Os estudos foram apoiados pelo NIH HL75503 para KP.

Todos os experimentos realizados em animais foram aprovados pelo Comitê da Universidade de Tennessee Health Science Center Animal Care Institucional e uso. 1. Tubulação de preparações perfusão Rat Lung <ol><li> Preparar um sistema de tubos com um tubo Tygon (# 18) para a perfusão de sangue como mostrado na Figura 1. Ligue transdutores de pressão (P23XL) e colocar o tubo na platina do microscópio. </li><li> Ajustar a temperatura do banho de água a 37 ° C. </li><li> Conecte a entrada e saída de pulmão temporariamente com Tygon tubulação. </li></ol><img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51552/51552fig1highres.jpg" width="500" /> Tubo de perfusão sanguínea Figura 1.. O esquema mostra a configuração tubulação utilizada para a circulação do sangue através da preparação de pulmão isolado. Também estão indicados os componentes associados, através do qual o tubo is roteado. Um diagrama esquemático do coração e pulmão está incluído para mostrar a sítios de ligação entre o tubo e da artéria pulmonar (PA), e atrial (LA) da cânula (azul linhas a tracejado) para a esquerda. 2. Preparação de isolado de rato Pulmões <ol><li> Anestesiar ratos machos (Sprague-Dawley; 250-300 g) com cetamina (80-100 mg / kg) e com xilazina (5-10 mg / kg). Depois de garantir um plano cirúrgico de anestesia, coloque o animal em decúbito dorsal. </li><li> Realizar traqueostomia, inserir uma cânula traqueal (PE-90) e prenda com sutura cirúrgica. </li><li> Infuse heparina (100-200 U) para o coração por punção cardíaca (21 G borboleta agulha), aguarde 60 segundos e sangue desangrar (~ 12-15 ml de sangue). </li><li> Prepare duas cânulas (Tygon tubulação, 3 mm de diâmetro, 4 cm de comprimento; queimado em uma das extremidades) e encher com solução salina. </li><li> Realize uma toracotomia. Faça uma incisão (3 mm) no ventrículo direito e deslize a extremidade alargada de uma cânula na incisão para a pulmonarartéria. Fixar a cânula da artéria pulmonar com suturas cirúrgicas. </li><li> Inciso (3 mm) a ponta do ventrículo esquerdo e deslizar a extremidade alargada da segunda cânula para a incisão. Orientar a cânula no átrio esquerdo. Segura cânula no ventrículo esquerdo com uma fita umbilical (largura 2 mm). </li><li> Dissecar qualquer tecido conjuntivo, e remover o pulmão e coração em conjunto com as cânulas anexas. Coloque pulmão e coração em uma placa de Petri. Reposicionar do pulmão de modo a que a superfície do diafragma está no topo. Os três cânulas deve enfrentar a mesma direção. </li><li> Coloque a preparação de pulmão em uma fase que pode ser manipulado na direcção XY. Qualquer etapa que vem com um microscópio pode ser modificada para manter o tubo ou uma fase feito por encomenda construída, se necessário. </li></ol> 3. Perfusão sanguínea dos pulmões isolados de ratos <ol><li> Misture o sangue sangrados com uma solução de albumina de volume igual (5%) e adicionar ao reservatório. Inicie a bomba peristáltica e definir fluxo de rComeram a 14 ml / min. Deixar o sangue para encher o tubo. </li><li> Com a derivação aberta, remova o conector de entrada-saída temporária de pulmão. Anexar a artéria pulmonar e da cânula do átrio esquerdo para a entrada e a saída do pulmão, respectivamente. Certifique-se de que não há bolhas de ar na tubulação. </li><li> Ligue a cânula traqueal a um transdutor de pressão terceiro (P23XL). Encha