Financiamento de Kidney Research UK, The Royal College of Surgeons de Edimburgo e da Sociedade Europeia de Transplante de Órgãos apoiou esse estudo.

Ética institucionais nacionais e locais apropriados devem estar no local antes de realizar experimentos com animais. Especificamente no Reino Unido os seguintes experimentos foram realizados sob os Animais (Scientific Procedures) Act 1986 Onde dois microsurgeons disponíveis para operar simultaneamente o cirurgião doador deve executar os passos 1,1-1,16 então 3.1 a 3.5, enquanto o cirurgião realiza destinatário 2,1-2,8 . Para um único operador os passos podem ser seguidos sequencialmente. 1. Doador Preparação Nota: O procedimento aqui apresentado é de um doador C57BL / 6 BM12 e destinatário C57BL / 6, machos, com idade entre 8 a 16 semanas de idade com um peso corporal superior a 20 g. No entanto, este procedimento pode ser realizado de forma reprodutível de uma variedade de estirpes de ratinho. Os dados apresentados na seção de resultados representativos foi obtido a partir de C57BL / 6, C57BL / 6 BM12 e BALB / c. <ol><li> Procedimentos de Conduta using instrumentos cirúrgicos estéreis e consumíveis (autoclave), com esforços para manter a área de operações tão estéril quanto possível. Realizar a preparação dos doadores em simultâneo com a preparação destinatário se dois cirurgiões estão disponíveis. </li><li> Anestesiar o rato com uma injecção intraperitoneal (agulha de 31G) de medetomidina (0,5 mg / kg) e cloridrato de cetamina (200 mg / kg). Isso resulta em um plano anestésico que é mantida durante 4 horas dando tempo suficiente para a totalidade do procedimento a ser realizado. Anestesia suplementar não é necessário. </li><li> Confirme se o mouse está anestesiado (sem resposta aos pés pitada). </li><li> Raspar o abdômen do rato e remover qualquer cabelo solto com fita adesiva. </li><li> Posicione o mouse sobre as suas costas em uma esteira aquecida esterilmente drapeado e vagamente imobilizar os membros com fita adesiva estéril. </li><li> Monitorar o mouse durante todo o procedimento para queimaduras térmicas. Se possível usar uma fonte de calor não-elétrico. </li><li> Aplique um lubrificante ocular e sanitize da parede abdominal com uma solução diluída de iodo. </li><li> Faça uma incisão na linha média do abdome para entrar na cavidade peritoneal e inserir um 3 centímetros Calibri afastador abdominal. </li><li> Aplicar soro fisiológico aquecido para manter os intestinos e área cirúrgica úmida e evitar a secagem desnecessária das vísceras. </li><li> Cubra o mouse com campos estéreis e mover os intestinos à esquerda do operador (direita do mouse) para expor a aorta, veia cava e rim esquerdo. </li><li> Aplique uma pinça reajustados para o estômago e puxar superiormente para expor os grandes vasos e rim esquerdo totalmente. Embalar amortecer zaragatoas esterilizadas (2 mm x 2 mm) no abdómen para retrair tecidos longe da área cirúrgica, tais como os lóbulos do fígado, vesículas seminais e do intestino. </li><li> Isolar o rim esquerdo de adventícia circundante, gordura e glândula adrenal esquerda na cavidade peritoneal por bruscamente dissecando o tecido conjuntivo usando uma pinça de ponta fina. Coloque as pontas fechadas da pinça entre as áreas que need para ser separados e, lentamente, permitir que as dicas fórceps para abrir a dissecar o espaço. </li><li> Isolar a veia renal esquerda por ligação e, em seguida, dividindo-se a veia supra-renal e veia gonadal esquerdo esquerda com 9 / ó de nylon. Coloque a sutura perto da veia renal. </li><li> Ligadura e dividir o ureter com 7 / S de seda de sutura para fechar a bexiga deixando o fio de sutura termina longo. Estas extremidades longo de sutura irá ser utilizado quando o rim é colhida e são necessárias para a anastomose