Name: rat heterotopic abdominal heart/single-lung transplantation in a volume-loaded configuration
Uploaded: 2015-05-29T13:00:00.000+00:00
Duration: 6 min 16 s
Description: Watch this Scientific Journal Video about Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration at JoVE.com

Mark J. Kearns recebe o apoio do Programa UBC Clínico Investigator (concedidos a partir do Royal College of Physicians e Surgeons of Canada), ea UBC 4YF Doutorado Fellowship. John H. Boyd é um Sanitorium Associação Nacional e Michael Smith Fundação para a Pesquisa de Saúde Escolar. Financiamento através CIHR. Os autores gostariam de agradecer ao Dr. M. Allard e Richard Wamboldt por sua assistência com a instalação e manutenção de equipamentos de perfusão.

Todos os animais foram alojados e tratados em conformidade com as diretrizes institucionais para o cuidado e uso de animais de laboratório e Nacional. Aprovação ética para este protocolo foi concedido pela Universidade do Comitê Animal Care da British Columbia. Macho, ratos Sprague-Dawley machos pesando entre 300-450 g foram usados ​​para este protocolo. 1. Doador de Operação <ol><li> Possui cerca de 100 ml de solução cardioplégica (RT) em um balão cilíndrico ligado ao longo intravenosa (IV) por um tubo de cateter torneira de 3 vias. Use um suporte para elevar o balão a cerca de 80 centímetros acima da superfície de trabalho, permitindo a entrega cardioplegia por gravidade. </li><li> Para visualizar estruturas adequadamente, use um par de lupas cirúrgicas ou um microscópio de dissecação. NOTA: Atualmente usamos um microscópio cirúrgico binocular com 3.4 - ampliação 21.3X. </li><li> Coloque o doador em uma câmara de anestésico e induzir a anestesia com 4-5% de isoflurano. </li&gt; <li> Transferir o rato a uma plataforma de operação e manutenção da anestesia através do nariz-cone com 1-2% de isoflurano. Aplicar veterinário pomada para os olhos do animal para prevenir o ressecamento. Administrar o midazolam (2 mg / kg), intraperitonealmente com uma agulha G 25. </li><li> Usando cortadores cirúrgicos, raspar o doador do processo xifóide do esterno a mandíbula. Raspar uma pequena região da virilha esquerda (para acesso subsequente IV). Aplicar um agente para depilar as superfícies operativas, esperar cerca de 5 min, e remover o cabelo com um pedaço de gaze. </li><li> Prepare os locais cirúrgicos com uma solução baseada em clorexidina iodopovidona ou (nós usamos somente clorexidina). Infiltrar nos sites incisional com 0,1-0,5% de lidocaína por via subcutânea. </li><li> Obtenha os membros dianteiros e membro esquerdo para a plataforma operacional com fita adesiva, deixando o membro posterior direito livre para monitoramento de profundidade anestésica e os sinais vitais. </li><li> Depois de assegurar a profundidade da anestesia adequada por pedal pitada, fazer uma incisão no sulco virilha esquerda usandoum bisturi 22-lâmina. Por dissecção romba, expor a veia femoral comum esquerda e obter acesso IV como se segue: <ol><li> Suavemente retrair o tecido que cobre o veia femoral, e canular a veia com um 24 g IV. Conecte o IV para um curto período de IV tubulação cheio de solução salina, e fixar o tubo no lugar com fita adesiva. NOTA: O procedimento de punção venosa femoral em ratos é descrito em outros lugares por Jespersen e colegas 14. </li><li> Ligar uma seringa de 10 ml cheio de solução salina para a tubagem IV, e suavemente aspirar sangue para garantir o posicionamento correcto do IV. </li><li> Injectar 300-500 UI de heparina não fraccionada através da tubagem IV, e, subsequentemente, lavar a tubagem com 3-5 ml de solução salina. </li></ol></li><li> Em seguida, o doador tracheotomize como se segue: <ol><li> Adicione uma incisão na linha média em tecidos moles entre o entalhe jugular e da mandíbula, utilizando um bisturi 22-lâmina. Penetrar a cápsula da glândula tireóide na linha média usando Metzentesoura Baum, e separados seus lóbulos usando dissecção romba. </li><li> Utilizando uma dissecção romba, separar os músculos do pescoço cinta na linha média para expor a superfície anterior da traqueia. </li><li> Use uma pinça curva sem rodeios para dissecar um plano circunferencial em torno da traquéia. Cercar a traqueia com um laço de seda 4-0. </li><li> Usando uma tesoura de íris, fazer uma incisão transversal na traquéia anterior, aproximadamente 5 mm inferior à cartilagem tireóide. Suavemente introduzir a cânula traqueal (a 14 G IV) e fixá-lo no lugar usando o laço de seda 4-0. </li><li> Conecte a cânula traqueal a um ventilador mecânico. Redirecionar o fluxo de oxigênio e isoflurano através do circuito do ventilador, e ventilar o doador a um ritmo e volume corrente previsto pelo seu peso 13. </li></ol></li><li> Realizar uma incisão na linha média do tórax (usando um bisturi 22-lâmina), estendendo-se a incisão na incisura jugular para um valor inferior a xipisternum. </li><li> Mantendo-se na linha média, Executar uma esternotomia mediana usando um cortador de osso. Retrair as bordas do esterno com uma válvula de auto-retenção. Digite o pericárdio e cavidade pleural. </li><li> Executar uma thymectomy. É mais fácil de dividir