Name: an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks
Uploaded: 2017-02-09T14:00:00.000+00:00
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Description: Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

This work was supported by National Institutes of Health Grant 5-P20GM103629 to WLM and the Tulane Center for Aging. We would like to thank Matthew Nice for his help with editing the protocol text.

Todos os experimentos e os procedimentos com animais foram aprovados pelo Comitê Cuidado e Uso da Universidade de Tulane Institucional Animal (IACUC). 1. Configuração Procedimento Cirúrgico <ol><li> instrumentos de autoclave, materiais cirúrgicos e fontes de cultura antes da cirurgia. material cirúrgico para cada rato incluem: 1 cortina, uma cortina com furo pré-cortados (0,5 pol x 1,5 pol) no centro, compressas de gaze, e um resguardo absorvente. Instrumentos cirúrgicos incluem: um bisturi com uma lâmina de número 10, 2 pares de pinças, e um par de tesouras finas. fontes de cultura incluem: uma cortina, um par de pinças, e preparado inserções placa de 6 poços com filtros de policarbonato. </li><li> Esterilizar uma plataforma de Plexiglass, uma fase cirúrgica e um espaço de bancada cirúrgica com 70% de etanol. Mantenha o estágio cirúrgico em um recipiente estéril até o uso. <ol><li> Criar uma fase cirúrgica através da perfuração de um a cerca de 2 em por em um furo no centro de uma placa de cultura de 100 milímetros. Em seguida, use uma lixa para suavizarquaisquer arestas vivas e adicionar uma camada de cola de silicone para as bordas do furo para criar uma superfície elevada para os tecidos. </li><li> Alternativamente, projetar o estágio cirúrgico utilizando software CAD e fazer por 3-D de impressão (Figura 1). </li></ol></li><li> Coloque um resguardo absorvente estéril para baixo e estabelecer uma plataforma de plexiglass em cima dela. Coloque a cortina, sem um orifício pré-cortado, uma almofada aquecida ao longo do lado do resguardo absorvente. </li><li> Pré-aquecido estéril soro fisiológico tamponado com fosfato (PBS), meios de comunicação e solução salina a 37 ° C. media de lugar e PBS em placas de cultura separados no topo da almofada de aquecimento e coloque soro fisiológico em um tubo de 50 mL cônico ao lado da configuração cirúrgico. </li><li> Certifique-se de todos os pacotes são abertos antes do início da cirurgia para garantir a manipulação estéril de todos os materiais. Uma lista completa das ferramentas comuns usados ​​neste procedimento são listados na Tabela de Materiais cirúrgicos específicos e ferramentas. </li></ol> 2. Mesentery colheita Tissue <ol><li> Use ratos Wistar machos adultos (350 ± 25 g; 6 - 8 semanas de idade). Outras tensões e as idades de ratos podem ser substituídos. </li><li> Anestesiar ratos por meio de uma injecção intramuscular de cetamina (80 mg / kg de peso corporal) e xilazina (8 mg / kg de peso corporal). Confirmar o rato está sob anestesia por beliscar entre os dedos para verificar se há uma resposta reflexa; não deve haver nenhum. analgesia preventiva para este procedimento terminal não é necessário. </li><li> Raspar a região abdominal e remover cabelo restante usando creme de depilação. Wipe pele abdominal duas vezes com álcool isopropílico a 70%, seguido de povidona-iodo. Para as toalhitas o cirurgião deve começar no centro do local cirúrgico e mover-se para o exterior da área preparada de uma maneira circular a fim de não se sobrepõem áreas que tenham sido previamente limpo com o mesmo pedaço de gaze estéril ou mecha de algodão estéril. Em seguida, transferir animal para a instalação cirúrgica esterilizada e coloque em cima da plat plexiglassFormato. </li><li> Usando uma lâmina de bisturi, fazer uma 0,75 em - 1,25 na incisão no intestino a partir de 1º de abaixo do esterno. Tenha cuidado para não perfurar o intestino ou mesentério (1 camada de pele, uma camada de tecido conjuntivo, e uma camada de músculo). </li><li> Coloque uma cortina com um buraco pré-cortada sobre a incisão e coloque um estágio cirúrgico esterilizado em cima da cortina. Assegurar as alinha abertura com a incisão. Use aplicadores com ponta de algodão estéreis para localizar e retirar o íleo através da abertura estágio cirúrgico. </li><li> Puxe 6 - 8 janelas mesentérica através do estágio usando aplicadores com ponta de algodão, e tenha cuidado para não tocar as janelas (Figura 1). Os tecidos são tipicamente colhidas a partir da região do ileo do intestino delgado começando perto do ceco. Mantenha tecidos expostos úmido com solução salina estéril aquecida, conforme necessário, utilizando uma seringa estéril para escorrer a solução. </li><li> Eutanásia do rato através de injecção intracardíaca de pentobarbital de sódio (0,2 mL por rato). Antes de retirar-mejanelas senteric, garantir o rato é sacrificado pela palpação do coração; não deve haver nenhum impulso. </li><li> Remover os tecidos mesentério desejados usando uma pinça para agarrar a almofada de gordura e uma tesoura fina para cortar a janela. Deixar uma fronteira de gordura (2 mm), em torno da janela. Lavar tecidos vez em aquecido PBS estéril e uma vez na mídia. </li><li> Voltar íleo exteriorizada para a cavidade abdominal e dispor de animais de acordo com as diretrizes institucionais. </li></ol> 3. Mesentery Cultura de Tecidos de Estudos de lapso de tempo <ol><li> Transferência de fontes autoclavados cultura (ver secção 1.1) e tecidos para uma capela de fluxo laminar estéril. </li><li> Use uma pinça para transferir cada tecido no topo de uma membrana filtro de policarbonato. tecidos garra pelas almofada de gordura para evitar danificar a vasculatura. </li><li> Rapidamente se espalhou o tecido usando a almofada de gordura, tomando cuidado para não tocar na janela. Inverta a inserção com o tecido para dentro da parte inferior de uma placa de 6 poços e cobrir com 3 ml de meio (Figura 1 </strong&gt;). meios típicos utilizados para este procedimento inclui mínima meio essencial (MEM) com 1% de Penicilina Estreptomicina (Penstrep) e 10% de Soro Fetal Bovino (FBS). Os meios podem ser suplementados com outros soros e / ou factores de crescimento para estimular a angiogénese. </li><li> Repita os passos de 3,2-3,3 para cada tecido e cultura em condições de incubadora padrão (5% de CO2, 37 ° C) durante até 5 dias. </li></ol> 4. Imagem Time-Lapse de Mesentery Tissue <ol><li> No dia da imagiologia, suplementar os meios em cada poço com conjugado BSI-LECTINA e incubar em condições padrão de cultura, durante 30 min. Lavar tecidos duas vezes com meio livre de lectina. BSI-LECTINA mancha permanecerá visível no tecido mesentério durante até 3 dias em cultura. </li><li> Transferir a placa para um estágio de microscópio. Identificar os vasos sanguíneos e linfáticos com base na sua morfologia e estrutura de rede. </li><li> Localize uma região de rede desejado em cada tecido e tomar imagens. Tome nota do imaginglocal para assegurar a mesma região será capturado para imagens subsequentes. Se estiver usando um palco motorizado, documentar as coordenadas. </li><li> Retorno tecidos para a incubadora e continuar a cultura até ponto final desejado. Repita os passos 4,1-4,3 conforme necessário dependendo pontos de tempo experimentais desejados. </li></ol> 5. Tissue imunomarcação <ol><li> BSI-lectina Labeling <ol><li> Incubar os tecidos durante 30 min a 37 ° C com 01:40 lectina conjugada com FITC em meios (2,5 ml solução de anticorpo por poço em placas de 6 poços) seguida de duas lavagens com meio. Para lavagens, adicionar mídia e substitua imediatamente. </li></ol></li><li> / Etiquetagem Morto Vivo <ol><li> Incubar os tecidos durante 10 min a 37 ° C com 1: 500 de etídio 2 mM homodímero-1 e 1: 500 calceína AM 1 mM em meio (2,5 ml solução de anticorpo por poço em placas de 6 poços) seguida de duas lavagens com meio. </li></ol></li><li> BSI-lectina / Etiquetagem NG2 <ol><li> tecidos espalhada sobre um microscópio deslizoue (1 - 2 tecidos / lâmina) e deixar secar. Retire o excesso de gordura com um bisturi, pressionando com firmeza para excisar a gordura. </li><li> Fix tecidos em metanol frio durante 30 min a -20 ° C. Lavar tecidos com PBS (3 x 10 min). </li><li> Para marcação com anticorpos primários incubar tecidos durante 1 h à temperatura ambiente com um anticorpo policlonal de NG2: 100 de coelho e soro de cabra normal a 5% (NGS). Lavar tecidos com PBS (3 x 10 min). </li><li> Para a marcação do anticorpo secundário tecidos incubar durante 1 h à temperatura ambiente com 1: 100 de cabra anti-coelho conjugado anticorpo-CY2 (GAR-CY2) e 5% de NGS. Lavar tecidos com PBS (3 x 10 min). </li><li> Incubar os tecidos durante 30 minutos à temperatura ambiente com lectina 01:40 conjugado com FITC em PBS seguido por duas lavagens com PBS. Para lavagens, adicione