uretérica. </li><li> Para mobilizar e dissecar totalmente a aorta ea veia cava inferior e superiormente à artéria e veia renal utilize uma pinça de ponta fina para dissecar sem rodeios vasos linfáticos e gordura da parte frontal e nas laterais dos vasos. </li><li> Encontre o plano tecido entre a aorta ea veia cava e lentamente se espalhou a pinça. Abra lentamente as pontas dos pinça fina cotado para espalhar o tecido com um mínimo de trauma. </li><li> Amarre uma solta 7 / O de seda em torno da aorta superior e inferior à artéria renal passando um bom ângulopinça de ponta em todo o volta dos vasos e desenho uma sutura através. Estas suturas serão mais rigorosas antes da recuperação renal para permitir perfusão retrógrada da Universidade de Wisconsin solução. </li></ol> 2. Destinatário Preparação De acordo com o acompanhamento preparação doador passos 1.2 a 1.8. <ol><li> Mova os intestinos à direita do operador (à esquerda do mouse) para expor a aorta, veia cava. Cubra os intestinos com uma solução salina estéril cotonete embebido. </li><li> Realize uma nefrectomia direita por ligadura de artéria renal direita e veia juntamente com 7/0 fio de seda e depois dividir. </li><li> Ligadura do ureter direito com 7 / O fio de seda e dividir. </li><li> Mobilizar e dissecar totalmente a aorta ea veia cava inferior à artéria e veia renal tal como descrito no passo 1.14 e 1.15. Certifique-se de dissecção completa entre a aorta ea veia cava. Tome o cuidado de preservar a artéria espermática interna que corre anterior à veia cava e aortum juntamente com o feixe linfático. </li><li> Identificar vasos lombares em execução no retroperitônio da veia cava e da veia para a coluna, ligar com o 9 / O de seda em continuidade, não há necessidade de dividir os vasos. </li><li> Identificar espaço suficiente para colocar grampos microvasculares com espaço entre para anastomoses vasculares. </li><li> Administrar heparina intravenosa (5 unidades) através da veia dorsal do pênis. </li></ol> 3. doador de rim Retrieval Apertar os 7 / S laços de seda que foram colocados em torno da aorta inferior e superior para isolar o rim a partir da circulação arterial. <ol><li> Infundir retrógradas frio 0,2-0,5 ml de solução da Universidade de Wisconsin, com uma agulha (31G) na aorta. </li><li> Dividir a aorta dentro das suturas e dividir a veia renal na sua junção com a veia cava para remover o rim e a artéria renal com um comprimento da aorta. Divide vasos lombares decorrentes da aorta, se estiverem presentes, wligadura ithout. </li><li> Inserir o rim em solução salina fria numa compressa estéril numa placa de cultura. </li><li> Eutanásia do camundongo doador por deslocamento cervical. </li></ol> 4. Transplante renal - Kidney Preparação <ol><li> Criar um remendo aórtico dividindo a parede da aorta longitudinalmente em frente à artéria renal. Identificar os lúmenes embarcação no patch que precisam ser ligados ou evitado ao realizar a anastomose arterial. </li><li> Coloque um fio de sutura 10 / O de fora para dentro do lúmen da veia renal superiormente e uma segunda sutura separada inferiormente. As suturas usadas para dividir a veia supra-renal e veia gonadal pode ser utilizado para orientar o recipiente. </li><li> Coloque o rim no flanco direito do destinatário (Figura 1.1) e garantir as suturas estão bem colocados para garantir anastomoses vasculares pode ser concluída sem as suturas fiquem entrelaçados em cotonetes ou outros instrumentos. </li></ol> 5. Kidney Transplantação - Vascular Anastomose Aplicar grampos microvasculares primeiro inferiormente então superiormente abrangendo a veia cava e aorta. <ol><li> Adicione uma ventomy com uma agulha (31G) por punção da parede anterior da veia cava. Lavar o sangue a partir da veia cava por injecção de cerca de 50 ul de 0,9% de