Reconocemos la labor del Dr. Adlah Sukkar, MD para ayudar con la colada del pulmón. Este trabajo ha sido financiado por el NHLBI, HL088005.

Todos los protocolos realizados en ratas han sido aprobados por el Cuidado de Animales Universidad Johns Hopkins y el empleo, y de conformidad con las directrices del NIH. Siempre que sea posible, el animal debe ser preparado quirúrgicamente en un área separada de la zona quirúrgica para minimizar la contaminación de la zona quirúrgica. 1. La anestesia / analgesia <ol><li> Rata lugar (ratas Sprague Dawley macho, 125-150 g; Harlan, Indianapolis, IN) en una cámara de inducción infundido con isoflurano 3%. </li><li> Coloque rata anestesiada a bordo cirugía unido al cono de la nariz y el ventilador con 3% de anestésico isoflurano. Utilice una técnica estéril para todos los procedimientos. Coloque rata para asegurar campo quirúrgico estéril. </li></ol> 2. Intubación <ol><li> Inmovilizar apéndices en posición supina con cinta quirúrgica. </li><li> Retire rata de cono de la nariz, extender la lengua con unas pinzas acolchadas. </li><li> El uso de calibre 14 intracath con estilete metálico embotado como guía. Deslice atrás paraNgue, en la tráquea. </li><li> Retire estilete metálico dejando intracath plástico blanco en la tráquea. Asegúrese de que la rata es la respiración y el aire fluye a través del tubo. </li><li> Reemplace cono de la nariz con el adaptador directo al catéter, conecte rata para ventilador (90 respiraciones / min; 8 ml / kg volumen corriente; Rodent Ventilator Modelo 683, Harvard Apparatus, Holliston, Massachusetts). </li><li> Aplicar Puralube (Butler Schein, Dublin, OH) ungüento veterinario en los ojos. </li></ol> 3. Toracotomía <ol><li> Lugar rata lado derecho hacia abajo, inmovilizar apéndices con cinta quirúrgica. </li><li> Afeitado zona izquierda caja torácica lateral. </li><li> Retire el exceso de piel con pequeña aspiradora. </li><li> Hacer campo estéril al lavarlo con alcohol seguido de bastoncillo con hisopo de povidona-yodo (Dynarex Corporation, Orangebur, NY). Repita este proceso dos veces más (para un total de tres exfoliantes). </li><li> Hacer incisión transversal con tijeras de disección estériles o estériles bisturí en el centro del campo. </li><li> Blunt diseccionar a través de layers de tejido y grasa a las costillas (última capa es una membrana delgada que cubre las costillas). </li><li> Cuente costillas para determinar 3 º espacio intercostal. </li><li> Usando estériles 45 ° Graefe forceps, hacer incisión contundente entre 3 ª y 4 ª costilla. </li><li> Inserte separadores costales, tire suavemente creando vacío abierto con la visualización completa del pulmón, cinta en lugar de suturas para mantener abierta. </li></ol> 4. Ligadura de la arteria pulmonar izquierda <ol><li> Con 90 ° fórceps Graefe, mueva pulmón izquierdo hacia atrás con la mano derecha. </li><li> Usando patrón Dumont # 5 pinzas rectas, coge la arteria pulmonar izquierda y la vía aérea con la mano izquierda. Arteria