This work was supported in part by 1R03DK096151.&#160;We acknowledge the UAB-UCSD O&#8217;Brien Center (NIH P30 DK079337) for providing initial technical assistance with setting up this model.

Todos los ratones fueron adquiridos de El Laboratorio Jackson (Bar Harbor, ME) y se mantuvieron en condiciones libres de patógenos en la Universidad de Colorado en Denver, Fondo Barbara Davis Centro Animal de acuerdo con las directrices del NIH y con la aprobación de la Universidad de Colorado en Denver IACUC. 1. donante de riñón Cosecha <ol><li> Esterilizar todos los instrumentos, usar guantes estériles durante todo el procedimiento y mantener un campo estéril. Realizar todas las cirugías con el uso de un microscopio operativo. </li><li> Quitar quirúrgicamente riñones de donantes de ratón a partir de (kg 60mg / IP) donantes anestesiado con pentobarbital. Determinar la profundidad de la anestesia por toe-pizca, y observar la frecuencia respiratoria. </li><li> Clip de la piel luego inmovilizar el ratón por 4 vías restricciones. Preparar la piel con povidona yodada y cubra el ratón de forma estéril. </li><li> Haga una incisión abdominal vertical de 2 cm de la línea media y entrar en la cavidad abdominal. Retraer el intestino superior y exteriorizarloen el pecho. Mantenga el intestino envuelto en una gasa húmeda estéril durante todo el procedimiento. </li><li> Identificar los grandes vasos abdominales y movilizarlos, identificar las ramas lumbares y cauterizar o ligar con 10/0 sutura de nylon. </li><li> Inmediatamente distal a los vasos renales izquierdos burlan de la vena cava inferior (VCI) y la aorta abdominal (AA) de separación mediante disección roma largo de aproximadamente 2 mm. Ligar con nylon 10/0 y dividir pequeña arteriales y venosos ramas de los vasos renales. </li><li> Ahora separar la vena renal de la arteria renal cuidadosamente por disección roma de estas estructuras. Esto permite la creación precisa de un parche de Carrel en el extremo proximal de la vena renal, que se utiliza para formar una anastomosis de extremo a lado a los destinatarios IVC. </li><li> Identificar, ligar y dividir los vasos suprarrenal izquierda. Esto permite el acceso a la aorta suprarrenal y una sutura de seda 6-0 se coloca alrededor de la aorta en la preparación para la ligación, pero no ligado en este momento. </li&gt; <li> Movilizar el riñón, los vasos y el uréter desde el salpicadero de los alrededores. Gire el riñón a la derecha y ligar / dividir las ramas posteriores. Luego devuelva el riñón a la izquierda. </li><li> Ahora dirigir la atención hacia el uréter. Sin perturbar el hilio renal liberar el uréter del salpicadero cuidando circundante para preservar los vasos ureterales. Divida el uréter a nivel de los conductos deferentes. El riñón ya está lista para la recuperación. </li><li> Inyecte lentamente 300 unidades de heparina en la vena cava inferior distal heparinización así el donante. Amarre la sutura de seda 6-0 colocado alrededor de la AA suprarrenal y perfundir el riñón izquierdo a través de la aorta abdominal distal con 0,8 ml de solución salina de heparina (100 U / ml). </li><li> Una vez que la perfusión ha dejado de crear un parche venoso Carrel inmediatamente para eliminar el reflujo en el riñón. Retraer la vena renal hacia el riñón revelando la AA y la arteria renal debajo. </li><li> Divida la aorta adyacente a la arteria renal y la creación de un talón ae manguito como se muestra en la Figura 1. extraer el riñón, los vasos y el uréter del donante. </li><li> Extraer el riñón directamente a un receptor pre-preparado (0 min tiempo de isquemia fría) o se almacena a 4 ° C en una solución de elección para un tiempo predeterminado hasta el momento de la implantación. Sacrificados los donantes por exsanguanation y dislocación cervical. El tiempo total para recuperar riñón del donante es de aprox. 15-20 min. </li></ol> 2. Riñón Implante Técnica <ol><li> Anestesiar el ratón receptor con pentobarbital (60 mg / kg dosis inicial IP, 25mg dosis suplementaria / kg IP si es necesario). Determinar la profundidad de la anestesia por toe-pizca, y observar la frecuencia respiratoria. Clip de la piel luego inmovilizar el ratón por 4 vías restricciones y aplicar pomada oftálmica para los ojos. Preparar la piel con povidona yodada y cubra el ratón de forma estéril. </li><li> Haga una incisión abdominal vertical de 2 cm de la línea media y entrar en la cavidad abdominal. Retraer el intestinosuperiormente y exteriorizar en el pecho. Mantenga el intestino envuelto en una gasa húmeda estéril durante todo el procedimiento. </li><li> En este momento realizar una nefrectomía derecha. Ligar la arteria renal y la vena con suturas de seda 6-0. Extirpar el riñón distal a las suturas. Ligar el uréter con seda 6-0 y luego dividir proximal al riñón. </li><li> Aislar la aorta abdominal y la vena cava inferior (IVC) por debajo de los vasos renales. Coloca 4-0 lazos de algodón alrededor de la aorta y vena cava inferior