M.-A. Deutsch is supported by Dr. Rusche Forschungsprojekt (2014) Deutsche Stiftung f&#252;r Herzforschung and Deutsche Gesellschaft f&#252;r Thorax- Herz- und Gef&#228;&#223;chirurgie. M. Krane is supported by Deutsche Stiftung f&#252;r Herzforschung (F/37/11), Deutsches Zentrum f&#252;r Herz Kreislauf Forschung (DZHK B 13-050A; DZHK B 14-013SE) and Deutsche Forschungsgemeinschaft (KR3770/7-1; KR3770/9-1)

Todos los experimentos fueron aprobados por el alemán Corazón Centro de Múnich y el Gobierno de Baviera, Alemania (Gz. 55.2-1-54-2532.3-17-13). Los animales fueron alojados en una instalación específica libre de patógenos y recibieron atención humana en el cumplimiento de los &quot;Principios de Laboratorio Animal Care&quot; formuladas por la Sociedad Nacional para la Investigación Médica y de la &quot;Guía para el Cuidado y Uso de Animales de Laboratorio&quot; proporcionada por la Academia Nacional de Ciencias y publicado por los Institutos Nacionales de Salud (NIH Publication no. 86-23, revisado 1985). NOTA: Un conjunto completo de instrumentos de microcirugía estériles, incluyendo dos pinzas curvas y dilatadores de los vasos, es necesario para el procedimiento (Figura 2a). La profilaxis antibiótica perioperatoria no es habitualmente necesaria, pero pueden estar indicados en animales inmunocomprometidos. Los animales fueron sacrificados por inhalación de isoflurano usando terminal. Donantes de Preparación y la cosecha del injerto &lt;ol&gt; <li> Anestesiar al animal mediante intraperitoneal (ip) de inyección de midazolam (5,0 mg / kg), medetomidina (0,5 mg / kg) y fentanilo (0,05 mg / kg de peso corporal). Coloque el ratón en una posición supina en el campo operatorio y confirmar la tolerancia quirúrgica por el reflejo de retirada pedal. </li><li> Desinfectar la piel tres veces con clorhexidina y realizar una incisión abdominal en la línea media. Retraer los intestinos con una gasa húmeda a la izquierda para exponer la vena cava inferior. </li><li> Inyectar 50 UI de heparina diluida en 0,4 ml de solución salina a través de la vena cava inferior para la anticoagulación. Incisión en el vaso 1 min después de heparinización. </li><li> Abra el diafragma y realizar una toracotomía bilateral para exponer el corazón. La pared torácica anterior se refleja en sentido superior y se mantiene en su lugar con una abrazadera. </li><li> Retire el timo y sin rodeos diseccionar el tejido conectivo entre la aorta y el tronco pulmonar. Incisión en la vena cava superior derecha. </li><li> Perforar la aorta ascendente movilizado a nivel de latronco braquiocefálico con una aguja de 30 G montado en una jeringa de 5 ml y lentamente perfundir el corazón retrogradually sin una presión excesiva con 3 ml de 4 ° C solución cardiopléjica fría. Evitar la entrada de aire en la circulación coronaria. Caída de solución salina enfriada con hielo en el injerto para mejorar aún más el paro cardiaco. </li><li> Seccionar la aorta proximal al origen del tronco braquiocefálico. </li><li> Ligar la vena inferior y ambas cavas superior cerca del corazón con 8-0 seda y seccionar los distalmente a las ligaduras. </li><li> Diseccionar libre el tronco pulmonar lo más distante posible. Ligar las arterias pulmonares cerca de la bifurcación con 8-0 seda y seccionar ellos distal. </li><li> Ligar las venas pulmonares todos juntos con una sola ligadura de 8-0 seda y seccionar ellos. </li><li> Quite el corazón del donante y almacenarlo en la solución cardiopléjica helada hasta la implantación. </li></ol> 2. Destinatario Preparación <ol><li> Prepararlos puños para anastomosis cortando el tubo de poliimida con un bisturí No. 11 o una tijera bajo el microscopio. El manguito se compone de un cuerpo cilíndrico y una extensión (1/3 de la circunferencia) para la colocación de la pinza vascular, cada uno de una longitud de 1 mm (figura 2b). </li><li> Anestesiar al animal y de confirmar la tolerancia quirúrgica como se describe anteriormente. Inyectar 0,25 ml de solución salina caliente por vía subcutánea (sc) para la reposición de líquidos. </li><li> Quite el pelo de la región cervical derecha usando una máquina de afeitar eléctrica y coloque el animal en posición supina sobre una almohadilla