PBS e depois substituir imediatamente. </li><li> Para montar os slides, cobrir tecidos com 50:50 PBS e solução de glicerol e lugar lamela em cima. Selar as bordas de slide usando unha polonês. </li></ol></li><li> LYVE-1 / Etiquetagem PECAM<ol><li> Espalhe tecidos numa lâmina de microscópio (1 - 2 tecidos / lâmina) e deixa-se secar. Retire o excesso de gordura com um bisturi, pressionando com firmeza para excisar a gordura. </li><li> Fix tecidos em metanol frio durante 30 min a -20 ° C. Lavar tecidos com PBS + 0,1% de saponina (3 x 10 min). </li><li> Para marcação com anticorpos primários incubar tecidos durante 1 h à temperatura ambiente com 1: anticorpo CD31 monoclonal biotinilado 200 rato e 1: policlonal LYVE-1 anticorpo 100 de coelho em PBS + 0,1% de saponina + albumina de soro bovino a 2% (BSA) + 5% de NGS . Lavar tecidos com PBS + 0,1% de saponina (3 x 10 min). </li><li> Para a marcação do anticorpo secundário, tecidos incubar durante 1 h à temperatura ambiente com 1: 500 de anticorpo de estreptavidina conjugada com Cy3 e 1: 100 GAR-CY2 em PBS + 0,1% de saponina + 2% de BSA + 5% de NGS. Lavar tecidos com PBS + 0,1% de saponina (3 x 10 min). </li><li> Para montar slides, tecidos de cobertura com 50:50 solução PBS e glicerol e colocar uma lamela em cima. Selar as bordas de slide usando unha polonês. </li></ol></li><li> BrdU / BSI-lectina Labeling <ol><li> Adicionar 1 mg / mL BrdU aos meios de comunicação e substituir a mídia de tecido com uma solução de BrdU. Incubar durante 2 horas a 37 ° C. </li><li> Espalhe tecidos numa lâmina de microscópio (1 - 2 tecidos / lâmina) e deixa-se secar. Retire o excesso de gordura com um bisturi, pressionando com firmeza para excisar a gordura. </li><li> Fix tecidos em metanol frio durante 30 min a -20 ° C. Lavar tecidos com PBS (3 x 10 min). </li><li> Desnaturação de DNA de tecidos em HCl 2 M durante 1 h a 37 ° C. Lavar tecidos em PBS + 0,1% de saponina (3 x 10 min). </li><li> Para marcação com anticorpos primários, tecidos incubar durante 1 h à temperatura ambiente com 1: 100 monoclonal de ratinho anti-BrdU em PBS + 0,1% de saponina + 2% de BSA + 5% de NGS. Lavar tecidos com PBS + 0,1% de saponina (3 x 10 min). </li><li> Para a marcação do anticorpo secundário, tecidos incubar durante 1 h à temperatura ambiente com 1: 100 de anticorpo de cabra anti-rato Cy3-conjugado (GAM-Cy3) em PBS + 0,1% de saponina + 2% de BSA + 5% de NGS. Lavar tecidos com PBS + 0,1% de saponina (3 x 10 min). </li><li&gt; tecidos Incubar durante 30 minutos à temperatura ambiente com lectina 01:40 conjugado com FITC em PBS seguido por duas lavagens com PBS. </li><li> Para montar slides, tecidos de cobertura com 50:50 PBS e solução de glicerol e lugar lamela em cima. Selar as bordas de slide usando unha polonês. </li></ol></li><li> BSI-lectina / rotulagem CD11b <ol><li> Espalhe tecidos numa lâmina de microscópio (1 - 2 tecidos / lâmina) e deixa-se secar. Retire o excesso de gordura com um bisturi, pressionando com firmeza para excisar a gordura. </li><li> Fix tecidos em metanol frio durante 30 min a -20 ° C. Lavar tecidos com PBS + 0,1% de saponina (3 x 10 min). </li><li> Para marcação com anticorpos primários tecidos incubar durante 1 h à temperatura ambiente com 1: 100 de murganho anti-rato de CD11b em PBS + 0,1% de saponina + 2% de BSA + 5% de NGS. Lavar tecidos com PBS + 0,1% de saponina (3 x 10 min). </li><li> Para a marcação do anticorpo secundário tecidos incubar durante 1 h à temperatura ambiente com 1: 100 GAM-Cy3 em PBS + 0,1% de saponina + 2% de BSA + 5% de NGS. Lavar tecidos com PBS+ 0,1% de saponina (3 x 10 min). </li><li> Incubar os tecidos durante 30 minutos à temperatura ambiente com lectina 01:40 conjugado com FITC em PBS seguido por duas lavagens com PBS. </li><li> Para montar slides, tecidos de cobertura com 50:50 PBS e solução de glicerol e lugar lamela em cima. Selar as bordas de slide usando unha polonês. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Este protocolo documenta um método para usar o modelo de cultura de mesentério de rato como uma ferramenta ex vivo para geração de imagens de lapso de tempo de crescimento da rede microvascular. Trabalhos anteriores em nosso laboratório estabeleceu a utilização do nosso modelo para 1) angiogênese 8, 2) lymphangiogenesis 8, 3) interações celulares pericyte-endotelial 8 e 4) teste de drogas anti-angiogênico 9.</su...

