Este trabalho foi financiado por uma doação do Instituto Nacional de Saúde, R01CA142928.

1. Injecção intravenosa de azul de Evans na veia da cauda do rato Lateral <ol><li> Prepara-se uma solução de 0,5% de azul de Evans estéril em PBS. Se necessário, esterilizar por filtração da solução para remover qualquer matéria particulada que não se dissolveu. </li><li> Aspirar 200 ul solução azul de Evans para uma seringa. Evitar as bolhas de ar que possam ter escapado para a seringa. </li><li> Coloque ratos que são de 8-12 semanas de idade para um dispositivo de retenção de modo a que o animal não seja livremente móvel, mas a cauda podem ser manipulados. </li><li> Colocar o dispositivo de retenção do rato sobre o seu lado, de modo a veia lateral da cauda é facilmente visível e está virada para cima. </li><li> Segure a cauda com a mão não-dominante entre o polegar eo dedo indicador. </li><li> Inserir a agulha (calibre pequeno, 27-30) a uma antecedência 10-15 graus de ângulo, bisel para cima, para dentro da veia lateral da cauda na direcção da cabeça. Manter o paralelo agulha e seringa para a cauda. </li><li> Do não aplicar pressão para trás para confirmar a colocação correta, pois isso poderia entrar em colapso da veia. </li><li> Lentamente injetar 200 ul Evans solução azul na veia da cauda do rato. </li><li> Observar a facilidade com que os avanços de êmbolo, tal como esta é a prova da correcta colocação da agulha na veia. </li><li> Coloque o mouse em sua gaiola e observá-lo durante 30 minutos. </li></ol> 2. Recolha de órgãos e Extração de azul de Evans dos Órgãos <ol><li> Sacrificar os animais por deslocamento cervical. Para fins de ensaio Miles deslocamento cervical é recomendada, uma vez que limita a interferência significativa com a permeabilidade vascular. Sacrificar todos os ratinhos, ao mesmo tempo, tão rápido quanto possível. Trabalhar com grupos de 6 ratinhos ou menos, porque logo após os vasos sanguíneos se tornam mais permeáveis ​​morte. </li><li> Coloque os ratos em suas costas e fixar os pés em uma placa branca. </li><li> Abra a cavidade abdominal e torácica para expor thorórgãos Acic e abdominal. </li><li> Tire fotos representativas para mostrar diferenças em Evans extravasamento Azul. Incluir todos os ratos no mesmo campo, a fim de ter condições de iluminação idênticas para todos os ratos. </li><li> Coletar órgãos de interesse e colocá-los em tubos de 1,5 ml </li><li> Pesar um tubo vazio e levar o valor do equilíbrio de zero. </li><li> Transfira a amostra de tecido e peso,. Repetir para todas as amostras de tecido. Os tecidos podem ser secos ao ar para eliminar a variabilidade teor de água entre os diferentes órgãos. </li><li> . Adicionar 500 ul de formamida a cada tubo de amostra de tecido. </li><li> Transferir todos os tubos para um banho de água 55 ° C ou bloco de calor. Incubar durante 24-48 h, para extrair Azul de Evans do tecido. </li></ol> 3. Quantificação de azul de Evans extravasado no tecido intersticial <ol><li> Centrifugar a formamida / azul de Evans mistura a pelota quaisquer fragmentos de tecido remanescentes. </li><li> Medir a absorvância a 610nm. Use 500 Formamida ul como em branco. </li><li> Calcular ng Azul de Evans extravasado por mg de tecido. </li><li> Traçar todos os dados em um gráfico. </li><li> Realizar a análise estatística para determinar diferenças significativas. </li></ol>

