Nós reconhecemos o trabalho do Dr. Adlah Sukkar, MD em ajudar com elenco do pulmão. Este trabalho foi financiado pelo NHLBI, HL088005.

Todos os protocolos realizados com ratos foram aprovados pelo Animal Care Universidade Johns Hopkins e do Comitê utilização e de acordo com as diretrizes do NIH. Sempre que possível, o animal deve ser cirurgicamente preparado numa zona separada da zona cirúrgica para minimizar a contaminação do local da cirurgia. 1. Anestesia / analgesia <ol><li> Rat lugar (ratos Sprague Dawley machos, 125-150 g; Harlan, Indianapolis, IN), numa câmara de indução infundida com 3% de isoflurano. </li><li> Coloque rato anestesiado a bordo cirurgia anexado ao cone do nariz e ventilador com 3% de anestésico isoflurano. Use a técnica estéril para todos os procedimentos. Drapejar rato para assegurar campo operacional estéril. </li></ol> 2. Intubação <ol><li> Imobilizar apêndices em posição supina, utilizando fita cirúrgica. </li><li> Remover rato de cone do nariz, estender a língua com uma pinça acolchoados. </li><li> Use 14 intracath bitola com Stilet de metal despontava como guia. Deslize para trás parangue, em traqueia. </li><li> Remover Stilet metálica deixando intracath plástico branco na traqueia. Verifique se o rato está respirando e ar flui através do tubo. </li><li> Substitua cone do nariz com adaptador direto ao cateter, ligue rato ao ventilador (90 respirações / min; 8 de volume ml / kg de maré; Rodent Ventilator modelo 683, Harvard Apparatus, Holliston, Massachusetts). </li><li> Aplicar Puralube (Butler Schein, Dublin, OH) pomada veterinária nos olhos. </li></ol> 3. Toracotomia <ol><li> Coloque o lado direito do rato para baixo, imobilizar apêndices com fita cirúrgica. </li><li> Shave área costelas do lado esquerdo. </li><li> Remover excesso de pele com pó pequeno. </li><li> Faça campo estéril, limpando-se com álcool seguido de iodo-povidona vareta com tampão (Dynarex Corporation, Orangebur, NY). Repetir este processo mais duas vezes (para um total de três esfrega). </li><li> Faça incisão transversal com tesoura estéril dissecando ou estéreis bisturi no centro de campo. </li><li> Blunt dissecar através layers de tecido e gordura para baixo para costelas (última camada é fina membrana que cobre as costelas). </li><li> Conte reforços para determinar 3 º espaço intercostal. </li><li> Usando estéreis 45 ° Graefe fórceps, fazer incisão brusco entre o 3 º e 4 ª costela. </li><li> Inserir separadores de costela, puxe-a delicadamente criando vazio aberto com visualização completa do pulmão, fita lugar em suturas para manter aberta. </li></ol> 4. Ligadura da artéria pulmonar esquerda <ol><li> Com 90 ° fórceps Graefe, mover pulmão esquerdo para trás com a mão direita. </li><li> Usando o padrão Dumont # 5 fórceps retas, pegue artéria pulmonar esquerda e vias aéreas com a mão esquerda. Artéria pulmonar esquerdo deitou em cima de vias aéreas. Certifique-se de que o fórceps são directamente perpendicular à mesa. Além disso, é mais eficaz para pegar a artéria pulmonar esquerda / brônquio principal esquerdo na posição mais distal (mais próxima de parênquima). Empurre o pulmão esquerdo de ventilação de lado com esta manobra mão esquerda. </li><li> Use Dumontpadrão # 5 45 ° fórceps curvos para separar a artéria pulmonar esquerda do brônquio principal esquerdo nas suas fronteiras naturais. Esta linha de separação parece fina e branca entre as duas estruturas individuais. </li><li> Separar diretamente sob artéria, sem passar pelo navio; suavemente deslizar as pontas fórceps mantidos juntos ao longo da separação. </li><li> Continue até dicas de pinças visivelmente separados da artéria pulmonar esquerda e brônquio principal esquerdo. Apenas um ponto pequeno de ponta precisa estar totalmente completamente. Se o sangue pode ser visualizado na ponta da pinça, então não é na sua totalidade através de ligação e não deve ser tentada. Depois de atravessar o espaço, a artéria pulmonar esquerda vai colocar na curva do fórceps. Segure nesta posição. </li><li> Gentilmente liberar reta brusco aperto fórceps (mão esquerda) e pegue um pedaço de fio de pré-corte (~ 2-3 polegadas, tamanho polipropileno sutura 6-0; Myco médicos, Cary, NC). </li><li> Abra fórceps curvos embalam artéria pulmonar