primeiro sem rodeios o timo na linha mediana, e então separá-lo de estruturas adjacentes usando uma combinação de dissecção brusca e precisa. NOTA: A origem das artérias torácicas internas pode ser lesado quando dissecando o timo longe das bordas do esterno superiores. Para prevenir o sangramento, clipes hemostáticos pode ser aplicado antes de retirar o timo nesses pontos. </li><li> Com uma tesoura Metzenbaum e / ou um Lauer afiada, dissecar peri-cava gordura longe da veia cava inferior (VCI). Certifique-se de que a VCI é relativamente livre de gordura e tecido conjuntivo a partir da junção cavo-atrial superiormente, para o diafragma inferiormente. </li><li> Usando um Lauer afiada, livre circunferencial da veia cava superior (SVC) e cercar-lo com um laço de seda 4-0. </li><li> Em seguida, dissecar odeixou veia cava livre de estruturas adjacentes, e ligar-lo proximal e distal com 4-0 gravatas de seda. Ressecar a parte interveniente da cava para expor a artéria subclávia esquerda. </li><li> Circunferencial livre os vasos do arco aórtico usando um Lauer afiado. Aplicar clips cirúrgicos proximal e distal à artéria inominada, e dividi-lo entre os clipes. Deixe a artéria carótida comum esquerda e un-cortado artéria subclávia esquerda. </li><li> Em seguida, canular a SVC com um cateter 24 G IV. Fixe o cateter com o anteriormente colocado 4-0 gravata de seda. </li><li> Prepare-se para uma colheita do enxerto expediente, mas suave. </li><li> Usando uma tesoura afiada, dividir o IVC apenas superior à superfície diafragmática. Divida o SVC superior ao seu sítio de punção, assegurando que as estruturas das vias aéreas próximas não estão feridos. </li><li> Desligue o ventilador e retire a cânula traqueal. Transecto a traqueia proximal. </li><li> Segure a traquéia seccionado com uma pinça, e remover o coraçãoe pulmões em blocos. Isso vai exigir tração suave com dissecção afiada como o enxerto de coração-pulmão é removido. Separa-se a traqueia a partir do esófago subjacente. Evitar ferir a aorta descendente, de modo que uma grande parte dele permanece intacto após a colheita. </li></ol> 2. Preparação do Coração-pulmão Graft NOTA: ao completar esta parte do procedimento, o assistente, deve ser anestesiar o roedor destinatário e se preparando para a implantação do enxerto. <ol><li> Inserir o enxerto de coração-pulmão de gaze cirúrgica com a superfície anterior do coração voltado para baixo. Usando dissecção afiada, remover qualquer esôfago residual, evitando prejuízo para as vias respiratórias posteriores. </li><li> Localize a aorta torácica descendente e inserir um 16 G de ponta romba cânula. Segurar a cânula no lugar com uma gravata de seda 4-0. </li><li> Localize os vasos do arco aórtico, e aplicar um grampo cirúrgico da artéria subclávia esquerda. Deixe a artéria carótida esquerda un-cortado para a subsequent de-aeração. </li><li> Administrar a primeira dose de cardioplegia, ligando o tubo de cardioplegia à cânula aórtica 16 L. A artéria carótida deve ser temporariamente fechada com uma pinça para garantir a entrega adequada de cardioplegia. NOTA: Uma vantagem de ter a cânula aórtica ligada é que cardioplegia pode ser administrada de forma contínua e / ou intermitente, como desejado. Para dosagem intermitente, que normalmente entregar cardioplegia cada 10 - 15 minutos em 5 ml bolus sobre 30 - 45 seg. </li><li> Com uma tesoura Metzenbaum e / ou um Lauer afiada, dissecar a gordura peri-aórtico longe da aorta. Certifique-se de que a aorta é relativamente livre de gordura e tecido conjuntivo a partir da artéria subclávia esquerda (arco distal) ao seu sítio de punção. </li><li> Em seguida, expor o brônquio fonte esquerdo usando um Lauer afiada, e ligar-lo proximamente com um laço de seda 4-0. Transecto a distal brônquio esquerdo para a ligadura da íris usando uma tesoura. Executar uma pneumonectomia esquerda ligando o pulmonar esquerdaartéria e veia com 4-0 gravatas de seda. Transecto distal à ligadura e remover o pulmão esquerdo. </li><li> Como acima, remover todos os outros lobos pulmonares com exceção dos lobos superior e médio direito. Evitar ferir o IVC no processo de realização de lobectomia. </li><li> Ligue a tubagem de cardioplegia à cânula aórtica e administrar cardioplegia contínua, enquanto o destinatário está sendo preparado. Inserir o enxerto de coração-pulmão, num recipiente estéril (por exemplo, um copo). </li></ol> 3. Destinatário Operação <ol><li> Anestesiar destinatário de acordo com a operação de dador, acima. Use veterinário pomada para proteger os olhos do animal de secura. Confirme anestesia com freqüência por pedal pitada. </li><li> Posicione o animal de acordo com a operação doador, desta vez deixando o membro anterior direito livre para monitorar os sinais vitais e profundidade anestésica. </li><li> Raspar o abdômen do processo xifóide do esterno ao pênis. Raspar uma pequena região da virilha esquerda (para acesso subsequente IV). Aplique umaagente para depilar as superfícies operativas, esperar cerca de 5 min, e remover o cabelo com um pedaço de gaze. </li><li> Prepare os locais cirúrgicos com um iodopovidona ou solução baseada clorexidina. Infiltrar