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. 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.

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

murine kidney transplant technique

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

Esta técnica 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...

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Murine Kidney Transplant Technique

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

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 ...

Research

JoVE Journal

Medicine

15.8K 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 Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Murino Cervical Modelo de coração Transplante usando uma técnica Cuff Modificado

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

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

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

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

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

Técnica de Subnormothermic<em&gt; Ex Vivo</em&gt; Fígado de Perfusão para o armazenamento, Avaliação e Reparação de enxertos de fígado Marginal

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

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

Técnica de transplante heterotópico Murino Coração

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

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

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

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

Técnica para as aquisições de fígado de porco e Transplante ortotópico usando um Active Porto-Caval Shunt

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

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 rato de rejeição vascular induzida por Aloimune e Transplante Arteriosclerose

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

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

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

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

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

Normotérmica<em&gt; Ex Vivo</em&gt; Perfusão rim para a Preservação do rim Os enxertos antes do transplante

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

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

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

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

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

Um &quot;All-a laser&quot; Endotelial Transplant

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

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

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

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

Modelo do rato para transplante de pâncreas, usando uma técnica modificada do manguito

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

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

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

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

Murine Modelo de Transplante de Aorta Cervical usando uma técnica de manguito não sutura modificada

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