superior, luego inferior al sitio de anastomosis. Identificar y ligar los vasos lumbares en el campo con 10-0 sutura de nylon. </li><li> Nudo de los lazos de algodón, primero el inferior seguido por el superior. De esta manera algo de sangre se retiene en la aorta haciendo que la aortotomía más fácil. </li><li> Forma la aortotomía con una aguja 30G para entrar en el lumen de la aorta. Extender la incisión con tijeras finas micro a una longitud de aproximadamente 2 mm. </li><li> Hacer fin a la anastomosis lateral del manguito aórtica donante punta tacón ala aorta destinatario de la siguiente manera. Coloque un nylon 10-0 estancia sutura puntada en la aorta del donante y para el ángulo inferior de la incisión en la aorta destinatario y corbata. </li><li> Coloque una segunda nylon 10-0 frente a la primera en la aorta del donante y la esquina superior de la incisión en la aorta abdominal y corbata. Hacer una línea de sutura continua de superior a inferior en la pared lateral de la aorta y corbata en contra de la estancia puntada colocado previamente. Entonces suturar la pared medial de manera corriente y corbata. </li><li> Hacer fin a la anastomosis lateral de la vena renal donante al receptor IVC de la siguiente manera. Perforar el IVC con una aguja 30G y extender la incisión con tijeras finas micro. Ate la vena renal del donante a la esquina inferior de la incisión en la vena cava inferior con nylon 10-0. Hacer una línea de sutura continua entre la vena renal y la vena cava inferior y corbata. NOTA: También es de vital importancia que todo el espesor pasa de la aguja de sutura incluyendo la adventit vascularIA y la íntima se logran. La eversión de los bordes también se asegura de que haya contacto-íntima a la íntima, que ayuda en el sellado y la cicatrización de las anastomosis. Mientras que un agente de coagulación hemostática puede ser útil para reducir las fugas, se recomienda que un cirujano en su lugar confiar en una buena técnica. </li><li> Asegúrese de que las anastomosis son &quot;limpia&quot;. Es decir, que las paredes opuestas no queden atrapados al colocar puntos de sutura. Esto hará que una constricción significativa para fluir que se traducirá en un injerto fracasado y en casos extremos a la parálisis de las extremidades posteriores. NOTA: Otro factor muy importante es asegurarse de que la tensión de las líneas de sutura anastomótica también es óptima. Demasiado flojo y no habrá fugas irreversible, demasiado apretado y estenosis a fluir resultará. Si en el lado arterial esto dará como resultado una pobre perfusión del injerto, si es en el lado venoso dará como resultado un riñón congestionado. </li><li> Suelte el flujo venoso 4-0 lazo de algodón restablecer distal. Una vez hemostasis del venonos anastomosis se ha observado gradualmente aflojar proximal el empate 4-0 de algodón y atienda la anastomosis arterial para la hemostasia. </li><li> Retire los lazos de algodón del ratón una vez que ambas anastomosis se consideran seguros. Pierce la vejiga con una aguja 20 G creando dos agujeros. </li><li> Pasar la punta de pinzas curvas través de los orificios y tire del uréter donante a través de la vejiga con el fórceps con el extremo proximal del uréter estar anclado a la pared de la vejiga con dos suturas de nylon de 10-0. Recorte el exceso de longitud del uréter que sobresale del segundo orificio que permite el uréter se retraiga dentro de la vejiga y cerrar el agujero con dos suturas de nylon 10-0. </li><li> Devuelva el intestino en el abdomen. Cierre de la pared abdominal en dos capas usando sutura de seda de 5-0 de una manera corriente. </li><li> Administrar un bolo de 1,0 ml, cálido solución salina normal estéril en el abdomen como la reanimación con líquidos al cierre, e inyectar 0,8 ml de solución salina normal por vía subcutánea después de la operación. Ningún otro supportiSe requieren medidas VE durante la cirugía. </li><li> Recuperar el animal en una manta térmica. Tiempo de implante total es de aprox. De 35-45 min. Administrar analgésicos tales como buprenorfina, 0,05 mg / kg, SC, 0,1-0,2 ml al comienzo del procedimiento y cada 6-12 hr durante 72 h post-op. </li></ol> 3. contralateral Nefrectomía <ol><li> Dependiendo de los requisitos de protocolo, varios días después del procedimiento de implante, llevan a cabo una nefrectomía contralateral bajo anestesia con isoflurano (5% de isoflurano inhalado para la inducción, 1.5-2.0% para mantenimiento). </li><li> Introduzca la cavidad abdominal y retraer con suavidad el intestino a la derecha del animal. Exponer el riñón izquierdo y la disección roma de la fascia de los alrededores. </li><li> Ligar la arteria renal, vena y uréter con suturas de seda 6-0 y luego escindir el riñón por encima de las suturas y extraer el riñón. Tiempo operatorio total es de aprox. 10 minutos. </li></ol> 4. Evaluación del injerto <ol><li> Medida creatinina séricay nueve utilizando el método de picrato alcalino (reacción de Jaffe). </li></ol>