esquerda e gsutura rab. Puxe suavemente sutura através do espaço entre a artéria pulmonar esquerda e brônquio principal esquerdo em um movimento ascendente em relação à curva do fórceps. </li><li> Amarrar ocluindo o pulmonar esquerda com um nó quadrado, restante cuidadosamente recorte de sutura. </li><li> Nervuras estreitas, utilizando fórceps rombas para segurar costela e duas vezes com sutura de polipropileno (monofilamento azul) 4-0 ligado ao tamanho de 19 mm, 3/8 reversa círculo agulha cortante (Myco Medical, Cary, NC) em uma pinça hemostática, tomando cuidado para não sutura da pele (apenas costelas). </li><li> Completar um nó frouxo quadrado, encha o pulmão, o lugar da pressão expiratória final positiva (PEEP; 2-5 cmH 2 O), hyperinflate nó de pulmão, então segura firmemente, e fazer outro nó completo antes snipping restante da sutura. Remover da PEEP e visualizar durante 30 segundos para garantir que o pulmão não se desmorone. Aplicar 5 gotas Bupivicaine (APP Pharmaceuticals, Schaumbur, IL). </li><li> Fechar pele colocando cola de tecido sobre a ferida e empurre a pele em conjunto, utilizando de volta end aplicador de ponta de algodão. Dar Bupivicaine (2,0 mg / kg por via subcutânea) no local da incisão posteriormente a cada 8 horas, durante 24 horas ou até que o animal retoma a actividade normal. À medida que a camada subcutânea nesta área é muito fina, não é fácil de suturar por conta própria. Quando a cola de tecidos é aplicado à pele e é pressionado em conjunto também encerra a camada subcutânea. </li><li> Desligue o gás isoflurano, mas continuam a ventilar o rato por 1-2 minutos em ar ambiente até a volta de movimento voluntário. Desligue o tubo traqueal do ventilador e garantir que o rato está respirando espontaneamente, antes de removê-lo. </li><li> Limpe Puralube de olhos com cotonete e movimento do monitor e recuperação. Injectar de cloridrato de buprenorfina (0,05 mg / kg por via intraperitoneal, Butler Schein, Dublin OH). Continuar a entrega de cada analgésico hr 12 para 48 horas após a cirurgia. </li></ol> 5. A canulação da artéria carótida esquerda Para avaliar a magnitude dos brônquios perfusion do pulmão esquerdo isquémica em pontos de tempo desejado, após a ligação da artéria pulmonar esquerda, injectar microesferas marcadas através da artéria carótida esquerda no arco aórtico. Prepare ratos como acima 1-2. <ol><li> Corte na linha média ao longo do pescoço, Blunt dissecar a revelar traquéia e artéria carótida esquerda (local da injeção de microesferas). </li><li> Inserção do cateter cheio de heparina ponta, solução salina sem corte de tubagem PE20 (Becton Dickinson, Sparks MD) para o vaso ligado à agulha de 25 g, de 4 vias torneira, uma seringa ml. </li><li> Garrafa lugar de microesferas (15 um carmesim microesferas fluorescentes de poliestireno, 1 x 10 6 / ml esferas; Invitrogen, Eugene, OR) em água ultra-sons durante 30 seg. </li><li> Remover vórtice garrafa, e elaborar 0,5 ml (500.000 microesferas) em um 1 ml de vidro seringa Hamilton (Hamilton Company, Reno, NV) através de uma agulha 20 g. </li><li> Anexar seringa Hamilton para 4 vias torneira e infundir microesferas com seringa bomba (taxa: 500 ul / min; Genie Plus, Kent Scientific, Torringtem, CT). </li><li> Remover Hamilton seringa e aparelho nivelado com 1 ml de solução salina heparized em 500 ul / min. </li><li> Toracotomia peito cheio e desangrar o rato, cortando a veia cava inferior. </li><li> Remover o pulmão esquerdo e de outros tecidos de interesse. </li><li> Para extrair microesferas a partir de tecido, tomar todo o pulmão esquerdo do rato, após sangria e colocá-lo em KOH 2 M (4-6 ml). Coloque em um banho de água 55 ° C e deixe durante a noite para a digestão do tecido. Adicionar Tween 80 (0,25%), para lavar as pérolas, vortex (10 seg) e centrifugação (2.000 rpm, 20 ° C durante 10 min). Remover o sobrenadante, adicionar acetato de 2-etoxietilo (1 ml), vortex e deixar repousar durante 1 hora. Vortex a suspensão e centrifuga-se (2000 rpm, (20 ° C durante 10 min) Remover a camada de acetato de 2-etoxietilo aquosa contendo a fluorescência lugar, numa cuvete e medida utilizando um Hitachi F-2500 espectrofotómetro de fluorescência (excitação 618 612/emission. ; Digilab, Holliston, MA). </li></ol>

