O projeto descrito foi apoiada por números Award HL064761 R01, R25 HL059868, 1SC1 HL099139 e P20 MD000175 forma National Institutes of Health. O conteúdo é da exclusiva responsabilidade dos autores e não representam, necessariamente, a posição oficial do National Institutes of Health.

1. Isolamento da artéria mesentérica de ratos pequenos <ol><li> Anestesiar animais com isoflurano em uma câmara fechada e limpe o abdômen com álcool. </li><li> Realizar uma laparotomia na linha média para expor cama mesentérica. </li><li> Com uma tesoura, retire cerca de 85 cm de intestino com a alimentação vasculatura com a artéria mesentérica superior. Corte a extremidade proximal da seção intestinal perto do piloro e da extremidade distal perto da junção íleo-coecal. Segmentos do intestino são isolados de ratos que estão profundamente anestesiados com isoflurano e submetidos à eutanásia por open-peito punção cardíaca. </li><li> Colocado seção extirpado numa placa de Petri revestidas contendo PSS e realizar a dissecção da artéria mesentérica em temperatura ambiente. </li><li> Pino para baixo a extremidade proximal do intestino no lado direito e pinos para fora o restante da direção intestineina sentido anti-horário (final ieproximal sobre a vasculatura alimentação deixou do outro lado do intestino). </li><li> Dissecar o ramo II e III segmentos juntos com um pedaço do segmento proximal. </li><li> Dissecar a veia e isolar a artéria (com V-shaped ponto de ramificação) e limpá-la através da remoção do tecido adiposo e conjuntivo. Evitar o contato direto com a artéria puxando delicadamente com uma pinça e corte através da membrana do tecido conjuntivo. </li></ol> 2. Montagem do navio <ol><li> Corte dois pequenos segmentos (≈ 4 cm) de um 40 mM de tungstênio sem fio de aço inoxidável e, com uma pinça fina, insira um para o lúmen de cada artéria tomando cuidado para não danificar o endotélio. Use a ponta do fio para abrir a luz, se necessário. Sangue escorrendo para fora da embarcação é um bom sinal o lúmen é aberto. </li><li> Encha a câmara myograph com solução salina fisiológica (PSS; mM: 115 NaCl, 4,7 KCl, 1,4 MgSO 4 .7 H 2 O, 5 NaHCO 3, 1,2 K 2 HPO 4, 1,1 Na 2 HPO 4, 1,0 CaCl 2, 20 e HEPES 5 de glicose, pH 7,4) com ácido ascórbico (100 mM) a 37 ° C e usando fórceps transferir cuidadosamente o segmento de rosca navio da placa de Petri na câmara à temperatura ambiente e transferir o segmento navio excisadas proximal para a câmara de myograph e puxe a final ao longo do fio para alimentá-lo dentro do vaso. Evitar esticar o navio. </li><li> Garantir o fim próximo do fio de sentido anti-horário sob o parafuso de fixação perto na mandíbula do lado direito ligado ao micrômetro. Pegar a extremidade livre do fio com uma pinça e prenda-o no sentido horário sob o parafuso de fixação muito na mandíbula do lado direito. Certifique-se que o segmento de navios ao longo do fio está situado no intervalo entre as mandíbulas sem fazer qualquer contato com a própria mandíbula. </li><li> Parafuso as mandíbulas separadas, e alinhar o segundo fio paralelo com o navio e inseri-lo na extremidade da luz. Suavemente alimentar o fio através do lúmen do segmento de navio em um movimento usando o fio já está montado como um guia. Segure o fio cerca de 1 cm do navio para evitar esticar-la durante a manobra e evite tocar o endotélio. </li><li> Parafuso as mandíbulas juntas e garantir que o segundo move fio debaixo de montagem o primeiro garantido na mandíbula do lado direito. </li><li> Garantir o fim próximo do segundo fio no sentido horário sob o parafuso de fixação perto da mandíbula do lado esquerdo ligado ao transdutor. </li><li> Assegurar a extremidade do fio sob o parafuso de fixação na mandíbula do lado esquerdo e aperte para esticar o fio. </li><li> Uma vez que a montagem está completa, reinicie o motor no &quot;Menu