NaCl. </li><li> Alargar a venotomia usando fórceps de ponta fina abrindo-os dentro do venotomia modo que o comprimento é equivalente ao diâmetro da veia renal doador. </li><li> Coloque a sutura superior, 10 / O (que já está na luz da veia renal) pela primeira vez no ápice da venotomia (Figura 1.2) e juntar-se a parede de trás da anastomose em sutura contínua até que o vértice inferior é alcançada (Figura 1.3). </li><li> Coloque a 10 / O sutura inferior e gravata pela primeira vez com um único nó (Figura 1.4), em seguida, amarre a sutura contínua da parede do fundo. </li><li> Usando fio de sutura inferior criar a parede frontall da anastomose (Figura 1.5), com uma sutura contínua e gravata para o final de sutura superior no ápice superiores (Figura 1.6). </li><li> Criar um Aortotomia por pegar a parede da aorta com uma pinça e corte um remendo elíptica com uma tesoura (aproximadamente aortotomia deve ser um quinto da circunferência da aorta e três vezes o lúmen da artéria renal de comprimento). </li><li> Coloque um 10 / S de sutura no ponto superior do remendo da aorta a partir do exterior para o interior (figura 1.7) e passar através da aorta para amarrar fora dos vasos no vértice superior do Aortotomia. </li><li> Criar a anastomose arterial com sutura contínua começando superiormente (Figura 1.8) anastomose o patch doador para o receptor aorta, tomar cuidado para não contrair a anastomose por amarrar a sutura muito apertado (Figura 1.9). </li><li> Retirar os grampos vasculares inferiores primeira então o grampo superior a reperfundir o rim (Figura 1.10 e 1.11). Peristaltismo visível do ureter talvez visto se o fornecimento de sangue adequado para o ureter é alcançado. </li></ol> 6. Transplante Renal - anastomose ureteral <ol><li> Divida os anexos da bexiga na parede abdominal. </li><li> Passar uma agulha (21G) a partir da esquerda para a direita da bexiga por meio de ambas as paredes. </li><li> Coloque dicas fórceps retas no lúmen da agulha e passar tanto de volta através da bexiga de tal forma que as pinças são agora passando da direita para a esquerda para fora do lado esquerdo da bexiga. </li><li> Desenhar a sutura no ureter dador através da bexiga tal que a uretra passa no defeito do lado esquerdo, em seguida, para fora da bexiga do lado direito. </li><li> Suturar a adventícia do ureter à adventícia da bexiga com três únicos pontos isolados ao redor do ponto de entrada com 9 / O nylon sutura em uma agulha round-bodied. </li><li> Corte o ureter proximal à ligadura, th abrindo assime do ureter para permitir que a urina e permitir que a extremidade do ureter para retrair para dentro do corpo da bexiga. Visible produção de urina pode ser observado junto com o sangramento de vasos peri-ureterais. </li><li> Feche a lesão da bexiga lado direito, com uma única interrupção 9 / O fio de seda. </li></ol> 7 Recuperação e cuidados pós-operatórios Substitua as vísceras gastrointestinal no abdômen na orientação original e fechar a parede abdominal, aproximando os músculos retos com 6 / O fio absorvível. <ol><li> Aproximar a pele com agrafos para pele de metal. </li><li> Pouco inverter a anestesia com uma injecção subcutânea de cloridrato de atipamazole (10 ul / g). </li><li> Administrar analgesia por cloridrato de buprenorfina por via subcutânea (0,05 mg / kg) e para a injecção de fluido de apoio de 1 ml de NaCl a 0,9% por via subcutânea. </li><li> Recuperar o mouse em um gabinete de aquecimento a 28 ° C por 24-48 horas. Observe o mouse para a doença até oconclusão da experiência. </li><li> Remova os clipes de pele de metal 7-10 dias pós-operatório. </li><li> Uma vez que a experiência é concluída a eutanásia por deslocamento cervical do rato. </li><li> Retire e recolher o rim contralateral e enxerto para análise histológica. </li></ol>