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Ligação da artéria pulmonar esquerda em todas as espécies testadas conduz a neovascularização sistémica robusta do pulmão isquêmico. Nós apresentamos os detalhes da abordagem cirúrgica num modelo de rato. Nossos resultados produzidos por vazamento vascular, histopatologia e na rotulagem vivo demonstram que artérias brônquicas proliferar e perfundir o parênquima pulmonar. Assim, os mecanismos de angiogénese brônquica pode ser estudado num modelo animal que se assemelha à condição huma...

Angiogénese sistémica no pulmão é bem reconhecida. Em estados patológicos tais como a asma 2, fibrose pulmonar intersticial, 3, 4 cancro, e o tromboembolismo pulmonar crónica 5, a vasculatura e sistémico em torno das prolifera pulmonares e invade o parênquima pulmonar. No entanto, os modelos animais para estudar esta ativação diferencial da sistêmica e não a circulação pulmonar são poucos. Talvez o modelo mais reprodutível da neovascularização sistémica no pulmão do mamífero adulto é a que ocorre após a indução de isquemia da artéria pulmonar crónica. A resposta à obstrução da artéria pulmonar em humanos 5-7, cães 8, 9 porcos, ovelhas 10, 11 cobaias, ratos 1, 12, 13, 14 e ratos é a rápida proliferação da artéria brônquica, bem como as artérias intercostais. Os mecanismos responsáveis ​​por neovasculari sistémicazação do pulmão após isquemia pulmonar são amplamente desconhecido e não tenham sido amplamente estudadas. O curso de tempo da angiogénese brônquica em ratos após obstrução da artéria pulmonar esquerda foi cuidadosamente descrito no trabalho histológico de Weibel 1. Estendendo este trabalho, no rato, o nosso laboratório tem-se centrado em ambos os factores de crescimento importantes neste processo, assim como o resultado fisiológico desta neovasculatura no pulmão. Os resultados demonstram a quimiocina CXC CINC-3 é elevado precoce após isquemia e tratamento de ratos com um anticorpo neutralizante para CXCR2, o receptor para o CINC-3, atenua a angiogénese 13. A vasculatura recém-criada brônquica 14 dias após o início da isquemia pulmonar mostrou-se anormal com permeabilidade da proteína aumentou significativamente 15. Função pulmonar esquerda não foi normal, mostrando a diminuição da capacidade de difusão e uma diminuição do volume pulmonar 15. Embora a neovasculatura pode ter contributed para a preservação do tecido pulmonar durante a isquemia pulmonar crónica, isto não parece ser normal e pode contribuir para uma redução sustentada da função pulmonar. Talvez um dos aspectos mais curiosos deste modelo refere-se a distribuição espacial da proliferação dos vasos sanguíneos. Apesar da liberação de fatores de crescimento dentro do parênquima pulmonar devido a isquemia, a neovascularização origina no montante relativamente grandes artérias brônquicas. A artéria brônquica normal surge como um pequeno ramo da aorta e invade a árvore da via aérea no carina. Assim, o mecanismo através do qual os factores de crescimento induzem a fase inicial da arteriogénese não é clara. Sugerimos que o rato, com uma anatomia vascular similar aos seres humanos, oferece uma oportunidade única de estudar os mecanismos responsáveis ​​pela angiogênese sistêmica durante a isquemia pulmonar. Apesar de obstrução completa da artéria pulmonar é uma ocorrência rara em seres humanos,aumento da vascularização brônquica parece ser igualmente induzidos em pacientes seja qual for o local e tamanho de obstrução da artéria pulmonar 16. Assim, fornecem uma descrição detalhada da abordagem cirúrgica para ligar a artéria pulmonar esquerda em ratos e um meio para quantificar a magnitude da angiogénese.

