Name: mouse kidney transplantation: models of allograft rejection
Uploaded: 2014-10-11T15:00:00.000+00:00
Duration: 16 min 15 s
Description: Watch this Scientific Journal Video about Mouse Kidney Transplantation: Models of Allograft Rejection at JoVE.com

La financiación procedente del riñón Research UK, el Colegio Real de Cirujanos de Edimburgo y de la Sociedad Europea de Trasplante de Órganos apoyó este estudio.

Ética institucionales nacionales y locales apropiados deben estar en su lugar antes de realizar los experimentos con animales. Específicamente en el Reino Unido se llevaron a cabo los siguientes experimentos bajo los Animales (Procedimientos Científicos) de la Ley de 1986 Cuando dos microcirujanos están disponibles para funcionar a la vez que el cirujano donante debe realizar los pasos 1.1 a 1.16 luego 3.1 a 3.5, mientras que el cirujano realiza receptor 2.1 a 2.8 . Para un solo operador los pasos pueden ser seguidos secuencialmente. 1. Donantes Preparación Nota: El procedimiento que aquí se presenta es un donante C57BL / 6 BM12 y el receptor C57BL / 6 ratones machos de edades comprendidas entre 8 y 16 semanas de edad con un peso corporal superior a 20 g. Sin embargo, este procedimiento se puede realizar de forma reproducible en una variedad de cepas de ratón. Los datos presentados en la sección de resultados representativos se obtuvieron de ratones C57BL / 6, C57BL / 6 y BM12, los cuales ratones BALB / c. <ol><li> Realizar procedimientos using instrumentos quirúrgicos estériles y consumibles (autoclave), con los esfuerzos para mantener la zona de trabajo lo más estéril posible. Realizar la preparación de donantes simultáneamente con la preparación destinatario si dos cirujanos están disponibles. </li><li> Anestesiar al ratón con una inyección intraperitoneal (aguja 31G) de medetomidina (0,5 mg / kg) y clorhidrato de ketamina (200 mg / kg). Esto resulta en un plano anestésico que se mantiene durante 4 horas dando tiempo suficiente para que todo el procedimiento a realizar. Anestesia suplementaria no es necesario. </li><li> Confirme que el ratón está anestesiado (sin respuesta a la pizca dedo del pie). </li><li> Afeitarse el abdomen del ratón y eliminar los pelos sueltos con cinta adhesiva. </li><li> Coloca el ratón sobre su espalda sobre una estera climatizada cubierta estéril y sin apretar inmovilizar las extremidades con cinta adhesiva estéril. </li><li> Monitorear el ratón durante todo el procedimiento para las quemaduras térmicas. Si es posible utilizar una fuente de calor no eléctrico. </li><li> Aplique un lubricante ocular y sanitize la pared abdominal con una solución de yodo diluido. </li><li> Hacer una incisión en el abdomen para entrar en la cavidad peritoneal e inserte un 3 cm Calibri retractor abdominal. </li><li> Aplicar solución salina calentada para mantener los intestinos y el área quirúrgica húmedo y evitar el secado innecesaria de las vísceras. </li><li> Cubra el ratón con paños estériles y mover los intestinos a la izquierda del operador (el derecho del ratón) para exponer la aorta, la vena cava y el riñón izquierdo. </li><li> Aplicar una pinza de trinquete hasta el estómago y tire superiormente para exponer los principales vasos y riñón izquierdo totalmente. Empaque amortiguar hisopos estériles (2 mm x 2 mm) en el abdomen para retraer los tejidos fuera de la zona de la cirugía, tales como los lóbulos hepáticos, vesículas seminales y de intestino. </li><li> Aislar el riñón izquierdo de adventicia que rodea, la grasa y la glándula suprarrenal izquierda en la cavidad peritoneal por rodeos disección del tejido conectivo usando pinzas de punta fina. Coloque las puntas cerradas de la pinza entre las áreas que NEed a separar y lentamente permita que las puntas de fórceps para abrir disecar el espacio. </li><li> Aislar la vena renal izquierda ligando y luego dividiendo la vena adrenal y la vena gonadal izquierda izquierda con 9 / O de nylon. Coloque la sutura cerca de la vena renal. </li><li> Ligar y dividir el uréter con 7 / O sutura de seda cerca de la vejiga dejando la sutura termina de largo. Estos