<ol>
	<li>Guild, W. R., Harrison, J. H., Merrill, J. P., Murray, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+homotransplantation+of+the+kidney+in+an+identical+twin.">Successful homotransplantation of the kidney in an identical twin.</a> Trans. Am. Clin. Climatol Assoc. 67, 167-173 (1955).</li><li>Wolfe, R. 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=Comparison+of+mortality+in+all+patients+on+dialysis,+patients+on+dialysis+awaiting+transplantation,+and+recipients+of+a+first+cadaveric+transplant.">Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant.</a> N. Engl. J. Med. 341, 1725-1730 (1999).</li><li>Nankivell, B. J., Alexander, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Rejection of the Kidney Allograft. N. Engl. J. Med. 363, 1451-1462 (2010).</li><li>Tse, G. H., Hughes, J., Marson, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+review+of+mouse+kidney+transplantation.">Systematic review of mouse kidney transplantation.</a> Transplant International. 26, 1149-1160 (2013).</li><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.+Transplant.">Kidney transplants between mice of graded immunogenetic diversity. Transplant.</a> Proc. 5, 721-725 (1973).</li><li>Zhang, Z., 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=Pattern+of+liver,+kidney,+heart,+and+intestine+allograft+rejection+in+different+mouse+strain+combinations.">Pattern of liver, kidney, heart, and intestine allograft rejection in different mouse strain combinations.</a> Transplantation. 62, 1267-1272 (1996).</li><li>Qi, F., 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=Depletion+of+cells+of+monocyte+lineage+prevents+loss+of+renal+microvasculature+in+murine+kidney+transplantation.">Depletion of cells of monocyte lineage prevents loss of renal microvasculature in murine kidney transplantation.</a> Transplantation. 86, 1267-1274 (2008).</li><li>Dang, Z., Mackinnon, A., Marson, L. P., Sethi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tubular+atrophy+and+interstitial+fibrosis+after+renal+transplantation+is+dependent+on+galectin-3.">Tubular atrophy and interstitial fibrosis after renal transplantation is dependent on galectin-3.</a> Transplantation. 93, 477-484 (2012).</li><li>Jabs, W. J., 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=Heterogeneity+in+the+Evolution+and+Mechanisms+of+the+Lesions+of+Kidney+Allograft+Rejection+in+Mice.">Heterogeneity in the Evolution and Mechanisms of the Lesions of Kidney Allograft Rejection in Mice.</a> Am. J. Transplant. 3, 1501-1509 (2003).</li><li>Lin, T., 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=Deficiency+of+C4+from+Donor+or+Recipient+Mouse+Fails+to+Prevent+Renal+Allograft+Rejection.">Deficiency of C4 from Donor or Recipient Mouse Fails to Prevent Renal Allograft Rejection.</a> Am. J. Pathol. 168, 1241-1248 (2006).</li></ol>