<ol>
	<li>Beck, K. 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=Inducible+NO+synthase:+role+in+cellular+signaling.">Inducible NO synthase: role in cellular signaling.</a> J. Exp. Biol. 202, 645-653 (1999).</li><li>Bertglia, S., Giusti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+nitric+oxide+in+capillary+perfusion+and+oxygen+delivery+regulation+during+systemic+hypoxia.">Role of nitric oxide in capillary perfusion and oxygen delivery regulation during systemic hypoxia.</a> Am. J. Physiol. Heart Circ. Physiol. 288, H525-H531 (2005).</li><li>Miles, A. A., Miles, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+reactions+to+histamine,+histamine-liberator+and+leutaxine+in+the+skin+of+guinea+pigs.">Vascular reactions to histamine, histamine-liberator and leutaxine in the skin of guinea pigs.</a> J. Physiol. (London). 118, 228-257 (1952).</li><li>Weis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability+in+cardiovascular+disease+and+cancer.">Vascular permeability in cardiovascular disease and cancer.</a> Curr. Opin. Hematol. 15, 243-249 (2008).</li><li>Kumar, P., Shen, Q., Pivetii, C. D., Lee, E. S., We, M. H., Yuan, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+endothelial+hyperpermeability:+implications+in+inflammation.">Molecular mechanisms of endothelial hyperpermeability: implications in inflammation.</a> Expert Rev. Mol. Med. 30, 11-19 (2009).</li><li>Viazzi, 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=Vascular+permeability,+blood+pressure,+and+organ+damage+in+primary+hypertension.">Vascular permeability, blood pressure, and organ damage in primary hypertension.</a> Hypertens. Res. 31, 873-879 (2008).</li><li>Scheppke, ., 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=Retinal+vascular+permeability+suppression+by+topical+application+of+a+novel+VEGFR2/Src+kinase+inhibitor+in+mice+and+rabbits.">Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits.</a> J. Clin. Invest. 118, 2337-2346 (2008).</li><li>Blanchet, M. R., 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=Loss+of+CD34+Leads+To+Exacerbated+Autoimmune+Arthritis+through+Increased+Vascular+Permeability.">Loss of CD34 Leads To Exacerbated Autoimmune Arthritis through Increased Vascular Permeability.</a> J. Immunol. 184, 1292-1299 (2010).</li><li>Dvorak, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell-derived+mediators+of+enhanced+microvascular+permeability,+vascular+permeability+factor/vascular+endothelial+growth+factor,+histamine,+and+serotonin,+cause+leakage+of+macromolecules+through+a+new+endothelial+cell+permeability+organelle,+the+vesiculo-vacuolar+organelle.">Mast cell-derived mediators of enhanced microvascular permeability, vascular permeability factor/vascular endothelial growth factor, histamine, and serotonin, cause leakage of macromolecules through a new endothelial cell permeability organelle, the vesiculo-vacuolar organelle.</a> Chem. Immunol. Allergy. 85, 185-204 (2005).</li><li>Le Guelte, A., Gavard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelial+cell-cell+junctions+in+endothelial+permeability.">Role of endothelial cell-cell junctions in endothelial permeability.</a> Methods Mol. Biol. 763, 265-279 (2011).</li><li>Martins-Green, M., Petreaca, M., Yao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+system+for+in+vitro+detection+of+permeability+in+human+"endothelium".">An assay system for in vitro detection of permeability in human "endothelium".</a> Methods Enzymol. 443, 137-153 (2008).</li></ol>

Os autores não têm nenhum conflito ou concorrentes interesses financeiros.

Permeabilidade vascular é um marcador para o estado fundamental do vaso sanguíneo. Aumento da permeabilidade vascular tem mostrado estar presente em várias doenças sistémicas, incluindo a diabetes, a hipertensão, e doenças auto-imunes 6,7,8. Aumento da permeabilidade vascular tem sido mostrado para ser mediada por tensão de corte, os factores de crescimento tais como o factor de crescimento endotelial vascular e factor de crescimento de fibroblastos, os mediadores inflamatórios tais como histamina, s...