os pulmões com 30% de oxigênio através da cânula traqueal e manter a pressão a 5 cm H 2 O. </li><li> Feche a derivação com uma braçadeira para começar a perfusão pulmonar. Manter artéria pulmonar e das pressões do átrio esquerdo em ~ 10 e 3 cm H 2 O, respectivamente. </li></ol> 4. Preparando Lung para microvascular Infusion <ol><li> Levantar a extremidade traseira do tubo de extensão para o cateter acima do nível do pulmão e seguro. </li><li> Preparar um cateter de infusão através da inserção de cerca de 30 cm de um tubo de PE10 40 cm de altura para um tubo de PE90 ~ 30 cm de comprimento. Ligar a extremidade exposta do tubo de PE10 de uma agulha (30 L) aapenso a uma seringa (1 cc). </li><li> Inserir a combinação de cateter para a extremidade posterior saliente do tubo de extensão. Orientar a combinação cateter através do átrio esquerdo e no pulmão até encontrar resistência. Em seguida, empurre o cateter PE10 só mais para o pulmão até encontrar resistência. Note-se que a aplicação de força excessiva durante a inserção do cateter vai prejudicar o pulmão. </li><li> Encha a seringa de 1 cc com solução de Ringer e anexar a uma bomba de seringa. Definir a taxa de infusão de 10 mL / min. </li><li> Local da infusão vai tornar-se pálida em comparação com resto do pulmão. </li><li> Umedeça o pulmão com soro fisiológico e cobrir o pulmão com filme plástico com um buraco grande o suficiente para deixar o local da infusão descoberto. </li><li> Pincelar algumas gorduras torneira no fundo de um O-ring (feito por encomenda) e colocar uma lamela de vidro (# 1.5, diâmetro de 22 mm) na parte inferior do anel de vedação. Suavemente posicionar o anel de vedação com a lamela no local da infusão. Fixe o O-ring com um hol tubo de ensaio padrãoder. A combinação lamela / O-ring não distorce as estruturas alveolares debaixo dela, como relatado recentemente 15. </li><li> Posicionar um objectivo (20X) de um microscópio de fluorescência acima do O-ring e focar na superfície do pulmão. </li></ol> 5. Imagem fluorescente Dextran trânsito através Microvasos <ol><li> Prepare o microscópio para FITC imagem florescimento. </li><li> Selecione uma região a ser trabalhada e adquirir imagens a uma taxa de 1/minuto usando o software de aquisição de imagem. </li><li> Comece a infusão de FITC-dextrano 20 kD (0,5 mg / ml) usando a bomba de seringa. Fluorescência nos vasos sanguíneos vai aumentar gradualmente e atingir um máximo. Continuar infusão por 60 minutos. </li><li> Então mude para a infusão de Ringer para lavar fluorescência luminal. Manter a infusão de mais de 10 min. Continuar a aquisição de imagens em 1/min durante a lavagem-off. </li></ol> 6. Image Analysis <ol><li> Abra o arquivo de aquisição de imagem. </li><li> Coloque regiõesde juros ao longo microvasos dentro da moldura da imagem. </li><li> Jogue o arquivo de imagem frame-by-frame e registrar a intensidade de fluorescência em cada região de interesse para todos os quadros. </li><li> Traça-se a alteração na intensidade da fluorescência para cada região de interesse, como uma função do tempo. </li><li> Quantificar a intensidade máxima da fluorescência e da intensidade de fluorescência residual após 10 min de washoff. </li><li> Calcula-se a razão entre a intensidade máxima de fluorescência residual em cada região de interesse para obter o índice de permeabilidade para os microvasos associados a essa região de interesse. </li></ol>

