Este trabajo fue apoyado en parte por una subvención del NIH (HL098228 SR1) de CKB.

Nota: Todos los animales procedimientos fueron aprobados por el Comité del Hospital Institucional Cuidado de Animales y Uso de los Niños de Nationwide. 1. Válvula pulmonar del corazón de la cosecha de un donante de la válvula del corazón del ratón <ol><li> Autoclave todas las herramientas quirúrgicas antes de la cirugía: 1x tijeras finas, 3x fórceps micro, pinzas vasculares micro de 2x, abrazadera 1x fórceps soliciten, sostenedor 1x micro aguja, tijeras de primavera 1x, 1x retractor. </li><li> Un 6-8 semanas de edad hembra C57BL / 6 de ratón se utiliza como un donante de válvula cardíaca pulmonar. Retire el ratón de su jaula y se pesa, y luego practicar la eutanasia con un cóctel de ketamina / xilazina (ketamina, 200 mg / kg y xilazina, 20 mg / kg, IP) sobredosis. </li><li> Clip de la zona del pecho y coloque el ratón en una posición de decúbito dorsal sobre una almohadilla. A continuación, realice la toracotomía. Exponer el corazón, hacer un pequeño corte en la aurícula derecha y el ventrículo izquierdo perfundir con hielo salina fría. </li><li> Francamente diseccionar la arteria pulmonar (AP) de la ascendente aorta. Corte la válvula pulmonar (PV), junto con 2 mm manguito de arteria pulmonar. Deseche el resto del corazón. </li><li> Guarde el PV en heparina frío y solución salina (100 unidades / ml). Nota: El PV se puede mantener en la solución durante dos horas antes del trasplante a el corazón del donante. </li></ol> 2. Preparación corazón del donante <ol><li> Un 6-8 semanas de edad hembra C57BL / 6 de ratón se utiliza como un donante de corazón. Retire el ratón de su jaula y se pesa, y luego practicar la eutanasia con un cóctel de ketamina / xilazina (ketamina, 200 mg / kg y xilazina, 20 mg / kg, IP) sobredosis. Este es un procedimiento terminal. </li><li> Clip de la zona del pecho y coloque el ratón en una posición de decúbito dorsal sobre una almohadilla. A continuación, realice la toracotomía. Francamente separar el corazón, la vena cava inferior (VCI), la vena cava superior (VCS), la aorta ascendente, PA, y la vena pulmonar. Perfundir la VCI con helado salina estéril. </li><li> Ligar el IVC, SVC, y la vena pulmonar con sutura de seda 6-0 a continuación, cortar superior a laligaduras. </li><li> Cortar la aorta y PA con 2 mm manguito. </li><li> Recorta el PV y disponer de ella. </li></ol> 3. Válvula de Trasplante Cardiaco en un donante de corazón <ol><li> Inmediatamente después de la etapa 2.5, colocar la válvula de corazón de la etapa 1.5 en el corazón del donante y orientar la válvula de corazón. </li><li> Asegure el PV con una puntada en el lado derecho de la válvula utilizando 10-0 monofilamento en las agujas afiladas y empezar a suturar continuamente con 5-6 puntos de sutura desde el otro lado de la PV. </li><li> Después de terminar el lado delantero, girar el corazón horizontalmente y empezar a suturar la parte trasera de la PV en el corazón del donante. </li><li> Almacenar el corazón en una solución de heparina / solución salina estéril fría. Nota: El corazón del donante se puede mantener en la solución durante dos horas antes de la implantación para el ratón receptor. </li></ol> 4. Heterotópico trasplante cardiaco a un destinatario Ratón <ol><li> Un 6-8 semanas de edad hembra C57BL / 6 de ratón se usó como una reciprocidadnatario. Retire el ratón de su jaula y se pesa, y luego anestesiados con cóctel de ketamina / xilazina (ketamina, 100 mg / kg y xilazina 10 mg / kg). Ketoprofeno (5 mg / kg) se usa como analgésico preanestesia. </li><li> Después de comprobar el nivel de sedación por la cola pellizcar, cortar el pelo en el pecho y el abdomen. Lubricar los ojos con un ungüento oftálmico estéril y colocar el ratón en una posición de decúbito dorsal sobre una almohadilla. Desinfectar el abdomen con almohadillas de betadine y alcohol. Luego cubra el ratón con un paño estéril y exponer sólo el área de la incisión. </li><li> Hacer una laparotomía de la línea media de la incisión por debajo de la xifoides a la región suprapúbica, e insertar un retractor de auto retención. Envuelva los intestinos en solución salina gasa humedecida. Francamente definir la aorta infrarrenal y la vena cava. </li><li> Coloque dos 6-0 suturas de seda proximal y distal alrededor de la aorta y la vena cava inferior para frenar la circulación sanguínea. </li><li> Coloque el corazón del donante en el lado derecho de la aorta abdominal y la cubrirá con gau estérilze. Hidratar con solución salina. </li><li> Hacer una aortotomía en la aorta abdominal con una aguja de 30 G y extender la abertura con tijeras para el tamaño de la aorta de donantes. </li><li> Realizar un extremo a lado anastomosis utilizando estériles suturas monofilamento 10-0 sobre agujas afiladas. Asegure la aorta del donante con una puntada en el extremo proximal de la abertura en la aorta abdominal y empezar a suturar continuamente con 4-5 puntos de sutura desde el extremo distal de la aorta abdominal. </li><li> Voltear el corazón hacia el lado izquierdo, cubrir con solución salina infundido gasa, y empezar a suturar continuamente con 4-5 puntos de sutura desde el extremo distal de la aorta abdominal. </li><li> Hacer una venotomía en la vena cava inferior usando una aguja de 30 G y extender la abertura con el tamaño de la arteria pulmonar del donante. </li><li> Lleve a cabo el fin de la anastomosis lateral con estériles suturas monofilamento 10-0 sobre agujas afiladas. Asegure el donante PA con una puntada en el extremo proximal de la abertura en la VCI y empezar a suturar continuamente con 4-5 puntos de sutura deel extremo distal de la vena cava inferior. Esta vez, debido a que la aorta es en la forma, asegúrese de que la sutura de la pared izquierda de PA del donante está en el interior de la vena cava inferior. </li><li> Enjuague el lumen de VCI con heparina y solución salina (100 unidades / ml). Cierre la pared de la derecha del donante y el receptor PA IVC mediante la sutura de forma continua hasta el extremo distal. </li><li> Retire la ligadura distal y controlar la hemorragia mediante la aplicación de un agente de hemostato absorbible estéril tópica. Cuando la hemorragia se detiene por completo, retire la sutura proximal y controlar la hemorragia de la misma manera. </li><li> Volver a los intestinos y cerrar la musculatura abdominal y la piel de dos capas usando una sutura monofilamento 6-0 poliamida negro. </li><li> Inyectar 0,5 ml de solución salina por vía subcutánea y colocar el ratón en una jaula de recuperación en una almohadilla térmica hasta que el ratón es completamente móvil. Después de la recuperación, devuelva el ratón a una nueva jaula con ropa de cama de papel. Déle medicina para el dolor (ibuprofeno, 30 mg / kg, el agua potable) durante 48 horas. HacerNo devuelva un animal que ha sido sometido a cirugía de la compañía de otros animales hasta que se recupere completamente. </li></ol>

