Mark J. Kearns recibe el apoyo del Programa UBC clínico Investigador (otorgado por el Colegio Real de Médicos y Cirujanos de Canadá), y el UBC 4YF Doctoral Fellowship. John H. Boyd es una Asociación Nacional sanatorio y Michael Smith Fundación para la Investigación de la Salud Escolar. La financiación a través de CIHR. Los autores desean agradecer al Dr. M. Allard y Richard Wamboldt por su ayuda con la instalación y mantenimiento de equipos de perfusión.

Todos los animales fueron alojados y atendidos de conformidad con las directrices institucionales para el cuidado y uso de animales de laboratorio y Nacional. Ética aprobación de este protocolo fue concedido por la Universidad de Comité de Cuidado de Animales de la Columbia Británica. Hombre, ratas Sprague-Dawley que pesaban entre 300-450 g se utilizaron para este protocolo. 1. Donante Operación <ol><li> Tienen aproximadamente 100 ml de cardioplegia (RT) en un matraz cilíndrico conectado a (IV) tubo de catéter intravenoso de largo por una llave de paso de 3 vías. Utilice un soporte para elevar el frasco aproximadamente 80 cm por encima de la superficie operativa, permitiendo la entrega cardioplejia por gravedad. </li><li> Para visualizar adecuadamente las estructuras, utilizar un par de lupas quirúrgicas o un microscopio de disección. NOTA: Actualmente usamos un microscopio quirúrgico binocular con 3,4 - magnificación 21.3X. </li><li> Coloque el donante en una cámara anestésica e inducir la anestesia con 4-5% isoflurano. </li&gt; <li> Traslado a la rata a una plataforma operativa y mantener la anestesia por la nariz de cono con 1 - 2% de isoflurano. Aplique un ungüento veterinario para los ojos del animal para evitar la sequedad. Administrar midazolam (2 mg / kg) por vía intraperitoneal con una aguja 25 G. </li><li> El uso de máquinas de cortar quirúrgicas, afeitarse el donante de xiphisternum de mandíbula. Shave una pequeña región de la ingle izquierda (para el acceso posterior IV). Aplicar un agente de depilación a las superficies operativas, espere aproximadamente 5 minutos, y quitar el pelo con un pedazo de gasa. </li><li> Prepara los sitios quirúrgicos con una solución a base de clorhexidina o povidona yodada (sólo usamos clorhexidina). Infíltrate en los sitios de incisión con 0,1 a 0,5% por vía subcutánea de lidocaína. </li><li> Asegure las extremidades anteriores y miembro posterior izquierdo de la plataforma operativa con cinta adhesiva, dejando la extremidad posterior derecha libre para monitorización de la profundidad anestésica y los signos vitales. </li><li> Después de asegurarse de la profundidad anestésica adecuada por pizca pedal, hacer una incisión en el pliegue de la ingle izquierda usandoun bisturí 22-cuchilla. Por disección roma, exponer la vena femoral común izquierda y obtener acceso IV de la siguiente manera: <ol><li> Retractar suavemente el tejido que recubre la vena femoral y canular la vena con un 24 G IV. Conecte el IV a un tramo corto de tubo IV lleno de solución salina, y asegurar el tubo en su lugar con cinta adhesiva. NOTA: El procedimiento para la canulación venosa femoral en ratas se describe en otro lugar por Jespersen y sus colegas 14. </li><li> Conectar una jeringa de 10 ml lleno de solución salina al tubo IV, y suavemente aspirar sangre para asegurar el correcto posicionamiento de la IV. </li><li> Inyectar 300 a 500 UI de heparina no fraccionada a través del tubo IV, y lavar posteriormente el tubo con de 3 - 5 ml de solución salina. </li></ol></li><li> A continuación, tracheotomize el donante como sigue: <ol><li> Hacer una incisión en los tejidos blandos entre la escotadura yugular y la mandíbula utilizando un bisturí 22-hoja. Penetrar la cápsula de la glándula tiroides en la línea media usando Metzentijeras Baum, y separados de sus lóbulos mediante disección roma. </li><li> Utilizando disección roma, separar los músculos de la correa del cuello en la línea media para exponer la superficie anterior de la tráquea. </li><li> Utilice unas pinzas curvas para diseccionar sin rodeos un plano circunferencial alrededor de la tráquea. Rodear la tráquea con una corbata de seda 4-0. </li><li> Usando tijeras iris, hacer una incisión transversal en la tráquea anterior, aproximadamente 5 mm por debajo del cartílago tiroides. Introducir suavemente la cánula traqueal (un 14 G IV) y fijarlo en su lugar con el empate 4-0 seda. </li><li> Conectar la cánula traqueal a un ventilador mecánico. Redirigir el flujo de oxígeno e isoflurano a través del circuito del ventilador, y ventilar el donante a una velocidad y volumen tidal predicho por su peso 13. </li></ol></li><li> Hacer una incisión en la línea media del pecho (utilizando un bisturí de hoja 22), que se extiende de la incisión en la muesca yugular a por debajo de la xipisternum. </li><li> Alojarse en la línea media, Realice una esternotomía media con un cortador de hueso. Retraer los bordes del esternón con un retractor de auto-retención. Introduzca el pericardio y cavidades pleurales. </li><li> Realice una timectomía. Es más fácil de dividir primero sin rodeos el timo en la línea media, y luego separarla de las estructuras circundantes utilizando una combinación de disección roma y cortante. NOTA: El origen de las arterias torácicas internas puede ser herido cuando diseccionando el timo de los bordes esternales superiores. Para prevenir el sangrado, clips hemostáticos se pueden aplicar antes de retirar el timo en estos puntos. </li><li> Con unas tijeras de Metzenbaum y / o un fuerte Lauer, diseccionar peri-cava grasa lejos de la vena cava inferior (VCI). Asegúrese de que el IVC está relativamente libre de grasa y tejido conectivo de la unión cavo fibrilación superiormente, al diafragma inferiormente. </li><li> Usando un fuerte Lauer, circunferencialmente libre de la vena cava superior (VCS) y rodear con un empate 4-0 seda. </li><li> A continuación, diseccionar ella izquierda de la vena cava, sin estructuras circundantes, y ligarlo proximal y distal con 4-0 corbatas de seda. Resecar la porción intermedia de cava para exponer la arteria subclavia izquierda. </li><li> Circunferencialmente gratis los vasos del cayado aórtico mediante un fuerte Lauer. Aplicar clips quirúrgicos proximal y distal a la arteria innominada, y dividirlo entre los clips. Deja la arteria carótida común izquierda y un-recortado la arteria subclavia izquierda. </li><li> A continuación, canular la vena cava superior con un catéter de 24 G IV. Asegure el catéter con el colocado previamente 4-0 corbata de seda. </li><li> Prepárese para una cosecha conveniente, pero suave injerto. </li><li> Con unas tijeras afiladas, divida el IVC simplemente superior a la superficie diafragmática. Divida la SVC superior a su sitio de canulación, asegurando que las estructuras de las vías respiratorias cercanos no están heridos. </li><li> Apague el ventilador y desconecte la cánula traqueal. Seccionar la tráquea proximal. </li><li> Sujete la tráquea seccionado con unas pinzas, y quitar el corazóny los pulmones en bloque. Esto requerirá una tracción suave con disección aguda como se retira el injerto de corazón-pulmón. Separar la tráquea desde el esófago subyacente. Evite lesionarse la aorta descendente, por lo que una buena parte de ella se mantiene intacta después de la cosecha. </li></ol> 2. Preparación del corazón-pulmón Graft NOTA: al completar esta parte del procedimiento, un asistente debe ser anestesiar el roedor destinatario y la preparación para la implantación del injerto. <ol><li> Coloque el injerto de corazón-pulmón en gasa quirúrgica con la superficie anterior del corazón hacia abajo. El uso de la disección aguda, eliminar cualquier esófago residual, evitando daños a las vías respiratorias posteriores. </li><li> Localice la aorta torácica descendente e insertar una cánula de punta roma 16 G. Fije la cánula en su lugar con un empate 4-0 seda. </li><li> Localice los vasos del cayado aórtico, y aplicar un clip quirúrgico a la arteria subclavia izquierda. Deja la arteria carótida izquierda un-recortado para subsecuente de-emisión. </li><li> Administrar la primera dosis de cardioplegia mediante la conexión de la tubería de cardioplegia a la cánula aórtica 16 G. La arteria carótida debe ser ocluida temporalmente con fórceps para asegurar la entrega adecuada de cardioplejía. NOTA: Una de las ventajas de tener la cánula aórtica adjunta es que cardioplejia se puede administrar de forma continua y / o de forma intermitente si lo deseas. Para la dosificación intermitente, que normalmente entregamos cardioplejia cada 10 - 15 min en 5 ml bolos sobre 30 - 45 seg. </li><li> Con unas tijeras de Metzenbaum y / o un fuerte Lauer, diseccionar la grasa peri-aórtica lejos de la aorta. Asegúrese de que la aorta es relativamente libre de grasa y tejido conectivo de la arteria subclavia izquierda (arco distal) a su sitio de canulación. </li><li> A continuación, exponer el bronquio principal izquierdo usando un fuerte Lauer, y ligarlo proximal con una corbata de seda 4-0. Seccionar el bronquio izquierdo distal a la ligadura usando tijeras iris. Realice una neumonectomía izquierda ligando el pulmonar izquierdaarteria y vena con 4-0 corbatas de seda. Seccionar distal a la ligadura y quitar el pulmón izquierdo. </li><li> Como anteriormente, eliminar todos los otros lóbulos pulmonares con la excepción de los lóbulos superior y medio derechos. Evite lesionar el IVC en el proceso de realizar lobectomías. </li><li> Conectar el tubo de cardioplegia a la cánula aórtica y administrar cardioplegia continua mientras se prepara el destinatario. Coloque el injerto de corazón-pulmón en un recipiente estéril (por ejemplo, un vaso de precipitados). </li></ol> 3. Receptor Operación <ol><li> Anestesiar el receptor como por el funcionamiento de los donantes, arriba. Use ungüento veterinario para proteger los ojos del animal de la sequedad. Confirme la anestesia frecuentemente por pizca pedal. </li><li> Coloque el animal como por la operación de los donantes, esta vez dejando la extremidad anterior derecha libre para monitorear los signos vitales y la profundidad anestésica. </li><li> Afeitarse el abdomen de xiphisternum al pene. Shave una pequeña región de la ingle izquierda (para el acceso posterior IV). Aplique unaagente de depilación a las superficies operativas, espere aproximadamente 5 minutos, y quitar el pelo con un pedazo de gasa. </li><li> Prepara los sitios quirúrgicos con povidona yodada o solución a base de clorhexidina. Infíltrate en los sitios de incisión con 0,1 a 0,5% por vía subcutánea de lidocaína. </li><li> Tracheotomize y ventilar el receptor como se indicó anteriormente. Alternativamente, mantener el receptor bajo anestesia nariz de cono. </li><li> Introducir un catéter en la vena femoral como se describe anteriormente. Inyectar 300 a 500 UI de heparina no fraccionada a través del tubo IV, y lavar posteriormente el tubo con de 3 - 5 ml de solución salina. </li><li> Realice una laparotomía haciendo una incisión abdominal en la línea media con un bisturí 22 palas de xiphisternum al pene. Retirar la pared abdominal utilizando un retractor auto-retención. A continuación, retraer los intestinos superior y al lado izquierdo de la rata. Envuelva en caliente gasa empapada en solución salina. </li><li> Exponer la aorta abdominal por IVC y bruscamente la disección a través de la suprayacente regrasa-tro peritoneal. </li><li> Tener una pinza vaso curvado disponible y listo. </li><li> Circunferencialmente liberar el IVC y la aorta proximal y distal y rodearlos con 4-0 corbatas de seda. No debe ser de aproximadamente 2-3 cm de espacio entre estos sitios. </li><li> Aplicar cuidadosamente la abrazadera curvada recipiente, asegurándose de que una porción suficiente de ambos IVC y la aorta están expuestas por encima de las mandíbulas de la pinza. </li><li> Hacer una incisión en la pared anterior de la IVC con una aguja 25 G conectada a una jeringa de 1 ml lleno de solución salina. Extender la incisión con tijeras Potts para que coincida con la longitud de la donante IVC orificio. </li><li> Retire el injerto de corazón-pulmón de su contenedor y desconéctelo de cardioplejía. Coloque el injerto en el abdomen del receptor en una posición óptima para realizar la anastomosis venosa. NOTA: El injerto de corazón-pulmón en última instancia, estar orientada ligeramente oblicua, con el vértice apuntando hacia la década de abdomen dejó cuadrante inferior. </li><li> Asegure el talóny dedo del pie, extremos de la anastomosis con sutura de nylon 9-0. Haga un nudo seguro en cada extremo, dejando la aguja unida a un brazo largo de la sutura, y un extremo corto de la sutura que estar atado a más tarde. Administrar una dosis de cardioplejía. </li><li> En el funcionamiento de la moda, completa la mitad de la línea de sutura y corbata para el brazo de sutura corto opuestas. Administrar una dosis de cardioplejía. </li><li> Complete la otra mitad de la línea de sutura y atarlo. La anastomosis venosa es completa. Administrar una dosis de cardioplejía </li><li> Oriente el injerto de corazón-pulmón con vértice apuntando del corazón hacia los del abdomen dejó cuadrante inferior. Asegúrese de que la anastomosis venosa no está doblado o retorcido. </li><li> Evaluar la longitud de la aorta del donante que se requiere para llegar a la aorta destinatario, manteniendo tanto IVC y anastomosis aórtica en configuraciones libres de tensión y un-retorcidas. </li><li> Divida la aorta descendente (distal a la arteria subclavia izquierda) con una tijera iris. </li><li> Hacer una incision en la pared anterior de la aorta destinatario con una aguja 25 G conectada a una jeringa de 1 ml lleno de solución salina. Extender la incisión con tijeras Potts para que coincida con la longitud del orificio aórtico donante. </li><li> Anclar el dedo del pie (cara superior) de la aorta donante al receptor aorta utilizando una sutura de nylon 9-0. A continuación, pasar la aguja con el aspecto medial de la aorta y completar la mitad de la anastomosis en el funcionamiento de la moda. </li><li> En el talón de la anastomosis aórtica, invertir la orientación de la línea de sutura (una transición en la dirección de la costura), y completar el medio lateral de la anastomosis en el funcionamiento de la moda. </li><li> Retire lentamente el catéter IV en la vena cava superior, y ligar la vena cava superior con una pinza quirúrgica. </li><li> De aire la aorta mediante la localización de la arteria carótida común-un recortado izquierda. Mantener abierta la arteria, en una posición vertical que permitirá que el aire sea evacuado libremente. </li><li> Abrir brevemente las mandíbulas de la pinza vaso curvo y permite la arteria carótida a sangrar (de-aire) por 2 - 3 seg. Vuelva a aplicar la pinza buque. </li><li> Aplicar un clip quirúrgico a la arteria carótida. Retire la abrazadera vaso curvo. </li><li> Compruebe si hay sangrado en las líneas de sutura. Si está presente, aplicar compresión suave con una gasa o la reparación quirúrgica con una longitud de sutura de nylon 9-0 (dependiendo de la gravedad de la hemorragia). </li><li> El corazón del donante debe reanudar superando en cuestión de minutos. </li><li> Preste mucha atención a los signos vitales del beneficiario, la profundidad de la anestesia, y administrar el volumen de reanimación, según sea necesario. </li><li> Dependiendo de los criterios de valoración experimentales, ya sea cerrar abdomen del animal y dejar que se recupere de la anestesia, o preparar el animal para la evaluación cardiovascular. NOTA: Los ejemplos de evaluación de injerto incluyen en las medidas in vivo de la hemodinámica dependiente de la carga y la independiente de la carga, las medidas in vitro de la función en Langendorff y modos de trabajo del corazón y (en la cirugía de la supervivencia) ecocardiográficos o investigaciones de resonancia magnética. </li><li> En elfinal de los experimentos terminales agudas, los animales son sacrificados por desangramiento. </li></ol>

<ol>
	<li>Abbott, C. P., Lindsey, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+heart+transplantation+in+the+rat.">A technique for heart transplantation in the rat.</a> Arch Surg. 89 (1964), 649-652 (1964).</li><li>Ma, Y., Wang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+2+heterotopic+heart+transplant+techniques+in+rats:+cervical+and+abdominal+heart.">Comparison of 2 heterotopic heart transplant techniques in rats: cervical and abdominal heart.</a> Exp Clin Transplant. 9 (2), 128-133 (2011).</li><li>Wiedemann, D., Boesch, F., Schneeberger, S., Kocher, A., Laufer, G., Semsroth, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft+function+after+heterotopic+rat+heart+transplant+with+an+isolated+reperfused+working+heart:+a+methodic+consideration.">Graft function after heterotopic rat heart transplant with an isolated reperfused working heart: a methodic consideration.</a> Exp Clin Transplant. 10 (2), 154-157 (2012).</li><li>Chen, Z. H., Xia, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+technique+of+heterotopic+heart-lung+transplantation+in+the+rat.">The technique of heterotopic heart-lung transplantation in the rat.</a> J Tongji Med Univ. 6 (2), 67-70 (1986).</li><li>Ibrahim, M., Navaratnarajah, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+abdominal+heart+transplantation+in+rats+for+functional+studies+of+ventricular+unloading.">Heterotopic abdominal heart transplantation in rats for functional studies of ventricular unloading.</a> J Surg Res. 179 (1), e31-e39 (2013).</li><li>Asfour, B., Hare, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+new+model+of+physiologically+working+heterotopic+rat+heart+transplantation+provides+hemodynamic+performance+equivalent+to+that+of+an+orthotopic+heart.">A simple new model of physiologically working heterotopic rat heart transplantation provides hemodynamic performance equivalent to that of an orthotopic heart.</a> J Heart Lung Transplant. 18 (10), 927-936 (1999).</li><li>Figueiredo, J. -. L., Nahrendorf, M., Sosnovik, D. E., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+of+a+novel+murine+working+heart+transplant+model.">MRI of a novel murine working heart transplant model.</a> Circ Heart Fail. 2 (3), 272-274 (2009).</li><li>Wen, P., Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+a+new+working+heterotopic+heart+transplantation+model+in+rats.">A simple technique for a new working heterotopic heart transplantation model in rats.</a> Transplant Proc. 45 (6), 2522-2526 (2013).</li><li>Didi&#233;, M., Biermann, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+left+ventricular+function+and+morphology+in+volume-loaded+versus+volume-unloaded+heterotopic+heart+transplants.">Preservation of left ventricular function and morphology in volume-loaded versus volume-unloaded heterotopic heart transplants.</a> A.Am J Physiol Heart Circ Physiol. 305 (4), H533-H541 (2013).</li><li>Gali&#241;anes, M., Zhai, X., Hearse, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+load+on+atrophy,+myosin+isoform+shifts+and+contractile+function:+studies+in+a+novel+rat+heart+transplant+preparation.">The effect of load on atrophy, myosin isoform shifts and contractile function: studies in a novel rat heart transplant preparation.</a> J Mol Cell Cardiol. 27 (1), 407-417 (1995).</li><li>Yokoyama, H., Ohmi, M., Murata, S., Nakame, T., Tabayashi, K., Mohri, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proposal+of+a+working+left+heart+model+with+a+heterotopic+transplantation+technique+in+rats.">Proposal of a working left heart model with a heterotopic transplantation technique in rats.</a> J Heart Lung Transplant. 14 (4), 706-712 (1995).</li><li>Maruyama, T., Swartz, M. T., McBride, L. R., Pennington, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Working+heart+model+of+heterotopic+heart-lung+transplantation+in+rats.">Working heart model of heterotopic heart-lung transplantation in rats.</a> J Thorac Cardiovasc Surg. 107 (1), 210-215 (1994).</li><li>Pacher, P., Nagayama, T., Mukhopadhyay, P., B&#225;tkai, S., Kass, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+cardiac+function+using+pressure-volume+conductance+catheter+technique+in+mice+and+rats.">Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats.</a> Nat Protoc. 3 (9), 1422-1434 (2008).</li><li>Jespersen, B., Knupp, L., Northcott, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femoral+arterial+and+venous+catheterization+for+blood+sampling,+drug+administration+and+conscious+blood+pressure+and+heart+rate+measurements.">Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements.</a> J Vis Exp. (59), 3496-3410 (2012).</li><li>Habertheuer, A., Kocher, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative,+simplified+orthotopic+lung+transplantation+in+rats).">Innovative, simplified orthotopic lung transplantation in rats).</a> J Surg Res. 185 (1), 419-425 (2013).</li></ol>

Los autores no tienen nada que revelar.

El éxito con la técnica descrita aquí se basa en varios factores. Clave entre ellos será garantizar la estabilidad tanto de los animales donantes y receptores, la adopción de la técnica quirúrgica meticulosa que es seguro y asociado con mínima pérdida de sangre, lo que garantiza la detención cardioplégica completo con un enfriamiento uniforme del injerto, lo que minimiza el tiempo de isquemia total y adecuadamente de-a transmitirse el injerto. Como ha reconocido anteriormente, la complejidad técnica de la t?...

The first rodent model of heterotopic abdominal heart transplantation (HAHT) was described by Abbott and colleagues in 19641. This technique, and subsequent modifications have been widely applied to characterize transplant graft function and immunologic status. The majority of HAHT techniques described involve a non-volume loaded heart2,3. Models of HAHT involving volume-loaded ventricles have been described, but they are frequently limited in one or more respects.
Heterotopic abdominal heart-lung transplantation (HAHLT) with a volume-loaded left ventricle (LV) has been described previously. Chen and colleagues4, and subsequently Ibrahim and colleagues5 described HAHLT with a single aorto-aortic (donor ascending to recipient abdominal aorta) anastomosis. The only volume load presented to the ventricle in this circulation is the coronary venous return. Asfour and colleagues described a HAHT technique in which the lung circuit was eliminated by anastomosing donor pulmonary artery (PA) to donor left atrium (LA)6. In this circulation, venous inflow to right ventricle (RV) occurs via a donor SVC to recipient IVC anastomosis, and the subsequent LV load is ejected into the aorto-aortic anastomosis. Cardiac function was partially assessed in vivo, and also in vitro using a Langendorff rig. Figueiredo and colleagues described a HAHLT model similar to our own7, but in mice. Venous inflow to the RV occurs via donor SVC to recipient IVC anastomosis. Blood subsequently passes through the single lung circulation and LV load is ejected into the aorto-aortic anastomosis. Cardiac function in their study was assessed by magnetic resonance imaging (MRI). Wen and colleagues described a unique HAHT technique in which the LV is loaded by means of a recipient aorta to donor LA anastomosis8. The LV, therefore, fills at systemic pressures. Cardiac function, and whether LV stroke volume is ejected antegradely in their model was not assessed.
Many of the techniques referenced above involve non-physiologic LV loading conditions, including the techniques whose partial LV load is represented only by coronary venous return. On the other hand, many techniques do approach physiologic LV loading. The majority of these techniques, as with the technique of Asfour and colleagues, omit the pulmonary circulation and utilize a donor PA to donor LA anastomosis6,9. The circulation described by Galinanes and colleagues10 employs a direct recipient cava to donor LA anastomosis, omitting the pulmonary circulation and the right heart. Yokoyama and colleagues achieve the same effect by ligating the donor PA and creating an interatrial communication in the donor heart (omitting donor lung and right heart circulations)11. The circulation of Maruyama and colleagues12 involves an anastomosis between donor left PA and recipient Ao, which permits LV filling via the pulmonary circulation as a conduit, but effectively excludes the right heart.
In cases where near physiologic loading conditions were met, we advance the technique of HAHLT in 2 major respects. First, to our knowledge, the exact configuration we report has not been described in rats. It is possibly the most versatile circulation for investigators wishing to study the physiology, structure, and immunology of the transplanted heart-lung graft. Second, we describe how the function of the transplant graft can be directly characterized in vivo. For this application, pressure-volume conductance catheters can be introduced directly into the LV apex of the transplant graft, which allows for complete cardiac functional characterization. 
The technique described here can be applied to both acute and chronic studies of transplant graft function, while the functional assessment may be performed either in vivo or in vitro. We present a model in which the loading conditions can be near physiologic, however the degree of ventricular loading may be manipulated both acutely and chronically by diverting venous return towards or away from the graft. Afterload conditions can also be manipulated. Because the lung and its airway are retained in this transplant configuration, investigators can re-ventilate the donor lung acutely. Uniquely, lung re-ventilation changes the composition of blood perfusing the transplant coronary arteries. Under non-ventilated conditions, blood ejected from the donor aorta is deoxygenated, and mixes with oxygenated blood in the recipient aorta. Under acutely ventilated conditions, ejected blood becomes oxygenated. Thus, transplant graft function can be compared under ventilated and non-ventilated conditions, and also under variably loaded conditions.
The protocol below describes important modifications to previously described HAHLT donor and recipient operations. It also describes an optimal technique for protecting the transplant graft throughout the period of ischemia (time between donor explant and recipient implant). Advantages of this technique include physiologic conditions potentially approaching that of an orthotopically transplanted graft, and a wide range of investigative applications. An important limitation is its technical complexity. With adequate mentoring and practice, the advantages of this technique will likely outweigh the challenges in adopting it.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Material/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Celsior Cardioplegic Soution</td>
			<td>Genzyme</td>
			<td></td>
			<td>The solution is kept on ice throughout the procedure. We prepare our own solution, with slight modifications.</td>
		</tr>
		<tr>
			<td>Rodent Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>Model 683</td>
			<td></td>
		</tr>
		<tr>
			<td>Vital Sign Monitor</td>
			<td>Nonin</td>
			<td>Model 9847V</td>
			<td>Displays SpO2 and heart rate.</td>
		</tr>
		<tr>
			<td>IV Cannulae</td>
			<td>Jelco</td>
			<td>3063</td>
			<td>24-26G x 3/4&#34; cannulae.</td>
		</tr>
		<tr>
			<td>IV Tubing</td>
			<td>CareFusion</td>
			<td>MP9259-C</td>
			<td>Short-length connector tubing (18cm).</td>
		</tr>
		<tr>
			<td>Surgical Clips</td>
			<td>Teleflex Medical</td>
			<td>001204</td>
			<td>Horizon titanium ligating clips.</td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Ethicon, Sharpoint</td>
			<td>LA54G, AK-0107</td>
			<td>3-0 silk reel, and 9-0 prolene suture (single-armed, DR5 needle).</td>
		</tr>
		<tr>
			<td>Surgical Instruments</td>
			<td>Not Applicable</td>
			<td>Not Applicable</td>
			<td>The instruments used are generic, and can be purchased from any surgical supply company.</td>
		</tr>
	</tbody>
</table>

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.

La técnica HAHLT descrito anteriormente es muy técnico y requiere mucha atención a los detalles. Tabla 1 se destacan algunos de los principales factores asociados con éxito frente a los procedimientos fallidos, y pueden ser utilizados como una guía para la solución de problemas dificultades técnicas. Después se suelta la aorta destinatario, las arterias coronarias del injerto deben ser vistos a llenarse de sangre oxigenada. En consecuencia, el miocardio se convierta ...

Watch this Scientific Journal Video about Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration at JoVE.com

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

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 ...

rat-heterotopic-abdominal-heartsingle-lung-transplantation-volume

Research

JoVE Journal

Medicine

8.3K vistas.  UBC James Hogg Research Center, St. Paul's Hospital. 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.

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

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Trasplante ortotópico de hígado en ratas

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Investigación

Medicina

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

Heterotópico Rata auxiliar Trasplante de Hígado Con Flujo regulado Portal Vein Arterialización en aguda Insuficiencia Hepática

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

Murino heterotópico Corazón Trasplante Técnica

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

El trasplante de la válvula pulmonar Utilizando un modelo de ratón de heterotópico trasplante cardiaco

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

Heterotópico cervical trasplante cardiaco en ratones

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

Reconstrucción Del Circuito Sanguíneo En Un Modelo De Trasplante De Corazón De Ratón Abdominal

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.

Un modelo simplificado para el trasplante heterotópico de válvulas cardíacas en roedores

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of ...

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

Un protocolo de trasplante de corazón heterotópico porcino para la administración de terapias a un aloinjerto cardíaco

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

Una técnica de manguito modificado para el modelo de trasplante cardíaco heterotópico cervical de ratón

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...

A Heterotopic Mouse Model for Studying Laryngeal Transplantation

Laryngeal heterotopic transplantation, although a technically challenging procedure, offers more scientific analysis and cost benefits compared to other animal models. Although first described by Shipchandler et al. in 2009, this technique is not widely used, possibly due to the difficulties in learning the microsurgical technique and time required to master it. This paper describes the surgical steps in detail, as well as potential pitfalls to avoid, in order to encourage effective use of this technique.
In this model, the bilateral carotid arteries of the donor larynx are anastomosed to the recipient carotid artery and external jugular vein, allowing for blood flow through the graft. Blood flow can be confirmed intraoperatively by the visualization of blood filling in the graft bilateral carotid arteries, reddening of the thyroid glands of the graft, and bleeding from micro vessels in the graft. The crucial elements for success include delicate preservation of the graft vessels, making the correct size arteriotomy and venotomy, and using the appropriate number of sutures on the arterial-arterial and arterial-venous anastomoses to secure vessels without leakage and prevent occlusion. 
Anyone can become proficient in this model with sufficient training and perform the procedure in approximately 3 h. If performed successfully, this model allows for immunologic studies to be performed with ease and at low cost.

The aim of this manuscript is to describe the microsurgical steps required to perform a heterotopic laryngeal transplant in mice. The advantages of this mouse model compared to other animal models of laryngeal transplantation are its cost-effectiveness and the availability of immunologic assays and data.

Un modelo heterotópico de ratón para estudiar el trasplante de laringe

Laryngeal heterotopic transplantation, although a technically challenging procedure, offers more scientific analysis and cost benefits compared to other ...