extremos largo de sutura se utilizarán cuando el riñón se cosecha y que sean necesarias para la anastomosis ureteral. </li><li> Movilizar y diseccionar plenamente la aorta y la vena cava superior y inferior a la arteria y vena renal utilizar fórceps de punta fina para diseccionar sin rodeos vasos linfáticos y grasa de la parte frontal y los lados de los vasos. </li><li> Encuentre el plano del tejido entre la aorta y la vena cava y propagarse lentamente las pinzas. Abra lentamente las puntas de la pinza de punta fina para extender el tejido con un mínimo trauma. </li><li> Ate un flojo 7 / O de seda alrededor de la aorta superior e inferior de la arteria renal pasando un buen ángulopinzas de punta alrededor de la parte posterior de los vasos y dibujo una sutura a través. Estas suturas se harán más estrictas antes de la recuperación del riñón para permitir la perfusión retrógrada de la Universidad de Wisconsin solución. </li></ol> 2. destinatario Preparación De acuerdo con el seguimiento de preparación de donantes pasos 1.2 a 1.8. <ol><li> Mover los intestinos a la derecha del operador (izquierda del ratón) para exponer la aorta, la vena cava. Cubra los intestinos con un bastoncillo empapado de solución salina estéril. </li><li> Realizar una nefrectomía derecha por ligación de la arteria renal derecha y la vena junto con 7/0 sutura de seda y luego dividir. </li><li> Ligar el uréter derecho con 7 / O sutura de seda y se dividen. </li><li> Movilizar y diseccionar plenamente la aorta y la vena cava inferior a la arteria y la vena renal como se describe en el paso 1.14 y 1.15. Asegurar la disección completa entre la aorta y la vena cava. Tenga cuidado para preservar la arteria espermática interna que se extiende por delante de la vena cava y aortA junto con el paquete linfático. </li><li> Identificar los vasos lumbares que se ejecutan en el retroperitoneo de la vena cava y la vena a la columna vertebral, se liga con 9 / O de seda en la continuidad, no hay necesidad de dividir los vasos. </li><li> Identificar suficiente espacio para colocar pinzas microvasculares con espacio para entre anastomosis vasculares. </li><li> Administrar heparina intravenosa (5 unidades) a través de la vena dorsal del pene. </li></ol> Recuperación de riñón 3. Donantes Apriete los lazos de seda 7 / S que se han colocado alrededor de la aorta inferior y superior para aislar el riñón de la circulación arterial. <ol><li> Infundir retrógradas frío desde 0,2 hasta 0,5 ml de solución de la Universidad de Wisconsin con una aguja (31G) en la aorta. </li><li> Divida la aorta dentro de las suturas y dividir la vena renal en su unión con la vena cava para extraer el riñón y la arteria renal con una longitud de aorta. Divida vasos lumbares derivados de la aorta, si están presentes, wligadura ithout. </li><li> Coloque el riñón en solución salina fría en un hisopo estéril en una placa de cultivo. </li><li> La eutanasia el ratón donante por dislocación cervical. </li></ol> 4. Trasplante de Riñón - Kidney Preparación <ol><li> Crear un parche aórtico dividiendo la pared de la aorta longitudinalmente justo enfrente de la arteria renal. Identificar los lúmenes de los vasos en el parche que necesitan ser ligado o evitar cuando se realiza la anastomosis arterial. </li><li> Colocar una sutura 10 de E / S del exterior al interior del lumen de la vena renal y superiormente una segunda sutura separada inferior. Las suturas utilizadas para dividir la vena adrenal y la vena gonadal se pueden utilizar para orientar la embarcación. </li><li> Coloque el riñón en el flanco derecho del receptor (Figura 1.1) y asegurar las suturas están en condiciones de garantizar las anastomosis vasculares se pueden completar sin las suturas se entrelaza en hisopos u otros instrumentos. </li></ol> 5. riñón Transplantación - Vascular Anastomosis Aplicar pinzas microvasculares primera inferiormente a continuación abarcan superiormente la vena cava y la aorta. <ol><li> Hacer un ventomy con una aguja (31G) por punción de la pared anterior de la vena cava. Flush sangre de la vena cava mediante la