pulmonar izquierda se colocará sobre la parte superior de las vías respiratorias. Asegúrese de que los fórceps son directamente perpendicular a la mesa. Adicionalmente, es más efectivo para recoger la arteria pulmonar izquierda / bronquio izquierdo mainstem en la posición más distal (la más cercana al parénquima). Empuje la ventilación pulmonar izquierdo a un lado con esta maniobra la mano izquierda. </li><li> Utilice Dumontpatrón # 5 45 ° pinzas curvas para separar la arteria pulmonar izquierda desde el bronquio principal izquierdo en sus fronteras naturales. Esta línea de separación aparece delgada y blanca entre las dos estructuras individuales. </li><li> Separe directamente debajo de la arteria sin pasar por el buque; suavemente deslice las puntas de las pinzas unidas a lo largo de la separación. </li><li> Continúe hasta que las puntas de pinzas han visiblemente separado de la arteria pulmonar izquierda y el bronquio principal izquierdo. Sólo un pequeño punto de la punta debe estar completamente a través. Si la sangre se puede visualizar en la punta de las pinzas, entonces no es completamente a través de la ligadura y no debe intentarse. Una vez atravesado el espacio, la arteria pulmonar izquierda se colocará sobre la curva de los fórceps. Mantener en esta posición. </li><li> Libere suavemente agarre pinza recta romo (mano izquierda) y tomar parte de una sutura precortada (~ 2.3 pulgadas, el tamaño de sutura de polipropileno 6-0; Myco médicos, Cary, NC). </li><li> Abra pinzas curvas acunando arteria pulmonar izquierda y grab sutura. Tire suavemente de sutura a través del espacio entre la arteria pulmonar izquierda y bronquio izquierdo mainstem en un movimiento hacia arriba respecto a la curva de los fórceps. </li><li> Ate ocluir la arteria pulmonar izquierda con un nudo cuadrado, el resto cuidadosamente recorte de sutura. </li><li> Cerrar costillas utilizando pinzas romas para mantener costilla y la sutura dos veces con polipropileno (monofilamento azul) tamaño 4-0 mm unida a un 19, 3/8 círculo inverso corte aguja (Myco Medical, Cary, NC) en una pinza hemostática, teniendo cuidado de no piel sutura (sólo costillas). </li><li> Completar un nudo flojo, inflar los pulmones, en lugar positiva al final de la espiración (PEEP; 2-5 cm H 2 O), hyperinflate nudo pulmón luego asegure con fuerza, y hacer otro nudo completo antes de cortar con tijeras resto de sutura. Eliminar de la PEEP y visualizar durante 30 segundos para asegurar que el pulmón no se colapsa. Aplicar 5 gotas bupivicaína (APP Pharmaceuticals, Schaumbur, IL). </li><li> Cerrar piel mediante la colocación de adhesivo tisular en heridas y empujar la piel junto con parte posterior end de aplicador de punta de algodón. Dar bupivicaína (2,0 mg / kg por vía subcutánea) en el sitio de incisión posteriormente cada 8 h durante 24 horas o hasta que el animal se reanuda la actividad normal. Como la capa subcutánea en esta zona es muy delgada, no es fácil de suturar en su propio. Cuando