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No tenemos nada que revelar.

La tasa de mortalidad de este procedimiento es cercana al 20%, que se debe principalmente a la hemorragia en el lugar de trasplante de PV y anastomosis en la aorta del donante a la aorta abdominal destinatario. En la mayoría de los casos, la tasa de mortalidad disminuye significativamente después de la cirugía 48 horas. Los ratones de supervivencia mostraron fuertes latidos del corazón y el flujo de sangre a través de la PV implantado. Todo el proceso tarda cuatro horas para un cirujano experimentado micro. Tomará...

Defectos cardiovasculares congénitos son una de las principales causas de mortalidad infantil en el mundo occidental 1,2. Entre ellos, la estenosis de la válvula pulmonar y defectos en las válvulas aórticas bicúspides son una forma que ocurre con frecuencia 3. La cirugía de reemplazo de válvulas cardíacas es una elección habitual de cirugías reconstructivas; Sin embargo, las complicaciones, incluyendo la estenosis y la calcificación de la válvula del corazón, y la dependencia de por vida con anticoagulantes son una fuente importante de enfermedad crónica y muerte 4-7. Por otra parte, la falta de potencial de crecimiento requiere cirugías de revisión, lo que aumenta aún más la mortalidad de los pacientes jóvenes 4,8,9. En un intento de desarrollar una válvula de corazón de sustitución funcional con potencial de crecimiento, Shinoka et al. Células autólogas sembradas en una válvula de corazón sintético biodegradable 8. La válvula sintética transformado en una válvula del corazón nativo como la estructura con potenciómetro crecimientoal. Los estudios en animales grandes preliminares demostraron la viabilidad de utilizar esta metodología para crear una válvula cardiaca funcional 10. Sin embargo, estudios de implantación a largo plazo demostraron poca durabilidad debido al engrosamiento progresivo de la neotejido válvula resulta en el estrechamiento de la válvula cardíaca. Trabaje desde Sodian et al. Utilizaron la metodología Shinoka, pero en última instancia, sustituyó a la matriz de la PGA con un elastómero biodegradable, que dio a las propiedades biomecánicas de la válvula de ingeniería tisular, la construcción de un perfil más fisiológico 9,11,12. En el estudio in vivo, a pesar del éxito de la implantación, no se formó un revestimiento de células endoteliales confluentes que podría limitar el éxito a largo plazo de este andamio 12. Con el fin de diseñar racionalmente una válvula de corazón sintética segunda generación mejorada, un modelo murino de trasplante de válvula de corazón fue creado para investigar los mecanismos celulares y moleculares underlyinla formación de neotejido g, engrosamiento de la válvula, y el desarrollo de estenosis. Los modelos murinos ofrecen una amplia gama de reactivos moleculares, incluyendo los transgénicos, que no son fácilmente disponibles en otras especies 7. En este modelo de trasplante de válvulas cardíacas, un vivo de reemplazo de válvula cardíaca pulmonar ex syngeneic se realizó en primer lugar; y luego el corazón con la válvula de corazón implantado se implantó heterotópicamente en un hospedador singénico utilizando una técnica de microcirugía. Este modelo permite el reemplazo de la válvula del corazón sin la necesidad de la derivación cardiopulmonar. En este trabajo, una explicación detallada de la cosecha de la válvula del corazón, preparaciones de corazón donante, trasplante de válvulas cardíacas y el trasplante cardíaco heterotópico se describe. Los resultados mostraron un latido continuo desde el corazón del donante, que era independiente de los latidos del corazón receptor. El flujo de sangre a través de la válvula pulmonar implantado se midió usando un sistema de ultrasonido de alta frecuencia con una onda pulsada hacerppler.

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			<td height="20" style="height:20px;width:176px;">DPBS</td>
			<td style="width:167px;">gibco</td>
			<td style="width:133px;">14190-144</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:176px;">Microscope</td>
			<td style="width:167px;">Leica</td>
			<td style="width:133px;">M80</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:176px;">C57BL/6J (H-2b), Female</td>
			<td style="width:167px;">Jackson Laboratories&#160;</td>
			<td style="width:133px;">664</td>
			<td style="width:168px;">8-12 weeks</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:176px;">Ketamine Hydrochloride Injection</td>
			<td style="width:167px;">Hospira Inc.</td>
			<td style="width:133px;">NDC 0409-2053</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Xylazine Sterile Solution</td>
			<td style="width:167px;">Akorn Inc.</td>