caliente. Asegure las piernas con hebras de cinta evitando estiramiento excesivo de las extremidades anteriores, ya que puede comprometer la respiración. Cubra los ojos utilizando pomada oftálmica para evitar la sequedad. Desinfectar el sitio quirúrgico tres veces con clorhexidina. </li><li> Hacer una incisión cutánea transversal desde el ángulo mandibular derecho a la muesca yugular. </li><li> Sin rodeos movilizar a la extvena yugular ernal. Cauterizar ramas laterales con unas pinzas bipolares y seccionar ellos. </li><li> Divida la vena yugular externa entre ligaduras en el nivel de la confluencia con la vena de la glándula submandibular. </li><li> Tire de la vena yugular externa a través del manguito y ocluir el vaso proximal mediante la colocación de una pinza vascular en la extensión del manguito. Retire la abrazadera y el riego de la luz del vaso con solución salina heparinizada 01:10. </li><li> Evert la pared del vaso sobre el manguito y fijarlo con una circunferencia 8-0 ligadura de seda preestablecido. </li><li> Retire la parte superficial del músculo esternocleidomastoideo. </li><li> Movilizar a la arteria carótida común derecha y seccionar el buque entre ligaduras por debajo de la bifurcación de la carótida. </li><li> Tire de la arteria carótida a través del manguito y ocluir el vaso proximal mediante la colocación de una pinza Yasargil en la extensión del manguito. Retire la abrazadera y el riego de la luz del vaso con solución salina heparinizada 01:10. </li><li> Suavemente dilate la luz arterial con un dilatador buque, Evert la pared del vaso sobre el manguito y arreglarlo con un circunferencial 8-0 ligadura de seda preestablecido. </li><li> Para ganar espacio suficiente para la implantación del injerto, quitar el lóbulo derecho de la glándula submandibular. </li><li> Mantenga el campo quirúrgico húmedo hasta la implantación. </li></ol> 3. Implantación del injerto <ol><li> Coloque el injerto en la región cervical receptor boca abajo con la aorta orientado medialmente y la arteria pulmonar lateralmente. </li><li> Tire de la aorta del injerto sobre el brazalete con la arteria carótida y asegurar la anastomosis con una ligadura circunferencial 8-0 seda. </li><li> Incisión en el tronco pulmonar en su cara anterior y tire de ella sobre el brazalete con la vena yugular externa. Una vez más asegurar la anastomosis con una ligadura circunferencial de 8-0 seda. </li><li> Retire la abrazadera en la vena yugular externa, siguiendo por el desamarre de la arteria carótida. Los corazones se llena inmediatamente con sangre. Para mejorar reperfusion caer 37 ° C una solución salina caliente en el injerto. Normalmente, el corazón se desarrolla el ritmo sinusal en un minuto. Retire la extensión del manguito en el sitio venosa. </li><li> Asegurar la posición correcta del injerto, anastomosis deben estar libre de tensiones y buques sin torsión. No vasos cauterizados de la piel ya que esto puede afectar la cicatrización de heridas. El sangrado generalmente se detiene espontáneamente. Cierre la piel con una sutura continua única de 8-0. </li></ol> 4. Cuidado posoperatorio <ol><li> Inyectar 0,25 ml cálida sc solución salina para la reposición de líquidos. </li><li> Para antagonizar narcóticos inyectan naloxona (1,2 mg / kg), flumazenil (0,5 mg / kg) y atipamezol (2,5 mg / kg de peso corporal) sc Izquierda del animal en la almohadilla caliente hasta que se haya recuperado el conocimiento suficiente para mantener decúbito esternal. Observar al animal durante al menos una hora adicional para registrar cualquier comportamiento anormal. No devuelva un animal que se ha sometido a una cirugía para la compañía de otros animales hasta que esté completamente Recoverojo. </li><li> Proporcionar analgesia preventiva con buprenorfina (0,05 mg / kg sc tres veces por día) o una analgesia alternativa de acuerdo con un protocolo institucional local para una duración mínima de 72 horas después del procedimiento. </li><li> El cirujano y un veterinario deben ver a los animales con regularidad. En caso de pérdida de peso&gt; 15%, la apatía, la hinchazón o animales de infección lado quirúrgicos son sacrificados mediante inhalación de isoflurano terminal. </li></ol>