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A forma mais bem descrito a anastomose arterial é usar a aorta distai do doador, com a artéria renal em continuação, de uma maneira extremo-a-lado para o aorta do receptor. Descreve-se o uso de um tapa-aórtica, semelhante ao espelhamento &#39;Carrell remendo &quot;que realizou no transplante de rim humano que acreditamos ser mais conveniente. Apesar de relatos na literatura de doador e receptor operatório tempo são escassos, acreditamos que a utilização de um remendo da aorta para aorta destinatário em vez de ...

Transplante renal bem sucedido para o tratamento da insuficiência renal foi descrita pela primeira vez em 1955 entre gêmeos monozigóticos 1, desde então, tornou-se um tratamento revolucionário para pacientes com insuficiência renal terminal em todo o mundo, oferecendo a melhoria da esperança e qualidade de vida 2. No entanto sobrevida do enxerto a longo prazo tem sido dificultado por uma multidão de processos patológicos, resultando em danos crônica do enxerto 3. A rejeição do rim transplantado em humanos continua a ser uma das principais causas de morbidade, apesar das melhorias significativas nos regimes immunosupporessive. O objetivo de desenvolver um modelo do rato de transplante renal é replicar de perto o processo e patologia encontrada no transplante renal humano 4. Skoskiewicz et al. Descreveu pela primeira vez o modelo do rato de transplante renal em 1973 5. Embora sejam necessárias técnicas de microcirurgia avançados, é um valioso tool por várias razões: o genoma de ratinho foi bem caracterizada e não existe uma grande variedade de métodos e técnicas experimentais disponíveis para estudos com ratinhos. Muitos grupos utilizam o modelo do rato de transplante renal têm usado o rim transplantado como um órgão de apoio à vida, no entanto, em outros estudos e na nossa metodologia descrita um dos rins nativa do rato destinatário é deixado no local durante todo o período do experimento 4. A vantagem é que o rato passa por uma única operação de anestesia e reduzindo desse modo a morbidade para o rato e o risco de morte de um segundo procedimento. Além disso, o mouse não sofrer com os efeitos adversos da insuficiência renal progressiva. Apesar de existirem modelos de rejeição alogênico em outros órgãos, como o coração e pele, estes nem sempre são diretamente relevantes para o transplante renal. Há evidências de que esses modelos suscitar diferentes modos e dynamics de rejeição, por exemplo, o curso de tempo de rejeição do enxerto cardíaco em enxerto renal e difere significativamente em determinadas combinações de cepa 6. Nós descrevemos padrões de rejeição do enxerto renal aguda em BALB doadores / c em não-transgênicos camundongos FVB / NJ, este modelo mostrou lesão celular mediada com acúmulo de células T e macrófagos 7. Alternativamente, também descrito um modelo de dano do aloenxerto crónica que apresenta fibrose intersticial e atrofia tubular, isto resulta a partir do transplante de um rim de ratinhos C57BL / 6 doadores BM12 em ratinhos C57BL / 6 receptores, uma vez que estes ratos são caracterizadas por uma única classe de MHC II mis loci -match 8. Vários aspectos do transplante, foram estudados utilizando o modelo do rato de transplante renal, incluindo a rejeição aguda, rejeição celular e humoral, isquemia lesão de reperfusão, e testando novos agentes terapêuticos. Nós modificamos o cirúrgico technique para reduzir o tempo de operação e melhorar a facilidade de operação. Particularmente descrevemos doador simultânea e preparação destinatário e uma técnica de anastomose vascular simplificado através da utilização de uma anastomose remendo aórtico contínuo. Este vídeo e manuscrito fornecerá pontos-chave para ajudar na criação desta técnica.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Blunt Dissecting Scissors</td>
			<td>Fine Science Tools</td>
			<td>&#160;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 &#8211; 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></td>
			<td></td>
			<td></td>
			<td>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></td>
			<td></td>
			<td></td>
			<td>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>&#160;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>
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		<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>
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			<td>Plain drapes</td>
			<td>Guardian</td>
			<td>CB03</td>
			<td>sterilised by autoclave</td>
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		<tr>
			<td>Cell culture dish 60mm x 15mm</td>
			<td>Corning Incorporated</td>
			<td>430166</td>
			<td></td>
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			<td>Dispensing Pin</td>
			<td>B-Braun</td>
			<td>DP3500L / 413501</td>
			<td>Used with NaCl 0.9%</td>
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		<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&#160;</td>
			<td>Orion Pharma</td>
			<td>Vm 06043/4003</td>
			<td>1mg/ml (dose 0.5mg/kg)</td>
		</tr>
		<tr>
			<td>(Vetergesic) Bupernorphine hydrochloride&#160;</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.

A rejeição do aloenxerto renal pode ser avaliada através da análise histológica de secções de tecido embebidos em parafina fixados com Methacarn do rim transplantado (Figura 2). Transplante de rins entre isoenxerto resultados ratos singï¿½icas em renal lesão de reperfusão isquêmica, porém por 4 semanas, os túbulos se recuperaram e são histologicamente comparável à rins nativos. A rejeição aguda pode ser modelado por C57BL / 6 em receptores de transplante de rim de BALB / c, no prazo ...

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.

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 ...

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

Research

JoVE Journal

Medicine

20.4K visualizações.  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.

Vídeo: 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.

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

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models.
Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2. We herein present in addition small animal positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4.

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.

Imagem não invasivo de rejeição do enxerto aguda após Rat Transplante Renal Usando<sup&gt; 18</sup&gt; F-FDG PET

The number of patients with end-stage renal  disease, and the number of kidney allograft recipients continuously increases.  Episodes of acute cellular ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

De alta frequência Ecografia de mouse linfonodos cervicais

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

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 ...

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.

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 ...

