The study was supported by research grants from the Roche Organ Transplant Research Foundation (ROTRF) and Astellas. Markus Selzner was supported by an ASTS Career Development Award. Matthias Knaak was supported by the Astellas Research Scholarship. We thank Uwe Mummenhoff and the Birmingham family for their generous support.

<ol>
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The authors have nothing to disclose.

Experimental suína transplante hepático ortotópico é um procedimento desafiador para um ambiente de pesquisa, sem os recursos de terapia intensiva de um cenário clínico. Complicações possíveis incluem instabilidade hemodinâmica, hemorragia, isquemia de órgãos, hipotermia, e metabólica, bem como respiratória, descompensação. Para qualquer grupo de pesquisa, formação processual suficiente da técnica cirúrgica 5, bem como a anestesia 14,15 porco é obrigatória a fim de alcançar resultados representativos e reprodutíveis. Muitos subtilezas técnicas têm sido descritos na literatura, especialmente em relação à fase de reconstrução vascular 5. O protocolo de transplante hepático ortotópico descrito acima fornece as informações necessárias para um modelo de substituição de cava que assemelha-se o transplante hepático ortotópico humano. Os resultados fornecidos demonstrar sobrevivência do animal confiável e recuperação do enxerto em ambos os modelos HBD e DCD. O protocolo é aplicável em cenários de sobrevivência de curto prazo utilizados na reperfusão do enxerto experiments, por exemplo, bem como em modelos de sobrevivência a longo prazo, tais como estudos de tolerância. Um grande obstáculo de suíno transplante hepático ortotópico é relativamente pobre tolerância de cava e da veia portal pinçamento. Congestão esplâncnica durante a fase anepática provoca hipertensão venosa capilar e danos que podem levar à isquemia intestinal major e instabilidade hemodinâmica, a ponto de um choque irreversível mesmo após reperfusão de órgãos 7. Desde a veia cava é completamente imersos em parênquima hepático, um procedimento piggy-back-preservando cava não é viável. A oclusão total da veia cava, durante a fase de reconstrução cava prejudica a estabilidade hemodinâmica do porco. Embora alguns relatos mostram que o transplante hepático ortotópico suína pode ser realizado durante a cava e da veia portal oclusão total de menos de 25 min 16,17, uma técnica de bypass porto-cava-jugular para o tempo de reconstrução vascular é a opção mais segura e mais prática 7- 9,18. Em thexperiência e dos autores, uma derivação porto-jugular passivo não é o ideal para manter o hemodinamicamente estável porco durante a fase anepática. O modelo de bypass, incluindo descompressão ativa de ambos cava infrahepatic e veia portal, permite uma fase de reconstrução calma das anastomoses cava e portal supra-hepáticas, mesmo com tempo de pinçamento prolongado devido a complicações imprevistas. Ao contrário do que relatórios anteriores 7, uma esplenectomia não é obrigatória quando o cateter de derivação portal é removido. Ambos artéria e veia esplênicas estão fechados sobre a meio caminho ao longo do comprimento do baço deixando a metade proximal suficientemente perfusão. Complicações como hemorragia ou embolia devido a desvio de desconexão são evitáveis, garantindo que o desvio é colocada com cuidado e protegido adequadamente. Em sobrevivência a longo prazo transplante hepático ortotópico experimentos, a anastomose do ducto biliar é considerado um ponto fraco, devido à sua alta taxa de complicações 19. O tecido biliar é muito frágil e needs cuidado especial quando está sendo manipulado. Muitas técnicas de anastomose diferentes têm sido descritas 5,19. Uma anastomose end-to-end é tecnicamente fácil e associado com o mínimo de complicações 19. Uma sutura contínua com uma agulha de não corte, incluindo grandes secções do tecido conjuntivo peribiliary parece ser superior a um sutura interrompida. O ducto biliar é colocado sob tensão desnecessária quando os pontos únicos de sutura interrompida estão atadas. Isso pode resultar em lágrimas de tecidos e vazamentos biliares consecutivos. O material de sutura - absorvível ou não absorvível - geralmente não é importante, dada a sua duração limitada até que o porco é encerrado. Para os modelos de sobrevivência a longo prazo ao longo de vários meses, suturas absorvíveis - como no transplante hepático ortotópico humano - são preferíveis. Cuidados específicos devem ser tomados com o pós-operatório follow-up. Alimentação suficiente e de fornecimento de fluido, um protocolo de alívio da dor de confiança, e uma imunossupressão adequadaregime são obrigatórios. Para as experiências de longo prazo, a imunossupressão parece particularmente importante. Em comparação com outros mamíferos, os porcos mostram uma surpreendentemente baixa taxa de rejeição imunológica após o transplante hepático ortotópico 20,21. Infiltrações de células redondas são máxima durante a segunda semana após o transplante e diminuir espontaneamente, mesmo sem imunossupressão. Rejeição é raramente a causa da morte após o transplante hepático ortotópico suíno 22. No entanto, mesmo com o protocolo de imunossupressão, envolvendo a administração de esteróides IV e inibidores da calcineurina po mencionado aqui, a rejeição do enxerto é indicado por um ligeiro aumento de transaminases a partir de cerca de 4 dias após o transplante hepático ortotópico e confirmada por aparente campo portal de infiltração de células redondas. Inibidores da calcineurina pode ser dada, quer po 23,24 ou iv 25,26; Ambos os métodos têm desvantagens. Mesmo com ajudas aplicação oral, a quantidade real de atingir o trato gastro-intestinal permanece indefinida. Por outro lado, a infusão intravenosa contínua, emcaneta de um porco com um animal ativo é difícil. Assim, a aplicação iv deve ser realizada como um bolus, o que resulta em picos de concentração de droga elevada, juntamente com os potenciais efeitos tóxicos. No entanto, ambos os métodos de aplicação parece permitir a sobrevivência a longo prazo. Semelhante a um ambiente clínico, recomenda a profilaxia pós-operatório úlcera de estresse. Sangramento pós-operatório de úlceras pépticas é um problema freqüente e pode estar relacionada com uma disfunção hepática 27. Depois de alguns casos de hemorragia digestiva em ambos os grupos transplante hepático ortotópico, os autores começaram a profilaxia regular com pantoprazol e não experimentar qualquer hemorragia gastrointestinal desde então. Manutenção rigorosa de condições estéreis no intra-operatório, comparáveis ​​com as condições de uma sala de cirurgia, clínica e consequentes profilaxia antibiótica, diminui o risco de complicações infecciosas. Em conclusão, este artigo pferece informações práticas para o estabelecimento de um programa de transplante hepático ortotópico suína em um ambiente de pesquisa. Dedicação suficiente, prática, e trabalho de equipa é importante, a fim de diminuir o período de aprendizagem, para produzir resultados fiáveis, e para reduzir os custos e o número de animais de pesquisa.

O transplante hepático (transplante hepático ortotópico) é a única opção de tratamento para pacientes com doença hepática em estágio final ou carcinoma hepatocelular avançado. Para os últimos 25 anos, o número de candidatos na lista de espera tem aumentado gradualmente e agora excede em muito o número de enxertos disponíveis. Na maioria das regiões de transplante, de 20 a 30% dos pacientes em lista de espera para transplante de fígado morrem sem receber um transplante de órgão ou são excluídos devido à progressão da doença. Estratégias para aumentar a lista de dadores e, assim, o número de enxertos disponíveis, são desesperadamente necessários. Critérios de alocação de órgãos estendidas, de preservação do enxerto prolongado, e de indução de tolerância imunológica ainda representam grandes desafios clínicos 1-3. Por isso, a investigação experimental transplante hepático ortotópico é fundamental, a fim de otimizar a prática clínica transplante hepático ortotópico. Porcina transplante hepático ortotópico é um modelo experimental bem estabelecido que se assemelha o transplante hepático ortotópico humana em muitos aspectos, incluindo a Ltamanho iver, anatomia, fisiologia e 4-6. Assim, tornou-se um método experimental padrão em campos de pesquisa, tais como técnicas cirúrgicas, fisiologia, imunologia, preservação e lesão de isquemia-reperfusão. Numerosas técnicas de aquisição de enxerto, hepatectomia destinatário e, em particular, a reconstrução vascular, foram descritos na literatura 5. A escolha da técnica apropriada varia de acordo com a preferência do pesquisador e capacidade técnica. Em contraste com a situação humana, congestão splanchnic durante a fase anepática representa um problema importante no transplante hepático ortotópico porcino. Isquemia intestinal subsequente e dano vascular congestiva pode causar instabilidade hemodinâmica grave, comprometendo a sobrevivência de porco e, assim, o sucesso da experiência 7-9. Portanto, a descompressão intestinal suficiente é obrigatória, especialmente em ambientes experimentais tecnicamente menos refinados. Nósing um ativo shunt porto-cava-jugular para a duração da fase de anepática é uma opção confiável para evitar o congestionamento intestinal. O sistema pode ser usado para as experiências iniciais de reperfusão, bem como cenários de sobrevivência a longo prazo. O protocolo a seguir contém todas as informações para um modelo estável e reprodutível transplante de fígado em suínos, incluindo os contratos fígado do doador, operação destinatário, incluindo hepatectomia e técnicas de reconstrução navio end-to-end, e cuidados pós-operatórios.

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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Propofol 1000mg/100mL</td>
			<td style="width:257px;">Pharmascience Inc.</td>
			<td align="right" style="width:201px;">2244379</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Protamine Sulfate 50mg/5mL</td>
			<td style="width:257px;">Pharmaceutical Partners of Canada Inc.</td>
			<td align="right" style="width:201px;">2139537</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Saline 0.9%, 1L</td>
			<td style="width:257px;">Baxter Corporation</td>
			<td align="right" style="width:201px;">60208</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Sodium Bicarbonate 50 mEq/50mL</td>
			<td style="width:257px;">Hospira Healthcare Corporation</td>
			<td align="right" style="width:201px;">261998</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Solu-Medrol 500mg</td>
			<td style="width:257px;">Pfizer Canada Inc.</td>
			<td align="right" style="width:201px;">2367963</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Tranexamic Acid 1000mg/10mL</td>
			<td style="width:257px;">Pfizer Canada Inc.</td>
			<td align="right" style="width:201px;">2064413</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">University of Wisconsin Solution, SPS-1</td>
			<td style="width:257px;">Organ Recovery Systms</td>
			<td style="width:201px;">SPS-1</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Xylocaine Endotracheal 10mg/50mL</td>
			<td style="width:257px;">AstraZeneca</td>
			<td align="right" style="width:201px;">2003767</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Appose ULC 35W skin stapler</td>
			<td style="width:257px;">Covidien Canada</td>
			<td align="right" style="width:201px;">803712</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Maxon, 1</td>
			<td style="width:257px;">Covidien Canada</td>
			<td align="right" style="width:201px;">606173</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Sofsilk, 0</td>
			<td style="width:257px;">Covidien Canada</td>
			<td style="width:201px;">S606</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Sofsilk, 2-0</td>
			<td style="width:257px;">Covidien Canada</td>
			<td style="width:201px;">S405</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Sofsilk, 3-0</td>
			<td style="width:257px;">Covidien Canada</td>
			<td style="width:201px;">S404</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Surgipro II, 4-0</td>
			<td style="width:257px;">Covidien Canada</td>
			<td style="width:201px;">VP581X</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Surgipro II, 5-0</td>
			<td style="width:257px;">Covidien Canada</td>
			<td style="width:201px;">VP725X</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Surgipro II, 6-0</td>
			<td style="width:257px;">Covidien Canada</td>
			<td style="width:201px;">VP733X</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;"></td>
			<td style="width:257px;"></td>
			<td style="width:201px;"></td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Catheter i.v, 18 G</td>
			<td style="width:257px;">BD Canada</td>
			<td align="right" style="width:201px;">381147</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Cook TPN catheter, 9.5Fr</td>
			<td style="width:257px;">Cook Medical Company</td>
			<td style="width:201px;">C-TPNS-9.5-90</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">PSI Kit for sheath catheter, 8.5Fr</td>
			<td style="width:257px;">Arrow International</td>
			<td style="width:201px;">ASK-09803-UHN</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Infusion Pump Line</td>
			<td style="width:257px;">Smith Medical ASD Inc.</td>
			<td style="width:201px;">21-0442-25</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Liver Admin Set (flush line)</td>
			<td style="width:257px;">CardioMed Supplies Inc</td>
			<td align="right" style="width:201px;">17175</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Mallinckrodt, Tracheal Tube, 6.5mm</td>
			<td style="width:257px;">Covidien Canada</td>
			<td align="right" style="width:201px;">86449</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Med-Rx Suction Connecting Tube</td>
			<td style="width:257px;">Benlan Inc.</td>
			<td style="width:201px;">70-8120</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Organ Bag</td>
			<td style="width:257px;">CardioMed Supplies Inc</td>
			<td align="right" style="width:201px;">2990</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Suction Tip</td>
			<td style="width:257px;">Tyco Healthcare Group LP</td>
			<td align="right" style="width:201px;">8888501023</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Valleylab, Cautery Pencil</td>
			<td style="width:257px;">Covidien Canada</td>
			<td style="width:201px;">E2515H</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Valleylab, Patient Return Electrode</td>
			<td style="width:257px;">Covidien Canada</td>
			<td style="width:201px;">E7507</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;"></td>
			<td style="width:257px;"></td>
			<td style="width:201px;"></td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Bypass Connector 3/8&#8221; x 1/4&#8220;</td>
			<td style="width:257px;">Raumedic AG</td>
			<td style="width:201px;">955083-001</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Bypass Connector 3/8&#8221; x 3/8&#8221; Luer Lock</td>
			<td style="width:257px;">Raumedic AG</td>
			<td style="width:201px;">955163-001</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Bypass Connector Y 3/8&#8221; x 3/8&#8221; x 1/4&#8221;</td>
			<td style="width:257px;">Raumedic AG</td>
			<td style="width:201px;">961360-002</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Bypass Tubing 1/4&#8221; x 1/16&#8221;</td>
			<td style="width:257px;">Raumedic AG</td>
			<td style="width:201px;">039505-010</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Bypass Tubing 3/8&#8221; x 3/32&#8221;</td>
			<td style="width:257px;">Raumedic AG</td>
			<td style="width:201px;">039535-005</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Rotaflow Cenrtifugal Pump</td>
			<td style="width:257px;">Maquet-Dynamed</td>
			<td style="width:201px;">HC 2821</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Stainless Steel Hose Clamp Ring, 5mm</td>
			<td style="width:257px;">Oetiker</td>
			<td align="right" style="width:201px;">16700007</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Abdominal Retractor</td>
			<td style="width:257px;">Medite GmbH</td>
			<td style="width:201px;">N/A</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">De Bakey &#8211; Beck, Infrahepatic Cava Clamp</td>
			<td style="width:257px;">Aesculap Inc.</td>
			<td style="width:201px;">FB519R</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Diethrich, Atraumaitc Clamp (Portal Vein)</td>
			<td style="width:257px;">Aesculap Inc.</td>
			<td style="width:201px;">FB525R</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Gregory Bull Dog Clamp, curved</td>
			<td style="width:257px;">Aesculap Inc.</td>
			<td style="width:201px;">FB382R</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Gregory Bull Dog Clamp, straight</td>
			<td style="width:257px;">Aesculap Inc.</td>
			<td style="width:201px;">FB381R</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Potts &#8211; De Martel, Scissors</td>
			<td style="width:257px;">Aesculap Inc.</td>
			<td style="width:201px;">BC648R</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Satinsky, Suprahepatic Cava Clamp</td>
			<td style="width:257px;">Aesculap Inc.</td>
			<td style="width:201px;">FB605R</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Symetrical Tubing Clamp</td>
			<td style="width:257px;">Codman Instruments</td>
			<td align="right" style="width:201px;">198010</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Anesthesia Machine, Optimax</td>
			<td style="width:257px;">Moduflex Anesthesia Equipment</td>
			<td style="width:201px;">SN5180</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Bypass Flow meter, HT 110</td>
			<td style="width:257px;">Transonic Systems Inc.</td>
			<td style="width:201px;">HT110B11106</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Flow meter probe, H6XL</td>
			<td style="width:257px;">Transonic Systems Inc.</td>
			<td style="width:201px;">H6Xl689</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Heat Therapy Pump, T/Pump</td>
			<td style="width:257px;">Gaymar Industries Inc</td>
			<td style="width:201px;">TP500-G89D19</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Infusion Pump 3000</td>
			<td style="width:257px;">SIMS Graseby LTD.</td>
			<td style="width:201px;">SN300050447</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Isoflurane Vapor 19.1</td>
			<td style="width:257px;">Draeger Medical Canada Inc.</td>
			<td style="width:201px;">N/A</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Monitor, Datex AS 3</td>
			<td style="width:257px;">Instrumentarium Corp./ Hitachi</td>
			<td style="width:201px;">D-VHC14-23-02</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Rotaflow Centrifugal Drive Unit</td>
			<td style="width:257px;">Marquet-Dynamed</td>
			<td align="right" style="width:201px;">952301</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Rotaflow Console</td>
			<td style="width:257px;">Marquet-Dynamed</td>
			<td align="right" style="width:201px;">706035</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Temperature Therapy Pad</td>
			<td style="width:257px;">Gaymar Industries Inc</td>
			<td style="width:201px;">TP26E</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Valleylab Force Tx</td>
			<td style="width:257px;">Valleylab Inc.</td>
			<td align="right" style="width:201px;">216151480</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Ventilator, AV 800</td>
			<td style="width:257px;">DRE Medical Equipment</td>
			<td style="width:201px;">40800AVV</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:276px;">Warm Touch, Patient Warming System</td>
			<td style="width:257px;">Nellcor/ Covidien Canada</td>
			<td style="width:201px;">5015300A</td>
			<td style="width:240px;"></td>
		</tr>
	</tbody>
</table>

technique of porcine liver procurement and orthotopic transplantation using an active porto-caval shunt

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

Em um primeiro estudo de transplante, um modelo de dador bater-coração (HBD, n = 5) foi comparado com um modelo TDC (n = 10) expostas a 45 min de isquemia quente in situ. Em ambos os grupos, os enxertos foram preservados em gelo durante 10 horas após a aquisição. No grupo HBD, 100% dos porcos receptores sobreviveu até ao fim do acompanhamento no dia 5 após o transplante. No grupo DCD, apenas 50% dos receptores sobreviveu porcos durante 5 dias, devido a problemas de coagulação ou descompensação metabólica, como resultado da diminuição da função do fígado pós-operatório. Todas as amostras de sangue foram coletadas a partir do cateter venoso central. Após centrifugação, as amostras de soro foram obtidas e analisadas para a lesão hepatocelular (aspartato aminotransferase, AST), a função biliar (bilirrubina e fosfatase alcalina total), e função hepática (INR). O curso de tempo de cada marcador é mostrado nas Figuras 2-5. <p class="jove_content"&gt; Níveis de AST atingiu um pico ao fim de 24 horas (1414 ± 538 U / L no grupo de HBD e 2.296 ± 1.313 U / L no grupo DCD, p = 0,13) e voltou a valores quase normais após 5 dias. Do mesmo modo, valores da fosfatase alcalina foram marcadamente aumentada após 36 horas no grupo DCD (224 ± 111 U / L) quando comparado com o grupo de HBD (162 ± 54 U / L, p = 0,27). Enquanto bilirrubina total era estável no grupo HBD (≤ 10 umol / L ao longo), é gradualmente aumentada no grupo DCD até ao dia 5 (23 ± 31 umol / L, p = 0,43). O grande desvio padrão dos valores de bilirrubina no grupo DCD heterogeneidade mostra uma lesão biliar peculiar deste grupo. INR como marcador de função hepática mostraram uma tendência semelhante aos valores de AST. Valores atingiu um pico de 24 horas e foram restauradas aos valores normais quase após 5 dias. O grupo HBD tinha valores mais baixos, com um pico a 1,47 ± 0,34 em comparação com o grupo DCD (pico de 1,70 ± 0,36, p = 0,32). Figura 1. Esquema do shunt porto-cava-jugular. O bypass é preenchido com solução de Ringer com lactato. Em seguida, a parte da veia cava é presa com uma pinça tubulação, as partes jugulares e esplênicos estão ligados aos cateteres pré-estabelecidos, o desvio é aberto, e da bomba centrífuga é iniciado após o pinçamento da veia porta. Após a ressecção do fígado, a parte da veia cava do bypass é inserido e fixado na infrahepatic coto da veia cava, cranial para as veias renais. A braçadeira de tubo é libertado para permitir a descompressão da veia cava, para além da descompressão porta. <img alt="Figura 2" src="/files/ftp_upload/52055/52055fig2.jpg" /> Figura 2. aspartato aminotransferase (AST) (HBD n = 5, n = 10 DCD). AST é um marcador sensível da lesão hepatocelular. A ré picoer 24 horas é inferior no HBD do que no grupo DCD, sugerindo lesão de reperfusão menos hepática; o menor desvio padrão mostra resultados mais homogêneos no grupo HBD. <img alt="Figura 3" src="/files/ftp_upload/52055/52055fig3.jpg" /> Figura 3. A bilirrubina total (HBD n = 5, n = 10 DCD). A bilirrubina total, como um marcador de depuração biliar e integridade do ducto biliar, mostra uma tendência estável e homogénea com valores inferiores a 10 mmol / L no grupo de HBD. A curva de bilirrubina no grupo DCD aumenta gradualmente ao longo do tempo e mostra um desvio-padrão elevado, sugerindo lesão biliar em apenas uma porção do grupo experimental. <img alt="Figura 4" src="/files/ftp_upload/52055/52055fig4.jpg" /> Figura 4. A fosfatase alcalina (HBD n = 5, n = 10 DCD). A fosfatase alcalina ié um indicador de lesão biliar. Os valores para o grupo de HBD são mais baixas do que a do grupo DCD, o que implica menos lesão biliar. <img alt="Figura 5" src="/files/ftp_upload/52055/52055fig5.jpg" /> Figura 5. INR (HBD n = 5, DCD n = 10). Um valor alto indica INR função hepatocelular diminuída devido à diminuição da liberação de fatores de coagulação. Em ambos os grupos HBD e DCD, os valores de INR regresso aos valores normais de 5 dias após o transplante, sugerindo que a recuperação da função do fígado. Os valores para o grupo de HBD aparecem inferior.

Watch this Scientific Journal Video about Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt at JoVE.com

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

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

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

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

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Research

JoVE Journal

Medicine

16.3K visualizações.  Toronto General Hospital. Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

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

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

Transplante de intestino delgado de ratos ortotópico

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Medicina

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Transplante hepático ortotópico experimental em ratos

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as studies on graft preservation and ischemia-reperfusion injury 1,2, immunological responses 3,4, hemodynamics 5,6, and small-for-size syndrome 7. The rat OLT is among the most difficult animal models in experimental surgery and demands advanced microsurgical skills that take a long time to learn. Consequently, the use of this model has been limited. Since the reliability and reproducibility of results are key components of the experiments in which such complex animal models are used, it is essential for surgeons who are involved in rat OLT to be trained in well-standardized and sophisticated procedures for this model.
While various techniques and modifications of OLT in rats have been reported 8 since the first model was described by Lee et al. 9 in 1973, the elimination of the hepatic arterial reconstruction 10 and the introduction of the cuff anastomosis technique by Kamada et al. 11 were a major advancement in this model, because they simplified the reconstruction procedures to a great degree. In the model by Kamada et al., the hepatic rearterialization was also eliminated. Since rats could survive without hepatic arterial flow after liver transplantation, there was considerable controversy over the value of hepatic arterialization. However, the physiological superiority of the arterialized model has been increasingly acknowledged, especially in terms of preserving the bile duct system 8,12 and the liver integrity 8,13,14.
In this article, we present detailed surgical procedures for a rat model of OLT with hepatic arterial reconstruction using a 50% partial graft after ex vivo liver resection. The reconstruction procedures for each vessel and the bile duct are performed by the following methods: a 7-0 polypropylene continuous suture for the supra- and infrahepatic vena cava; a cuff technique for the portal vein; and a stent technique for the hepatic artery and the bile duct.

Orthotopic liver transplantation in rats is an indispensable experimental model for biomedical research. Here we present our surgical procedures for orthotopic rat liver transplantation with hepatic arterial reconstruction using a 50% partial graft.

Procedimentos cirúrgicos para um modelo de rato de Transplante de Fígado parcial ortotópico com reconstrução arterial hepática

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Usando Quantitative PCR em tempo real para determinar enxerto célula dadora num modelo murino de osso Competitiva Marrow Transplantation

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Orthotopic Hind Limb Transplantation in the Mouse

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

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

Ortotópico Hind Limb Transplantation no mouse

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

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

Usando a técnica de manga em um modelo do rato da transplantação da aorta - um vídeo instrutivo

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

A Rat Model of Orthotopic Liver Transplantation Using a Novel Magnetic Anastomosis Technique for Suprahepatic Vena Cava Reconstruction

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep learning curve. The introduction of the cuff technique for anastomosis of the portal vein (PV) and infrahepatic vena cava (IHVC) has significantly simplified the transplant procedure in rats. However, due to the short anterior wall of the recipients' suprahepatic vena cava (SHVC), the cuff technique is very difficult to use for the reconstruction of the SHVC. Most researchers in this field still use the hand-suture technique for SHVC reconstruction, which makes it the bottleneck step in rat orthotopic liver transplantation. The magnetic anastomosis technique (i.e., magnamosis) is a method of connecting two vessels using the attractive force between two magnets. Our recent study has shown that the magnetic anastomosis technique is superior to the hand-suture technique for SHVC reconstruction in rats. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using the novel magnetic anastomosis technique. In this model, the reconstruction of the PV and IHVC was performed by the standard cuff technique, while the reconstruction of the bile duct (BD) was performed by a stent technique. The hepatic re-arterialization was not performed. The magnetic anastomosis technique made SHVC reconstruction much easier and significantly shortened the anphepatic phase. After a reasonable learning curve, even researchers without advanced microsurgical skills can produce reliable and reproducible results using this rat model of OLT.

The reconstruction of the suprahepatic vena cava (SHVC) remains a difficult step in rat orthotopic liver transplantation. In this article, we show a step-by-step protocol for SHVC reconstruction in rats using a novel magnetic anastomosis technique.

Um modelo do rato de ortotópico transplante hepático, usando uma técnica de anastomose magnético romance para reconstrução de veia Cava Suprahepatic

The rat model of orthotopic liver transplantation (OLT) is essential for transplant research. It is a very sophisticated animal model and requires a steep ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Um modelo pré-clínico porcino de transplante cardíaco ortotópico

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

Reduced Complications after Arterial Reconnection in a Rat Model of Orthotopic Liver Transplantation

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation of human liver transplantation due to the absence of arterial reconnection. Described here is a modified transplantation procedure that includes the incorporation of hepatic artery (HA) reconnection, leading to a marked improvement in transplant outcomes. With a mean anhepatic time of 12 min and 14 s, HA reconnection results in improved perfusion of the transplanted liver and an increase in long-term recipient survival from 37.5% to 88.2%. This protocol includes the use of 3D-printed cuffs and holders to connect the portal vein and infrahepatic inferior vena cava. It can be implemented for studying multiple aspects of liver transplantation, from immune response and infection to technical aspects of the procedure. By incorporating a simple and practical method for arterial reconnection using a microvascular technique, this modified rat OLT protocol closely mimics aspects of human liver transplantation and will serve as a valuable and clinically relevant research model.

The goal of this study is to modify the rat orthotopic liver transplant model to better represent human liver transplantation and improve recipient survival. The presented method reestablishes hepatic arterial inflow by connecting the donor liver&#39;s common hepatic artery to the recipient liver&#39;s proper hepatic artery.

Complicações reduzidas após reconexão arterial em um modelo de rato de transplante de fígado ortotópico

The rat orthotopic liver transplantation (OLT) model is a powerful tool to study acute and chronic rejection. However, it is not a complete representation ...

Orthotopic Kidney Auto-Transplantation in a Porcine Model Using 24 Hours Organ Preservation And Continuous Telemetry

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for kidney transplantation (KT). Even though, the use of allografts from extended criteria donors (ECD) could partially ease the shortage of organ donors, ECD organs carry a potentially higher risk for inferior outcomes and postoperative complications. Dynamic organ preservation techniques, modulation of ischemia-reperfusion and preservation injury, and allograft therapies are in the spotlight of scientific interest in an effort to improve allograft utilization and patient outcomes in KT.
Preclinical animal experiments are playing an essential role in translational research, especially in the medical device and drug development. The major advantage of the porcine orthotopic auto-transplantation model over ex vivo or small animal studies lies within the surgical-anatomical and physiological similarities to the clinical setting. This allows the investigation of new therapeutic methods and techniques and ensures a facilitated clinical translation of the findings. This protocol provides a comprehensive and problem-oriented description of the porcine orthotopic kidney auto-transplantation model, using a preservation time of 24 hours and telemetry monitoring. The combination of sophisticated surgical techniques with highly standardized and state-of-the-art methods of anesthesia, animal housing, perioperative follow up, and monitoring ensure the reproducibility and success of this model.

Large animal models play an essential role in preclinical transplantation research. Due to its similarities to the clinical setup, the porcine model of orthotopic kidney auto-transplantation described in this article provides an excellent in vivo setting for the testing of organ preservation techniques and therapeutic interventions.

Auto-transplante ortotópico de rim em um modelo suíno usando preservação de órgãos 24 horas e telemetria contínua

In the present era of organ transplantation with critical organ shortage, various strategies are employed to expand the pool of available allografts for ...