<ol>
	<li>Corry, R. J., Winn, H. J., Russell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+transplantation+in+congenic+strains+of+mice.">Heart transplantation in congenic strains of mice.</a> Transplant Proc. 5, 733-735 (1973).</li><li>Chen, Z. H. <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+of+cervical+heterotopic+heart+transplantation+in+mice.">A technique of cervical heterotopic heart transplantation in mice.</a> Transplantation. 52, 1099-1101 (1991).</li><li>Martins, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+graft+function+in+rodent+models+of+heart+transplantation.">Assessment of graft function in rodent models of heart transplantation.</a> Microsurgery. 28, 565-570 (2008).</li><li>Schramm, R., 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=The+subepicardial+microcirculation+in+heterotopically+transplanted+mouse+hearts:+an+intravital+multifluorescence+microscopy+study.">The subepicardial microcirculation in heterotopically transplanted mouse hearts: an intravital multifluorescence microscopy study.</a> J Thorac Cardiovasc Surg. 134, 210-217 (2007).</li><li>Liu, F., Kang, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+heart+transplantation+in+mice.">Heterotopic heart transplantation in mice.</a> J Vis Exp : JoVE. 238, (2007).</li><li>Gong, W., 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=Introduction+of+modified+cervical+cardiac+transplant+model+in+mice.">Introduction of modified cervical cardiac transplant model in mice.</a> Exp Clin Transplant. 10, 158-162 (2012).</li><li>Matsuura, A., Abe, T., Yasuura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+mouse+cervical+heart+transplantation+using+a+cuff+technique.">Simplified mouse cervical heart transplantation using a cuff technique.</a> Transplantation. 51, 896-898 (1991).</li><li>Chen, H., Zhang, Y., Zheng, D., Praseedom, R. K., Dong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+kidney+transplantation+in+mice:+technique+using+cuff+for+renal+vein+anastomosis.">Orthotopic kidney transplantation in mice: technique using cuff for renal vein anastomosis.</a> PloS one. 8, e77278 (2013).</li><li>Maglione, 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+novel+technique+for+heterotopic+vascularized+pancreas+transplantation+in+mice+to+assess+ischemia+reperfusion+injury+and+graft+pancreatitis.">A novel technique for heterotopic vascularized pancreas transplantation in mice to assess ischemia reperfusion injury and graft pancreatitis.</a> Surgery. 141, 682-689 (2007).</li><li>Sucher, R., 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=Orthotopic+hind-limb+transplantation+in+rats.">Orthotopic hind-limb transplantation in rats.</a> J Vis Exp : JoVE. , (2010).</li><li>Kamada, N., Calne, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+the+rat.+Technique+using+cuff+for+portal+vein+anastomosis+and+biliary+drainage.">Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage.</a> Transplantation. 28, 47-50 (1979).</li><li>Oldani, G., Lacotte, S., Morel, P., Mentha, G., Toso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+rats.">Orthotopic liver transplantation in rats.</a> J Vis Exp : JoVE. , (2012).</li><li>Okazaki, 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+mouse+model+of+orthotopic+vascularized+aerated+lung+transplantation.">A mouse model of orthotopic vascularized aerated lung transplantation.</a> Am J Transplant. 7, 1672-1679 (2007).</li><li>Suzuki, H., Fan, L., Wilkes, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+obliterative+bronchiolitis+in+a+murine+model+of+orthotopic+lung+transplantation.">Development of obliterative bronchiolitis in a murine model of orthotopic lung transplantation.</a> J Vis Exp : JoVE. , (2012).</li><li>Zou, Y., 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=Mouse+model+of+venous+bypass+graft+arteriosclerosis.">Mouse model of venous bypass graft arteriosclerosis.</a> Am J Pathol. 153, 1301-1310 (1998).</li><li>Zhou, Y., Gu, X., Xiang, J., Qian, S., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+on+suture+versus+cuff+anastomosis+in+mouse+cervical+cardiac+transplant.">A comparative study on suture versus cuff anastomosis in mouse cervical cardiac transplant.</a> Exp Clin Transplant. 8, 245-249 (2010).</li><li>Niimi, M. <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+for+heterotopic+cardiac+transplantation+in+mice:+experience+of+3000+operations+by+one+surgeon.">The technique for heterotopic cardiac transplantation in mice: experience of 3000 operations by one surgeon.</a> J Heart Lung Transplant. 20, 1123-1128 (2001).</li><li>Arras, M., Autenried, P., Rettich, A., Spaeni, D., Rulicke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+intraperitoneal+injection+anesthesia+in+mice:+drugs,+dosages,+adverse+effects,+and+anesthesia+depth.">Optimization of intraperitoneal injection anesthesia in mice: drugs, dosages, adverse effects, and anesthesia depth.</a> Comp Med. 51, 443-456 (2001).</li></ol>

