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><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>Micro-dissecting scissors, straight, sharp, long</td><td></td><td>11.602.11</td><td></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&amp;rdquo; x 6 &amp;frac12;&amp;rdquo; x &amp;frac34;&amp;rdquo;</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>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>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>Syringes</td><td></td><td>Cotton applicators</td><td></td></tr><tr><td>B-D 1cc insulin, #329424</td><td></td><td>Fisher-brand, #23-400-100</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>4/0 Cotton ties</td><td></td><td></td><td></td></tr><tr><td>Domestic cotton autoclaved with instruments</td><td></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...

Watch this Scientific Journal Video about Murine Kidney Transplant Technique at JoVE.com

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

16.0K 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 Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Procedimiento murino Trasplante Renal

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Biología

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

Aislamiento y Trasplante de diferentes Aged murinos tímicos injertos.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Inmunología e Infección

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Ratón Trasplante de Riñón: Modelos de rechazo del aloinjerto

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

El destino de la asignación de células estaminales embrionarias humanas mediante la formación de teratoma

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

Heterotópico renal autotrasplante en un modelo porcino: Un Protocolo de Paso a Paso

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

Production of Humanized Mouse via Thymic Renal Capsule Grafting, CD34+ Cells Injection, and Cytokine Delivery

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation of human systems, thereby allowing for a more accurate study of pathogenesis, biomarkers, and many other scientific queries. In this method described, immune-deficient NOD-scid IL2R&#947;null (NSG) mice are implanted with autologous thymus, injected with liver-derived CD34+ cells followed by a series of injected cytokine deliveries. In contrast to other models of a similar nature, the model described here promotes an improved reconstitution of immune cells by delivering cytokines and growth factors via transgenes encoded in AAV8 or pMV101 DNA-based vectors. Moreover, it offers long-term stability with reconstituted mice having an average lifespan of 30 weeks after CD34+ injections. Through this model, we hope to provide a stable and impactful method of studying immunotherapy and human disease in a murine model, thus demonstrating the need for predictive preclinical models.

<meta charset="utf8"></head><body>Producción de ratón humanizado mediante injerto de cápsula renal tímica, inyección de células CD34+ y administración de citocinas

Humanized mouse models provide a more accurate representation of the human immune microenvironment. This manuscript describes the process in which these models are created through a renal graft of human thymus, injection of human CD34+ cells, and the targeted delivery of human cytokine transgenes to promote CD34+ cell proliferation and differentiation.

Producción de ratón humanizado mediante injerto de cápsula renal tímica, inyección de células CD34+ y administración de citocinas

Animal models provide a vital translation between in vitro and in vivo biomedical research. Humanized mouse models provide a bridge in the representation ...

A Modified Surgical Technique for Kidney Transplantation in Mice

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this operation. Therefore, this article introduces the technique and points out the surgical caveats associated with this operation. In addition, important modifications in comparison to the conventional procedure are demonstrated. Firstly, a patch of the abdominal aorta is cut and prepared so that the proximal bifurcations of the renal artery, including the ureteral artery are transected together with the donor kidney en bloc. This reduces the risk of a ureter necrosis and avoids the development of a urinary tract occlusion. Secondly, a new method of the vascular anastomosis is demonstrated that allows the operator to flexibly increase or decrease the size of the anastomosis after renal transplant reperfusion has already been initiated. This avoids the development of vessel strictures and intraabdominal bleeding. Thirdly, a technique that enables the anastomosis of the delicate donor ureter and the recipient bladder that does not cause a trauma is shown. Adopting this protocol can shorten the operation time and reduces the damage to the recipient's bladder, thereby significantly increasing the operation success rate for the recipient mice.

This protocol presents a new surgical technique of mouse kidney transplantation focusing on a modified arterial anastomosis strategy. A vascular suture technique including a simple and safer ureter-bladder anastomosis method is also presented. These modifications shorten the operation time and improve the success rate of the mouse kidney transplantation procedure.

Una técnica quirúrgica modificada para el trasplante de riñón en ratones

Kidney transplantation in mice is a complicated and challenging surgery procedure. There are very few publications demonstrating the key steps of this ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of renal replacement therapy. Over the past few decades, the rat kidney transplantation model has been used to study the immunological phenomena of rejection and tolerance. This model has become an indispensable tool to test new immunomodulatory pharmaceuticals and regimens prior to proceeding with expensive preclinical large animal studies.
This protocol provides a detailed overview of how to reliably perform orthotopic kidney transplantation in rats. This protocol includes three distinctive steps that increase the probability of success: perfusion of the donor kidney by flushing through the portal vein and the use of a cuff system to anastomose the renal veins and ureters, thereby decreasing cold and warm ischemia times. Using this technique, we have achieved survival rates beyond 6 months with normal serum creatinine in animals with syngeneic or tolerant kidney transplants. Depending on the aim of the study, this model can be modified by pre- or posttransplant treatments to study the acute, chronic, cellular, or antibody-mediated rejection. It is a reproducible, reliable, and cost-effective animal model to study different aspects of kidney transplantation.

The purpose of this manuscript and protocol is to explain and demonstrate in detail the surgical procedure of orthotopic kidney transplantation in rats. This method is simplified to achieve the correct perfusion of the donor kidney and shorten the reperfusion time by using the venous and ureteral cuff anastomosis technique.

Trasplante de riñón de rata ortotópica: un enfoque quirúrgico novedoso y simplificado

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...