nos sites incisional com 0,1-0,5% de lidocaína por via subcutânea. </li><li> Tracheotomize e ventilar o destinatário como indicado acima. Como alternativa, manter o destinatário sob anestesia nariz-cone. </li><li> Introduzir um cateter na veia femoral como descrito acima. Injectar 300-500 UI de heparina não fraccionada através da tubagem IV, e, subsequentemente, lavar a tubagem com 3-5 ml de solução salina. </li><li> Executar uma laparotomia fazendo uma incisão na linha média abdominal com um bisturi 22-blade de processo xifóide do esterno ao pênis. Retrair a parede abdominal utilizando um afastador auto-retenção. Em seguida, retirar as entranhas superiormente e para o lado esquerdo do rato. Envolvê-los em, gaze embebida com solução salina quente. </li><li> Expor a aorta abdominal IVC e por acentuadamente dissecando através da re sobrepostagordura tro-peritoneal. </li><li> Ter uma braçadeira navio curvo disponível e pronto. </li><li> Circunferencial libertar a VCI e aorta proximal e distal e cercar-los com laços de seda 4-0. Não deve ser de aproximadamente 2-3 cm de espaço entre estes sítios. </li><li> Cuidadosamente aplicar o grampo vaso curvo, assegurando que uma porção suficiente de ambos VCI e aorta são expostos por cima das maxilas da peça de aperto. </li><li> Realizar uma incisão na parede anterior da VCI com uma agulha de 25 G ligada a uma seringa de 1 ml cheio de solução salina. Estender a incisão com tesoura de Potts para corresponder ao comprimento do dador VCI orifício. </li><li> Remova o enxerto de coração-pulmão de seu recipiente e desconecte-o cardioplegia. Coloque o enxerto no abdômen do destinatário em uma posição ideal para realizar a anastomose venosa. NOTA: O enxerto de coração-pulmão acabará por ser orientada ligeiramente oblíqua, com o ápice apontando para as abdômen do quadrante inferior esquerdo. </li><li> Prenda o calcanhare dedo do pé termina da anastomose com 9-0 nylon de sutura. Dê um nó segura em cada extremidade, deixando a agulha ligada a um braço longo do fio de sutura e uma extremidade curta do fio de sutura para ser ligada a mais tarde. Administrar uma dose de cardioplegia. </li><li> Na gestão de moda, completa a metade da linha de sutura e gravata para o braço adversária sutura curto. Administrar uma dose de cardioplegia. </li><li> Complete a outra metade da linha de sutura e amarrá-lo para baixo. A anastomose venosa é completa. Administrar uma dose de cardioplegia </li><li> Orient o enxerto de coração-pulmão com apontando o ápice do coração em direção aos abdômen do quadrante inferior esquerdo. Certifique-se de que a anastomose venosa não está dobrada ou torcida. </li><li> Avaliar o comprimento da aorta do doador, que será necessária para alcançar a aorta do receptor, mantendo tanto IVC e anastomose da aorta em configurações livre de tensão e desdobrado. </li><li> Divida a aorta descendente (distal à artéria subclávia esquerda) com uma tesoura de íris. </li><li> Realizar uma incision na parede anterior da aorta do receptor com uma agulha de 25 G ligada a uma seringa de 1 ml cheio de solução salina. Estender a incisão com tesoura de Potts para corresponder ao comprimento do orifício aórtico dador. </li><li> Ancorar o dedo do pé (aspecto superior) da aorta dador até ao receptor aorta utilizando uma sutura 9-0 nylon. Em seguida, passe a agulha para a face medial da aorta e completar metade da anastomose na execução de moda. </li><li> No calcanhar da anastomose da aorta, inverter a orientação da linha de sutura (uma transição na direcção de costura), e concluir a metade lateral da anastomose no funcionamento da forma. </li><li> Lentamente remover o cateter IV na SVC, e ligar o SVC com um grampo cirúrgico. </li><li> De-ar da aorta, localizando a artéria carótida comum esquerda un-cortado. Segurar a artéria aberta, em posição vertical, que vai permitir que o ar a ser evacuado livremente. </li><li> Resumidamente abrir as maxilas do grampo vaso curvo e permitir que a artéria carótida para sangrar (de-air) para 2-3 sec. Reaplicar o grampo navio. </li><li> Aplique um grampo cirúrgico à artéria carótida. Remover o grampo vaso curvo. </li><li> Verifique se há sangramento nas linhas de sutura. Se estiver presente, aplicar compressão suave com gaze cirúrgica ou de reparação com um comprimento de 9-0 sutura de nylon (dependendo da gravidade do sangramento). </li><li> O coração do doador deve retomar batendo em poucos minutos. </li><li> Preste atenção aos sinais vitais do destinatário, profundidade da anestesia, reanimação e administrar o volume conforme necessário. </li><li> Dependendo dos endpoints experimentais, quer fechar abdômen do animal e deixá-lo recuperar da anestesia, ou preparar o animal para avaliação cardiovascular. NOTA: Exemplos de avaliação de enxerto incluem em medidas in vivo de hemodinâmica dependente da carga e independente de carga, in vitro medidas de função em Langendorff e modos coração de trabalho, e (em cirurgia sobrevivência) ecocardiográficos ou inquéritos de ressonância magnética. </li><li> Nofinal de experiências agudas de terminais, os animais são sacrificados por exsanguinação. </li></ol>

