Este trabalho foi financiado pelo Conselho Nacional de Saúde e Pesquisa Médica (NHMRC) da Austrália Projeto Grant 1029205, pela Fundação Microsearch da Austrália, e pela Fundação Myee Codrington Pesquisa Médica.

Antes do início das experiências, obter aprovação do Comitê de Ética Animal Care da instituição relevante para os experimentos planejados. Manter os ratos na conformidade com os requisitos da sua instituição. O protocolo a seguir foi aprovado pelos comitês Universidade de Sydney e Royal Prince Alfred Hospital. Antes de iniciar a cirurgia, todos os instrumentos devem ser cuidadosamente limpos e esterilizados por imersão em etanol 80%. Algumas instituições estipular autoclavagem no entanto isso pode danificar os instrumentos de microcirurgia multa no longo prazo. Sempre que possível o uso do equipamento descartável estéril. 1. Anestesia <ol><li> Anestesiar rato com isoflurano em um recipiente fechado, em seguida, colocá-lo em decúbito dorsal em uma placa de funcionamento, de forma rápida conexão anestésico cone do nariz. Teste de adequação da anestesia por beliscar a pata traseira para garantir que não haja reflexo. </li><li> Raspar a pele com uma lâmina cirúrgica eesterilizar com etanol a 80%. Além disso, os 2% de clorexidina em 70% de álcool isopropílico pode ser utilizado para a esterilização da pele. Durante a indução da anestesia isoflurano a concentração é de 3%, mas reduzir isso para 1-1,5% para a manutenção. Faça pequenos ajustes para esta concentração para manter a respiração regular e freqüência cardíaca. Mantenha a temperatura do animal em uma almofada de aquecimento. </li></ol> 2. Operação Donor <ol><li> Fazer uma toracotomia para expor o coração e os vasos cortando o peito por meio de ambos os lados da caixa torácica a partir da borda nervura até à axila seguido de um corte transversal ao nível do processo xifóide, para formar uma aba da parede torácica. Levante essa aba para cima ao lado da cabeça e fixá-lo ao conselho operacional. Rasgue pericárdio para expor o coração e os vasos. </li><li> Levante a veia cava inferior com uma pinça em uma mão e com a outra injetar 1 ml proximal salina heparinizada frio ao coração através da veia cava inferior, em seguida, colocar uma braçadeira pequena artéria no IVC para prevençãofluxo t de solução de perfusão de volta pelo buraco da agulha. </li><li> Usando gaze e um cotonete, retirar o coração para baixo para expor a aorta ascendente e artéria pulmonar. Passe uma lâmina de um par de microtesoura através do (seio transverso) posterior canal para o pacote de aorta e da artéria pulmonar e cortou a aorta ea artéria pulmonar em conjunto, tanto distal quanto possível para garantir comprimento suficiente para anastomose. </li><li> Laço e dividir o IVC, veia cava superior direita (SVC), deixou SVC e veias pulmonares utilizando 6-0 fio de seda. Amarre o IVC e SVC direito separadamente, em seguida, colocar uma única linha posterior ao coração para cercar o SVC esquerda junto com veias pulmonares e amarrá-lo. Colha o coração da área doadora, cortando os vasos distal para os laços, em seguida, armazená-lo em solução salina estéril fria a 4 ° C até o transplante. Isso resulta em morte do doador por sangria. </li></ol> 3. Operação Destinatário <ol><li> Anestesiar odestinatário como acima (seção 1). Raspar cuidadosamente abdômen para evitar a irritação, em seguida, fazer uma laparotomia por incisão mediana do púbis ao processo xifóide e retrair usar clipes de papel dobrado para formar retratores. Enrole o intestino em solução salina estéril gaze embebida quente e retrair a parte superior direita do abdômen. </li><li> Para expor a aorta infra-renal e IVC, libertar os segmentos agrupados de aorta e veia cava inferior da artéria renal esquerda e veia para a bifurcação ilíaca, dividindo-as dos vasos lombares usando um cautério. Tome cuidado para que o cautério está na temperatura correta e usada por um período suficiente para dividir e selar as pontas dos vasos. </li><li> Aplicar pequenas pinças atraumáticas embarcação para a aorta e