O crescimento da rede microvascular e remodelação são denominadores comuns para a função do tecido, cicatrização de feridas, e patologias múltiplas e um processo chave é a angiogénese, definido como o crescimento de novos vasos sanguíneos a partir dos existentes 1, 2. Para Engenharia de Tecidos novos navios ou projetar terapias baseadas angiogénicos, compreendendo a importância da dinâmica celulares envolvidos na angiogênese é crítica. No entanto, este processo é complexo. Ela pode variar em locais específicos dentro de uma rede microvascular e envolve vários tipos de células (isto é, células endoteliais, células musculares lisas, pericitos, macrófagos, as células estaminais) e múltiplos sistemas (redes linfáticas e redes neurais). Embora os modelos in vitro têm contribuído enormemente para examinar a relação entre diferentes células envolvidas na angiogênese 3, a sua relevância fisiológica pode ser prejudicada devido à thei<html> r complexidade limitada eo fato de que eles não refletem de perto um cenário in vivo. Para superar estas limitações, os sistemas de cultura tridimensionais 3, ex vivo modelos de tecido 4, sistemas de microfluidos 5, 6, 7 e modelos computacionais foram desenvolvidos e introduzidos nos últimos anos. No entanto, ainda existe uma necessidade para um modelo com capacidade de lapso de tempo para investigar a angiogénese em redes microvasculares intactas ex vivo. A criação de novos modelos de lapso de tempo para estudos de angiogênese com esse nível de complexidade irá fornecer uma ferramenta inestimável para compreender os mecanismos subjacentes que regulam a angiogênese e para melhorar terapias. Um modelo de potencial que permite a investigação ex vivo da angiogênese através de uma rede microvascular intacta é o modelo de cultura de mesentério de rato&gt; 8. Em trabalho recente, temos demonstrado que o sangue e redes microvasculares linfáticos permanecer viável após o cultivo. Mais importante, o modelo de cultura de mesentério de rato pode ser usado para investigar as interações funcionais pericyte-endotelial de células, sangue e conexões das células endoteliais linfáticas e de imagem time-lapse. O objetivo deste trabalho é fornecer nosso protocolo para o método de imagem time-lapse. Os resultados representativos documentar os vários tipos de células que permanecem viáveis ​​após a estimulação de angiogénese com soro e oferecem exemplos da utilização deste método para a quantificação de respostas angiogénicas específicas dos tecidos, bem como estudos de acompanhamento de células endoteliais.