de montagem&quot; e começar a normalização do navio. </li></ol> 3. Normalização O Auto Myograph fio duplo System-510A tem uma função de normalização automatizado, que é avaliada a partir da &quot;normalização&quot; no menu e permite que o navio a ser esticado a uma circunferência normalizado interna por um procedimento padronizado de acordo com o protocolo do fabricante, após equilíbrio por 30 min a 37 ° C. Uma curva exponencial é então ajustada aos dados de pressão interna circunferência. O procedimento define o diâmetro do lúmen (d 100) que a artéria teria in vivo quando relaxado e sob uma pressão transmural de 100 mmHg 1 Os parâmetros de normalização para artéria mesentérica de ratos são as seguintes.: <ol><li> Pressão transmural alvo = 13,3 kPa (100 mm Hg). </li><li> Tempo = 60 seg; Duração de cada uma das etapas de normalização. </li><li> IC 1 / IC 100 = 0,9 (IC 1 = circunferência normalizado interna, IC 100 = circunferência interna correspondente à pressão alvo. </li><li> Calibração ocular 2 * Δ (mm / ocular divisão); 2 delta é por razões de programação. </li></ol> Meça o comprimento da artéria mesentérica montado usando o microscópio de olho peça leituras quando os fios são mais as extremidades longe e de perto do navio montado segment. Resumidamente, o comprimento do segmento de navio montada é medida a ampliação máxima com uma ocular calibrada olho peças no microscópio de dissecação. A leitura do olho peças com cabelo-line sobre a extremidade do segmento (a 1) e perto do final do segmento (a 2) nas divisões ocular são medidos e registados. Estes valores, com as leituras micrômetro, antes e após o alongamento da embarcação são registrados e entrou em menus para o programa para calcular a curva ajustada eo diâmetro interno correspondente à uma meta de pressão transmural de 100 mm Hg Em nossos estudos, as artérias foram criados para o diâmetro do lúmen do d 1 = 0,9 xd 100, onde o desenvolvimento força ativa é máxima. Desenvolvimento da força ativa de ≥ 10 mN em artérias mesentéricas de ratos é considerada ideal para experimentos para prosseguir. Tecidos com menor força ativa foram descartados. 4. Medição de respostas Após a normalização, as propriedades mecânicas e funcionais dos vasos são re-ativado por realizar uma &quot;start-padrão&quot;, que envolve vasos desafiador, com aplicações repetidas (normalmente 2 ou 3) de 5 mM de fenilefrina (PE) para obter contrações reprodutível. A &quot;start padrão&quot; típico em nosso laboratório consiste de uma série de estímulos e os períodos de washout da seguinte forma: <ol><li> Substituir câmara myograph cobrir e começar a aeração com ar de 95% e 5% CO 2. </li><li> Encher a câmara com PSS fresco (ácido ascórbico, contendo 100 mM) </li><li> Navio contrato com 5 mM PE por 5 min e lavar 4 vezes com tampão de PSS. </li><li> Após a última lavagem, recarga com câmara de PSS e esperar 3 min antes de repetir os passos (ii) e (iii). </li></ol> Após o início padrão, a artéria está pronto para o experimento. Relaxamento dos vasos contratados são avaliados pela adição cumulativa de concentrações crescentes de CaCl 2 (0,5-4 mM). Embarcações, quando os inibidores são usados ​​são pré-incubadas com os compostos na câmara myograph por 20 min e presente durante os ensaios. 5. Análise de Dados A &quot;normalização&quot; dos dados para os navios e os traçados força, diretamente convertido em dados de texto, foram analisadas com o programa de 11,0 SigmaPlot gráficos (Systat Software, Point Richmond, CA) e plotados como mostrado na Figura 1. Concentração-resposta de dados, calculados a partir dos traçados, foram analisados ​​através da determinação EC 50 valores a partir de dados experimentais instalados em uma função logística quatro parâmetros no menu de Farmacologia do programa. Comparações entre grupos e dentro dos grupos foram realizadas por uma análise de variância (ANOVA) e as diferenças com 0,05 p &lt;são considerados significativos. 