segmentos VCI distal e proximal. Fazer uma incisão na parede frontal da aorta pelo primeiro perfurar um furo com uma agulha 30 G; em seguida, cortado de uma incisão vertical com microtesouras para coincidir com o tamanho da aorta do doador. Lave o lúmen da aorta com solução salina heparinizada para remover qualquer blcoágulos ood. </li><li> Traga o coração do doador para o site receptor coberto com gaze embebida em solução salina fria e coloque-o para o lado direito do abdômen. Certifique-se a aorta do doador é posicionado ao lado da incisão sobre a aorta do receptor e a artéria pulmonar do doador posicionado ao lado do receptor da VCI. </li><li> Anastomose lado end-to-the aorta do doador para a aorta destinatário usando execução 10-0 suturas de nylon a partir do canto proximal e sutura no lado esquerdo até chegar ao primeiro canto distal, em que fase o animal é girada em 180 °. Suavemente mover o coração de dador para o lado esquerdo do abdómen para expor o lado direito da aorta e continuar a sutura através do lado direito da parede da aorta a partir da extremidade distal para a extremidade proximal. Antes de fechar a anastomose da aorta, lave delicadamente o lúmen com solução salina heparinizada para remover qualquer coágulo e do ar. </li><li> Anastomosar a artéria pulmonar do doador (PA) de ponta-a-lado com o destinatário da VCI. Faça uma incisão vercamente com microtesouras na parede frontal da VCI em um local, em conformidade com a anastomose arterial. Anastomosar dador PA aos receptores com CIV executando 10-0 suturas de nylon a partir da extremidade distal da parede esquerda dentro do lúmen da veia cava inferior. Depois de atingir a extremidade proximal, suturas continuar ao longo da parede lateral direita da frente até a extremidade distal para completar a anastomose. Antes de fechar a anastomose, lave delicadamente a luz para remover quaisquer coágulos e ar. </li><li> Antes de liberar a embarcação grampos, coloque pedaços de Gelfoam em torno dos locais de anastomose, e aplicar uma leve pressão com um aplicador de algodão até que a hemostasia seja alcançado. No momento da revascularização, libertar a pinça distal em primeiro lugar, seguido pela braçadeira proximal. </li><li> Após a revascularização, aplicar soro fisiológico quente a 37 ° C ao enxerto externamente para ajudar na sua recuperação. O enxerto geralmente começa imediatamente atrial e reverte espontaneamente para o ritmo sinusal em poucos minutos. Injetar 0,6 ml quentessubcutaneamente salina para manter a hidratação do destinatário. Injetar buprenorfina subcutânea para analgesia antes da conclusão da cirurgia </li><li> Feche a ferida abdominal com um 5-0 sutura contínua absorvível para ambas as camadas. Comece por completar a camada interna e continuar ao longo da pele. </li></ol> 4. Recuperação e Monitorização do Enxerto <ol><li> Injectar a ampicilina para a profilaxia da infecção e colocar o recipiente em um bloco de aquecimento a 37 ° C durante a recuperação. A maioria dos animais recuperar-se rapidamente e são geralmente beber e comer muitas vezes dentro de 3 horas. Se o mouse mostra sinais de angústia, examinar de perto para determinar a causa. Se nenhuma causa óbvia, tratar com buprenorfina e acompanhar de perto. Consulte um veterinário se os sintomas forem graves ou persistir mais de 8 horas e considerar a eutanásia. Se necessário, dar continuidade 12 injeções por hora de buprenorfina até os sintomas desaparecerem. Se o mouse mostra sinais de angústia após 8 horas, dar continuidade 12injecções horárias de buprenorfina até que resolver. Consulte o veterinário se os sintomas persistirem por mais de 48 horas e sacrificar, se necessário. </li><li> Monitorar enxerto batimento cardíaco por palpação abdominal direto e registrar a força da batida como + + + + para um enxerto saudável + para fraco batimento devido à rejeição avançada e - como não bater devido à completa rejeição do enxerto. Monitorar ratinhos diariamente para os primeiros 10 dias, depois 3x por semana durante a duração da experiência. </li></ol>

<ol>
	<li>Aramaki, O., 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=Interleukin-10+but+not+transforming+growth+factor-beta+is+essential+for+generation+and+suppressor+function+of+regulatory+cells+induced+by+intratracheal+delivery+of+alloantigen.">Interleukin-10 but not transforming growth factor-beta is essential for generation and suppressor function of regulatory cells induced by intratracheal delivery of alloantigen.</a> Transplantation. 79, 568-576 (2005).</li><li>Chen, R. H., Bushell, A., Fuggle, S. V., Wood, K. J., Morris, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+granzyme+A+and+perforin+in+mouse+heart+transplants+immunosuppressed+with+donor-specific+transfusion+and+anti-CD4+monoclonal+antibody.">Expression of granzyme A and perforin in mouse heart transplants immunosuppressed with donor-specific transfusion and anti-CD4 monoclonal antibody.</a> Transplantation. 61, 625-629 (1996).</li><li>Poulin, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-9+promotes+eosinophilic+rejection+of+mouse+heart+allografts.">Interleukin-9 promotes eosinophilic rejection of mouse heart allografts.</a> Transplantation. 76, 572-577 (2003).</li><li>Ono, K., 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=Improved+technique+of+heart+transplantation+in+rats.">Improved technique of heart transplantation in rats.</a> J. Thorac. Cardiovasc. Surg. 57, 225-229 (1969).</li><li>Corry, R. J., Winn, H. J., Russell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primarily+vascularized+allografts+of+heart+in+mice.">Primarily vascularized allografts of heart in mice.</a> Transplantation. 16, 343-350 (1973).</li><li>Larsen, C. P., 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=CD40-gp39+interactions+play+a+critical+role+during+allograft+rejection.+Suppression+of+allograft+rejection+by+blockade+of+the+CD40-gp39+pathway.">CD40-gp39 interactions play a critical role during allograft rejection. Suppression of allograft rejection by blockade of the CD40-gp39 pathway.</a> Transplantation. 61, 4-9 (1996).</li><li>Saitovitch, D., Bushell, A., Mabbs, D. W., Morris, P. J., Wood, K. 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Immunol. 24, 149-156 (2011).</li><li>Liu, F., Kang, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+Heart+Transplantation+in+Mice.">Heterotopic Heart Transplantation in Mice.</a> J. Vis. Exp. , (2007).</li><li>Mottram, P. L., 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=Electrocardiographic+monitoring+of+cardiac+transplants+in+mice.">Electrocardiographic monitoring of cardiac transplants in mice.</a> Cardiovasc. Res. 22, 315-321 (1988).</li></ol>

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Transplante cardíaco Mouse é um método de microcirurgia exigente, que requer habilidade cirúrgica considerável para dominar. O aspecto mais difícil é o pequeno diâmetro dos vasos. Além disso, é necessário limitar o tempo de funcionamento do destinatário e sangramento. A técnica de transplante cardíaco do rato foi primeiramente descrita por Corry et al. em 1973 e, posteriormente, por Mottram et al 12. Nossos modificações incluem os seguintes pontos. Em primeiro lugar, a perfus...

Os modelos animais de transplante de órgãos pode fornecer informações valiosas para melhorar o tratamento de pacientes transplantados clínicos. Modelos de mouse são particularmente úteis para o estudo do mecanismo imunológico da rejeição de transplantes de órgãos ou de aceitação, devido à ampla gama de ratos e reagentes específicos para os ratos que não estão disponíveis para outros modelos animais geneticamente modificados 1-3. Um desafio com modelos do rato do transplante é a pequena dimensão dos doadores e receptores que requer habilidade técnica considerável para alcançar resultados satisfatórios. A técnica foi descrita pela primeira vez para o transplante heterotópico de coração de ratos 4, que foi posteriormente adaptado para transplante cardíaco do rato por Corry et al 5. Esta técnica envolveu a preparação do receptor antes da operação de dador e sem perfusão do coração de dador, ambos os quais são de natureza a comprometer a sobrevivência do transplante cardíaco ou tele destinatário. O procedimento tem sido amplamente utilizado como descrito originalmente para examinar os mecanismos de rejeição de transplantes e de tolerância de 6-8. Outros têm adaptado o procedimento original coração de rato transplante de Ono e Lindsey para transplantes cardíacos em ratos 9,10. Mais recentemente, uma técnica foi publicada por transplante de coração rato que superou alguns dos problemas associados com o método de Corry et al 11. O protocolo aqui descrito incorpora nossas modificações, com base no método de Mottram et al, 12, os quais incluem: perfusão in situ com solução salina heparinizada a frio imediatamente após toracotomia e realizar a operação de dador antes da operação de receptor para receptor minimizar o tempo de operação. Além disso, podemos usar grampos embarcação atraumáticas pequenas em vez de gravatas de seda 6-0. Embora grampos vasos têm a desvantagem de tomar mais espaço que eles são mais fáceis de controlar do que os laços, que não deve ser muito apertado ou soltoe são menos fáceis de remover do que as braçadeiras. O nosso método utiliza um único fio de sutura de corrida para a anastomose navio, embora na aprendizagem inicial estágios uma alternativa é a utilização de suturas de ancoragem na extremidade proximal e cantos distais para assegurar uniformidade de suturas e, portanto, a permeabilidade da anastomose.

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			<td style="width:247px;">Leica, Heerbrugg, Switzerland</td>
			<td style="width:124px;">M651</td>
			<td style="width:167px;">10x - 25x magnification</td>
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			<td height="21" style="height:21px;">Anesthetic machine&#160;</td>
			<td>Vet Quip Pty Ltd, Sydney, Australia</td>
			<td style="width:124px;">Vett3</td>
			<td>Capable of delivering a mixture of isoflurane and oxygen in air&#160;</td>
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			<td height="21" style="height:21px;width:293px;">Operating board&#160;</td>
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			<td>Dense cork or synthetic capable of taking pins</td>
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			<td height="23" style="height:23px;width:293px;">Heparinised saline (5 U/ml, 4oC)</td>
			<td>Pfizer, USA</td>
			<td style="width:124px;">FW25</td>
			<td>In 1ml syringe with 23 gauge needle on ice</td>
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			<td height="23" style="height:23px;width:293px;">Normal saline (37oC)</td>
			<td style="width:247px;">AstraZeneca Pty Ltd, Australia</td>
			<td style="width:124px;">4538</td>
			<td>In 1ml syringe with 23 gauge needle on warm pad</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Sutures 10- nylon, 5-0 Vicryl</td>
			<td>Ethicon, Inc. NJ, USA</td>
			<td style="width:124px;">2870G/J421H</td>
			<td></td>
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			<td height="21" style="height:21px;">Buprenorphine (0.05mg/kg in 0.1ml saline)</td>
			<td>Reckitt Benckiser, Sydney, Australia</td>
			<td style="width:124px;"></td>
			<td>In 1ml syringe with 23 gauge needle on ice</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ampcillin (5mg/kg in 0.1 ml saline)&#160;</td>
			<td>Aspen, Sydney, Australia</td>
			<td style="width:124px;"></td>
			<td>In 1ml syringe with 23 gauge needle on ice</td>
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			<td height="21" style="height:21px;">Gel Foam&#160;</td>
			<td>Pharmacia &#38; Upjohn Co. USA</td>
			<td style="width:124px;">801289304</td>
			<td>Cut into small pieces</td>
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			<td height="21" style="height:21px;">High temperature cautery device&#160;</td>
			<td>Medtronic, USA</td>
			<td style="width:124px;">8444000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Microsurgery instruments:</td>
			<td colspan="2">Shanghai Medical Instruments Co. Ltd.,&#160;&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-needleholders</td>
			<td style="width:247px;">Shanghai, China</td>
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			<td>&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">WT1020</td>
			<td></td>
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			<td height="21" style="height:21px;width:293px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-forceps (straight tip)</td>
			<td style="width:247px;">&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">WA3010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-forceps (curved tip)</td>
			<td>&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">WA3020</td>
			<td></td>
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			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-mosquito clamps (1 pair)</td>
			<td style="width:247px;">&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">W40350</td>
			<td></td>
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			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160;&#160;&#160;&#160; micro-vessel atraumatic clamps (1 pair)</td>
			<td>&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#34;</td>
			<td style="width:124px;">W40130/W40150</td>
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			<td height="42" style="height:42px;width:293px;">Heating Pad/Right Temp</td>
			<td style="width:247px;">Kent Scientific Corporation, Turrington, CT 06790</td>
			<td style="width:124px;"></td>
			<td></td>
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a modified method for heterotopic mouse heart transplantion