<table border="1"><tbody><tr><td> Reagentes: </td><td> Companhia </td><td></td><td></td></tr><tr><td> de cloridrato de buprenorfina, Puralube </td><td> Butler Schein </td><td></td><td></td></tr><tr><td> bupivicaine </td><td> APP Pharmaceuticals </td><td></td><td></td></tr><tr><td> Povidona-iodo vareta com tampão </td><td> Dynarex Corporação </td><td></td><td></td></tr><tr><td> polipropileno tamanho sutura 6-0, 3/8 círculo agulha reversa corte </td><td> Myco Médica </td><td></td><td></td></tr><tr><td> PE20 tubulação </td><td> Becton Dickinson </td><td></td><td></td></tr><tr><td> 15 um carmesim fluoroesferas de poliestireno </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> 1 ml Hamilton seringa de vidro </td><td> Hamilton Company </td><td> &lt;/ Td&gt; <td></td></tr><tr><td> Equipamento: </td><td></td><td></td><td></td></tr><tr><td> Genie bomba de seringa Além </td><td> Kent Científico </td><td></td><td></td></tr><tr><td> Espectrofotômetro de fluorescência </td><td> Digilab </td><td></td><td></td></tr><tr><td> Roedor ventilador modelo 683 </td><td> Harvard Apparatus </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tabela 1. Tabela de reagentes e equipamentos específicos. </td></tr></tbody></table>

angiogenesis in the ischemic rat lung

O pulmão adulto é perfundido por tanto arterial sistémica brônquica e o retorno venoso inteiro que flui através das artérias pulmonares. Na maioria das patologias do pulmão, é a mais pequena da vasculatura sistémica que responde a uma necessidade de perfusão pulmonar melhorada e mostra neovascularização robusto. Isquemia vascular pulmonar induzida pela obstrução da artéria pulmonar demonstrou resultar na angiogénese arterial sistémica rápida no homem, bem como em vários modelos animais. Embora a avaliação histológica do curso de tempo da proliferação artéria bronquial em ratos foi cuidadosamente descrito por Weibel 1, os mecanismos responsáveis ​​para este crescimento organizado de novos vasos não são claras. Fornecemos detalhes cirúrgicos de induzir isquemia da artéria pulmonar esquerda no rato que conduz a neovascularização brônquica. A quantificação da extensão da angiogénese apresenta um desafio adicional, devido à presença dos dois leitos vasculares no interior do pulmão. Métodospara determinar a angiogénese funcional com base em injecções de microesferas marcadas são fornecidos.

Fundido vascular: Resultados dos efeitos da isquemia da artéria pulmonar esquerda no rato são apresentadas na Figura 1. É mostrado um molde de metacrilato da vasculatura brônquica e da vascularização extensa da árvore da via aérea esquerdo 28 dias após LPAL. Para obter esse elenco, a vasculatura sistêmica foi injetado com uma mistura de metacrilato (vermelho), retrógrada para a aorta descendente ea traquéia foi canulada e injetada com um material de silicone branco baseado. Este molde vascul...

Watch this Scientific Journal Video about Angiogenesis in the Ischemic Rat Lung at JoVE.com

Angiogenesis in the Ischemic Rat Lung

O pulmão é perfundida, tanto arterial sistémica brônquica e artérias pulmonares. Na maioria das patologias do pulmão, é a mais pequena da vasculatura sistémica que mostra a neovascularização robusto. Cessação do fluxo sangüíneo pulmonar promove a angiogênese brônquica vivo. Nós fornecemos detalhes cirúrgicos de induzir isquemia da artéria pulmonar esquerda que promove neovascularização brônquica.

Angiogênese no pulmão de rato Isquêmica

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

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15.5K visualizações.  Johns Hopkins University. O pulmão é perfundida, tanto arterial sistémica brônquica e artérias pulmonares. Na maioria das patologias do pulmão, é a mais pequena da vasculatura sistémica que mostra a neovascularização robusto. Cessação do fluxo sangüíneo pulmonar promove a angiogênese brônquica vivo. Nós fornecemos detalhes cirúrgicos de induzir isquemia da artéria pulmonar esquerda que promove neovascularização brônquica.