			<td style="width:133px;">NADA# 139-236</td>
			<td style="width:168px;"></td>
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		<tr height="36">
			<td height="36" style="height:36px;width:176px;">ketoprofen</td>
			<td style="width:167px;">Fort Dodge Animal Health</td>
			<td style="width:133px;">NDC 0856-4396-01</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:176px;">Ibuprofen</td>
			<td style="width:167px;">PrecisionDose</td>
			<td style="width:133px;">NDC 68094-494-59</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:176px;">Heparin Sodium</td>
			<td style="width:167px;">Sagent Pharmaceticals</td>
			<td style="width:133px;">NDC 25021-400</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:176px;">Saline solution (Sterile 0.9% Sodium Chloride)</td>
			<td style="width:167px;">Hospira Inc.</td>
			<td style="width:133px;">NDC 0409-0138-22</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:176px;">0.9% Sodium Chloride Injection</td>
			<td style="width:167px;">Hospira Inc.</td>
			<td style="width:133px;">NDC 0409-4888-10</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:176px;">Petrolatum Ophthalmic Ointment</td>
			<td style="width:167px;">Dechra Veterinary Products</td>
			<td style="width:133px;">NDC 17033-211-38</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Iodine Prep Pads</td>
			<td style="width:167px;">Triad Disposables, Inc.</td>
			<td style="width:133px;">NDC 50730-3201-1</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Alcohol Prep Pads</td>
			<td style="width:167px;">McKesson Corp.</td>
			<td style="width:133px;">NDC 68599-5805-1</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Cotton tipped applicators</td>
			<td style="width:167px;">Fisher Sientific</td>
			<td style="width:133px;">23-400-118</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:176px;">Fine Scissor</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">14028-10</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:176px;">Micro-Adson Forcep</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">11018-12</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:176px;">Clamp Applying Forcep</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">00072-14</td>
			<td style="width:168px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:176px;">S&#38;T Vascular Clamp</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">00396-01</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Spring Scissors</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">15008-08</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Colibri Retractors</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">17000-04</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Dumont #5 Forcep</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">11251-20&#160;</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Dumont #7 - Fine Forceps</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">11274-20</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Dumont #5/45 Forceps</td>
			<td style="width:167px;">FST</td>
			<td style="width:133px;">11251-35</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Tish Needle Holder/Forceps</td>
			<td style="width:167px;">Micrins</td>
			<td style="width:133px;">MI1540</td>
			<td style="width:168px;"></td>
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		<tr height="38">
			<td height="38" style="height:38px;width:176px;">Black Polyamide Monofilament Suture, 10-0</td>
			<td style="width:167px;">AROSurgical Instruments Corporation</td>
			<td style="width:133px;">TI638402</td>
			<td style="width:168px;">For sutureing the graft</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:176px;">Black Polyamide Monofilament Suture, 6-0</td>
			<td style="width:167px;">AROSurgical Instruments&#160;</td>
			<td style="width:133px;">SN-1956</td>
			<td style="width:168px;">For musculature and skin closure</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Non-Woven Songes</td>
			<td style="width:167px;">McKesson Corp.</td>
			<td style="width:133px;">94442000</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:176px;">Absorbable hemostat</td>
			<td style="width:167px;">Ethicon</td>
			<td style="width:133px;">1961</td>
			<td style="width:168px;"></td>
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			<td height="24" style="height:24px;width:176px;">1 ml Syringe</td>
			<td style="width:167px;">BD</td>
			<td style="width:133px;">309659</td>
			<td style="width:168px;"></td>
		</tr>
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transplantation of pulmonary valve using a mouse model of heterotopic heart transplantation