Ratos são muitas vezes utilizados como doadores de transplante de coração e receptores em estudos de imunologia do transplante devido à ampla gama de camundongos transgênicos e reagentes disponíveis. A dificuldade é apresentada devido ao pequeno tamanho do animal e os desafios técnicos consideráveis ​​da microcirurgia envolvidos no transplante de coração. Em particular, uma alta taxa de falha técnica logo após o transplante pode resultar da morte do destinatário e complicações pós-operatórias, como a paralisia dos membros posteriores ou um coração sem bater. Aqui, a técnica completa para o coração transplante heterotópico rato é demonstrado, envolvendo a colheita do coração do doador e sua posterior implantação em um rato destinatário. O coração de dador é colhido imediatamente após perfusão in situ com solução salina heparinizada a frio e a transecção da aorta ascendente e da artéria pulmonar. A operação envolve a preparação de receptor a aorta abdominal e veia cava inferior (IVC), seguido pelaanastomose término-lateral da aorta do doador com a aorta do receptor através de um único corredor 10-0 microsuture e uma anastomose semelhante da artéria pulmonar do dador com o destinatário da VCI. Na sequência da operação, o animal é injectado com 0,6 ml de solução salina normal subcutaneamente e deixados a recuperar numa almofada de aquecimento de 37 ° C. Os resultados de 227 transplantes de coração do rato são resumidas com uma taxa de sucesso de 48 horas de 86,8%. Das falhas 13,2% dentro de 48 horas, 5 (2,2%) apresentaram paralisia dos membros posteriores, 10 (4,4%) tinha um coração não bater devido ao enxertar lesão isquêmica e / ou trombose, enquanto 15 (6,6%) morreram dentro de 48 horas .

Depois de um período de formação inicial, foram analisados ​​227 casos de rato transplante cardíaco heterotópico em nosso grupo. A taxa de sucesso nas primeiras 24 horas foi de 90,3% e em 48 horas foi de 86,8%. Dos 30 (13,2%) falhas dentro de 48 horas, 5 (2,2%) apresentaram paralisia dos membros posteriores e teve que ser sacrificado, 10 (4,4%) tinha um coração não bater devido a lesão de isquemia do enxerto e / ou trombose, enquanto 15 (6,6%) morreram dentro de 48 horas. Alguns sobreviventes de enxertos ex...

Watch this Scientific Journal Video about A Modified Method for Heterotopic Mouse Heart Transplantion at JoVE.com

A Modified Method for Heterotopic Mouse Heart Transplantion

A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

Um método modificado para heterotópico Rato coração transplante

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

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Research

JoVE Journal

Medicine

11.9K visualizações.  University of Sydney. A technique is demonstrated for the microsurgical procedure for heterotopic transplantation of hearts in mice, including simplified methods for donor harvesting and recipient vessel anastomosis.

Vídeo: A Modified Method for Heterotopic Mouse Heart Transplantion

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

Um modelo de transplante heterotópico Modificado Swine Hind Limb para Translational vascularizado Composite Alotransplante (VCA) Pesquisa

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

Medicina

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

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

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

Murino Cervical Modelo de coração Transplante usando uma técnica Cuff Modificado

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

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 ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

O transplante de válvula pulmonar Usando um modelo do rato de Heterotópica Transplante de Coração

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

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.

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 ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

Heterotopic Cervical Transplante Cardíaco em Ratos

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

A Microsurgical Modelo Novel para heterotópico, em bloco da parede torácica, Thymus, e Transplante Cardíaco em Ratos

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

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 Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

Um Protocolo de Transplante de Coração Heterotópico Porcino para Entrega de Terapêutica a um Aoenxerto Cardíaco

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...

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 ...