Formação e manutenção de barreiras permeáveis ​​selectivos são essenciais para o desenvolvimento do órgão apropriado e 1,2 desempenho. Linha de células endoteliais do lúmen do vaso sanguíneo e formar uma barreira semi-permeável, que é essencial para o transporte selectivo entre o sangue e o espaço intersticial de todos os órgãos. Uma barreira de permeabilidade adequada é mantida através apertados célula-a-célula junções que são estritamente controladas por factores de crescimento, citoquinas e moléculas de stress outros produtos 3. O rompimento da barreira de células endoteliais pode resultar em aumento da permeabilidade vascular e de fuga. Estes efeitos são observados em vários estados de doença e a compreensão da sinalização sublinhado molecular requer métodos multidisciplinares 4,5. Neste artigo, descreve um método in vivo para medir a permeabilidade vascular utilizando um modelo de rato. O ensaio que se descreve, também conhecido como ensaio de Miles, é uma bem establtada método para testar a permeabilidade vascular in vivo. O ensaio baseia-se no facto de que, sob condições fisiológicas basais, a albumina não atravessa a barreira endotelial. Azul de Evans, um corante azóico com elevada afinidade para a albumina, é injectado na corrente sanguínea de um animal experimental e, sob condições fisiológicas, é esperado para ser restringida dentro dos vasos sanguíneos. Quando um estímulo permeabilidade vascular é adicionado, quer por aplicação tópica ou sistemicamente, os vasos sanguíneos começar a vazar proteína e, assim, também o azul de Evans, que se liga à albumina. Isto resulta numa coloração rápida azulada dos tecidos que têm vasos permeáveis. Injecção com êxito do corante na veia da cauda do rato lateral é crítica para o bom resultado do experimento. Técnica de injecção da veia da cauda requer prática extensiva e deverá ser dominadas antes de se iniciar a experiência. Permeabilidade vascular é altamente dependente da idade e do peso do umImal, então quando comparando diferentes linhagens de camundongos, é imperativo que os ratos ou outros assuntos de teste têm perto de datas de nascimento e peso idênticos. Outros factores que influenciam a permeabilidade da barreira endotelial são condições ambientais, tais como temperatura, humidade, e muito importante, o stress manuseamento do rato. Devido ao grande número de fatores que podem influenciar o resultado do experimento, é sempre aconselhável que o experimento é repetido pelo menos três vezes e análise estatística realizada. Este ensaio pode ser utilizado para comparar a permeabilidade ou vaso de ratos que foram geneticamente modificadas, bem como os ratos com diferentes antecedentes genéticos. A permeabilidade pode ser determinada na presença ou na ausência de um estímulo, dependendo da função do gene, que é modulada. Este ensaio também pode ser usado ou para testar o efeito de diferentes compostos sobre a permeabilidade do vaso.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> REAGENTES </td></tr><tr><td> Azul de Evans </td><td> SIGMA </td><td> E2129 </td><td></td></tr><tr><td> FORMAMIDA </td><td> INVITROGEN </td><td> 15515-026 </td><td></td></tr><tr><td> PBS </td><td></td><td></td><td> Fosfato 0,2 M 1,5 M NaCl pH 7,4 </td></tr><tr><td></td><td></td><td></td><td> EQUIPAMENTOS </td></tr><tr><td> ESPECTROFOTÔMETRO </td><td> EPPENDORF </td><td> 952000006 </td><td></td></tr><tr><td> Dispositivo de retenção MOUSE </td><td> Harvard Apparatus </td><td> 340012 </td><td></td></tr><tr><td> SERINGA </td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> AGULHAS </td><td> BD </td><td> 305106 </td><td> O calibre da agulha depende do tamanho do animal. </td></tr><tr><td> BALANÇO </td><td> Denver Instrument </td><td> TP-64 </td><td></td></tr></tbody></table>

an in vivo assay to test blood vessel permeability

Este método baseia-se na injecção intravenosa de azul de Evans nos ratinhos como modelo animal de teste. Azul de Evans é um corante que se liga a albumina. Sob condições fisiológicas o endotélio é impermeável à albumina, de modo do azul de Evans encadernados restos de albumina restrito dentro dos vasos sanguíneos. Em condições patológicas que promovem o aumento da permeabilidade vascular células endoteliais parcialmente perder seus contatos próximos e com o endotélio torna-se permeável a pequenas proteínas, como a albumina. Esta condição permite o extravasamento de azul de Evans dos tecidos. Um endotélio saudável impede o extravasamento do corante nos tecidos vizinhos vascularizados. Órgãos com aumento da permeabilidade irá mostrar um aumento significativo em comparação com coloração azul órgãos com endotélio intacto. O nível de permeabilidade vascular pode ser avaliado por simples visualização ou por medição quantitativa do corante incorporado por miligrama de tecido de controlo versus animal experimental / tecido. Dois powerful aspectos deste ensaio são a simplicidade e características quantitativas. O Evans Blue, podem ser extraídos a partir de tecidos por incubação de uma quantidade específica de tecido em formamida. Evans Blue é a absorvância máxima a 620 nm e a absorvência é mínima a 740 nm. Utilizando uma curva padrão para o azul de Evans, medições da densidade óptica pode ser convertida em miligrama de corante capturado por miligrama de tecido. A análise estatística deve ser usado para avaliar diferenças significativas na permeabilidade vascular.