6. Resultados representativos: <table border="1"><tbody><tr><th> Parâmetros </th><th> Wistar </th><th> Dahl sensíveis ao sal </th><th> Dahl sal-resistente </th></tr><tr><td> L 100 (mm) </td><td> 83,26 ± 2,33 </td><td> 91,52 ± 4,67 </td><td> 117,45 ± 8,43 * </td></tr><tr><td> X 1 </td><td> 604,21 ± 41,97 </td><td> 752,24 ± 85,25 </td><td> 745,84 ± 110,09 </td></tr><tr><td> r 2 </td><td> 0,99 ± 0,003 </td><td> 0,97 ± 0,008 </td><td> 0,97 ± 0,007 </td></tr></tbody></table> * A diferença entre os valores para L 100 Wistar e DR são estatisticamente significativas. Shapiro-Wilk teste de normalidade passado (p = 0,588), Teste de Igualdade de Variância passado (p = 0,237). L 100 = diâmetro interno do segmento montado correspondente à pressão transmural alvo. X 1 = A configuração do posicionador micro necessário para esticar o navio montado em sua circunferência interna normalizado (ou seja, o valor da leitura micrômetro em que os experimentos foram realizados). r 2 = coeficiente de regressão para ajuste de (X i, Y i) para uma curva exponencial. NB: O procedimento detalhado está descrito no manual para o Sistema Myograph 510A 6. Tabela 1. Sample Leia-Out dos parâmetros de normalização para os segmentos de artéria mesentérica Wistar, DS e DR de programa básico mostrado no &quot;Menu normalização&quot;. Os valores são médias (± SEM) de 5 animais. * As diferenças entre os valores para L 100 Wistar e DS são estatisticamente significativas (p &lt;0,05). <table border="1"><tbody><tr><th> Wistar </th><th> DS </th><th> DR </th></tr><tr><td> 14,2 ± 0,6 </td><td><strong&gt; 25,8 ± 2,7 * </td><td> 20,9 ± 1,3 * </td></tr></tbody></table> Tabela 2. Tensões Desenvolvidos (mN) em artérias mesentéricas isoladas de Wistar, DS e ratos DR, na sequência de pedidos de 5 mM PE para cada navio. Valores reportados são meios (± SEM) de 6-8 animais. * Significativamente diferente do Wistar controles (p &lt;0,05). <img alt="Figura 1" src="/files/ftp_upload/3119/3119fig1.jpg" /> Figura 1. Ca 2 + e relaxamento induzido de uma artéria mesentérica PE-contratados a partir de um rato Wistar montado em um myograph fio em solução salina fisiológica contendo 1 mM Ca 2 +. A artéria foi equilibrada por 30 min a 37 ° C com aeração constante e respostas para Ca 2 + e determinado. Uma força representativa de rastreamento em adição de 5 mM PE seguido por adições acumulada de Ca 2 + é mostrada. <img alt="Figura 2" src="/files/ftp_upload/3119/3119fig2.jpg" /> Figura 2. Ca 2 + e relaxamento induzido de uma artéria mesentérica PE-contratados a partir de um rato DS montado em um myograph fio em solução salina fisiológica contendo 1 mM Ca 2 +. A artéria foi equilibrada por 30 min a 37 ° C com aeração constante e respostas para Ca 2 + e determinado. Uma força representativa de rastreamento em adição de 5 mM PE seguido por adições acumulada de Ca 2 + é mostrada. Ca 2 + relaxamento foi severamente comprometida neste tecido em comparação com aqueles de ratos Wistar e DR. <img alt="Figura 3" src="/files/ftp_upload/3119/3119fig3.jpg" /> Figura 3. Ca 2 + e relaxamento induzido de uma artéria mesentérica PE-contratados a partir de um rato DR montado em um myograph fio em solução salina fisiológica contendo 1 mM Ca 2 +. A artéria foi equilibrada por 30 min a 37 ° C com aeração constante e respostas para Ca 2 + e determined.A força representativa de rastreamento em adição de 5 mM PE seguido por adições acumulada de Ca 2 + é mostrada. <img alt="Figura 4" src="/files/ftp_upload/3119/3119fig4.jpg" /> Figura 4. A. [Ca 2 +] e-resposta curvas. B. O gráfico de barras mostrando EC 50 valores para o relaxamento determinado pelo ajuste dos dados para um quatro expressão curve.C.CaR parâmetro logística em arcadas mesentéricas isoladas de ratos DR e DS.

