dos fármacos , Animais , Plaquetas/metabolismo , Movimento Celular , Endotélio Vascular/metabolismo , Hipercolesterolemia/fisiopatologia , Leucócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxidantes/biossíntese , Selectina-P/metabolismo , Adesividade Plaquetária , Receptor Tipo 1 de Angiotensina/sangue , Receptor Tipo 1 de Angiotensina/metabolismo , Trombose/etiologia , Vasculite/etiologia , Vênulas/metabolismo
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