inyección de aproximadamente 50 l de 0,9% de NaCl. </li><li> Ampliar el venotomía usando fórceps de punta fina mediante la apertura de ellos dentro de la venotomía manera que la longitud es equivalente al diámetro de la vena renal del donante. </li><li> Coloque la sutura superiores 10 de E / S (que ya está en el lumen de la vena renal) primero en el vértice de la venotomía (Figura 1.2) y unirse a la pared posterior de la anastomosis en una sutura continua hasta que se alcanza el ápice inferior (figura 1.3). </li><li> Coloque el 10 de E / S de sutura inferior y atar primero con un solo nudo (Figura 1.4) luego atar a la sutura que va desde la pared trasera. </li><li> Uso de la sutura inferior crear el Wal frentel de la anastomosis (Figura 1.5) con una sutura continua y atar al extremo de sutura superior en el vértice superior, (Figura 1.6). </li><li> Crear una aortotomía recogiendo la pared de la aorta con una pinza y corte un parche elíptico con tijeras (aproximadamente el aortotomía debe ser una quinta parte de la circunferencia de la aorta y tres veces la luz de la arteria renal de longitud). </li><li> Coloque una sutura de 10 E / S en el punto superior del parche aórtico del exterior al interior (Figura 1.7) y pasar a través de la aorta para empatar fuera de los vasos en el vértice superior de la aortotomía. </li><li> Cree la anastomosis arterial con una sutura continua de comenzar superiormente (Figura 1.8) anastomosando el parche de donantes a la aorta destinatario, tenga cuidado de no constreñir la anastomosis mediante la vinculación de la sutura demasiado apretado (Figura 1.9). </li><li> Retire las abrazaderas vasculares inferiores primero y luego la pinza superior a reperfundir el riñón (Figura 1.10 y 1.11). Peristaltismo visible del uréter tal vez visto si se logra el suministro adecuado de sangre al uréter. </li></ol> 6. Trasplante de Riñón - Anastomosis ureteral <ol><li> Divida los archivos adjuntos de la vejiga de la pared abdominal. </li><li> Pasar una aguja (21G) de izquierda a derecha de la vejiga a través de ambas paredes. </li><li> Coloque puntas de fórceps rectos en el lumen de la aguja y pasar tanto a través de la vejiga de tal manera que las pinzas están pasando ahora de derecha a izquierda de la parte izquierda de la vejiga. </li><li> Dibujar la sutura en el uréter donante a través de la vejiga de tal manera que el uréter pasa en el defecto de la vejiga izquierda y luego fuera del lado derecho. </li><li> Suturar la adventicia del uréter a la adventicia de la vejiga con tres suturas interrumpidas simples alrededor del punto de entrada con 9 / O sutura de nylon en una aguja de cuerpo redondo. </li><li> Cortar el uréter proximal a la ligadura, abriendo así ªe uréter para permitir que la orina fluya y permitir que el extremo uréter se retraiga en el cuerpo de la vejiga. Visible producción de orina se puede observar a lo largo de con hemorragia de los vasos peri-ureteral. </li><li> Cierre el defecto de la vejiga lado derecho con un solo interrumpido 9 / O sutura de seda. </li></ol> 7. Recuperación y Cuidado Postoperatorio Vuelva a colocar las vísceras gastrointestinal en el abdomen en su orientación original y cierre de la pared abdominal mediante la aproximación de los músculos rectos con 6 E / S de sutura absorbible. <ol><li> Aproximar la piel con clips de la piel metálica. </li><li> Parcialmente invertir la anestesia con una inyección subcutánea de hidrocloruro de atipamazol (10 l / g). </li><li> Administrar analgesia por vía subcutánea hidrocloruro de buprenorfina (0,05 mg / kg) y para la inyección de fluido de soporte 1 ml de NaCl al 0,9% por vía subcutánea. </li><li> Recuperar el ratón en un armario de calentamiento a 28 ° C durante 24 a 48 horas. Observe el ratón para la enfermedad hasta elfinalización del experimento. </li><li> Retire los clips de la piel metálica 7 - 10 días después de la operación. </li><li> Una vez que el experimento se ha completado la eutanasia el ratón por dislocación cervical. </li><li> Retire y recoger el riñón contralateral y aloinjerto para el análisis histológico. </li></ol>