<ol>
	<li>Ware, L. B., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> N Engl J Med. 342, 1334-1349 (2000).</li><li>Matthay, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> J Clin Invest. 122, 2731-2740 (2012).</li><li>Bhattacharya, J., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+repair+of+the+alveolar-capillary+barrier+in+acute+lung+injury.">Regulation and repair of the alveolar-capillary barrier in acute lung injury.</a> Annu Rev Physiol. 75, 593-615 (2013).</li><li>Parthasarathi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+43+mediates+spread+of+Ca2+-dependent+proinflammatory+responses+in+lung+capillaries.">Connexin 43 mediates spread of Ca2+-dependent proinflammatory responses in lung capillaries.</a> J Clin Invest. 116, 2193-2200 (2006).</li><li>Parthasarathi, K., Bhattacharya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+Acid+instillation+by+a+wedged-catheter+method+reveals+a+role+for+vascular+gap+junctions+in+spatial+expansion+of+Acid+injury.">Localized Acid instillation by a wedged-catheter method reveals a role for vascular gap junctions in spatial expansion of Acid injury.</a> Anat Rec (Hoboken). 294, 1585-1591 (2011).</li><li>Gorin, A. B., Stewart, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+permeability+of+endothelial+and+epithelial+barriers+to+albumin+flux.">Differential permeability of endothelial and epithelial barriers to albumin flux.</a> J Appl Physiol Respir Environ Exerc Physiol. 47, 1315-1324 (1979).</li><li>Boutoille, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FITC-albumin+as+a+marker+for+assessment+of+endothelial+permeability+in+mice:+comparison+with+125I-albumin.">FITC-albumin as a marker for assessment of endothelial permeability in mice: comparison with 125I-albumin.</a> Exp Lung Res. 35, 263-271 (2009).</li><li>Thorball, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FITC-dextran+tracers+in+microcirculatory+and+permeability+studies+using+combined+fluorescence+stereo+microscopy,+fluorescence+light+microscopy+and+electron+microscopy.">FITC-dextran tracers in microcirculatory and permeability studies using combined fluorescence stereo microscopy, fluorescence light microscopy and electron microscopy.</a> Histochemistry. 71, 209-233 (1981).</li><li>Stevens, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+molecular+heterogeneity+of+pulmonary+endothelial+cells.">Functional and molecular heterogeneity of pulmonary endothelial cells.</a> Proc Am Thorac Soc. 8, 453-457 (2011).</li><li>Ofori-Acquah, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+barrier+function+in+the+lung+reflects+diversity+in+endothelial+cell+junctions.">Heterogeneity of barrier function in the lung reflects diversity in endothelial cell junctions.</a> Microvasc Res. 75, 391-402 (2008).</li><li>Kandasamy, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+reveals+endothelium-mediated+leukocyte+retention+in+LPS-treated+lung+microvessels.">Real-time imaging reveals endothelium-mediated leukocyte retention in LPS-treated lung microvessels.</a> Microvasc Res. 83, 323-331 (2012).</li><li>Kandasamy, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+induces+endoplasmic+store+Ca2+-dependent+inflammatory+responses+in+lung+microvessels.">Lipopolysaccharide induces endoplasmic store Ca2+-dependent inflammatory responses in lung microvessels.</a> PloS One. 8, (2013).</li><li>Qiao, R. L., Bhattacharya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmental+barrier+properties+of+the+pulmonary+microvascular+bed.">Segmental barrier properties of the pulmonary microvascular bed.</a> J Appl Physiol. 71, 2152-2159 (1991).</li><li>Parthasarathi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+connexin43+mediates+acid-induced+increases+in+pulmonary+microvascular+permeability.">Endothelial connexin43 mediates acid-induced increases in pulmonary microvascular permeability.</a> Am J Physiol Lung Cell Mol Physiol. 303, (2012).</li><li>Wu, Y., Perlman, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+methods+for+assessing+alveolar+mechanics.">In situ methods for assessing alveolar mechanics.</a> J Appl Physiol 1985. 112, 519-526 (2012).</li><li>Kuebler, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+signaling+mechanism+between+gas+and+blood+compartments+of+the+lung.">A novel signaling mechanism between gas and blood compartments of the lung.</a> Journal Clin Invest. 105, 905-913 (2000).</li></ol>

