Este trabalho foi apoiado em parte por 1R03DK096151.

Todos os ratinhos foram adquiridos da The Jackson Laboratory (Bar Harbor, ME) e foram alojados em condições livres de patógenos na Universidade do Colorado Denver, Facility Barbara Davis Centro de animal de acordo com diretrizes do NIH e com a aprovação da University of Colorado Denver IACUC. 1. doador de rim Colheita <ol><li> Esterilizar todos os instrumentos, usar luvas estéreis durante todo o processo e manter um campo esterilizado. Executar todas as cirurgias com a utilização de um microscópio de operação. </li><li> Remover cirurgicamente rins de doadores de rato anestesiado-pentobarbital (60mg / kg IP) doadores. Verificar profundidade da anestesia por toe-pitada, e observar a freqüência respiratória. </li><li> Clipe da pele, em seguida, imobilizar o rato por apoios de 4-way. Prepare a pele com iodopovidona e armar o mouse de forma estéril. </li><li> Adicione a 2 cm da linha média incisão abdominal vertical e entrar na cavidade abdominal. Retrair o intestino superiormente e exteriorizá-lopara o peito. Mantenha o intestino envolto em gaze estéril úmido durante todo o procedimento. </li><li> Identificar os grandes vasos abdominais e mobilizá-los, identificar quaisquer ramos lombares e ou cauterizar ou ligá-los com 10/0 nylon de sutura. </li><li> Imediatamente distal para os vasos renais esquerda provocar a veia cava inferior (IVC) e aorta abdominal (AA) por dissecção romba para além ao longo de aproximadamente 2 mm. Ligadura com 10/0 nylon e dividir pequena arterial e ramos venosos dos vasos renais. </li><li> Agora separar a veia renal a partir da artéria renal cuidadosamente por dissecção romba destas estruturas. Isto permite a criação de um emplastro precisas Carrel na extremidade proximal da veia renal, que vai ser utilizado para formar uma anastomose extremidade-a-lado para os destinatários IVC. </li><li> Identificar, ligadura e dividir os vasos adrenal esquerda. Isto permite que o acesso à aorta supra-renal e uma sutura de seda 6-0 é colocado em torno da aorta em preparação para a ligação, mas não amarrado neste momento. </li&gt; <li> Mobilizar o rim, ureter e vasos do tablier envolvente. Gire o rim para a direita e ligar / dividir quaisquer ramos posteriores. Em seguida, a rim de regresso para a esquerda. </li><li> Agora dirija a atenção para o ureter. Sem perturbar o hilo renal libertar o ureter a partir da envolvente tablier tendo o cuidado de preservar os vasos ureterais. Divida o ureter no nível do canal deferente. O rim está agora pronto para recuperar. </li><li> Lentamente injetar 300 unidades de heparina para a VCI distal heparinizar assim o doador. Atar o fio de sutura de seda 6-0 colocado em torno do AA supra-renal e perfundir o rim esquerdo via a aorta abdominal distai com 0,8 ml de solução salina de heparina (100 U / mL). </li><li> Uma vez que a perfusão cessou criar um remendo Carrel venosa imediatamente para eliminar o refluxo para o rim. Retrair a veia renal em direção ao rim revelando a AA ea artéria renal abaixo. </li><li> Dividir a aorta adjacente à artéria renal, criando um calcanhar ee braçadeira como mostrado na Figura 1. Remover o rim e ureter, vasos do dador. </li><li> Remover o rim directamente a um receptor pré-preparado (0 min de tempo de isquemia fria) ou armazenado a 4 ° C numa solução de escolha para um tempo pré-determinado, até que o tempo de implantação. Eutanasiados os doadores por exsanguanation e deslocamento cervical. O tempo total para recuperar doador de rim é de aprox. 15-20 min. </li></ol> 2. Rim Técnica de Implante <ol><li> Anestesiar o mouse destinatário com pentobarbital (60 mg / kg dose inicial IP, 25mg / kg IP dose suplementar se necessário). Verificar profundidade da anestesia por toe-pitada, e observar a freqüência respiratória. Clipe da pele, em seguida, imobilizar o rato por apoios de 4 vias e aplicar pomada oftálmica para os olhos. Prepare a pele com iodopovidona e armar o mouse de forma estéril. </li><li> Adicione a 2 cm da linha média incisão abdominal vertical e entrar na cavidade abdominal. Retrair o intestinosuperiormente e exteriorizar para o peito. Mantenha o intestino envolto em gaze estéril úmido durante todo o procedimento. </li><li> Neste momento realizar uma nefrectomia direita. Ligar a artéria e veia renal com 6-0 seda. Extirpar o rim distal para as suturas. Ligadura do ureter com seda 6-0 e depois dividir proximal do rim. </li><li> Isolar a aorta abdominal e veia cava inferior (IVC) abaixo dos vasos renais. Coloque 4-0 laços de algodão em torno da aorta e, em seguida, IVC superiores inferior ao local da anastomose. Identificar e ligar quaisquer vasos lombares dentro