<ol>
	<li>Abbott, C. P., Lindsey, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+for+heart+transplantation+in+the+rat.">A technique for heart transplantation in the rat.</a> Arch Surg. 89 (1964), 649-652 (1964).</li><li>Ma, Y., Wang, G. <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+2+heterotopic+heart+transplant+techniques+in+rats:+cervical+and+abdominal+heart.">Comparison of 2 heterotopic heart transplant techniques in rats: cervical and abdominal heart.</a> Exp Clin Transplant. 9 (2), 128-133 (2011).</li><li>Wiedemann, D., Boesch, F., Schneeberger, S., Kocher, A., Laufer, G., Semsroth, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft+function+after+heterotopic+rat+heart+transplant+with+an+isolated+reperfused+working+heart:+a+methodic+consideration.">Graft function after heterotopic rat heart transplant with an isolated reperfused working heart: a methodic consideration.</a> Exp Clin Transplant. 10 (2), 154-157 (2012).</li><li>Chen, Z. H., Xia, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+technique+of+heterotopic+heart-lung+transplantation+in+the+rat.">The technique of heterotopic heart-lung transplantation in the rat.</a> J Tongji Med Univ. 6 (2), 67-70 (1986).</li><li>Ibrahim, M., Navaratnarajah, M., 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=Heterotopic+abdominal+heart+transplantation+in+rats+for+functional+studies+of+ventricular+unloading.">Heterotopic abdominal heart transplantation in rats for functional studies of ventricular unloading.</a> J Surg Res. 179 (1), e31-e39 (2013).</li><li>Asfour, B., Hare, J. M., 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=A+simple+new+model+of+physiologically+working+heterotopic+rat+heart+transplantation+provides+hemodynamic+performance+equivalent+to+that+of+an+orthotopic+heart.">A simple new model of physiologically working heterotopic rat heart transplantation provides hemodynamic performance equivalent to that of an orthotopic heart.</a> J Heart Lung Transplant. 18 (10), 927-936 (1999).</li><li>Figueiredo, J. -. L., Nahrendorf, M., Sosnovik, D. E., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+of+a+novel+murine+working+heart+transplant+model.">MRI of a novel murine working heart transplant model.</a> Circ Heart Fail. 2 (3), 272-274 (2009).</li><li>Wen, P., Wang, X., 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=A+simple+technique+for+a+new+working+heterotopic+heart+transplantation+model+in+rats.">A simple technique for a new working heterotopic heart transplantation model in rats.</a> Transplant Proc. 45 (6), 2522-2526 (2013).</li><li>Didi&#233;, M., Biermann, D., 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=Preservation+of+left+ventricular+function+and+morphology+in+volume-loaded+versus+volume-unloaded+heterotopic+heart+transplants.">Preservation of left ventricular function and morphology in volume-loaded versus volume-unloaded heterotopic heart transplants.</a> A.Am J Physiol Heart Circ Physiol. 305 (4), H533-H541 (2013).</li><li>Gali&#241;anes, M., Zhai, X., Hearse, D. 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O sucesso com a técnica descrita aqui vai ser em função de diversos factores. O principal entre eles será assegurar a estabilidade de ambos os animais doadores e receptores, adotando a técnica operatória meticulosa que é seguro e associado com a perda de sangue mínima, garantindo parada cardioplégica completo com arrefecimento uniforme enxerto, minimizando o tempo de isquemia total, e de forma adequada de-arejar o enxerto. Como reconheceu acima, a complexidade técnica da técnica é a sua limitação chefe. </...