<table><tbody><tr><td>Drape</td><td>Cardinal Health</td><td>4012</td><td>12&amp;rdquo; x 12&amp;rdquo; Bio-Shield Regular Sterilization Wraps</td></tr><tr><td>Scalpel Handle</td><td>Roboz Surgical Instrument</td><td>RS-9843</td><td>Scalpel Handle, #3; Solid; 4&quot; Length</td></tr><tr><td>Sterile Surgical Blade</td><td>Cincinnati Surgical</td><td>0110</td><td>Stainless Steel; Size 10</td></tr><tr><td>Culture Dish (60 mm)</td><td>Thermo Scientific</td><td>130181</td><td>10/Sleeve</td></tr><tr><td>Graefe Forcep (curved tweezers)</td><td>Roboz Surgical Instrument</td><td>RS-5135</td><td>Micro Dissecting Forceps; Serrated; Slight Curve; 0.8 mm Tip Width; 4&quot; Length</td></tr><tr><td>Graefe Forcep (straight tweezers)</td><td>Roboz Surgical Instrument</td><td>RS-5130</td><td>Micro Dissecting Forceps; Serrated, Straight; 0.8 mm Tip Width; 4&quot; Length</td></tr><tr><td>Noyes Micro Scissor</td><td>Roboz Surgical Instrument</td><td>RS-5677</td><td>Noyes Micro Dissecting Spring Scissors;&lt;br/&gt; Straight, Sharp-Blunt Points; 13 mm Cutting Edge; 0.25 mm Tip Width, 4 1/2&quot; Overall Length</td></tr><tr><td>Gauze Pads</td><td>FisherBrand</td><td>13-761-52</td><td>Non-Sterile Cotton Gauze Sponges; 4&quot; x 4&quot; 12-Ply</td></tr><tr><td>Cotton-Tippled Applicators</td><td>FisherBrand</td><td>23-400-124</td><td>6&quot; Length; Wooden Shaft; Single Use Only</td></tr><tr><td>6-Well Plate</td><td>Fisher Scientific</td><td>08-772-49</td><td>Flat Bottom with Low Evaporation Lid; Polystyrene; Non-Pyrogenic</td></tr><tr><td>Sterile Syring 5 mL</td><td>Fisher Scientific</td><td>14-829-45</td><td>Luer-Lok Tip</td></tr><tr><td>Sterile Bowl</td><td>Medical Action Industries Inc.</td><td>01232</td><td>32 oz. Peel Pouch; Blue; Sterile Single Use</td></tr><tr><td>6-Well Plate Inserts (CellCrown Inserts)</td><td>SIGMA</td><td>Z681792-3EA</td><td>6-Well Plate Inserts; Non-Sterile</td></tr><tr><td>Polycarbonate Filter Membrane</td><td>SIGMA</td><td>TMTP04700</td><td>Isopore Membrane Filter; Polycarbonate; Hydrophilic; 5.0&amp;nbsp;&amp;micro;m, 47&amp;nbsp;mm, White Plain</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>Beuthanasia</td><td>Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply)</td><td>MWI #: 011168</td><td>Active Ingredient: Per 100 mL, 390 mg pentobarbital sodium, 50 mg phenytoin sodium&amp;nbsp;</td></tr><tr><td>Ketamine</td><td>Fort Dodge Animal Health (Ordered from MWI Veterinary Supply)</td><td>MWI #: 000680</td><td>Kateset 100 mg/mL</td></tr><tr><td>Xylazine</td><td>LLOYD. Inc. (Ordered from MWI Veterinary Supply)</td><td>MWI #: 000680</td><td>Anased 100 mg/mL</td></tr><tr><td>Saline</td><td>Baxter</td><td>2F7122</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14040-133</td><td></td></tr><tr><td>MEM</td><td>Invitrogen</td><td>11095080</td><td></td></tr><tr><td>PenStrep</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>16000-044</td><td></td></tr><tr><td>BSA</td><td>Jackson ImmunoResearch</td><td>001-000-162</td><td></td></tr><tr><td>Saponin&amp;nbsp;</td><td>SIGMA</td><td>S7900-100G</td><td></td></tr><tr><td>Isopropyl Alcohol</td><td>Fisher Scientific</td><td>S25372</td><td></td></tr><tr><td>Povidone-Iodine</td><td>Operand</td><td>82-226</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>SIGMA</td><td>320331</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>67-56-1</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>56-81-5</td><td></td></tr><tr><td>FITC-conjugated Lectin</td><td>SIGMA</td><td>L9381-2MG</td><td></td></tr><tr><td>Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody</td><td>SIGMA</td><td>AB5320</td><td></td></tr><tr><td>PECAM (CD31) Antibody</td><td>BD Biosciences</td><td>555026</td><td></td></tr><tr><td>LYVE-1 Antibody</td><td>AngioBio Co.</td><td>11-034</td><td></td></tr><tr><td>Goat Anti-Rabbit Cy2-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>111-585-144</td><td></td></tr><tr><td>Goat Anti-Mouse Cy3-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>115-227-003</td><td></td></tr><tr><td>Streptavidin Cy3-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>016-160-084</td><td></td></tr><tr><td>Live/Dead Viability/Cytotoxicity Kit</td><td>Invitrogen</td><td>L3224</td><td></td></tr><tr><td>Normal Goat Serum&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>005-000-121</td><td></td></tr><tr><td>5-Bromo-2&#039;-Deoxyuridine</td><td>SIGMA</td><td>B5002</td><td></td></tr><tr><td>Monoclonal Mouse Anti-Bromodeoxyuridine&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Clone Bu20a</td><td>Dako</td><td>M074401-8</td><td></td></tr><tr><td>Mouse Anti-Rat CD11b&amp;nbsp;</td><td>AbD Serotec</td><td>MCA275R</td><td></td></tr></tbody></table>

