Agradecemos He de Qi de Wei de Dr. (Universidade de Soochow, Suzhou, China) e Dr. Yan Ning Qiao (Shaanxi Normal University, Xi ' an, China) pela assistência técnica. Este trabalho foi apoiado pela Fundação Nacional de ciências naturais da China (Grant, 31272311, 81373295 e 81473420) e o projeto financiado pela prioridade acadêmica programa desenvolvimento de Jiangsu instituições de ensino superior (Grant no. ysxk-2016).

Manipulações de animais foram aprovadas pelo cuidado institucional do Animal e Comissão de utilização (IACUC) da Universidade modelo Animal Research Center de Nanjing.
1. preparação da solução
<ol><li>Preparar a solução de Tyrode-HEPES (H-T) usando 137,0 mM NaCl, 2,7 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2∙6H2O, 5,6 mM D-glicose e 10 mM HEPES, pH 7.3-7.4.</li>
	<li>Preparar a solução de Tyrode HEPES sem cálcio (Ca2 +-livre H-T) usando 140,6 mM NaCl, 2,7 mM KCl, 1 mM MgCl2∙6H2O, 5,6 mM D-glicose e 10 mM HEPES, pH 7.3-7.4.</li>
	<li>Preparar a solução de Tyrode HEPES usando 124 mM KCl (alta K+) usando 15,7 mM NaCl, 124,0 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2∙6H2O, pH de 5,6 mM D-glicose e 10 mM HEPES, 7.3-7.4.</li>
</ol>2. preparação do experimento
<ol><li>Pré-aqueça o H-T e High-K+ soluções usando banho maria a 37 ° C.</li>
	<li>Ligue o sistema de myograph, o hardware de aquisição de dados e o computador.</li>
	<li>Com cuidado, encha todas as câmaras de myograph com 5 mL de solução de H-T cada.</li>
	<li>Preencher dois pratos de Petri com 20 mL de 4 ° C H-T e Ca2 +-livre soluções H-T, respectivamente e armazenar no gelo.</li>
	<li>Encha um prato de Petri 10 cm revestido com 20 mL de solução de H-T e mantê-lo em temperatura ambiente.</li>
</ol>3. Mouse dissecação de artéria mesentérica
<ol><li>Eutanásia em um 8-12-week-old C57BL/6J masculino ou feminino do mouse por deslocamento cervical. Segure o mouse com o abdômen voltado para cima.</li>
	<li>Umedeça o abdômen com etanol a 70%. Em seguida, cortar a pele com a tesoura ao longo da linha mediana ventral da virilha e fazer incisões desde o início da primeira incisão para baixo para as pernas em ambos os lados. Puxamos a pele para trás em ambos os lados; Faça incisões semelhantes para abrir o peritônio.</li>
	<li>Com uma tesoura, corte o esófago, cólon e outros tecidos conectivos para isolar completamente o trato gastrointestinal com alimentação vasculatura do corpo.</li>
	<li>Com fórceps, mova o segmento isolado na cápsula contendo o frio H-T preparado no passo 2.4 e delicadamente, lave o tecido na solução H-T várias vezes para lavar o sangue.</li>
	<li>Transferir o segmento isolado para o prato de Petri revestida, preparado na etapa 2.5 e faz dissecação da artéria mesentérica em temperatura ambiente.</li>
	<li>Suavizar o estômago, jejuno, íleo e ceco no sentido horário, e fixar o estômago e o ceco no lado esquerdo e direito, respectivamente.</li>
	<li>Esticar a cama da vasculatura mesentérica e corrigir o intestino com pinos para expor as artérias mesentéricas dissecadas. Nota: Nestas condições, as artérias estão no topo das veias.</li>
	<li>Ligue a fonte de transmissão de luz de um microscópio estereoscópico e dissecar as artérias sob o microscópio. Certifique-se de que o tecido inteiro está imerso na solução.</li>
	<li>Prenda os tecidos adiposo em torno das artérias com fórceps e isolar as artérias cortando todos os tecidos conjuntivos com tesoura de dissecação. Evite ferir as artérias.</li>
</ol>4. arterial montagem
<ol><li>Transferência e imergir a árvore de artéria mesentérica o frio Ca2 +-livre solução do H-T (preparada na etapa 2.4), fixando as artérias em excesso com a pinça.</li>
	<li>Corte uma parte de 1,4 mm da artéria proximal à parede intestinal de uma arcada mesentérica e usar duas pinças para abrir ambos os lados deste segmento de artéria cuidadosamente.</li>
	<li>Prepare-se dois segmentos de 2,5 cm de comprimento de fio de aço inoxidável e colocá-los no mesmo prato.</li>
	<li>Delicadamente, prenda uma extremidade da artéria usando fórceps e Introduza cuidadosamente os dois fios no lúmen da artéria um por um com a ajuda de outra pinça. Certifique-se que os fios são mantidos em linha reta e não toque o endotélio.</li>
	<li>Usando duas pinças, prenda os dois fios de aço fora do navio roscado simultaneamente e transferir cuidadosamente o navio do prato de Petri a uma câmara de myograph anteriormente preenchida com solução de H-T (passo 2.3).</li>
	<li>Que se lixe os mordentes separados para fazer espaço para montagem. Prenda os dois lados de um dos dois fios inseridos usando duas pinças e coloque o recipiente na abertura mandíbula (figura 1A).</li>
	<li>Envolva ambos os lados do fio preso por parafusos da mandíbula conectado para o micrômetro (figura 1B).</li>
	<li>Fixe o parafuso esquerdo girando no sentido horário. Endireitar o fio usando fórceps da direita e, em seguida, fixar o parafuso da direita girando no sentido horário (Figura 1). Certifique-se de que o navio está sempre dentro da abertura da mandíbula, mas não toque no queixo para evitar danos.</li>
	<li>Feche as duas mandíbulas usando o micrômetro (Figura 1). Certifique-se que as duas garras estão perto o suficiente, mas que não tocam uns aos outros e que o fio não fixado na parte superior do fio fixo.</li>