Foi utilizado um ensaio in vivo para testar a permeabilidade vazamento navio em ratinhos de 8-12 semanas de idade. Este teste é útil para comparar a permeabilidade vascular relativa entre animais de fundo genético diferente ou em uma única estirpe de ratos submetidos a tratamentos que afectam a vasculatura. Os nossos resultados mostram que a estirpe de murganhos geneticamente modificados, criados no nosso laboratório tem um endotélio mais permeável em comparação com ratinhos de tipo selvagem. ...

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An in vivo Assay to Test Blood Vessel Permeability

Estamos apresentando um<em&gt; In vivo</em&gt; Ensaio para testar a permeabilidade dos vasos sanguíneos. Este ensaio baseia-se na injecção intravenosa de uma visualização do corante e subsequente da sua difusão para os espaços intersticiais.

Um<em&gt; In vivo</em&gt; Ensaio para ensaio de permeabilidade dos vasos sangüíneos

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

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Medicine

An in vivo Assay to Test Blood Vessel Permeability

64.4K visualizações.  Fox Chase Cancer Center. Estamos apresentando um In vivo Ensaio para testar a permeabilidade dos vasos sanguíneos. Este ensaio baseia-se na injecção intravenosa de uma visualização do corante e subsequente da sua difusão para os espaços intersticiais.

Vídeo: An in vivo Assay to Test Blood Vessel Permeability

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Ensaios In Vitro para Avaliar Formação e Perturbação de Embarque de Barreira de Sangue-Cérebro

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Medicina

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Na Vivo barreira hemato - encefálica permeabilidade ensaio nos ratos usando fluorescente etiquetado traçadores

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Neurociência

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

Um método In Vivo para avaliar a barreira de sangue do intestino e metabolismo hepático da Microbiota produtos

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

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

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

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 ...

Biologia

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 ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

No estudo in vivo da Human Interaction Endotelial-pericyte usando o plug Ensaio Matrix Gel no mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

Ensaio de Transcitose de Células Endoteliais como Modelo In Vitro para Avaliar a Permeabilidade da Barreira Interna Sangue-Retina

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

Avaliação de agentes de indução Hyperpermeability vasculares na pele com o ensaio de milhas

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the endothelial barrier dysfunction is related to many salivary gland secretory disorders. The present protocol describes an in vivo paracellular permeability detection method to evaluate the function of endothelial tight junctions (TJs) in mouse submandibular glands (SMG). First, fluorescence-labeled dextrans with different molecular weights (4 kDa, 40 kDa, or 70 kDa) were injected into the angular veins of mice. Afterward, the unilateral SMG was dissected and fixed in the customized holder under a two-photon laser-scanning microscope, and then images were captured for blood vessels, acini, and ducts. Utilizing this method, the real-time dynamic leakage of the different-sized tracers from blood vessels into the basal sides of the acini and even across the acinar epithelia into the ducts was monitored to evaluate the alteration of the endothelial barrier function under physiological or pathophysiological conditions.

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

In the present protocol, the endothelial barrier function of the submandibular gland (SMG) was evaluated by injecting different molecular weighted fluorescent tracers into the angular veins of test animal models in vivo under a two-photon laser-scanning microscope.

In vivo Detecção de Permeabilidade Vascular em Glândula Submandibular de Camundongo

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the ...

Simplificando os estudos de angiogênese com um ensaio de tampão de gel de matriz modificada

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

Autor em destaque: Investigando a angiogênese e a permeabilidade dos vasos por meio de um ensaio de tampão de gel de matriz modificada

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

Ensaio de plugue de gel de matriz in vivo modificado para estudos de angiogênese

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...

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