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La manera más bien descrito para realizar la anastomosis arterial es utilizar la aorta distal del donante, con la arteria renal en la continuación, de una manera de extremo a lado a la aorta destinatario. Se describe el uso de un parche de aorta, similar a la creación de reflejo &#39;Carrell parche&#39; que lleva a cabo en el trasplante de riñón humano que creemos que es más conveniente. Aunque los informes en la literatura de donante y receptor operativo de tiempo son escasos creemos que la utilización de un par...

El trasplante renal con éxito para el tratamiento de la insuficiencia renal fue descrita por primera vez en 1955 entre los gemelos monocigóticos 1, desde entonces se ha convertido en un revolucionario tratamiento para los pacientes con insuficiencia renal terminal en todo el mundo, ofreciendo mejoras en duración y calidad de vida 2. Sin embargo la supervivencia del injerto a largo plazo se ha visto obstaculizada por una multitud de procesos patológicos que resultan en daños crónica del injerto 3. El rechazo del riñón trasplantado en el ser humano sigue siendo una causa importante de morbilidad, a pesar de las mejoras significativas en los regímenes immunosupporessive. El objetivo de desarrollar un modelo murino de trasplante renal es replicar de cerca el proceso y la patología que se encuentra en el trasplante renal humano 4. Skoskiewicz et al. Describe primero el modelo murino de trasplante renal en 1973 5. Aunque se requieren habilidades de microcirugía avanzados, es una valiosa tool por varias razones: el genoma del ratón ha sido bien caracterizada y hay una gran variedad de métodos y técnicas disponibles para los estudios del ratón experimentales. Muchos grupos utilizando el modelo de ratón de trasplante renal han utilizado el riñón trasplantado como un órgano que sustenta la vida, sin embargo, en otros estudios y en nuestra metodología descrita uno de los riñones naturales del ratón receptor se deja in situ durante la duración del experimento 4. El beneficio es que el ratón se somete a una única operación de la anestesia y reduciendo así la morbilidad para el ratón y el riesgo de muerte a partir de un segundo procedimiento. Además, el ratón no sufren de los efectos adversos de la insuficiencia renal progresiva. Aunque existen modelos de rechazo alogénico en otros órganos tales como el corazón y la piel, estos no siempre son directamente relevantes para el trasplante renal. Hay evidencia de que estos modelos provocan diferentes modos y dymica de rechazo, por ejemplo, el curso de tiempo de rechazo de aloinjerto cardíaco y en aloinjerto renal difiere significativamente en ciertas combinaciones de deformación 6. Hemos descrito patrones de rechazo de aloinjerto renal aguda en donantes BALB / c en ratones FVB / NJ no transgénicos, este modelo mostró la lesión mediada celular con acumulación de células T y macrófagos 7. Alternativamente también hemos descrito un modelo de daño crónico del aloinjerto que exhibe la fibrosis intersticial y atrofia tubular, esto resulta de trasplante de un riñón de ratones C57BL / 6 BM12 donantes C57BL / 6 en los destinatarios, ya que estos ratones se caracterizan por una sola MIS loci MHC de clase II -match 8. Múltiples aspectos del trasplante han sido estudiados utilizando el modelo murino de trasplante renal, incluyendo rechazo agudo, rechazo celular y humoral, lesión de reperfusión de isquemia, y probando nuevos agentes terapéuticos. Hemos modificado el t quirúrgicaechnique para reducir el tiempo de operación y mejorar la facilidad de la cirugía. Particularmente hemos descrito donante simultánea y preparación de receptor y una técnica de anastomosis vascular simplificada mediante la utilización de una anastomosis aórtica parche continuo. Este video y manuscrito proporcionarán puntos clave para ayudar en el establecimiento de esta técnica.