do campo com nylon 10-0 sutura. </li><li> Atar os laços de algodão, o primeiro inferior seguido pelo superior. Deste modo algum sangue é retida na aorta fazendo o aortotomy mais fácil. </li><li> Formar o aortotomy com uma agulha de 30G para introduzir o lúmen da aorta. Estender a incisão com tesoura micro finas para um comprimento de cerca de 2 mm. </li><li> Faça um fim a anastomose lado do manguito doador aórtica punta-taco paraa aorta do receptor da seguinte maneira. Coloque um nylon 10-0 sutura estadia ponto na aorta do doador e ao ângulo inferior da incisão na aorta destinatário e gravata. </li><li> Coloque um segundo nylon 10-0 frente ao primeiro lugar na aorta do doador e do canto superior da incisão na aorta abdominal e gravata. Faça uma linha de sutura contínua de superior para inferior na parede lateral da aorta e empate contra o ponto estadia previamente colocado. Em seguida, sutura da parede medial de uma forma correndo e gravata. </li><li> Faça um fim-anastomose da veia renal dador até ao receptor IVC da seguinte forma. Perfurar o IVC com uma agulha de 30G e estender a incisão com tesoura micro finas. Amarre a veia renal de doadores para o canto inferior da incisão na VCI com nylon 10-0. Faça uma linha de sutura contínua entre a veia renal e da VCI e gravata. NOTA: É também de vital importância que a espessura total passa da agulha de sutura incluindo o adventit vascularIA e da íntima são alcançados. Eversão das bordas também assegura que não há contacto íntima-a-íntima, que ajuda na cura e selagem das anastomoses. Enquanto um agente de coagulação hemostática pode ser útil para reduzir vazamentos, recomendamos que um cirurgião sim contar com boa técnica. </li><li> Certifique-se de que as anastomoses são &quot;limpos&quot;. Isto é, que as paredes opostas não são capturados ao colocar pontos. Isto fará com que uma constrição significativa ao fluxo que irá resultar numa falha do enxerto e em casos extremos, à paralisia dos membros posteriores. NOTA: Outro factor extremamente importante é assegurar que a tensão das linhas de sutura anastomóticos também é óptima. Muito solto e haverá vazamento irreversível, muito apertado e estenose ao fluxo resultará. Se no lado arterial isto irá resultar em má perfusão do enxerto, se no lado venoso de um rim congestionado vai resultar. </li><li> Solte o fluxo venoso 4-0 tie algodão restabelece distal. Uma vez que a hemostasia do venonos anastomose tem sido observado gradualmente afrouxar o laço de algodão 4-0 proximal e observar a anastomose arterial para hemostasia. </li><li> Retire os laços de algodão do mouse uma vez ambas as anastomoses são consideradas seguras. Pierce da bexiga com uma agulha de 20 G a criação de dois furos. </li><li> Passar as pontas das pinças curvas através dos orifícios e puxar o ureter dador através da bexiga com a pinça com a extremidade proximal do ureter sendo ancorada à parede da bexiga com duas suturas de nylon 10-0. Aparar o excesso de comprimento do ureter saliente a partir do segundo furo permitindo do uréter se retraia dentro da bexiga e fechar o buraco com duas suturas de nylon 10-0. </li><li> Retorno do intestino para o abdômen. Feche a parede abdominal em duas camadas usando 5-0 sutura de seda de forma a funcionar. </li><li> Administrar um bolus de 1,0 ml de solução salina esterilizada quente no abdômen como a reanimação com líquidos em cima do fecho, e injectar 0,8 ml de solução salina normal subcutaneamente no pós-operatório. Nenhum outro supportisão necessárias medidas ve durante a cirurgia. </li><li> Recuperar o animal em uma manta térmica. Tempo de implante total é de aprox. 35-45 min. Administrar analgésicos, tais como a buprenorfina, de 0,05 mg / kg, SC, 0,1-0,2 ml no início do procedimento e cada 6-12 horas, durante 72 h pós-op. </li></ol> 3. A nefrectomia contralateral <ol><li> Dependendo dos requisitos do protocolo, vários dias após o procedimento de implante, uma nefrectomia contralateral executar sob anestesia com isoflurano (5% de isoflurano inalado por indução, 1,5-2,0% para manutenção). </li><li> Digite a cavidade abdominal e retrair delicadamente o intestino para a direita do animal. Expor o rim esquerdo e dissecar romba da fáscia circundantes. </li><li> Ligar a artéria renal, veia e do ureter com 6-0 suturas de seda e, em seguida, o rim acima excisar as suturas e remover o rim. Tempo total do procedimento é de aprox. 10 min. </li></ol> 4. Avaliação do Enxerto <ol><li> Medida creati soronove utilizando o método do picrato alcalino (Jaffe reacção). </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. 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The authors have nothing to disclose.

Dominar esta técnica de transplante é difícil, mas uma vez conseguido isso é uma ferramenta de pesquisa muito poderoso. O cirurgião paciente / pesquisador será recompensado pela atenção aos detalhes e consistência da técnica, que é a chave para dominar qualquer procedimento cirúrgico, ainda mais em pequenos modelos animais. As dificuldades técnicas de dominar o transplante de rim do rato são muitas dobras, e é altamente provável que a experiência em outros modelos pequenos transplante animal deve ser ad...

Desde 1973, o modelo de transplante renal em ratos tem sido um valioso instrumento de pesquisa, mas problemas técnicos têm dificultado seu uso generalizado. Ao longo dos anos, vários trabalhos têm sido publicados detalhando melhorias / aperfeiçoamentos a este procedimento. Como um modelo de transplante de órgãos sólidos, principalmente vascularizado este procedimento é, provavelmente, perdendo apenas para o modelo de transplante cardíaco heterotópico, que também foi concebido pelo laboratório Russell em 1973 6. Ambos os modelos se prestam à investigação sobre respostas de rejeição alogênicos, o desenvolvimento da função retardada do enxerto e lesão por isquemia-reperfusão. Um dos problemas mais comuns que devem ser comunicadas com transplante renal é a relativamente alta incidência de trombose arterial 4,5,7 que também experiente em nosso laboratório. Por isso nos propusemos a realizar uma revisão da literatura sobre a formação de trombos e, possivelmente, encontrar a causa deste problema técnico e também para elaborar umsolução possível. A causa mais provável de trombose é o caminho tortuoso tanto o sangue toma da aorta do receptor, para a aorta do doador renal em seguida, para a artéria renal dador. Este caminho provoca turbulência na artéria renal, o que pode conduzir à activação de plaquetas e formação de trombos. Com base nas observações recentes e uma pesquisa da literatura relevante 14/08 que surgiu com uma nova técnica que reduziu trombose para 0%. A técnica descrita aqui varia de técnicas previamente relatados na formação de um arterial punta-taco manguito que as instalações melhora a circulação sanguínea e reduz significativamente a formação de trombos. A braçadeira é formada pela divisão da aorta infra-renal através da face do óstio da artéria renal com um ângulo inferior a 45 ° em relação ao eixo longitudinal da aorta (Figura 1A e 1B). Isto resulta numa braçadeira aproximadamente 2 mm de comprimento. Um patch Carrel venosa é formado por transecting o renal veia para a VCI aumentando assim o diâmetro da braçadeira. A infra-renal de doadores abdominal aorta punta-taco manguito é de ponta a lado anastomosada à aorta abdominal destinatário ea veia renal doador / patch IVC é de ponta a lado anastomosado ao destinatário abdominal veia cava inferior (VCI) . O ureter é então introduzida e ancorada à bexiga tal como descrito por Han et al 3. Para este estudo transplantes não tratados apenas com tempo de isquemia quente (ie., Sem isquemia fria) são comparados. Neste caso isquemia quente refere-se ao tempo da cessação do fluxo sangüíneo através do rim de doador (passo 1.11 abaixo) e reperfusão do enxerto no receptor (passo 2.11 abaixo). Isquemia fria refere-se ao tempo que o rim não perfundido é e é mantido em armazenamento a frio, até ao início do procedimento 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 cirúrgica permite tanto para estudos simples sobrevivência de enxerto / rejeição, ou protocolos experimentais bastante complexas. Nas figuras abaixo demonstramos as vantagens de usar esse melhor técnica de anastomose arterial. Usando esta técnica, reduzimos significativamente a incidência de trombose arterial de 35% para 0%, aumentando assim a produtividade. Temos utilizado esta técnica para mais de um ano com o mesmo resultado de 0% a trombose mantida. A Figura 1 descreve o métod...

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.

Rim murino técnica de transplante

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

16.0K visualizações.  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. 

Vídeo: 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.

Um método para isolamento de ilhotas murino e transplante renal subcapsular

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

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.

Murino Procedimento de Transplante Renal

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

Biologia

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.

Isolamento e Transplante de diferentes envelhecidas murino tímicos enxertos.

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

Imunologia e Infecção

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.

Rato Transplante Renal: Modelos de rejeição de enxertos

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.

Mapeamento de destino de células estaminais embrionárias humanas por formação de teratomas

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 autotransplante em um modelo suíno: Um Protocolo Passo-a-Passo

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>Produção de camundongo humanizado por meio de enxerto de cápsula renal tímica, injeção de células CD34+ e entrega 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.

Produção de camundongo humanizado por meio de enxerto de cápsula renal tímica, injeção de células CD34+ e entrega 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.

Uma técnica cirúrgica modificada para transplante de rim em camundongos

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.

Transplante de rim de rato ortotópico: uma abordagem cirúrgica nova e simplificada

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