The first rodent model of heterotopic abdominal heart transplantation (HAHT) was described by Abbott and colleagues in 19641. This technique, and subsequent modifications have been widely applied to characterize transplant graft function and immunologic status. The majority of HAHT techniques described involve a non-volume loaded heart2,3. Models of HAHT involving volume-loaded ventricles have been described, but they are frequently limited in one or more respects.
Heterotopic abdominal heart-lung transplantation (HAHLT) with a volume-loaded left ventricle (LV) has been described previously. Chen and colleagues4, and subsequently Ibrahim and colleagues5 described HAHLT with a single aorto-aortic (donor ascending to recipient abdominal aorta) anastomosis. The only volume load presented to the ventricle in this circulation is the coronary venous return. Asfour and colleagues described a HAHT technique in which the lung circuit was eliminated by anastomosing donor pulmonary artery (PA) to donor left atrium (LA)6. In this circulation, venous inflow to right ventricle (RV) occurs via a donor SVC to recipient IVC anastomosis, and the subsequent LV load is ejected into the aorto-aortic anastomosis. Cardiac function was partially assessed in vivo, and also in vitro using a Langendorff rig. Figueiredo and colleagues described a HAHLT model similar to our own7, but in mice. Venous inflow to the RV occurs via donor SVC to recipient IVC anastomosis. Blood subsequently passes through the single lung circulation and LV load is ejected into the aorto-aortic anastomosis. Cardiac function in their study was assessed by magnetic resonance imaging (MRI). Wen and colleagues described a unique HAHT technique in which the LV is loaded by means of a recipient aorta to donor LA anastomosis8. The LV, therefore, fills at systemic pressures. Cardiac function, and whether LV stroke volume is ejected antegradely in their model was not assessed.
Many of the techniques referenced above involve non-physiologic LV loading conditions, including the techniques whose partial LV load is represented only by coronary venous return. On the other hand, many techniques do approach physiologic LV loading. The majority of these techniques, as with the technique of Asfour and colleagues, omit the pulmonary circulation and utilize a donor PA to donor LA anastomosis6,9. The circulation described by Galinanes and colleagues10 employs a direct recipient cava to donor LA anastomosis, omitting the pulmonary circulation and the right heart. Yokoyama and colleagues achieve the same effect by ligating the donor PA and creating an interatrial communication in the donor heart (omitting donor lung and right heart circulations)11. The circulation of Maruyama and colleagues12 involves an anastomosis between donor left PA and recipient Ao, which permits LV filling via the pulmonary circulation as a conduit, but effectively excludes the right heart.
In cases where near physiologic loading conditions were met, we advance the technique of HAHLT in 2 major respects. First, to our knowledge, the exact configuration we report has not been described in rats. It is possibly the most versatile circulation for investigators wishing to study the physiology, structure, and immunology of the transplanted heart-lung graft. Second, we describe how the function of the transplant graft can be directly characterized in vivo. For this application, pressure-volume conductance catheters can be introduced directly into the LV apex of the transplant graft, which allows for complete cardiac functional characterization. 
The technique described here can be applied to both acute and chronic studies of transplant graft function, while the functional assessment may be performed either in vivo or in vitro. We present a model in which the loading conditions can be near physiologic, however the degree of ventricular loading may be manipulated both acutely and chronically by diverting venous return towards or away from the graft. Afterload conditions can also be manipulated. Because the lung and its airway are retained in this transplant configuration, investigators can re-ventilate the donor lung acutely. Uniquely, lung re-ventilation changes the composition of blood perfusing the transplant coronary arteries. Under non-ventilated conditions, blood ejected from the donor aorta is deoxygenated, and mixes with oxygenated blood in the recipient aorta. Under acutely ventilated conditions, ejected blood becomes oxygenated. Thus, transplant graft function can be compared under ventilated and non-ventilated conditions, and also under variably loaded conditions.
The protocol below describes important modifications to previously described HAHLT donor and recipient operations. It also describes an optimal technique for protecting the transplant graft throughout the period of ischemia (time between donor explant and recipient implant). Advantages of this technique include physiologic conditions potentially approaching that of an orthotopically transplanted graft, and a wide range of investigative applications. An important limitation is its technical complexity. With adequate mentoring and practice, the advantages of this technique will likely outweigh the challenges in adopting it.