Os autores não têm nada a revelar.

A preparação isolada de perfusão pulmonar com sangue combinado com imagens em tempo real fornece uma ferramenta simples para a determinação de alterações de permeabilidade em microvasos pulmonares individuais. Aplicamos esse método para definir alterações de permeabilidade em resposta à infusão de LPS. Os nossos dados sugerem claramente que a infusão de LPS causou um aumento da permeabilidade microvascular. Além disso, os dados também indicam que as alterações de permeabilidade induzida por LPS foram si...

O aumento da permeabilidade microvascular nos pulmões leva ao desenvolvimento de edema alveolar e troca gasosa comprometida e é uma das principais características da lesão pulmonar aguda (LPA) 1-3. Assim, estimativas da permeabilidade vascular são importante na definição do grau de lesão dos pulmões e a eficácia das intervenções terapêuticas propostas. Análise gravimétrica como proporção wet-a-seco de pulmão livre sangue e coeficiente de filtração microvascular são amplamente utilizados métodos para estimar a permeabilidade 4,5. Outros métodos incluem a quantificar a retenção de sondas radioactivas ou fluorescentes no tecido pulmonar 6-8. No entanto, os métodos descritos acima requerem processamento postexperiment de amostras de tecido do pulmão para elucidar os dados de permeabilidade. Além disso, uma vez que um animal pode ser usado somente para um único protocolo de tratamento, pode ser necessário um grande número de animais para o estudo completo. Uma característica comum dos métodos acima é que eles determinam a permeabilidade vascular significativo paratodos os vasos sanguíneos no interior da amostra de tecido. No entanto, é bem estabelecido que o micro e macro-vasos pulmonares são fenotipicamente diferente 9. Por isso, as respostas de permeabilidade pode ser heterogênea entre os diversos segmentos de navios, bem 9,10. Assim, a quantificação da permeabilidade média de todos os vasos pulmonares em uma amostra de tecido pode não refletir adequadamente esta heterogeneidade. Na preparação isolada de perfusão pulmonar de sangue, dos vasos sanguíneos na superfície do pulmão podem ser visualizados por um microscópio vertical 4,11,12. Isto permite que caracterizam as respostas em vasos individuais e, assim, tratar qualquer heterogeneidade na resposta 13. Além disso, através da utilização de imagens de fluorescência de microvasos, ensaios de fluorescência com base pode ser incorporada. Além disso, um microcateter atrial esquerda pode ser utilizado para fornecer agentes e as sondas de fluorescência nos vasos sanguíneos 11,14. O microcatheter limita a entrega a uma região de pulmão de pequenas, assim, exposando apenas os vasos sanguíneos na região, para os agentes infundidos e fluoróforos. Isto permite que várias pequenas regiões dentro do mesmo pulmão a ser utilizado para experiências em separado, levando a uma redução global de animais necessários para um estudo. Imagens em tempo real permite a captura de mudanças dinâmicas na vascular e fluorescência extravascular de microvasos individuais da preparação de pulmão isolado. Assim, para cada um dos microvasos dentro de um campo de imagem, as alterações na fluorescência durante a infusão de fluoróforos e washoff pode ser gravada, e quantificado desligada 14. Usando valores de fluorescência máximo e residual vascular, um índice de permeabilidade para cada microvasos no campo de imagem pode ser determinada. Para determinar as alterações de permeabilidade na resposta aos agentes inflamatórios ou prejudiciais, o agente desejado, pode ser administrado em primeiro lugar e, em seguida, o índice de permeabilidade determinada. Além disso, o campo de imagem pode ser colocado em qualquer lugar dentro da região do pulmão infundida pelamicrocatheter, permitindo assim um elevado grau de flexibilidade na escolha da rede vascular desejado. Assim, a preparação isolada de perfusão pulmonar com sangue em conjunto com imagens em tempo real fornece um modelo experimental atraente para quantificar permeabilidade em microvasos pulmonares individuais.

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tygon Tubing</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:297px;">#18</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pressure Transducer</td>
			<td style="width:108px;">Data Sciences International</td>
			<td style="width:119px;">P23XL</td>
			<td>Need quantity 3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Butterfly Needle</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td align="right" style="width:119px;">450081</td>
			<td>21G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peristaltic pump</td>
			<td style="width:108px;">Cole Parmer</td>
			<td style="width:119px;">Masterflex L/S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-90 tubing</td>
			<td style="width:108px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">427421</td>
			<td>30 cm needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PE-10 tubing</td>
			<td style="width:108px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">427401</td>
			<td>40 cm needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syringe Pump</td>
			<td style="width:108px;">Braintree Scientific</td>
			<td style="width:119px;">BS8000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">O-ring</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>Custom made with a 20 mm diamter hole&#160; and a handle&#160; to secure o-ring to holder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Upright fluorescence microscope</td>
			<td style="width:108px;">Olympus America</td>
			<td style="width:119px;">BX61WI</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Image Acquisition Software</td>
			<td style="width:108px;">Molecular Devices</td>
			<td style="width:119px;">Metamorph</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC Dextran 20KD</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>0.5 mg/ml (A dextran of different molecular size can be selected, if trial experiments indicate its suitability based on the calculated permeability index values)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lipopolysaccharide</td>
			<td style="width:108px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>Serotype 0111:B4</td>
		</tr>
	</tbody>
</table>

quantifying single microvessel permeability in isolated blood-perfused rat lung preparation