The authors have nothing to disclose.

Hemos completado más de 200 trasplantes cardíacos con una tasa de éxito superior al 90% en varios parámetros experimentales. Con la técnica descrita aquí, el procedimiento completo se puede hacer dentro de los 60 min por un cirujano experimentado. Mediante la preparación de los vasos cervicales antes de la obtención de órganos, el tiempo total de isquemia del injerto puede ser limitado a 20 min. Tiempo para la implantación (tiempo de isquemia caliente) es significativamente más corto usando la técnica del ma...

El modelo de trasplante de corazón se utiliza con frecuencia para investigar I / R-lesión, rechazo de aloinjerto, vasculopatía post-trasplante y la eficiencia de los agentes inmunomoduladores. Los modelos de ratón se combinan muchas ventajas como el inmunológico conocido y antecedentes genéticos, la disponibilidad de muchas cepas puras y transgénicos y los costos experimentales relativamente bajos. En 1973, una técnica de trasplante de corazón abdominal en ratones fue descrita por primera vez por Corry et al 1. En este modelo injertos se revascularizados antes de fin de a lado anastomosis de la aorta ascendente a la aorta abdominal del receptor y de la principal arteria pulmonar a la vena cava inferior. En 1991, una técnica de sutura para el trasplante de corazón cervical en ratones fue descrito por Chen et al 2. En este modelo una perfusión coronaria retrógrada se establece por la anastomosis de la arteria carótida del destinatario a la aorta ascendente del grpopa. Los drenajes de sangre venosa a través del seno coronario en la aurícula derecha, el ventrículo derecho y se expulsa a través de la arteria pulmonar en el receptor vena yugular externa (Figura 1). En comparación con la implantación del injerto abdominal el modelo de cuello uterino es menos invasiva y la localización superficial del injerto permite un acceso muy fácil para los exámenes de seguimiento adicionales (por ejemplo, la palpación, la ecocardiografía, la microscopía de fluorescencia intravital) 3,4. A pesar de las mejoras técnicas 5,6, un uso más generalizado de este modelo ha sido limitado debido a dificultades técnicas para realizar las anastomosis suturadas de vasos pequeños con una alta incidencia de fugas de anastomosis y la trombosis. En 1991, Matsuura et al. introdujo la técnica de manguito en la que la pared del vaso es evertido sobre un cilindro sintético, que facilita en gran medida el modelo de trasplante de corazón 7 cervical. La técnica fue further adoptado para varios modelos experimentales de trasplante renal incluyendo 8, 9 de páncreas, la integridad física 10, 11,12 hepática, pulmonar 13,14 y el trasplante vascular 15. En comparación con la técnica de sutura el tiempo requerido para las anastomosis y, por lo tanto, el tiempo de isquemia caliente es más corta y hay complicaciones vasculares significativamente menos usando la técnica del manguito 16. Además, permite a los cirujanos con poca formación en microcirugía para realizar la operación con una alta tasa de éxito. Una desventaja de la técnica del manguito es el requisito previo de la ligadura de la arteria carótida común, que puede alterar la perfusión cerebral. En este trabajo de vídeo presentamos una técnica de manguito mejorado y simplificado para el trasplante de corazón cervical en ratones.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Forceps JFC-7.2</td>