<table><tbody><tr><td colspan="4">Surgical Instruments</td></tr><tr><td>Blunt Dissecting Scissors</td><td>Fine Science Tools</td><td>&amp;nbsp;14072-10</td><td>For skin cutting</td></tr><tr><td>Curved Castoviejo scissors</td><td>Fine Science Tools</td><td>15017-10</td><td>For tissue cutting</td></tr><tr><td>Spring Scissors &amp;ndash; straight</td><td>Fine Science Tools</td><td>15000-08</td><td>For suture cutting</td></tr><tr><td>Toothed forceps 1x2 teeth</td><td>Fine Science Tools</td><td>11021-12</td><td></td></tr><tr><td>2 x Fine Tip forceps (Dumont No.5)</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Angled Fine Tip forceps (Dumont No. 5/45)</td><td>Fine Science Tools</td><td>11253-25</td><td>For blunt dissecting</td></tr><tr><td>Curved Fine Tip forcep (Dumont No.7)</td><td>Fine Science Tools</td><td>11273-22</td><td>Useful to pass around vessels</td></tr><tr><td>Curved Crile Haemostat</td><td>Fine Science Tools</td><td>1300-04</td><td></td></tr><tr><td>Micro clip applicator with lock</td><td>Fine Science Tools</td><td>18056-14</td><td></td></tr><tr><td>2 x Micro serrefines spring width 2mm, jaw length 4mm</td><td>Fine Science Tools</td><td>18055-04</td><td>Microvascular clamps</td></tr><tr><td>2 x Colibri 3cm wire retractor</td><td>Fine Science Tools</td><td>17000-03</td><td></td></tr><tr><td>Castroviejo needle holder with lock</td><td>Fine Science Tools</td><td>120660-01</td><td></td></tr><tr><td>Wound clip applicator</td><td>Fine Science Tools</td><td>12031-07</td><td></td></tr><tr><td>7mm wound clips</td><td>Fine Science Tools</td><td>12032-07</td><td>Remove 7 to 10 days after surgery</td></tr><tr><td colspan="4">Equipment</td></tr><tr><td>OPMI pico microscope</td><td>Carl Zeiss</td><td>S100</td><td></td></tr><tr><td>Thermal cautery unit with fine tip</td><td>Geiger</td><td>150A</td><td></td></tr><tr><td>Heat electronic pad</td><td>Cozee Cumfort</td><td>n/a</td><td></td></tr><tr><td>Euroklav 23-S</td><td>Melag</td><td>n/a</td><td>Autoclave</td></tr><tr><td colspan="4">Disposable equipment</td></tr><tr><td>7/O Silk braided suture</td><td>Pearsall</td><td>30514</td><td></td></tr><tr><td>10/O Dafilon (polyamide) suture</td><td>B-Braun</td><td>&amp;nbsp;G1118099</td><td></td></tr><tr><td>6/O Vicryl (plygalectin)</td><td>Ethicon</td><td>W9537</td><td></td></tr><tr><td>Regular bevel needle, 1 inch, 21G</td><td>Bection, Dickinson and Company</td><td>305175</td><td>For ureteric anastamosis</td></tr><tr><td>Regular bevel needle, 5/8 inch, 25G</td><td>Bection, Dickinson and Company</td><td>305122</td><td></td></tr><tr><td>Regular bevel needle, 1/2 inch, 30G</td><td>Bection, Dickinson and Company</td><td>304000</td><td></td></tr><tr><td>Insulin needle 1ml, 29G</td><td>Bection, Dickinson and Company</td><td>324827</td><td></td></tr><tr><td>Insulin needle 0.3ml, 30G</td><td>Bection, Dickinson and Company</td><td>324826</td><td></td></tr><tr><td>1 ml syringe slip tip</td><td>Bection, Dickinson and Company</td><td>300184</td><td></td></tr><tr><td>5 ml syringe slip tip</td><td>Bection, Dickinson and Company</td><td>302187</td><td></td></tr><tr><td>Wypall paper swabs</td><td>Kimberley-Clark</td><td>L40</td><td>sterilised by autoclave</td></tr><tr><td>Cotton wool buds</td><td>Johnson and Johnson</td><td>n/a</td><td>sterilised by autoclave</td></tr><tr><td>Plain drapes</td><td>Guardian</td><td>CB03</td><td>sterilised by autoclave</td></tr><tr><td>Cell culture dish 60mm x 15mm</td><td>Corning Incorporated</td><td>430166</td><td></td></tr><tr><td>Dispensing Pin</td><td>B-Braun</td><td>DP3500L / 413501</td><td>Used with NaCl 0.9%</td></tr><tr><td>Re-agents and Drugs</td><td></td><td></td><td></td></tr><tr><td>(Lacri-Lube) White soft paraffin 57.3%, mineral oil 42.5% and lanolin alcohols 0.2%</td><td>Allergan Ltd</td><td>21956GB10X</td><td></td></tr><tr><td>(Videne) Povidone-iodine 10%</td><td>Ecolab Ltd</td><td>PL 04509/0041</td><td></td></tr><tr><td>(Vetalar V) Ketamine hydrochloride</td><td>Pfizer Animal Health</td><td>Vm 42058/4165</td><td>100mg/ml solution (dose 200mg/kg)</td></tr><tr><td>(Domitor) Medetomidine hydrochloride&amp;nbsp;</td><td>Orion Pharma</td><td>Vm 06043/4003</td><td>1mg/ml (dose 0.5mg/kg)</td></tr><tr><td>(Vetergesic) Bupernorphine hydrochloride&amp;nbsp;</td><td>Alsto Animal Health</td><td>Vm 00063/4002</td><td>0.3mg/ml (dose 0.05mg/kg)</td></tr><tr><td>(Antisedan) Atipamezole hydrochoride</td><td>Orion Pharma</td><td>Vm 06043/4004</td><td>5mg/ml (dose 2mg/kg)</td></tr><tr><td>University of Wisconsin Solution</td><td>Belzer Bridge to Life</td><td>n/a</td><td>dose approximately 500 microlitres/mouse</td></tr><tr><td>NaCl 0.9%</td><td>Baxter</td><td>FKE1323</td><td></td></tr><tr><td>Heparin Sulphate</td><td>non-proprietary</td><td>n/a</td><td>5000units/ml (dose 5units/mouse)</td></tr></tbody></table>