el pegamento se aplica el tejido y la piel se empuja juntos también cierra la capa subcutánea. </li><li> Apague el gas isoflurano, pero siguen ventilar la rata durante 1-2 min en aire ambiente hasta que se recupere el movimiento voluntario. Desconecte el tubo endotraqueal del ventilador y asegúrese de que la rata está respirando espontáneamente antes de retirarlo. </li><li> Limpie Puralube de los ojos con un hisopo de algodón y movimiento del monitor y la recuperación. Inyectar hidrocloruro de buprenorfina (0,05 mg / kg intraperitoneal, Butler Schein, Dublin OH). Continuar la entrega de analgésico cada 12 horas durante 48 horas después de la cirugía. </li></ol> 5. La canulación arteria carótida Para evaluar la magnitud de perfus bronquialesion del pulmón izquierdo isquémico en puntos de tiempo deseados después de la ligadura de la arteria pulmonar izquierda, inyectar microesferas marcadas a través de la arteria carótida izquierda en el arco aórtico. Preparar ratas como arriba 1-2. <ol><li> Cortar a lo largo de la línea media del cuello, embotar diseccionar a revelar tráquea y la arteria carótida izquierda (sitio de la inyección de microesferas). </li><li> Insertar catéter lleno con solución salina heparinizada punta, romo de PE20 tubo (Becton Dickinson, Sparks MD) en el recipiente conectado a la aguja 25 g, 4-way llave de paso, jeringa de 1 ml. </li><li> Coloque la botella de microesferas (15 micras carmesí poliestireno microesferas fluorescentes, 1 x 10 6 esferas / ml; Invitrogen, Eugene, OR) en sonicador de agua durante 30 seg. </li><li> Extraiga la botella y agitar y preparar 0,5 ml (500.000 microesferas) en un 1 ml jeringa de vidrio Hamilton (Hamilton Company, Reno, NV) a través de una aguja de 20 g. </li><li> Conecte una jeringa Hamilton de llave de paso de cuatro vías e infundir microesferas con bomba de jeringa (velocidad: 500 l / min; Genie Plus, Kent Científico, Torringten, CT). </li><li> Retire la jeringa Hamilton y aparato ras con 1 ml de solución salina heparized a 500 l / min. </li><li> Realizar toracotomía pecho lleno y exsanguinate la rata por la ruptura de la vena cava inferior. </li><li> Retire el pulmón izquierdo y en los tejidos de interés. </li><li> Para extraer microesferas a partir de tejido, tomar todo el pulmón izquierdo de la rata después de la exanguinación y colocarlo en KOH 2M (4-6 ml). Colocar en un baño de 55 ° C el agua y dejar toda la noche para la digestión del tejido. Añadir Tween 80 (0,25%) para lavar las perlas, vórtice (10 segundos) y se centrifuga (2.000 rpm, 20 ° C durante 10 min). Eliminar el sobrenadante, añadir acetato de 2-etoxietilo (1 ml) y agitar y dejar reposar durante 1 hora. Vortex la suspensión y se centrifuga (2.000 rpm; (20 ° C durante 10 min) Remover la capa de acetato de 2-etoxietilo acuosa que contiene la fluorescencia, lugar en una cubeta y medida utilizando un Hitachi F-2500 espectrofotómetro de fluorescencia (excitación 618 612/emission. ; Digilab, Holliston, MA). </li></ol>

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No hay conflictos de interés declarado.

La ligadura de la arteria pulmonar izquierda en todas las especies estudiadas conduce a la neovascularización robusto sistémica del pulmón isquémica. Hemos presentado los detalles de la técnica quirúrgica en un modelo de rata. Nuestros resultados vascular producido por fundición, histopatología, y en el etiquetado in vivo demuestran que las arterias bronquiales proliferan y perfundir el parénquima pulmonar. Por lo tanto, los mecanismos de la angiogénesis bronquial puede ser estudiada en un mo...

Angiogénesis sistémica en el pulmón está bien reconocida. En estados de enfermedad tales como el asma 2, fibrosis pulmonar intersticial 3, 4 cáncer y tromboembolismo pulmonar crónico 5, la vasculatura sistémica en y alrededor de los prolifera pulmonares e invade el parénquima pulmonar. Sin embargo, los modelos animales para el estudio de esta activación diferencial de la sistémico en lugar de la circulación pulmonar son pocos. Quizás el modelo más reproducible de la neovascularización sistémica en el pulmón del mamífero adulto es el que ocurre después de la inducción de isquemia crónica de la arteria pulmonar. La respuesta a la obstrucción de la arteria pulmonar en seres humanos 5-7, 8 perros, cerdos, ovejas 9 10, 11 conejillos de indias, ratas 1, 12, 13, 14 y ratones es la rápida proliferación de la arteria bronquial, así como las arterias intercostales. Los mecanismos responsables de neovascularización sistémicación del pulmón después de la isquemia pulmonar son en gran parte desconocido y no se han estudiado ampliamente. El curso temporal de la angiogénesis bronquial en la rata después de la obstrucción de la arteria pulmonar se ha descrito cuidadosamente en el trabajo histológico de Weibel 1. La extensión de este trabajo en la rata, nuestro laboratorio ha centrado tanto en los factores de crecimiento importantes en este proceso, así como el resultado fisiológico de este neovasculatura en el pulmón. Los resultados demuestran la quimiocina CXC CINC-3 se eleva temprano después de la isquemia y el tratamiento de ratas con un anticuerpo neutralizante para CXCR2, el receptor de CINC-3, atenúa la angiogénesis 13. La vasculatura recientemente establecida bronquial 14 días después de la aparición de la isquemia pulmonar ha demostrado ser anormal con permeabilidad proteína aumentó significativamente 15. La función pulmonar izquierda no mostraba normal de disminución de la capacidad de difusión y una disminución en el volumen pulmonar 15. Aunque la neovasculatura puede tener contributed a la preservación de tejido pulmonar durante la isquemia pulmonar crónica, parece no ser normal y puede contribuir a una disminución sostenida de la función pulmonar. Quizás uno de los aspectos más curiosos de este modelo se refiere a la distribución espacial de la proliferación de vasos sanguíneos. A pesar de la liberación de factores de crecimiento dentro del parénquima pulmonar por isquemia, la neovasculatura origina en relativamente grandes arterias bronquiales aguas arriba. La arteria bronquial normal surge como una pequeña rama de la aorta e invade el árbol de la vía aérea en la carina. Así, el mecanismo por el cual los factores de crecimiento inducen la fase inicial de la arteriogénesis no está claro. Sugerimos que la rata, con una anatomía vascular similares a los humanos, proporciona una oportunidad única para estudiar los mecanismos responsables de la angiogénesis pulmonar durante la isquemia sistémica. Aunque la obstrucción completa de la arteria pulmonar izquierda es un fenómeno poco frecuente en sujetos humanos,aumento de la vascularidad bronquial parece estar inducida en los pacientes de manera similar cualquiera que sea el sitio y el tamaño de la obstrucción de la arteria pulmonar 16. Por lo tanto, ofrecemos una descripción detallada de la técnica quirúrgica para ligar la arteria pulmonar izquierda en ratas y un medio para cuantificar la magnitud de la angiogénesis.

<table border="1"><tbody><tr><td> Reactivos: </td><td> Empresa </td><td></td><td></td></tr><tr><td> hidrocloruro de buprenorfina, Puralube </td><td> Mayordomo Schein </td><td></td><td></td></tr><tr><td> bupivicaína </td><td> APP Pharmaceuticals </td><td></td><td></td></tr><tr><td> Povidona Yodada bastoncillo con hisopo </td><td> Dynarex Corporación </td><td></td><td></td></tr><tr><td> polipropileno tamaño de la sutura 6-0, 3/8 círculo inverso aguja de corte </td><td> Myco Médico </td><td></td><td></td></tr><tr><td> PE20 tubo </td><td> Becton Dickinson </td><td></td><td></td></tr><tr><td> 15 micras fluoroesferas de poliestireno carmesí </td><td> Invitrogen </td><td></td><td></td></tr><tr><td> 1 ml vidrio de la jeringa Hamilton </td><td> Hamilton Company </td><td> &lt;/ Td&gt; <td></td></tr><tr><td> Equipamiento: </td><td></td><td></td><td></td></tr><tr><td> Genie Plus bomba de jeringa </td><td> Kent Científico </td><td></td><td></td></tr><tr><td> Espectrofotómetro de Fluorescencia </td><td> Digilab </td><td></td><td></td></tr><tr><td> Rodent Ventilator Modelo 683 </td><td> Harvard Apparatus </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Tabla 1. Tabla de reactivos y equipos específicos. </td></tr></tbody></table>

angiogenesis in the ischemic rat lung

El pulmón adulto se perfunde por tanto la arteria sistémica bronquial y el retorno venoso todo fluye a través de las arterias pulmonares. En la mayoría de las patologías de pulmón, que es más pequeño de la vasculatura sistémica que responde a una necesidad de mejorar la perfusión pulmonar y muestra neovascularización robusto. Isquemia vascular pulmonar inducido por la obstrucción de la arteria pulmonar se ha demostrado que resulta en una rápida angiogénesis arterial sistémica en el hombre, así como en varios modelos animales. Aunque la evaluación histológica de la evolución temporal de la proliferación de la arteria bronquial en ratas se ha descrito cuidadosamente por Weibel 1, los mecanismos responsables de este crecimiento organizado de nuevos vasos no son claras. Ofrecemos detalles quirúrgicos de la inducción de isquemia arteria pulmonar en la rata que lleva a la neovascularización bronquial. La cuantificación de la extensión de la angiogénesis constituye un desafío adicional debido a la presencia de los dos lechos vasculares en el pulmón. Métodospara determinar la angiogénesis funcional basado en inyecciones de microesferas marcadas se proporcionan.

Reparto vascular: Resultados de los efectos de la isquemia arteria pulmonar izquierda en la rata se muestra en la Figura 1. Se muestra un reparto de metacrilato de la vasculatura bronquial y la vascularización extensa del árbol de las vías respiratorias izquierda 28 días después de LPAL. Para obtener este molde, la vasculatura sistémica se inyectó con una mezcla de metacrilato (rojo), retrógrada en la aorta descendente y de la tráquea se canuló y se inyecta con un material a base de silicona b...

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Angiogenesis in the Ischemic Rat Lung

El pulmón se perfundieron por tanto la arteria sistémica bronquial y las arterias pulmonares. En la mayoría de las patologías de pulmón, que es más pequeño de la vasculatura sistémica que muestra neovascularización robusto. La cesación del flujo sanguíneo pulmonar promueve la angiogénesis bronquial a paso ligero. Ofrecemos detalles quirúrgicos de la inducción de isquemia arteria pulmonar que promueve la neovascularización bronquial.

La angiogénesis en el pulmón de rata isquémico

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 vistas.  Johns Hopkins University. El pulmón se perfundieron por tanto la arteria sistémica bronquial y las arterias pulmonares. En la mayoría de las patologías de pulmón, que es más pequeño de la vasculatura sistémica que muestra neovascularización robusto. La cesación del flujo sanguíneo pulmonar promueve la angiogénesis bronquial a paso ligero. Ofrecemos detalles quirúrgicos de la inducción de isquemia arteria pulmonar que promueve la neovascularización bronquial.

Video: 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.

Multimodal de imágenes de la angiogénesis en un modelo de rata desnuda de las metástasis óseas del cáncer de pecho utilizando imágenes de resonancia magnética, tomografía computarizada y la ecografía volumétrica

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

Investigación

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.

Desprendimiento de Retina modelo en roedores por inyección subretiniana de Hialuronato de Sodio

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.

El implante de gel de fibrina en pulmón de ratón para el Estudio de la angiogénesis pulmón específico

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 aislados<em&gt; Ex Vivo</em&gt; Perfusión pulmonar en un modelo de rata: Lecciones Aprendidas de Desarrollo de un Programa EVLP Rata

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.

Rata Modelo de neuropatía óptica isquémica posterior inducida por Fotoquímicamente

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.

Rata heterotópico abdominal Corazón / Trasplante de un solo pulmón en una configuración de volumen cargado

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.

Demostración de la rata isquémica de la piel herida Modelo

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 Angiogénesis Coronaria

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.

Un modelo de trasplante de pulmón de rata de lesión por isquemia/reperfusión caliente: optimizaciones para mejorar los 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>Monitorización en tiempo real de la dinámica celular en el trasplante de pulmón de ratón

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 destacado: Avance de la inmunología del trasplante de pulmón a través de imágenes intravitales

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.

Microscopía intravital de dos fotones del pulmón de ratón trasplantado

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