<ol>
	<li>Skoskiewicz, M., Chase, C., 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=Kidney+transplants+between+mice+of+graded+immunogenetic+diversity.">Kidney transplants between mice of graded immunogenetic diversity.</a> Transplant Proc. 5 (1), 721-725 (1973).</li><li>Kalina, S. L., Mottram, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microsurgical+technique+for+renal+transplantation+in+mice.">A microsurgical technique for renal transplantation in mice.</a> Aust N Z J Surg. 63 (3), 213-216 (1993).</li><li>Han, W. R., Murray-Segal, L. J., Mottram, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+technique+for+kidney+transplantation+in+mice.">Modified technique for kidney transplantation in mice.</a> Microsurgery. 19 (6), 272-274 (1999).</li><li>Ge, F., Gong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+successfully+establishing+a+kidney+transplant+in+a+mouse+model.">Strategies for successfully establishing a kidney transplant in a mouse model.</a> Exp Clin Transplant. 9 (5), 287-294 (2011).</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=Learning+curve,+surgical+results+and+operative+complications+for+kidney+transplantation+in+mice.">Learning curve, surgical results and operative complications for kidney transplantation in mice.</a> Microsurgery. 26 (8), 590-593 (2006).</li><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=Primarily+vascularized+allografts+of+hearts+in+mice.+The+role+of+H-2D,+H-2K,+and+non-H-2+antigens+in+rejection.">Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection.</a> Transplantation. 16 (4), 343-350 (1973).</li><li>Zhang, Z., Chen, H., Qian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+Transplantation+in+Mice.">Kidney Transplantation in Mice.</a> Experimental Organ Transplantation. , 45-64 (2013).</li><li>Zhang, L., Moskovitz, M., Piscatelli, S., Longaker, M. T., Siebert, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+study+of+different+angled+end-to-side+anastomoses.">Hemodynamic study of different angled end-to-side anastomoses.</a> Microsurgery. 16 (2), 114-117 (1995).</li><li>Liu, Q., Mirc, D., Fu, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+mechanisms+of+thrombosis+in+intact+bent+microvessels+of+rat+mesentery.">Mechanical mechanisms of thrombosis in intact bent microvessels of rat mesentery.</a> J Biomech. 41 (12), 2726-2734 (2008).</li><li>Chesnutt, J. K., Han, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tortuosity+triggers+platelet+activation+and+thrombus+formation+in+microvessels.">Tortuosity triggers platelet activation and thrombus formation in microvessels.</a> J Biomech Eng. 133 (12), 121004 (2011).</li><li>Han, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessel+buckling+within+soft+surrounding+tissue+generates+tortuosity.">Blood vessel buckling within soft surrounding tissue generates tortuosity.</a> J Biomech. 42 (16), 2797-2801 (2009).</li><li>Gutierrez-Diaz, J. 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=Intraluminal+thrombus+and+neointimal+hyperplasia+after+microvascular+surgery.">Intraluminal thrombus and neointimal hyperplasia after microvascular surgery.</a> Surg Neurol. 24 (2), 153-159 (1985).</li><li>Khouri, R. K., Cooley, B. C., Kenna, D. M., Edstrom, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombosis+of+microvascular+anastomoses+in+traumatized+vessels:+fibrin+versus+platelets.">Thrombosis of microvascular anastomoses in traumatized vessels: fibrin versus platelets.</a> Plast Reconstr Surg. 86 (1), 110-117 (1990).</li><li>Johnson, P. C., 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=Initial+platelet+deposition+at+the+human+microvascular+anastomosis:+effect+on+downstream+platelet+deposition+to+intact+and+injured+vessels.">Initial platelet deposition at the human microvascular anastomosis: effect on downstream platelet deposition to intact and injured vessels.</a> Plast Reconstr Surg. 90 (4), 650-658 (1992).</li></ol>