<table><tbody><tr><td>Celsior Cardioplegic Soution</td><td>Genzyme</td><td></td><td>The solution is kept on ice throughout the procedure. We prepare our own solution, with slight modifications.</td></tr><tr><td>Rodent Ventilator</td><td>Harvard Apparatus</td><td>Model 683</td><td></td></tr><tr><td>Vital Sign Monitor</td><td>Nonin</td><td>Model 9847V</td><td>Displays SpO&lt;sub&gt;2&lt;/sub&gt; and heart rate.</td></tr><tr><td>IV Cannulae</td><td>Jelco</td><td>3063</td><td>24 - 26 G x 3/4&quot; cannulae.</td></tr><tr><td>IV Tubing</td><td>CareFusion</td><td>MP9259-C</td><td>Short-length connector tubing (18 cm).</td></tr><tr><td>Surgical Clips</td><td>Teleflex Medical</td><td>001204</td><td>Horizon titanium ligating clips.</td></tr><tr><td>Sutures</td><td>Ethicon, Sharpoint</td><td>LA54G, AK-0107</td><td>3-0 silk reel, and 9-0 prolene suture (single-armed, DR5 needle).</td></tr><tr><td>Surgical Instruments</td><td>Not Applicable</td><td>Not Applicable</td><td>The instruments used are generic, and can be purchased from any surgical supply company.</td></tr></tbody></table>