A preparação isolada de perfusão pulmonar com sangue é amplamente usado para visualizar e definir a sinalização em microvasos individuais. Juntando esta preparação com imagens em tempo real, torna-se viável para determinar alterações da permeabilidade em microvasos pulmonares individuais. Aqui nós descrevemos etapas para isolar pulmões de ratos e perfundem-los com sangue autólogo. Em seguida, descrevemos as etapas para infundir fluoróforos ou agentes através de um microcatheter em uma região de pulmão de pequenas. Utilizando estes procedimentos descritos, determinou-se o aumento da permeabilidade em microvasos de pulmão de rato em resposta a infusões de lipopolissacarídeo bacteriano. Os dados revelaram que lipopolissacarídeo aumento de fugas de fluido em ambos os segmentos venulares e microvasos capilar. Assim, este método faz com que seja possível comparar as respostas de permeabilidade entre os segmentos vasculares e, assim, definir qualquer heterogeneidade na resposta. Enquanto os métodos comumente utilizados para definir a permeabilidade pulmonar requerem pós-processamento de amostras de tecido do pulmão, ouso de imagens em tempo real elimina esta exigência, como é evidente a partir do presente método. Assim, a preparação de pulmão isolado combinado com a imagem em tempo real oferece várias vantagens em relação aos métodos tradicionais para determinar a permeabilidade microvascular pulmão, ainda é um método simples para desenvolver e implementar.

Uma preparação isolada de perfusão pulmonar de sangue ligado ao sistema de perfusão e afins é mostrado na Figura 2. Para fins de demonstração, foi utilizado um rato Sprague Dawley, embora os procedimentos aqui descritos podem ser utilizados com qualquer espécie de rato. Infusões com um microcateter atrial esquerdo atingir apenas uma pequena região do pulmão. A região infundido pode ser identificado pela descoloração induzida por infusão (Figura 3). A preparação de pulm?...

Watch this Scientific Journal Video about Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation at JoVE.com

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

A preparação isolada de perfusão pulmonar de sangue faz com que seja possível visualizar redes microvasos na superfície do pulmão. Aqui nós descrevemos uma abordagem para quantificar a permeabilidade de microvasos de solteiro em pulmões isolados tempo real usando imagens de fluorescência.

Quantificar Único microvasos Permeabilidade em perfusão isolada-Sangue Rat Lung Preparação

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

quantifying-single-microvessel-permeability-isolated-blood-perfused

Research

JoVE Journal

Biology

9.4K visualizações.  The University of Tennessee Health Science Center.  A preparação isolada de perfusão pulmonar de sangue faz com que seja possível visualizar redes microvasos na superfície do pulmão. Aqui nós descrevemos uma abordagem para quantificar a permeabilidade de microvasos de solteiro em pulmões isolados tempo real usando imagens de fluorescência. 

Vídeo: Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

Preparação padronizado de uma única célula Suspensões do tecido pulmonar do rato usando o Dissociator gentleMACS

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

Biologia

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

Caracterização do isolado, ventilado, Lung Mouse e Instrumentados perfundidos com fluxo pulsátil

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

O Ex vivo Preparação microvasos isolada esquelético de Investigação da reatividade vascular

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Parâmetro Multi-Medição da abertura dos poros transição de permeabilidade em mitocôndrias isoladas de coração Rato

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Medindo o Coeficiente de Permeabilidade osmótica de água (P<sub&gt; F</sub&gt;) De células esféricas: protoplastos planta isolada como um exemplo

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels.

Changes in the intracellular calcium levels in the podocytes are one of the most important means to control the filtration function of glomeruli. Here we explain a high-throughput approach that allows detection of real-time calcium handling and single ion channels activity in the podocytes of the freshly isolated glomeruli.

Single-channel Análise e cálcio Criação de imagem do Podócitos do recentemente isolados glomérulos

Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperf Técnica para Investigar Regulamento de microvasos Permeabilidade de Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Isolamento de CD146<sup&gt; +</sup&gt; Células Residente Lung estromais mesenquimais de rato Pulmões

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

A técnica de rim perfundidos rato isolado

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Um ensaio de quimiotaxis melhorado para a identificação rápida de quimioattractants rizobacterianos em Exudates radiculares

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...