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.

Rechazo de aloinjerto renal puede ser evaluada por análisis histológico de parafina embebido en secciones de tejido fijadas con methacarn del riñón trasplantado (Figura 2). El trasplante de riñones entre isoinjerto resultados ratones singénicos en la lesión por reperfusión isquémica renal, sin embargo por 4 semanas, los túbulos se han recuperado y son histológicamente comparables a los riñones nativos. El rechazo agudo puede ser modelado por C57BL / 6 trasplante de riñón en receptores BALB...

Watch this Scientific Journal Video about Mouse Kidney Transplantation: Models of Allograft Rejection at JoVE.com

Mouse Kidney Transplantation: Models of Allograft Rejection

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 ...

mouse-kidney-transplantation-models-of-allograft-rejection

Research

JoVE Journal

Medicine

20.5K vistas.  The University of Edinburgh. 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.

Video: Mouse Kidney Transplantation: Models of Allograft Rejection

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 Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

Trasplante de piel murina

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Biología

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Murino Trasplante corneal: un modelo para estudiar la forma más común de trasplante de órganos sólidos

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

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 ...

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.

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 ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

In Vitro e In Vivo la evaluación de la actividad supresora T, B y las células mieloides y las respuestas humorales de los receptores de trasplante

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Inmunología e Infección

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

Ortotópico de la extremidad posterior de trasplante en el ratón

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

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 ...

A Rat Orthotopic Renal Transplantation Model for Renal Allograft Rejection

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential model to investigate the mechanism of renal allograft rejection in pre-clinical studies and could aid in the development of novel approaches to improve the long-term survival of renal allografts. Donor kidney implantation in rat orthotopic renal transplantation is commonly performed by end-to-side anastomosis to recipients' aorta and inferior vena cava. In this model, the donor's kidney was implanted using end-to-end anastomosis to the recipients' renal artery and renal vein. The donor's ureter was anastomosed to the recipient's bladder in an end-to-side 'tunnel' method. This model contributes to better healing of ureter-bladder anastomosis and increases the recipients' survival by avoiding interference with blood supply and venous reflux of the lower body. This model can be used to investigate the mechanisms of acute and chronic immune and pathologic rejection of renal allografts. Here, the study describes the detailed protocols of this orthotopic renal transplantation between rats.

The rat orthotopic renal transplantation model contributes to investigating the mechanism of renal allograft rejection. The current model increases the recipients' survival without interference with blood supply and venous reflux of the lower body using an end-to-end anastomosis of kidney implantation and an end-to-side "tunnel" method of ureter-bladder anastomosis.

Un modelo de trasplante renal ortotópico de rata para el rechazo del aloinjerto renal

Renal allograft rejection limits the long-term survival of patients after renal transplantation. Rat orthotopic renal transplantation is an essential ...