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 ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function in diabetic nephropathy and podocyte-specific VEGF-A knock-out mice; therefore, this protocol describes the full kidney work-up used in our lab to assess these mouse models of glomerular disease, enabling a vast amount of information regarding kidney and glomerular function to be obtained from a single mouse. In comparison to alternative methods presented in the literature to assess glomerular function, the use of the method outlined in this paper enables the glomerular phenotype to be fully evaluated from multiple aspects. By using this method, the researcher can determine the kidney phenotype of the model and assess the mechanism as to why the phenotype develops. This vital information on the mechanism of disease is required when examining potential therapeutic avenues in these models. The methods allow for detailed functional assessment of the glomerular filtration barrier through measurement of the urinary albumin creatinine ratio and individual glomerular water permeability, as well as both structural and ultra-structural examination using the Periodic Acid Schiff stain and electron microscopy. Furthermore, analysis of the genes dysregulated at the mRNA and protein level enables mechanistic analysis of glomerular function. This protocol outlines the generic but adaptable methods that can be applied to all mouse models of glomerular disease.

This protocol describes a full kidney work-up that should be carried out in mouse models of glomerular disease. The methods allow for detailed functional, structural, and mechanistic analysis of glomerular function, which can be applied to all mouse models of glomerular disease.

Avaliação da função renal em modelos do rato da doença Glomerular

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...

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 ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) imaging. They have received great attention in all research areas for the potential to analyze microscopic multicellular structures that extend over macroscopic distances. Given that kidney tubules, vasculature, nerves, and glomeruli extend in many directions, which have been only partially captured by traditional two-dimensional techniques so far, tissue clearing also opened up many new areas of kidney research. The list of optical clearing methods is rapidly growing, but it remains difficult for beginners in this field to choose the best method for a given research question. Provided here is a simple method that combines antibody labeling of thick mouse kidney slices; optical clearing with cheap, non-toxic and ready-to-use chemical ethyl cinnamate; and confocal imaging. This protocol describes how to perfuse kidneys and use an antigen-retrieval step to increase antibody- binding without requiring specialized equipment. Its application is presented in imaging different multicellular structures within the kidney, and how to troubleshoot poor antibody penetration into tissue is addressed. We also discuss the potential difficulties of imaging endogenous fluorophores and acquiring very large samples and how to overcome them. This simple protocol provides an easy-to-setup and comprehensive tool to study tissue in three dimensions.

The combination of antibody labeling, optical clearing, and advanced light microscopy allows three-dimensional analysis of complete structures or organs. Described here is a simple method to combine immunolabeling of thick kidney slices, optical clearing with ethyl cinnamate, and confocal imaging that enables visualization and quantification of three-dimensional kidney structures.

Esclarecimento ótico e imagem latente do tecido Immunolabeled do rim

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...

Digital Home-Monitoring of Patients after Kidney Transplantation: The MACCS Platform

The MACCS (Medical Assistant for Chronic Care Service) platform enables secure sharing of key medical information between patients after kidney transplantation and physicians. Patients provide information such as vital signs, well-being, and medication intake via smartphone apps. The information is transferred directly into a database and electronic health record at the kidney transplant center, which is used for routine patient care and research. Physicians can send an updated medication plan and laboratory data directly to the patient app via this secure platform. Other features of the app are medical messages and video consultations. Consequently, the patient is better-informed, and self-management is facilitated. In addition, the transplant center and the patient's local nephrologist automatically exchange notes, medical reports, laboratory values, and medication data via the platform. A telemedicine team reviews all incoming data on a dashboard and takes action, if necessary. Tools to identify patients at risk for complications are under development. The platform exchanges data via a standardized secure interface (Health Level 7 (HL7), Fast Healthcare Interoperability Resources (FHIR)). The standardized data exchange based on HL7 FHIR guarantees interoperability with other eHealth solutions and allows rapid scalability to other chronic diseases. The underlying data protection concept is in concordance with the latest European General Data Protection Regulation. Enrollment started in February 2020, and 131 kidney transplant recipients are actively participating as of July 2020. Two large German health insurance companies are currently funding the telemedicine services of the project. The deployment for other chronic kidney diseases and solid organ transplant recipients is planned. In conclusion, the platform is designed to enable home monitoring and automatic data exchange, empower patients, reduce hospitalizations, and improve adherence, and outcomes after kidney transplantation.

The MACCS platform&#160;is a comprehensive telemedicine concept aiming at better outcomes after kidney transplantation by sharing key medical information between patients and physicians. A telemedicine team reviews incoming data to detect potential complications and to improve adherence in kidney transplant recipients to achieve better long-term outcomes.