Vídeo: Angiogenesis in the Ischemic Rat Lung

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multi-modal de imagem de angiogênese em um modelo de rato nu de Câncer de mama utilizando a ressonância magnética, tomografia computadorizada volumétrica e ultra-som

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Medicina

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Descolamento de Retina modelo em roedores por injeção sub-retiniana de hialuronato de sódio

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Implantação de fibrina Gel no tapete do pulmão para o Estudo de angiogênese Lung-specific

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

Método de Isolado<em&gt; Ex Vivo</em&gt; Perfusão pulmonar em um modelo de rato: Lições aprendidas com o desenvolvimento de um Programa PPEV Rat

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Rat Model of Photochemically-Induced Posterior Ischemic Optic Neuropathy

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history have&#160;remained poorly understood. Recently, we developed a reliable, reproducible animal model of PION and tested the treatment effect of some neurotrophic factors in this model1. The purpose of this video is to demonstrate our photochemically induced model of posterior ischemic optic neuropathy, and to evaluate its effects with retrograde labeling of retinal ganglion cells. Following surgical exposure of the posterior optic nerve, a photosensitizing dye, erythrosin B, is intravenously injected and a laser beam is focused onto the optic nerve surface. Photochemical interaction of erythrosin B and the laser during irradiation damages the vascular endothelium, prompting microvascular occlusion mediated by platelet thrombosis and edematous compression. The resulting ischemic injury yields a gradual but pronounced retinal ganglion cell dieback, owing to a loss of axonal input - a remote, injury-induced and clinically relevant outcome. Thus, this model provides a novel platform to study the pathophysiologic course of PION,&#160;and can be further optimized for testing therapeutic approaches for optic neuropathies as well as other CNS ischemic diseases.

The goal of this protocol is to photochemically induce ischemic injury to the posterior optic nerve in rat. This model is critical to studies of the pathophysiology of posterior ischemic optic neuropathy, and therapeutic approaches for this and other optic neuropathies, as well as of other CNS ischemic diseases.

Modelo de Rato Induzida fotoquimicamente posterior neuropatia óptica isquémica

Posterior Ischemic optic neuropathy (PION) is a sight-devastating disease in clinical practice. However, its pathogenesis and natural history ...

Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft involves anastomosis of donor inferior vena cava (IVC) to recipient IVC, and donor ascending aorta (Ao) to recipient abdominal Ao. The right upper and middle lung lobes are preserved and function as conduits for blood flow from right heart to left heart.
There are several advantages to using this technique, and it lends itself to a broad range of applications. Because the graft is transplanted in a configuration that allows for dyamic volume-loading, cardiac function may be directly assessed in vivo. The use of pressure-volume conductance catheters permits characterization of load-dependent and load-independent hemodynamic parameters. The graft may be converted to a loaded configuration by applying a clamp to the recipient&#8217;s infra-hepatic IVC. We describe modified surgical techniques for both donor and recipient operations, and an ideal myocardial protection strategy. Depending on the experimental aim, this model may be adapted for use in both acute and chronic studies of graft function, immunologic status, and variable ventricular loading conditions. The conducting airways to the transplanted lung are preserved, and allow for acute lung re-ventilation. This facilitates analysis of the effects of the mixed venous and arterial blood providing coronary perfusion to the graft.
A limitation of this model is its technical complexity. There is a significant learning curve for new operators, who should ideally be mentored in the technique. A surgical training background is advantageous for those wishing to apply this model. Despite its complexity, we aim to present the model in a clear and easily applicable format. Because of the physiologic similarity of this model to orthotopic transplantation, and its broad range of study applications, the effort invested in learning the technique is likely to be worthwhile.

We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft.

Rat heterotópico abdominal coração / pulmão Transplante Single-carregado em uma configuração-Volume

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

Demonstração da Wound modelo de pele de rato Isquêmico

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Modelo In Vitro de Angiogênese Coronariana

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

Modelo de Lesão de Isquemia/Reperfusão de Pulmão de Rato em Transplante de Pulmão de Rato: Otimizações para Melhorar os Resultados

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...

<meta charset="utf8"></head><body>Monitoramento em tempo real da dinâmica celular em transplante de pulmão de camundongo

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

<meta charset="utf8"></head><body>Autor em destaque: Avançando na imunologia do transplante de pulmão por meio de imagens intravitais

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Microscopia intravital de dois fótons do pulmão de camundongo transplantado

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...