The authors have nothing to disclose.

Mastering this transplant technique is difficult, but once accomplished it is a very powerful research tool. The patient surgeon/researcher will be rewarded by attention to detail and consistency of technique, which is the key to mastering any surgical procedure, even more so in small animal models. The technical difficulties of mastering the mouse kidney transplant are many folds, and it is highly probable that experience in other small animal transplant models must be gained before tackling this procedure.
<p class...

Desde 1973 el modelo de trasplante de riñón en ratones ha sido una valiosa herramienta de investigación, pero los problemas técnicos han impedido su uso generalizado. A través de los años, varios trabajos han sido publicados detallando mejoras / mejoras a este procedimiento. Como modelo de trasplante de órganos sólidos principalmente vascularizado este procedimiento es, probablemente, sólo superada por el modelo de trasplante heterotópico de corazón que también fue ideado por el laboratorio Russell en 1973 6. Ambos modelos se prestan a la investigación de las respuestas de rechazo alogénico, el desarrollo de la función retardada del injerto y la lesión por reperfusión de la isquemia. Uno de los problemas más comunes que se informaron con el trasplante de riñón es la relativamente alta incidencia de trombosis arterial 4,5,7 que también hemos experimentado en nuestro laboratorio. Por lo tanto, nos propusimos realizar una revisión de la literatura de la formación de trombos y posiblemente encontrar la causa de este problema técnico y elaborar también unsolución posible. La causa más probable de la trombosis es el camino algo tortuoso toma la sangre desde la aorta destinatario, en la aorta renal donante luego a la arteria renal del donante. Este camino provoca turbulencia en la arteria renal que puede conducir a la activación plaquetaria y la formación de trombos. Sobre la base de las recientes observaciones y una búsqueda de la literatura relevante 8-14 se nos ocurrió una nueva técnica que ha reducido la trombosis a 0%. La técnica descrita aquí varía de técnicas previamente reportados en la formación de un manguito de punta tacón arterial que las instalaciones mejoraron el flujo sanguíneo y reduce significativamente la formación de trombos. El manguito se forma dividiendo la aorta infra-renal través de la cara del ostium arterial renal en un ángulo de menos de 45 o al eje longitudinal de la aorta (Figura 1A y 1B). Esto se traduce en un manguito de aproximadamente 2 mm de longitud. Un parche de Carrel venosa está formado por transección de la rvena enal en el IVC aumentando así el diámetro del manguito. El donante abdominal aorta manguito punta tacón infra-renal es de extremo a lado anastomosa a la aorta abdominal destinatario y la vena renal del donante / parche IVC es de extremo a lado anastomosa al destinatario abdominal vena cava inferior (VCI) . El uréter se introduce entonces en y anclado a la vejiga como se ha descrito por Han et al 3. Para este estudio los trasplantes no tratados con sólo tiempos de isquemia caliente (es decir., Sin isquemia fría) se comparan. En este caso de isquemia caliente se refiere al tiempo desde el cese del flujo de sangre a través del riñón del donante (paso 1.11 a continuación) y la reperfusión del injerto en el receptor (paso 2.11 a continuación). Isquemia fría se refiere al tiempo que el riñón no se perfunde y se mantiene en almacenamiento en frío hasta el comienzo del procedimiento de implante.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Instrument</td>
			<td>Roboz #</td>
			<td>Fine Science Tools #</td>
			<td>Arosurgical #</td>
		</tr>
		<tr>
			<td>Straight micro-dissecting forcep #5</td>
			<td>RS-5015</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved micro-dissecting forcep #7</td>
			<td>RS-5047</td>
			<td>11297-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved serrated forcep</td>
			<td>RS-5137</td>
			<td>11052-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas micro-dissecting scissors, short</td>
			<td>RS-5610</td>
			<td></td>
			<td>09.140.08</td>
		</tr>
		<tr>
			<td colspan="2">Micro-dissecting scissors, straight, sharp, long</td>
			<td></td>
			<td>11.602.11</td>
		</tr>
		<tr>
			<td>Micro spring handle needle holder</td>
			<td></td>
			<td></td>
			<td>11.549.15</td>
		</tr>
		<tr>
			<td>Straight mosquito forcep</td>
			<td></td>
			<td>91308-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors, straight, blunt</td>
			<td>RS-5962</td>
			<td>14078-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors, curved, blunt</td>
			<td>RS-5981</td>
			<td>14079-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro retractor</td>
			<td>RS-6540</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Instrument tray, 10&#8221; x 6 &#189;&#8221; x &#190;&#8221;</td>
			<td>RT-1350S</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silk suture, 5/0, 22.5m spool</td>
			<td></td>
			<td>18020-50</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10/0 nylon</td>
			<td></td>
			<td></td>
			<td>T4A10Q07</td>
		</tr>
		<tr>
			<td>5/0 silk</td>
			<td></td>
			<td></td>
			<td>E19A05N</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td></td>
			<td>Drapes</td>
			<td></td>
		</tr>
		<tr>
			<td>Biogel from Medex Supply</td>
			<td></td>
			<td>Precept, #64-9012-9</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td></td>
			<td>Cotton applicators</td>
			<td></td>
		</tr>
		<tr>
			<td>B-D 1cc insulin, #329424</td>
			<td></td>
			<td colspan="2">Fisher-brand, #23-400-100</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-Iodine swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PDI, #B40600</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4/0 Cotton ties</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Domestic cotton autoclaved with instruments</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