an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks

A angiogénese, definido como o crescimento de novos vasos sanguíneos a partir de vasos pré-existentes, envolve células endoteliais, pericitos, células musculares lisas, células do sistema imunológico, bem como a coordenação com vasos linfáticos e nervos. A multi-celular, as interacções multi-sistema exige a investigação de angiogénese num ambiente fisiologicamente relevante. Assim, enquanto o uso de modelos de cultura celular in vitro proporcionaram conhecimentos mecanicistas, uma crítica comum é que eles não recapitular a complexidade associada com uma rede microvascular. O objetivo deste protocolo é o de demonstrar a capacidade de fazer comparações de lapso de tempo de redes microvasculares intactas antes e após a estimulação da angiogénese em tecidos mesentério de rato cultivadas. tecidos cultivados contêm redes microvasculares que mantêm a sua hierarquia. imunohistoquímica confirma a presença de células endoteliais, células musculares lisas, pericitos, vasos sanguíneos e vasos linfáticos. Em umddition, rotulando tecidos com BSI-lectina permite a comparação de lapso de tempo de regiões de rede local antes e após a estimulação fator de soro ou crescimento caracterizada pelo aumento da germinação capilar e densidade de vasos. Em comparação com os modelos de cultura de células comuns, este método proporciona uma ferramenta para estudos de linhagens de células endoteliais e avaliação de medicamentos angiogénica do tecido específico em redes microvasculares fisiologicamente relevantes.

Após 3 dias em cultura, os tecidos foram marcados com um kit de viabilidade / citotoxicidade Live / morto para demonstrar a viabilidade da microcirculação no mesentério de ratos modelo de cultura (Figura 2A). A maioria das células presentes no mesentério permaneceram viáveis ​​na cultura em que as células endoteliais foram identificados com base na sua localização em segmentos microvasculares. Proliferação de células endoteliais foi também confirmada po...

Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

A<em&gt; Ex Vivo</em&gt; Método for Imaging Time-Lapse of cultivadas Rato Mesentéricas microvasculares Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

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Research

JoVE Journal

Developmental Biology

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

6.8K visualizações.  Tulane University. Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis. 

Vídeo: An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In Vitro e In Vivo modelo para estudar a adesão bacteriana para o Vaso de parede em condições de escoamento

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Imunologia e Infecção

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imagiologia neutrófilos e monócitos em Mesenteric Veins por Microscopia intravital em ratos anestesiados em Tempo Real

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

Rat Assay Angiogenesis Mesentery

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Biologia

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Exteriorização Rat Mesentério: Um modelo para investigar a dinâmica celulares envolvidos na angiogênese

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

Avaliação dos mecanismos de controlo Vascular utilizando a microscopia das artérias de resistência isolada de ratos

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Medicina

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Rato Fetal Whole Intestino Sistema de Cultura para<em&gt; Ex Vivo</em&gt; Manipulação de vias de sinalização e tridimensional Imagens ao vivo de Desenvolvimento Villus

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

Medição dinâmica e imagem de capilares, arterioles e pericias em coração de rato

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

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

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

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 ...

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 ...

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 ...

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