	<li>Usando a pinça direita, cuidadosamente dobre o fio retirado no canto da mandíbula conectado para forçar o transdutor e envolvê-la no sentido horário em torno do parafuso do lado direito (Figura 1E). Em seguida, fixe o parafuso. Repita essa etapa no lado esquerdo do fio e fixar o parafuso do lado esquerdo (Figura 1F).</li>
	<li>Mova a mandíbula ligeiramente afastada girando cuidadosamente o micrômetro (Figura 1). Evite esticar o navio. Use a pinça para mover o fio no lado de micrômetro para o plano horizontal do fio ao lado do transdutor. Gire o micrômetro cuidadosamente para que a lacuna entre as duas garras só pode acomodar os dois fios.</li>
	<li>Repita as etapas 4.2 – 4.11 para montar as artérias para as outras câmaras. Conecte todas as câmaras para o equipamento, cobrir as câmaras, anexar o fornecimento de oxigênio a 100% e uma sonda de temperatura e começar aquecendo-se a 37 ° C. Abra o software de gráfico e pressione o botão Iniciar na janela de Visualização de gráfico para iniciar a gravação.</li>
	<li>Equilibrar-se por cerca de 20 min.</li>
</ol>5. normalização
Nota: A fim de padronizar as condições experimentais e obter a capacidade de resposta fisiológica confiável dos navios, um processo de normalização é necessário15. De acordo com a relação entre a força activa e a circunferência interna do navio, o sistema de myograph do fio tem um programa de normalização de padrão para avaliar a circunferência interna (IC) do navio montado5,8, 9. Brevemente, calcular IC (µm), ler o micrômetro e o valor de entrada como o valor de X e o transdutor de saída de força, ou seja, descansando a tensão parede (mN/mm), como o valor de Y. O programa irá retornar uma curva equipada de (X, Y) e calcular o IC correspondente à pressão transmural de 100 mmHg (IC100). A embarcação é definida como a circunferência interna normalizada (IC1) quando a capacidade de resposta ativa é máxima.
<ol><li>Conjunto de forças para zero para todos os canais no dispositivo e equilibrar por mais 1-2 minutos.</li>
	<li>Selecione configurações de normalização do "menu DMTe configurar os parâmetros da seguinte forma: Calibração de ocular (mm/div): 0,36; Alvo de pressão (kPa): 13,3; IC1/IC100: 0,9; On-line com média de tempo (segundos): 2; Atraso de tempo (segundos): 60. Clique no botão para fechar a janela de Configurações de normalização de DMT Okey .</li>
	<li>Selecione o canal de interesse do menu de DMT para abrir uma janela de normalização de DMT para o canal correspondente. Inserir os valores constantes na janela da seguinte forma: a1 de pontos de extremidade de tecido: 0.1; A2 de pontos de extremidade de tecido: 4; Diâmetro de fio (µm): 40. A janela exibe o comprimento do navio calculado como 1,40 mm.</li>
	<li>Leia o micrômetro da câmara de tecido apropriado. Digite o valor na caixa de leitura do micrômetro e clique o botão Adicionar ponto . Esse valor é o valor inicial de X (X0). Depois de um tempo de atraso 60 s, a janela exibe a força e a pressão eficaz (ERTP) correspondente a esse valor de micrômetro. Simultaneamente, a caixa de leitura micrômetro se torna ativa.</li>
	<li>Estica o navio sendo normalizado girando o micrômetro no sentido anti-horário. Digite o valor de micrômetro na caixa de leitura do micrômetro e clique o botão Adicionar ponto . Esperar por um tempo de atraso de 60 s novamente.</li>
	<li>Repita a etapa 5.5, continuar a esticar o navio e adicionar valores de micrômetro até a janela exibe o valor de "micrômetro X1", que é a configuração de micrômetro calculado necessária para esticar a embarcação para sua IC1.</li>
	<li>Defina o micrômetro para X o valor1 . Nota: A tensão normalizada é geralmente 1-2 mN.</li>
</ol>6. artéria contração gravação
Nota: Todas as soluções, incluindo o H-T e High-K+ solução usado nesta seção, foram preparadas na etapa 2.1.
<ol><li>Após a normalização, equilibrar o navio na câmara para 15-20 min. Nota: Estas é nenhuma necessidade de mudar a solução nesta etapa.</li>
	<li>Desafie o recipiente com solução High-K+ duas vezes.<ol>
<li>Para desafiar o navio, substitua a solução H-T com 5 mL de solução de High-K+ para induzir a contração por 10 min, seguido por lavagem com 5 mL de solução de H-T 3 - 4 vezes. Nota: Contração típica tem uma força máxima sobre 3 mN e uma força constante e sustentada em torno de 2,5 mN12. Se o primeiro desafio gera uma força máxima abaixo de 2,5 mN ou a força sustentada diminui com o tempo ou o segundo desafio gera uma força muito menor do que a dose pela primeira vez, o navio é Descartado e não será usado para investigação futura.</li>
	</ol>
</li>
	<li>Desafie o vaso com 5 mL de solução de High-K+ para induzir a contração. Depois de 5 min, adicione 0,5 µ l de solução-mãe (10mm em DMSO) neoliensinine14 na câmara para relaxar o navio em uma concentração final de 1 µM de neoliensinine.</li>
	<li>Quando a força é estável (isto geralmente leva vários minutos), adicionar outra 0,5 μL de solução-mãe de neoliensinine na câmara para aumentar a concentração de µ l 2 µM. adicionar 1 da solução-mãe cada vez para aumentar a concentração de 4, 6, 8 e 10 µM para gerar t curva de dose-resposta. Nota: O estoque e as concentrações de trabalho variam entre drogas.</li>
</ol>