murine kidney transplant technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. 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 using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

Esta técnica quirúrgica permite, ya sea para estudios de supervivencia / rechazo del injerto simples, o protocolos experimentales bastante complejas. En las figuras siguientes se demuestra las ventajas de utilizar esta técnica anastomosis arterial mejorada. Usando esta técnica, hemos reducido significativamente la incidencia de la trombosis arterial de 35% a 0% aumentando así la productividad. Hemos utilizado esta técnica durante más de un año con el mismo 0% Resultado trombosis mantenido. La figura 1 de...

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Murine Kidney Transplant Technique

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

Riñón murino Trasplante Técnica

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Research

JoVE Journal

Medicine

15.8K vistas.  University of Colorado, Denver. The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis. 

Video: Murine Kidney Transplant Technique

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Un método para el aislamiento de los islotes murinos y Trasplante Renal subcapsular

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

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 ...

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

Técnica de Subnormothermic<em&gt; Ex Vivo</em&gt; Perfusión hepática para el Almacenamiento, Evaluación y Reparación de marginales injertos hepáticos

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

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 ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

Técnica de Adquisiciones de hígado porcino y trasplante ortotópico usando un Active Porto-Caval derivación

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

Modelo de ratón de rechazo vascular inducida por aloinmune y Arteriosclerosis Trasplante

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

Normotérmica<em&gt; Ex Vivo</em&gt; Perfusión renal para la Preservación de riñón Injertos antes del trasplante

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

An &#8220;All-laser&#8221; Endothelial Transplant

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an intended depth, and a near infrared diode laser to weld the corneal tissue. The proposed technique enables to reach the three main goals in endothelial keratoplasty: a precise control in the thickness of the donor tissue; its easy insertion in the recipient bed and a reduced risk of donor lenticule dislocation. The donor cornea thickness is measured in the surgery room with optical coherence tomography (OCT), in order to correctly design the donor tissue dimensions. A femtosecond laser is used to cut the donor cornea. The recipient eye is prepared by manual stripping of the descemetic membrane. The donor endothelium is inserted into a Busin-injector, the peripheral inner side is stained with a proper chromophore (a water solution of Indocyanine Green) and then it is pulled in the anterior chamber. The transplanted tissue is placed in the final and correct location and then diode laser welding is induced from outside the eyeball. The procedure has been performed on more than 15 patients evidencing an improvement in surgery performances, with a good recovery of visual acuity and a reduced donor lenticule dislocation event.

An &amp;#8220;All-laser&amp;#8221; Endothelial Transplant

An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

Un &quot;All-láser&quot; Trasplante endotelial

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Modelo murino de trasplante de páncreas mediante una técnica modificada del pun ¢ o

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

Modelo de trasplante de aórtica cervical murino utilizando una técnica de manguito no sutura modificada

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...