rat heterotopic abdominal heart/single-lung transplantation in a volume-loaded configuration

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft involves anastomosis of donor inferior vena cava (IVC) to recipient IVC, and donor ascending aorta (Ao) to recipient abdominal Ao. The right upper and middle lung lobes are preserved and function as conduits for blood flow from right heart to left heart.
There are several advantages to using this technique, and it lends itself to a broad range of applications. Because the graft is transplanted in a configuration that allows for dyamic volume-loading, cardiac function may be directly assessed in vivo. The use of pressure-volume conductance catheters permits characterization of load-dependent and load-independent hemodynamic parameters. The graft may be converted to a loaded configuration by applying a clamp to the recipient&#8217;s infra-hepatic IVC. We describe modified surgical techniques for both donor and recipient operations, and an ideal myocardial protection strategy. Depending on the experimental aim, this model may be adapted for use in both acute and chronic studies of graft function, immunologic status, and variable ventricular loading conditions. The conducting airways to the transplanted lung are preserved, and allow for acute lung re-ventilation. This facilitates analysis of the effects of the mixed venous and arterial blood providing coronary perfusion to the graft.
A limitation of this model is its technical complexity. There is a significant learning curve for new operators, who should ideally be mentored in the technique. A surgical training background is advantageous for those wishing to apply this model. Despite its complexity, we aim to present the model in a clear and easily applicable format. Because of the physiologic similarity of this model to orthotopic transplantation, and its broad range of study applications, the effort invested in learning the technique is likely to be worthwhile.

A técnica HAHLT descrito acima é altamente técnica e requer muita atenção aos detalhes. Tabela 1 destaca alguns dos principais fatores associados ao sucesso contra procedimentos mal sucedidos, e pode ser usado como um guia para a resolução de problemas dificuldades técnicas. Após a aorta do receptor está desapertada, as artérias coronárias de enxerto deve ser visto a encher com sangue oxigenado. Assim, o miocárdio deve tornar-se rosa e bem perfundido. Em experim...

Watch this Scientific Journal Video about Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration at JoVE.com

Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft.

Rat heterotópico abdominal coração / pulmão Transplante Single-carregado em uma configuração-Volume

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft ...

rat-heterotopic-abdominal-heartsingle-lung-transplantation-volume

Research

JoVE Journal

Medicine

8.3K visualizações.  UBC James Hogg Research Center, St. Paul's Hospital. We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft.

Vídeo: Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

Transplante cardíaco heterotópico em ratos

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Biologia

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

Heterotópico Rat auxiliar Liver Transplantation com fluxo regulado Portal Vein Arterialização na Insuficiência Hepática Aguda

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

Medicina

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 ...

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.

We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.

Um modelo imunológico para transplante heterotópico de células cardíacas e cardíacas em ratos

Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications ...

Imunologia e Infecção

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

Reconstrução do circuito sanguíneo em um modelo de transplante de coração de rato abdominal

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.

Um modelo simplificado para transplante de válvula cardíaca heterotópica em roedores

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

Técnica de Cuff Modificada para Modelo de Transplante Cardíaco Heterotópico Cervical:

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...