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A hipertensão é uma das principais causas de morbidade cardiovascular, renal e cerebral / mortalidade. A ocorrência de hipertensão é alta na população e hipertensão sal-sensível é particularmente elevada na população idosa, e mais prevalente em negros do que brancos. Este é acreditado para ser devido à tendência dos negros para reter sódio em seus rins 9. Sal-sensibilidade é um fator importante que contribui para a doença renal e está associada à disfunção endotelial, mas o mecanismo nã...

<table border="1"><tbody><tr><th> Nome de equipamentos / reagentes </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Auto Myograph fio duplo System-510A DMT-EUA, Inc. </td><td> Atlanta, GA. </td><td> 100151 </td><td></td></tr><tr><td> PowerLab/4SP Sistema de Aquisição de Dados </td><td> ADInstruments, Colorado Springs, CO </td><td> ML750 </td><td> Novos modelos com 4-16 canais de entrada estão disponíveis. </td></tr><tr><td> Dell Dimension XPS Gen 4 Computador </td><td> Dell </td><td></td><td></td></tr><tr><td> Stemi SV II (Apo) microscópio de dissecção com Ocular </td><td> Carl Zeiss International. </td><td></td><td></td></tr><tr><td> Wistar, Dahl sensíveis ao sal, Dahl sal-resistentes ratos </td><td> Harlan Sprague Dawley, Indianapolis, IN. </td><td></td><td></td></tr><tr><td> Para roedores </td><td> Harland Tekland, Madison, WI. </td><td></td><td></td></tr><tr><td> Fenilefrina </td><td> Sigma-Aldrich, St. Louis, MO. </td><td></td><td></td></tr><tr><td> Outros produtos químicos </td><td></td><td></td><td> Todos os produtos químicos utilizados foram da mais pura notas disponíveis comercialmente. </td></tr></tbody></table>

mesenteric artery contraction and relaxation studies using automated wire myography

Vasos de resistência proximal, tais como as artérias mesentéricas, contribuem substancialmente para a resistência periférica. Esses pequenos vasos entre 100-400 mM em função de diâmetro principalmente no direcionamento do fluxo de sangue para vários órgãos de acordo com os requisitos gerais do corpo. A artéria mesentérica de ratos tem um diâmetro superior a 100 mm. A técnica Miografia, primeiramente descrita por Mulvay e Halpern 1, foi baseada no método proposto por Bevan e Osher 2. A técnica fornece informações sobre navios de pequeno porte em condições isométricas, onde o encurtamento substancial da preparação muscular é impedida. Desde produção de força e sensibilidade de navios para diferentes agonistas é dependente da extensão do trecho, de acordo com a relação tensão-comprimento ativo, é essencial a realização de estudos de contração isométrica em condições para impedir o cumprimento dos fios de montagem. Fios de aço inoxidável são preferíveis aos fios de tungstênio por causa da oxidação deste último, que afeta as respostas registradas 3. A técnica permite a comparação de agonista-induzida contrações dos vasos montados para obter provas para a função normal de receptores vascular das células musculares lisas. Temos demonstrado em vários estudos que artérias mesentéricas isoladas que são contratados com phenylyephrine relaxar com a adição de concentrações cumulativas de cálcio extracelular (Ca 2 + e). As descobertas levaram-nos a concluir que os nervos sensoriais perivascular, que expressam a proteína G acoplada Ca 2 + receptor-sensing (CaR), mediar esta resposta vasorelaxamento. Utilizando um método automatizado Miografia fio, mostramos aqui que artérias mesentéricas de Wistar, Dahl sensíveis ao sal (DS) e Dahl sal-resistentes (DR) ratos respondem diferentemente ao Ca 2 + e. Tecidos de ratos Wistar mostrou Ca 2 + maior sensibilidade em comparação com os de DR e DS. CaR expressão reduzida em artérias mesentéricas de ratos DS correlaciona-se com Ca 2 + reduzida de relaxamento e-induzida de isoladas, pré-contraídos artérias. Os dados sugerem que o carro é necessário para o relaxamento das artérias mesentérica sob tônus ​​adrenérgico aumentado, como ocorre na hipertensão, e indicar um defeito inerente na via de sinalização CaR em animais Dahl, que é muito mais grave no DS. O método é útil para determinar a reatividade vascular ex vivo nas artérias mesentéricas de resistência e similares pequenos vasos sanguíneos e comparações entre diferentes agonistas e / ou antagonistas podem ser facilmente e constantemente avaliada lado a lado 6,7,8.

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Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Um método automatizado para medições Miografia vigor em artérias mesentéricas isoladas é descrito. Ele emprega um Mulvany-Halpern Auto Myograph fio duplo 510A para determinar as respostas à fenilefrina e cálcio extracelular. O método permite a determinação consistente de respostas isométrica aos agonistas em pequenos vasos de diâmetros de 60 - 300 mm, de forma independente.

A contração da artéria mesentérica e Estudos Relaxamento Usando Miografia Fio Automated

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

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JoVE Journal

Medicine

28.4K visualizações.  North Carolina Central University, Durham. Um método automatizado para medições Miografia vigor em artérias mesentéricas isoladas é descrito. Ele emprega um Mulvany-Halpern Auto Myograph fio duplo 510A para determinar as respostas à fenilefrina e cálcio extracelular. O método permite a determinação consistente de respostas isométrica aos agonistas em pequenos vasos de diâmetros de 60 - 300 mm, de forma independente.

Vídeo: Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

Avaliando Miogênica Resposta e vasoactividade em artérias mesentéricas de resistência Usando Miografia Pressão

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Medicina

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

Isolando e com cortes de bovinos artéria e veia mesentérica como Bioassay teste para vasoactividade no intestino delgado

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Modelo murino de artéria femoral Injury fio com a implantação de uma droga patch Entrega perivascular

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

Um modelo murino de Arterial Restenosis: Aspectos Técnicos do Femoral Injury Fio

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Indução de Aterosclerose Acelerada em Camundongos: O Modelo de "Lesão por Fio"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

Modelo de Doença Cardíaca Isquêmica e Comparação Baseada em Vídeo de Contração de Cardiomiócitos usando cardiomiócitos derivados do hiPSC

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Mechanical Control of Relaxation Using Intact Cardiac Trabeculae

Diastolic dysfunction is a common phenotype across cardiovascular disease presentations. In addition to elevated cardiac stiffness (elevated left ventricular end-diastolic pressure), impaired cardiac relaxation is a key diagnostic indicator of diastolic dysfunction. While relaxation requires the removal of cytosolic calcium and deactivation of sarcomeric thin filaments, targeting such mechanisms has yet to provide effective treatments. Mechanical mechanisms, such as blood pressure (i.e., afterload), have been theorized to modify relaxation. Recently, we showed that modifying the strain rate of a stretch, not afterload, was both necessary and sufficient to modify the subsequent relaxation rate of myocardial tissue. The strain rate dependence of relaxation, called the mechanical control of relaxation (MCR), can be assessed using intact cardiac trabeculae. This protocol describes the preparation of a small animal model, experimental system and chamber, isolation of the heart and subsequent isolation of a trabecula, preparation of the experimental chamber, and experimental and analysis protocols. Evidence for lengthening strains in the intact heart suggests that MCR might provide new arenas for better characterization of pharmacological treatments, along with a method to assess myofilament kinetics in intact muscles. Therefore, studying the MCR may elucidate a path to novel approaches and new frontiers in the treatment of heart failure.