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Hipertensão é um desafio da saúde pública generalizada devido a suas complicações graves, incluindo cardiovascular e renal doenças16. Compreender a patogênese da hipertensão e explorar mais anti-hipertensivos drogas tornou-se uma tarefa urgente neste campo. Pressão arterial é gerado e mantido pela resistência periférica da circulação. De acordo com a lei de Poiseuille, as relativamente pequenas artérias geram uma grande proporção de resistência circulatória e servem como o prod...

O sistema de myograph de pequeno vaso usado aqui foi para medir a contração isométrica dos vasos de resistência pequena com diâmetro interno que variam de 100 a 400 µm. isolados pequenos vasos (cerca de 2 mm de comprimento) foram inseridos por dois fios de diâmetro de 40 µm e foram então montado sobre as garras do micrômetro-lado e lado do transdutor sequencialmente. Esta técnica de myograph foi sugerida primeiramente em 19721 e então desenvolvida principalmente por Mulvany e seus colegas2,3,4,5,6. Agora é uma técnica madura com equipamento estável, desempenho fácil e um padrão de normalização procedimento7,8,9. Utilizamos este método com algumas modificações para medições na artéria mesentérica de rato.
Músculo liso vascular as linhas das paredes de quase todos os vasos sanguíneos. Sua função fundamental é gerar forças através da contração em resposta a vários estímulos. A contratilidade normal do músculo liso vascular é essencial para a regulação da pressão de sangue e nutrição complementar10. Regulamento anormal da pressão arterial resulta em uma variedade de doenças, incluindo hipertensão, insuficiência cardíaca e isquemia. Vários estudos têm sugerido que a pressão anormal de sangue sempre é associada com disfuncional músculo liso vascular contratilidade7,11,12,13. O método myograph permite investigação da contratilidade isométrica dos navios de rato induzida por vários estímulos, incluindo vasoconstritores, inibidores e drogas. Medições bem sucedidas de contração nos ajudará a compreender os mecanismos de manutenção da pressão arterial e a patogênese de doenças associadas a músculo liso vasculares e explorar novas abordagens terapêuticas.
Muitas ervas chinesas têm sido amplamente utilizadas para tratamento clínico de doenças vasculares; no entanto, seus ingredientes eficazes geralmente permanecem desconhecidos. Assim, o isolamento e identificação dos componentes eficazes é muito importante para o desenvolvimento de novos medicamentos. Tecnologia multi fio myograph oferece uma abordagem simples para a seleção de componentes ativos em ervas. Nós temos relatado vários estudos utilizando o sistema de myograph de pequeno vaso para investigar a contração de artéria mesentérica de rato e identificados compostos naturais com atividade anti-hipertensão12,13,14. Aqui, descrevemos o detalhada do protocolo para o método de myograph e avaliar o efeito relaxante do neoliensinine isolada de embriões de sementes de Lótus (Nelumbo nucifera Gaertn.) 14.

<table>
	<tbody>
		<tr>
			<td>Multi wire myograph system</td>
			<td>DMT</td>
			<td>610-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel wire</td>
			<td>DMT</td>
			<td>400447</td>
			<td></td>
		</tr>
		<tr>
			<td>Geuder dissection scissor</td>
			<td>DMT</td>
			<td>400431</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps</td>
			<td>DMT</td>
			<td>300413</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab/8SP</td>
			<td>ADInstruments</td>
			<td>ML785</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>ADInstruments</td>
			<td>LabChart 5</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>SigmaAldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>SigmaAldrich</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>SigmaAldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>SigmaAldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>SigmaAldrich</td>
			<td>G6152</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sangon Biotech</td>
			<td>A100511-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>SigmaAldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>SigmaAldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
	</tbody>
</table>

isometric contractility measurement of the mouse mesenteric artery using wire myography

A técnica de myograph do fio é usada para avaliar a contratilidade da musculatura de Lisa vascular em resposta à despolarização, GPCR agonistas/inibidores e drogas. É amplamente utilizado em muitos estudos sobre as funções fisiológicas do músculo liso vascular, a patogênese de doenças vasculares, como hipertensão e desenvolvimento de fármacos relaxante músculo liso. O rato é um animal modelo amplamente utilizado com uma grande piscina de doença modelos e variedades geneticamente modificadas. Nós introduzimos esse método para medir a contração isométrica de artéria mesentérica de rato em detalhe. Um segmento de 1,4 mm da artéria mesentérica resistência de rato foi isolado e montado em uma câmara de myograph, passando dois fios de aço por meio de seu lúmen. Após etapas de equilíbrio e normalização, o segmento de navio foi potencializado por uma solução de alta-K+ duas vezes antes do ensaio de contração. Como um exemplo da aplicação desse método no desenvolvimento de drogas, Nós medimos o efeito relaxante de uma substância natural romance, neoliensinine, isolado de uma erva chinesa, embriões da semente de Lótus (Nelumbo nucifera Gaertn.) no mouse mesentérico artérias. Os segmentos de embarcação montados na câmara de myograph foram estimulados com uma solução de alta-K+ . Quando a força de tensão atingiu uma fase estável e sustentada, doses cumulativas de neoliensinine foram adicionadas para a câmara. Encontramos que essa neoliensinine teve um efeito relaxante dose-dependente na contração do músculo liso, sugerindo assim, que tem atividade potencial contra a hipertensão. Além disso, como o segmento de navio pode sobreviver pelo menos 4 horas após a montagem e manter a contratilidade induzida pela solução high-K+ por muitas vezes, sugerimos que o sistema de myograph do fio pode ser utilizado para o moroso processo de despistagem de drogas.

Podemos medir a contratilidade isométrica de artéria mesentérica de rato usando um sistema multi fio myograph e avaliou o efeito relaxante da neoliensinine purificado a partir de embriões de sementes de Lótus (Nelumbo nucifera Gaertn.) 14. a artéria mesentérica resistência do rato foi isolada, limpo dos tecidos conjuntivos e cortada em segmentos de 1,4 mm. O segmento de artéria foi inserido por dois fios de aço em Ca2 +-livre solução...

Watch this Scientific Journal Video about Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography at JoVE.com

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

A técnica de myograph do fio é usada para investigar as funções do músculo liso vascular e tela novas drogas. Nós relatamos um protocolo detalhado para medir a contratilidade isométrica da artéria mesentérica de rato e para a seleção de novos relaxantes do músculo liso vascular.

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

isometric-contractility-measurement-mouse-mesenteric-artery-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

14.6K visualizações.  Nanjing University. A técnica de myograph do fio é usada para investigar as funções do músculo liso vascular e tela novas drogas. Nós relatamos um protocolo detalhado para medir a contratilidade isométrica da artéria mesentérica de rato e para a seleção de novos relaxantes do músculo liso vascular.

Vídeo: Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Modelo do rato de Oclusão da Artéria Cerebral Média

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Medicina

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

A aplicação de ligadura da artéria Cerebral Média Permanente no mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

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 ...

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Medida da Pressão Intracraniana peridural em ratos, utilizando um transdutor de pressão de fibra óptica

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

Na medição in vivo da camada superficial do rato pulmonar endotelial

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

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 ...

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 ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

Medição da curva pressão-volume no mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

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. ...

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.

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. ...