Ingeniería tisular las válvulas del corazón, válvulas especialmente descelularizado, están empezando a cobrar impulso en el uso clínico de la cirugía reconstructiva con resultados mixtos. Sin embargo, los mecanismos celulares y moleculares de la neotejido desarrollo, engrosamiento de la válvula, y el desarrollo de estenosis no son investigados extensivamente. Para responder a estas preguntas, hemos desarrollado un modelo murino heterotópico de corazón Válvula de trasplante. Una válvula de corazón fue cosechada de un ratón donante y se trasplanta a la válvula de un ratón donante corazón. El corazón con una nueva válvula se trasplantó heterotópicamente a un ratón receptor. El corazón trasplantado mostró su propio latido del corazón, independiente de los latidos del corazón del destinatario. El flujo de sangre se cuantificó usando un sistema de ultrasonido de alta frecuencia con un Doppler pulsado. El flujo a través de la válvula pulmonar implantado mostró delante de flujo con regurgitación mínimo y el flujo pico fue cerca de 100 mm / seg. Este modelo murino de trasplante de válvulas cardíacas es highly versátil, por lo que puede ser modificado y adaptado para proporcionar diferentes entornos hemodinámicas y / o se puede utilizar con varios ratones transgénicos para estudiar el desarrollo neotejido en una válvula de corazón de ingeniería tisular.

La Figura 1 ilustra los esquemas de la modelo de trasplante de válvula de corazón usando el trasplante heterotópico de corazón. La válvula de corazón fue cosechada de un corazón de un donante y se implanta en un corazón de un segundo ratón donante. Entonces el corazón con la nueva válvula cardíaca se implanta en el abdomen de un ratón receptor. La figura 2 muestra una ilustración del corazón implantado en el espacio abdominal (A), justo después de traspl...

Watch this Scientific Journal Video about Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation at JoVE.com

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

Con el fin de comprender los mecanismos celulares y moleculares que subyacen a la formación de neotejido y el desarrollo de estenosis en las válvulas del corazón de la ingeniería tisular, se desarrolló un modelo murino de trasplante heterotópico de válvula cardiaca. Una válvula cardíaca pulmonar fue trasplantado al receptor mediante la técnica de trasplante cardíaco heterotópico.

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 ...

transplantation-pulmonary-valve-using-mouse-model-heterotopic-heart

Research

JoVE Journal

Medicine

18.8K vistas.  Nationwide Children's Hospital.  Con el fin de comprender los mecanismos celulares y moleculares que subyacen a la formación de neotejido y el desarrollo de estenosis en las válvulas del corazón de la ingeniería tisular, se desarrolló un modelo murino de trasplante heterotópico de válvula cardiaca. Una válvula cardíaca pulmonar fue trasplantado al receptor mediante la técnica de trasplante cardíaco heterotópico. 

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

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

Un modelo de ratón En el útero Trasplante

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Investigación

Medicina

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Murino Cervical Trasplante Cardiaco Modelo Usando una técnica de Cuff Modificado

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

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 ...

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 ...

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 ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

Un microquirúrgica Modelo Novela de heterotópico, en bloque, pared torácica, el timo y el trasplante cardiaco en ratones

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Un modelo preclínico porcino de trasplante ortotópico de corazón

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

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 ...