A Heterotopic Rat Heart Transplantation Model using Circulatory Death Donor Hearts

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There are two setups for this protocol: heart donor setup and recipient setup. In the heart donor setup, Sprague Dawley rats are anesthetized, endotracheally intubated, and ventilated. The right carotid artery is cannulated to deliver heparin and the paralytic agent vecuronium-bromide. The DCD process is initiated by terminating the ventilation. After 20 min, the heart is exposed and the aorta distal to the brachiocephalic branch is clamped. At 25 min from terminating the ventilator, ice-cold University of Wisconsin (UW) solution is perfused through the carotid catheter to flush the heart. The heart is procured by dividing the aorta, pulmonary artery, venae cavae, and pulmonary veins and stored in UW solution for implantation. In the recipient setup, the Lewis rat is anesthetized with isoflurane. Slow-release buprenorphine is administered subcutaneously to facilitate a smooth postoperative recovery. Through a midline abdominal incision, the infra-renal aorta and the inferior vena cava are isolated and clamped with an atraumatic vascular clamp. The donor heart aorta and pulmonary artery are sutured to the recipient abdominal aorta and vena cava, respectively, with a running 8-0 Prolene. The vascular clamp is removed to reperfuse the heart. The abdominal wall is closed and the rat is recovered. After a set interval (24 h to 2 weeks), the recipient rat is anesthetized, the transplanted heart is exposed, and a balloon-tip-catheter is inserted into the left ventricle via the apex to record developed pressure and dP/dt using a data acquisition system. The heart tissue is collected for histology, immunology, or molecular analysis. A successful DCD donor rat heart transplantation model will allow further studies on the cardioprotective approaches to improve heart transplantation outcomes from DCD donors.

Here, we describe the preparation and technical details of murine heterotopic heart transplantation utilizing a circulatory death donor heart.

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There ...

Rat Model of Normothermic Ex-Situ Perfused Heterotopic Heart Transplantation

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the number of heart failure patients waiting for transplantation is still increasing. The normothermic ex situ preservation technique has been established as a comparable method to the conventional static cold storage technique. The main advantage of this technique is that donor hearts can be preserved for up to 12 h in a physiologic condition. Moreover, this technique allows resuscitation of the donor hearts after circulatory death and applies required pharmacologic interventions to improve donor function after implantation. Numerous animal models have been established to improve normothermic ex situ preservation techniques and eliminate preservation-related complications. Although large animal models are easy to handle compared to small animal models, it is costly and challenging. We present a rat model of normothermic ex situ donor heart preservation followed by heterotopic abdominal transplantation. This model is relatively cheap and can be accomplished by a single experimenter.

Here, we present an assessment protocol of a heterotopically implanted heart after normothermic ex situ preservation in the rat model.

Modelo de Transplante Cardíaco Heterotópico Perfundido Normothermic Ex-Situ em Rato

Heart transplantation is the most effective therapy for end-stage heart failure. Despite the improvements in therapeutic approaches and interventions, the ...

Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical technique. Modifying the transplantation procedure to strengthen the myocardial protection could prolong the ischemia time while preserving the donor&#39;s cardiac function. This technique&#39;s key points are as follows: transecting the donor&#39;s abdominal aorta before harvesting to unload the donor&#39;s heart; perfusing the donor&#39;s coronary arteries with a cold cardioplegic solution; and topical cooling of the donor&#39;s heart during the anastomosis procedure. Consequently, since this procedure prolongs the acceptable ischemia time, beginners can easily perform it and achieve a high success rate.
Moreover, a new aortic regurgitation (AR) model was established in this work using a technique different from the existing one,&#160;which is created by inserting a catheter from the right carotid artery and puncturing the native aortic valve under continuous echocardiographic guidance. A heterotopic abdominal heart transplantation was performed using the novel AR model. In the protocol, after the donor&#39;s heart is harvested, a stiff guidewire is inserted into the donor&#39;s brachiocephalic artery and advanced toward the aortic root. The aortic valve is punctured by pushing the guidewire further even after the resistance is felt, thus inducing AR. It is easier to damage the aortic valve using this method than with the procedure described in the conventional AR model. Additionally, this novel AR model does not contribute to the recipient&#39;s circulation; therefore, this method is expected to produce a more severe AR model than the conventional procedure.

This study demonstrates a reproducible heterotopic abdominal heart transplantation technique in rats that beginners can learn and perform. Additionally, a novel aortic regurgitation model in rats is generated by performing heterotopic abdominal heart transplantation and damaging the donor's aortic valve using a guidewire after harvesting.

Transplante Cardíaco Heterotópico Abdominal Modificado e um Novo Modelo de Regurgitação Aórtica em Ratos

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical ...