Agradecemos ao Dr. William Cashdollar, ea Northwestern Mutual Foundation Imaging Center do Instituto de Pesquisa Wisconsin sangue para obter ajuda com os estudos de imagem. Agradecemos também o Dr. Daria Ilatovskaya para a leitura crítica deste manuscrito e discussão útil. Este estudo foi financiado pelo National Institutes of Health Grants HL108880 (como) e DP2-OD008396 (a AMG).

Os procedimentos experimentais descritos abaixo foram aprovados pelo cuidado e uso Comitê Institucional Animal (IACUC) da Faculdade de Medicina de Wisconsin e estavam de acordo com os Institutos Nacionais de Saúde Guia para o Cuidado e Uso de Animais de Laboratório. 1. Isolamento da aorta de rato <ol><li> Anestesiar ratos com isoflurano (5% de indução, de 1,5 a 2,5% de manutenção) ou outro método aprovado IACUC. </li><li> Coloque o rato em uma posição supina. A fim de expor os órgãos abdominais, fazer uma pequena incisão na linha média ventral, através da pele e do tecido abdominal subcutâneo. Usando compressas de gaze desviar ligeiramente os intestinos para expor a aorta e veia cava como se mostra na Figura 1. </li><li> Resumidamente, sem corte dissecar a aorta a partir do tecido conjuntivo e da veia cava. Usando fio de sutura, amarrar a aorta tão próximo quanto possível para o rim. Dissecar cerca de 1 - 2 cm de aorta e colocá-lo em um cônico de 15 ml tuser fisiológico normal contendo solução salina (PSS; em mM: 140 de NaCl, 1,2 MgCl2, 2 CaCl2, 4,5 de KCl, 6 de glucose, 10 de HEPES) em gelo. </li><li> Uma vez que a aorta é isolado, sacrificar o animal de acordo com os protocolos aprovados IACUC. Após a conclusão de todos os não-sobrevivência procedimentos sacrificar animais profundamente anestesiados por toracotomia induzir pneumotórax para assegurar o desaparecimento humano do animal. 3 </li><li> Usando um microscópio de dissecação, uma pinça de ponta fina, e microtesoura dissecar a gordura e tecido conjuntivo fora da aorta. Uma vez que a aorta é limpo, cortado longitudinalmente da aorta e colocá-lo em PSS em gelo durante mais tarde. </li></ol> 2. Dye carregar e Incubação <ol><li> Pipetar 7 ul de 1 mM de Fluo-04:00 corante, que é fornecido em solução à base de DMSO, em 500 ul tubos individuais que foram cobertos com folha de alumínio. Armazenar no congelador a -20 ° C para preservar as propriedades de corante ao longo do tempo. Nota: estes aliquotados assimluções deve ser estável durante pelo menos seis meses. </li><li> Antes do uso, permitir que as soluções de tintura-se aquecer até à temperatura ambiente antes da abertura. Preparar as soluções de trabalho imediatamente antes da utilização. Não guarde o reagente diluído para uso posterior. </li><li> Adicionar 7 ul de 1 mM de Fluo-04:00 corante pronta dissolvido em solução de DMSO a 450 ul de PSS contendo nenhum cálcio. Adicionar 40 ul de solução de DMSO não com base 10% de ácido Pluronic ao cocktail de carregamento para ajudar a dispersar os grupos acetoximetilo (AM) e ésteres de melhorar o carregamento do corante de cálcio. </li><li> Coloque as aortas dissecados para os tubos 500 ul contendo o cocktail de carregamento (como descrito em 2.3), durante 1 h. Cobrir todos os tubos com papel alumínio e coloque em um agitador rotativo a RT. </li><li> Para monitorizar a produção de NO na vasculatura, usar corante diacetato DAF-FM. Incubar a aorta em uma solução contendo 450 uL PSS, 40 ul de ácido plurónico e 5 ul de 10 mM de FAD FM-diacetato. Semelhante ao protocolo de incubação de cálcio (descrito em 2.3), colocar a aortanum tubo de 500 ul contendo a solução sem corante coberta na folha e lugar num agitador rotativo durante 30 minutos a 4 C seguindo-se próximo de 30 min à TA. </li><li> Quando a produção de NO é medido, utilizar a NO-sintase endotelial bloqueador, L-NAME, para bloquear a produção de NO como um controlo, como mostrado na Figura 4B. Para esse procedimento, adicione a cocktail (como descrito em 2.5) 100 nM L-NAME para os últimos 30 minutos de incubação (em RT). </li></ol> 3. Laser Scanning Dois Protocolo photon Microscopia <ol><li> Após a 1 h de incubação, lava-se a aorta de 2 - 3 vezes em PSS limpo para remover o corante extracelular. </li><li> Transferir a aorta para uma placa revestida com silicone contendo ou PSS normal ou tampão livre de Ca 2+ (depende do protocolo experimental). Pin da aorta para baixo para o revestimento de silicone com a adventícia em contacto com o silicone (ou seja, lúmen endoteliais expostas à abertura prato) utilizando pinos em forma de μ. Como alternativa, use uma fatia Anchorgrade ou cola para fixar o tecido. Nota: Para reduzir o estresse e possíveis artefatos mecânicos, transferência e corrigir aorta em Ca 2 + livre solução e esperar 5 - 10 min antes do início do experimento. </li><li> Conectar uma bomba peristáltica ou uma seringa para a placa revestida com silicone para aplicação automática ou manual de drogas ou para modificar a solução extracelular. </li><li> Coloque o prato sob a parte do nariz dos retos microscópio de dois fótons, e instalar e posicione a 25 × (NA 1,05 e trabalhando distância 2 mm) de imersão em água lente objetiva acima do espécime. Nota: Se esta lente específica não está disponível, use um objectivo fabricados especificamente para dois fótons de imagem, porque pode aumentar a resolução do sinal dramaticamente em comparação com um objectivo regular. </li><li> Ative o laser de dois fótons usando o software (o laser começa a bomba e ligue). Sintonizar o laser de excitação a 820 nm. </li><li> Usando epifluorescência, localize a aorta e focar o objetivo ema superfície endotelial usando ajuste primário. </li><li> Alterne o microscópio de varredura a laser em modo de dois fótons, garantindo que o laser de excitação é modo bloqueado, os detectores não descanned estão envolvidos e os filtros de emissão adequadas para os espectros de emissão esperada estão instalados. Nota: Aqui, foram utilizados o FV10-MRL / R (495-540 nM). </li><li> Iniciar a digitalização vivo utilizando aproximadamente 5% da potência do laser e finamente concentrar o objectivo, a fim de visualizar as células endoteliais. Nota: As células endoteliais exibirão um sinal fluorescente forte quando incubadas em PSS normal. Este será visivelmente mais fraco quando os vasos são incubadas em tampão isento de cálcio. As células endoteliais estará presente na parte superior da aorta aberta e as células do músculo liso pode ser visto abaixo das células endoteliais (ver Figura 2). Porque os vasos sanguíneos não são nem lisa nem mesmo, haverá locais onde ambas as células de músculo liso e células endoteliais estão presentes. &lt;/ Li&gt; <li> Uma vez que as células estão em foco, o programa de software microscópio para coletar imagens sequenciais no modo de varredura rápida (varredura raster, 512 x 512 pixels de janela com uma frequência coleção de quadros de 1,1 s). Em paralelo com Fluo-04:00 fluorescência, recolher transmitido imagens de luz, a fim de detectar alterações na contração dos vasos e visualizar melhor as condições experimentais. </li><li> Após a coleta de imagens de sinal de linha de base, altere a concentração de íons de Ca2 + na solução externa ou lentamente aplicar agentes farmacológicos de estímulo usando a bomba peristáltica, enquanto imagem. Observar o aumento de sinal fluorescente dentro das células carregadas. Nota: As células endoteliais responder às mudanças no fluxo, portanto, recomenda-se usar aplicações de baixa laminares e ensaio do veículo antes da aplicação de activadores farmacológicos de Ca 2+ ou NO sinalização. Para recalcular cálcio intracelular para valores de concentração nm de embarcações carregadas com Fluo-4 (constante de dissociação para Fluo-4 is 345 nM), a intensidade de fluorescência pode ser gravada no início e após adição de ionomicina e MnCl2 4. </li></ol> 4. Processamento de Imagem e Cálculos <ol><li> Baixe e instale Fiji pacote de processamento de imagem. </li><li> Abra o arquivo de imagem em Fiji. Após importar o arquivo, dividir os canais (luz transmitida e de sinal fluorescente) quando solicitado. Utilize apenas o sinal fluorescente para a análise de dados. </li><li> Dentro do programa de Fiji, clique na guia analisar e desça até a opção ferramentas. Sob a opção de ferramentas, encontrar a aba que diz o gerente de ROI e clique sobre ele; uma nova janela aparecerá. </li><li> Após isso, clique em medições definido na guia analisar. Selecione as medições específicas de interesse. Para as medidas transitórias de cálcio, use a opção valor médio cinza. </li><li> Com a ferramenta de rastreio circular, começam a traçar as regiões de interesse (ROI) e clique em &quot;adicionar&quot; na tela do gerenciador de ROI para cada ROI. Uma vez que todas as células de interesse foram rastreados, na janela do gerenciador de ROI, selecione multi Medida sob o &quot;mais&quot; guia. Lembre-se de salvar os medições diretamente ou copiar e colar todas essas medidas em uma planilha. </li><li> Abrir arquivo * .txt ou planilha do Excel de Fiji e transferir os números para uma nova planilha de Origem. Nota: Como alternativa, use um programa como o Sigma Plot ou Prism para análise posterior. </li><li> Dentro do programa de Origem, use Plot → menu de Linha + Símbolo de observar uma visão geral de todas as respostas transientes. Desmarque as células não respondem. </li><li> Recalcular o número de rastreamento para escala de tempo (eixo X), use Coluna → Definir valores de coluna e multiplicar a coluna de número de rastreamento para a freqüência de coleta de imagem (no nosso caso 1.1s). </li><li> Use Analysis → Estatísticas sobre linhas para todas as gravações selecionadas para calcular Média (eixo Y) e rastreamento de erro padrão. Traçar a curva final para grupo atual de células Traçar → Linha + Symbol. </li><li> O mean cálcio transiente cálculo é descrito como se segue. <ol><li> Para total liberação de cálcio dentro do programa de Origem, clique no gráfico, em seguida, usar o menu Análise → Cálculo → Integrar. Integrante total de área sob a curva aparece no registro de resultado. </li><li> Para cinética transitórios, clique no gráfico, em seguida, use o menu: Curva Análise → não-linear Fit → Selecionar Conjunto de Dados (escolher eixo X variam de máximo até o final da resposta transitória). Comece Fitting → Select → Função Exponencial Decay 1 → Concluído. Nota: encaixe exponencial aparece na janela Graph e Resultado Log. Os dados obtidos (valores) representam quantidade total de cálcio liberado, e cinética dos canais mediada a resposta transitória. </li></ol></li></ol>

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Visão geral Experimental. Para melhor compreender a contribuição de cálcio e NÃO a fisiologia vascular, um novo método foi desenvolvido para medir [Ca2 +] i e NÃO dentro do músculo liso e células endoteliais de aortas intactas isoladas. Juntos, este protocolo é composto por estes passos críticos: 1) o isolamento mecânico e preparação (digestão enzimática não) da embarcação. É importante manter o tecido saudável e intacta, tanto quanto possível obter gravações fisiol...

O cálcio é um segundo mensageiro fundamental dentro da células vasculares tais como células endoteliais e do músculo liso. É o estímulo primário para a contracção vascular e desempenha um papel importante na dilatação vascular, incluindo os seus efeitos através de nenhuma geração dentro do endotélio. Devido às limitações da tecnologia de imagem, tem sido praticamente impossível observar manuseamento de cálcio no interior do vaso intacto. O desenvolvimento de dois sistemas de imagem de fótons e da criação de novos cálcio ou sem corantes de rotulagem, torna possível a imagem a uma profundidade e resolução suficiente para começar a entender a dinâmica de cálcio e produção de NO na vasculatura. Dois microscopia de fotões foi recentemente aplicada em tecidos, órgãos e estudos em animais inteiros, mesmo devido à sua superior capacidade para penetrar profundamente tecidos com baixa fluorescência de fundo e a sensibilidade do sinal de alta 1,2. O espectro estreito de dois fotões de excitação no Illuminatião ponto focal e a utilização de detectores não-descanned são as razões pelas quais microscopia dois fotões é superior a microscopia confocal tradicional. A microscopia confocal não pode produzir imagens de alta qualidade a uma profundidade de tecido necessária devido à auto-fluorescência e dispersão de luz fora de foco no pinhole confocal. Por conseguinte, temos desenvolvido um método usando um microscópio de dois fotões para medir a [Ca2 +] i de sinalização e a produção de NO em células dos vasos sanguíneos individuais intactas com alta resolução e baixa relação de sinal-para-ruído.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DAF-FM</td>
			<td>Life Technologies</td>
			<td>D-23842</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo 4 AM</td>
			<td>Life Technologies</td>
			<td>F14217</td>
			<td>500&#181;l in DMSO</td>
		</tr>
		<tr>
			<td>Pluronic F-68 solution</td>
			<td>Sigma-Aldrich</td>
			<td>P5556</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Name</td>
			<td>Tocris</td>
			<td>0665</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus upright microscope</td>
			<td>Olympus</td>
			<td>Fluoview FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>MaiTai HP DeepSee-OL&#160;</td>
			<td>Spectra Physics</td>
			<td></td>
			<td>Ti:sapphire laser 690nm &#8212; 1040nm</td>
		</tr>
		<tr>
			<td>Filters</td>
			<td>Olympus</td>
			<td>FV10-MRL/R&#160;</td>
			<td>495 to 540 nm&#160;</td>
		</tr>
		<tr>
			<td>25&#215; water-immersion objective lens</td>
			<td>Olympus</td>
			<td>XLPL25XWMP</td>
			<td>N.A. 1.05 and working distance 2 mm</td>
		</tr>
		<tr>
			<td>Slice Anchor grid&#160;</td>
			<td>Warner Instrument&#160;</td>
			<td>SHD-27LH</td>
			<td></td>
		</tr>
		<tr>
			<td>Other basic reagents&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

two-photon imaging of intracellular ca2+ handling and nitric oxide production in endothelial and smooth muscle cells of an isolated rat aorta

O cálcio é um regulador muito importante de diversos processos fisiológicos em tecidos vasculares. A maioria das funções endoteliais e do músculo liso dependem muito alterações de cálcio intracelular ([Ca 2+] i) e óxido nítrico (NO). A fim de entender como [Ca2 +] i, NO e moléculas jusante são manipulados por um vaso sanguíneo em resposta a vasoconstritores e vasodilatadores, desenvolvemos uma nova técnica que se aplica a rotulagem de cálcio (ou NO-rotulagem) tinge com microscopia de dois fótons para medir a manipulação de cálcio (ou nenhuma produção) nos vasos sanguíneos isolados. Aqui descrito é um procedimento passo-a-passo que demonstra como isolar uma aorta de um rato, etiqueta de cálcio ou NO nas células endoteliais ou de músculo liso, e imagem transientes de cálcio (ou a produção de NO), usando um microscópio de dois fotões seguinte fisiológico ou estímulos farmacológicos. As vantagens da utilização do método são multi-dobra: 1) é possível simultânealy medir transientes de cálcio em ambas as células endoteliais e células musculares lisas em resposta a diferentes estímulos; 2) que permite que uma imagem de células endoteliais e células do músculo liso na sua configuração nativa; 3) este método é muito sensível ao cálcio intracelular ou nenhumas mudanças e gera imagens de alta resolução para medições precisas; e 4) abordagem descrita pode ser aplicada para a medição de outras moléculas, tais como espécies de oxigénio reactivas. Em resumo, a aplicação de microscopia de emissão de laser de dois fotões para monitorar transientes de cálcio e a produção de NO nas células endoteliais e do músculo liso de um vaso sanguíneo isolado forneceu dados quantitativos de alta qualidade e promoveu a nossa compreensão dos mecanismos que regulam a função vascular.

A fim de avaliar com precisão a contribuição de cálcio a fisiologia vascular (vasodilatação e vasoconstrição), um protocolo foi concebido para carregar corantes de cálcio em ambas as células endoteliais e células do músculo liso na aorta isolada intacta. A experimental geral definido em cima representada na Figura 1, mostra a estratégia básica para o isolamento e preparação do vaso antes de imagem. Resumidamente, após o isolamento da aorta do rato, ele deve ser limpo de gordura e tecido...

Watch this Scientific Journal Video about Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta at JoVE.com

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Dois fótons de imagem de cálcio intracelular<sup&gt; 2+</sup&gt; Manuseio e produção de óxido nítrico no endotélio e músculo liso células de uma aorta de rato isolada

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

two-photon-imaging-intracellular-ca2-handling-nitric-oxide-production

Research

JoVE Journal

Biology

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

11.2K visualizações.  Medical College of Wisconsin. Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta. 

Vídeo: Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Dois fótons axotomia e lapso de tempo de imagem confocal em embriões de peixe-zebra ao vivo

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Biologia

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

Focal Ca 2 + Detecção de transitórios no músculo liso

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

Isolamento da Human Umbilical células musculares arteriais (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

A medição da função do músculo liso nas Isolado tecido de banho-aplicativos para Farmacologia Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imagem local Ca<sup&gt; 2+</sup&gt; Sinais em células de mamíferos em cultura

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Face Detection En de óxido nítrico e superóxido na camada endotelial das artérias intactas

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

Medição das flutuações cálcio dentro do Sarcoplasmic retículo de células musculares lisas cultivadas utilizando FRET baseado em imagem confocal

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Isolamento de Murino vasculares coronários células musculares lisas

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Desenvolvimento e Caracterização de<em&gt; In Vitro</em&gt; Rede de microvasos e medições quantitativas de endotelial [ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; E óxido nítrico Produção

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Lesão por esmagamento aórtico murino: um eficiente<em&gt; In Vivo</em&gt; Modelo de proliferação de células musculares lisas e função endotelial

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...