Rapid myocardial and cardiac relaxation is essential for normal physiology. Mechanical relaxation mechanisms are now known to be dependent on strain rate. This protocol provides an overview of the acquisition and analysis of experiments to further study the mechanical control of relaxation.

Controle mecânico do relaxamento utilizando trabéculas cardíacas íntegras

Diastolic dysfunction is a common phenotype across cardiovascular disease presentations. In addition to elevated cardiac stiffness (elevated left ...

Multimodality Diagnosis of Mesenteric Ischemia

Early diagnosis of mesenteric ischemia remains challenging because mesenteric ischemia presents with no key symptoms or physical findings, and no laboratory data specifically indicates intestinal tissue ischemic status before necrosis develops. While computed tomography is the standard for diagnostic imaging, there are several limitations: (1) repeated assessments are associated with increased radiation exposure and risk of renal damage; (2) the computed tomography findings can be misleading because necrosis occasionally occurs despite opacified mesenteric arteries; and (3) computed tomography is not necessarily available within the golden time of salvaging the intestines for those patients in the operating room or at a place far from the hospital. This article describes a challenge to overcome such limitations using ultrasonography and near-infrared light, including clinical studies. The former is capable of providing not only morphologic and kinetic information of the intestines but also perfusion of the mesenteric vessels in real-time without transferring the patient or exposing them to radiation. Transesophageal echocardiography enables precise assessment of mesenteric perfusion in the OR, ER, or ICU. Representative findings of mesenteric ischemia in seven aortic dissection cases are presented. Near-infrared imaging with indocyanine green helps visualize the perfusion of vessels and intestinal tissues although this application requires laparotomy. Findings in two cases (aortic aneurysm) are shown. Near-infrared spectroscopy demonstrates oxygen debt in the intestinal tissue as digital data and can be a candidate for early detection of mesenteric ischemia without laparotomy. The accuracy of these assessments has been confirmed by intraoperative inspections and postoperative course (prognosis).

This article presents a multimodal approach that aims to overcome the limitations of traditional methods in detecting mesenteric ischemia and preventing bowel necrosis. The presented technique offers a promising solution by combining state-of-the-art ultrasonography with cutting-edge near-infrared light technologies.

Diagnóstico Multimodalidade da Isquemia Mesentérica

Early diagnosis of mesenteric ischemia remains challenging because mesenteric ischemia presents with no key symptoms or physical findings, and no ...

Rodent Model of Intestinal Ischemia-Reperfusion Injury via Occlusion of the Superior Mesenteric Artery

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, such as gastric dilatation volvulus (GDV), mesenteric torsion, and colic, are observed in animals such as dogs and horses. An initial interruption of blood flow causes tissues to become ischemic. Although necessary to salvage viable tissue, subsequent reperfusion can induce further injury. The main mechanism responsible for IRI is free radical formation upon reperfusion and reintroduction of oxygen into damaged tissue, but there are many other components involved. The resulting local and systemic effects often impart a poor prognosis. 
Intestinal IRI has been the subject of extensive research over the past 50 years. An in vivo rodent model in which the base of the superior mesenteric artery (SMA) is temporarily ligated is currently the most common method used to study intestinal IRI. Here, we describe a model of intestinal IRI utilizing isoflurane anesthesia in 21% O2 medical air that yields reproducible injury, as demonstrated by consistent histopathology of the small intestines. Tissue injury was also assessed in the colon, liver, and kidneys.

We describe how to generate a widely used surgical model of intestinal ischemia-reperfusion injury (IRI) in rodents. The procedure involves occlusion of the superior mesenteric artery followed by the restoration of blood flow. This model is useful for studies investigating occlusive causes of intestinal IRI in both veterinary and human medicine.

Modelo de lesão de isquemia-reperfusão intestinal por roedores via oclusão da artéria mesentérica superior

Intestinal ischemia-reperfusion injury (IRI) is associated with a myriad of conditions in both veterinary and human medicine. Intestinal IRI conditions, ...