			<td>S&#38;T</td>
			<td>P-00036</td>
			<td>Curved tip 0.20 mm</td>
		</tr>
		<tr>
			<td>Vessel dilatator D-5a</td>
			<td>S&#38;T</td>
			<td>S-00124</td>
			<td></td>
		</tr>
		<tr>
			<td>Adventita scissor</td>
			<td>S&#38;T</td>
			<td>S-00102</td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular clamp B-1V</td>
			<td>S&#38;T</td>
			<td>S-00396</td>
			<td></td>
		</tr>
		<tr>
			<td>Yasargil Clip</td>
			<td>Peter Lazic</td>
			<td>65,097</td>
			<td>3.5 mm lenght</td>
		</tr>
		<tr>
			<td>Bipolar forceps</td>
			<td>Micromed</td>
			<td>148-100-011</td>
			<td>tip 0.25 mm</td>
		</tr>
		<tr>
			<td>Polyimide tubes</td>
			<td>River Tech Medical</td>
			<td></td>
			<td>19-24 gauge</td>
		</tr>
		<tr>
			<td>8.0 silk ligatures</td>
			<td>Catgut</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Custodiol HTK solution</td>
			<td>Essential Pharmaceuticals</td>
			<td></td>
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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.

Nuestro equipo podría realizar con éxito la técnica descrita después de aproximadamente 20 cirugías de formación. La parte más difícil es eversión la pared vascular de la arteria carótida sobre el brazalete, ya que fácilmente voltea hacia atrás y múltiples intentos puede conducir a la rotura de la pared. Después del período de entrenamiento, el tiempo quirúrgico total media fue de 60 ± 8 min. Se requieren alrededor de 20 minutos para la cosecha de donantes, 25 min para la preparación de los vasos cervi...

Watch this Scientific Journal Video about Heterotopic Cervical Heart Transplantation in Mice at JoVE.com

Heterotopic Cervical Heart Transplantation in Mice

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 ...

heterotopic-cervical-heart-transplantation-in-mice

Research

JoVE Journal

Medicine

11.1K vistas.  German Heart Center Munich, Technische Universität München. 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.

Video: Heterotopic Cervical Heart Transplantation in Mice

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

Controlado lesiones laceración cervical en ratones

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

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 ...

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 ...

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 ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

Cateterismo cardíaco en ratones para medir la relación de volumen Presión: Investigar el efecto Bowditch

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

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 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 ...