Name: partial heterotopic hindlimb transplantation model in rats
Uploaded: 2021-06-09T14:45:00
Description: Watch this Scientific Journal Video about Partial Heterotopic Hindlimb Transplantation Model in Rats at JoVE.com

Este trabalho foi apoiado pelo Escritório do Secretário Adjunto de Defesa para Assuntos de Saúde através do Programa de Pesquisa Médica Dirigido pelo Congresso sob o Prêmio Nº. W81XWH-17-1-0680. Opiniões, interpretações, conclusões e recomendações são dos autores e não são necessariamente endossadas pelo Departamento de Defesa.

Todos os animais receberam cuidados humanos de acordo com o Guia do NIH para o Cuidado e Uso de Animais de Laboratório. O Comitê Institucional de Cuidados e Uso de Animais (IACUC-protocol 2017N000184) e o Escritório de Revisão de Cuidados e Uso animal (ACURO) aprovaram todos os protocolos animais. Ratos machos de raça Lewis (250-400 g) foram usados para todos os experimentos.
1. Cirurgia
<ol><li>Anestesiar os ratos de Lewis usando inalação de isoflurano. Induzir anestesia com 5% de is...

<ol>
	<li>Lanteiri, 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=Feasibility,+reproducibility,+risks+and+benefits+of+face+transplantation:+a+prospective+study+of+outcomes.">Feasibility, reproducibility, risks and benefits of face transplantation: a prospective study of outcomes.</a> American Journal of Transplantation. 11 (2), 367-378 (2011).</li><li>Park, S. H., Eun, S. C., Kwon, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hand+transplantation:+current+status+and+immunologic+obstacles.">Hand transplantation: current status and immunologic obstacles.</a> Experimental and Clinical Transplantation. 17 (1), 97-104 (2019).</li><li>Cetrulo, C. 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=Penis+transplantation:+first+US+experience.">Penis transplantation: first US experience.</a> Annals of Surgery. 267 (5), 983-988 (2018).</li><li>Grajek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+complex+allotransplantation+of+neck+organs:+larynx,+trachea,+pharynx,+esophagus,+thyroid,+parathyroid+glands,+and+anterior+cervical+wall:+a+case+report.">First complex allotransplantation of neck organs: larynx, trachea, pharynx, esophagus, thyroid, parathyroid glands, and anterior cervical wall: a case report.</a> Annals of Surgery. 266 (2), 19-24 (2017).</li><li>Pribaz, J. J., Caterson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+limitations+of+conventional+autologous+reconstruction+of+the+head+and+neck.">Evolution and limitations of conventional autologous reconstruction of the head and neck.</a> Journal of Craniofacial Surgery. 24 (1), 99-107 (2013).</li><li>Lipson, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+limb+transplantation+in+the+rat.+I.+Results+with+syngeneic+grafts.">Vascularized limb transplantation in the rat. I. Results with syngeneic grafts.</a> Transplantation. 35 (4), 293-299 (1983).</li><li>Lipson, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+limb+transplantation+in+the+rat.+II.+Results+with+allogeneic+grafts.">Vascularized limb transplantation in the rat. II. Results with allogeneic grafts.</a> Transplantation. 35 (4), 300-304 (1983).</li><li>Adamson, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+model+of+hindlimb+osteomyocutaneous+flap+for+the+study+of+tolerance+to+composite+tissue+allografts.">A modified model of hindlimb osteomyocutaneous flap for the study of tolerance to composite tissue allografts.</a> Microsurgery. 27 (7), 630-636 (2007).</li><li>Arav, A., Friedman, O., Natan, Y., Gur, E., Shani, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+hindlimb+cryopreservation+and+transplantation:+a+step+toward+"organ+banking".">Rat hindlimb cryopreservation and transplantation: a step toward "organ banking".</a> American Journal of Transplantation. 17 (11), 2820-2828 (2017).</li><li>Gok, E., 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=Development+of+an+ex-situ+limb+perfusion+system+for+a+rodent+model.">Development of an ex-situ limb perfusion system for a rodent model.</a> ASAIO Journal. 65 (2), 167-172 (2019).</li><li>Gok, E., Rojas-Pena, A., Bartlett, R. H., Ozer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+skeletal+muscle+metabolomic+changes+associated+with+static+cold+storage.">Rodent skeletal muscle metabolomic changes associated with static cold storage.</a> Transplantation Proceedings. 51 (3), 979-986 (2019).</li><li>Brandacher, G., Grahammer, J., Sucher, R., Lee, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+basic+and+translational+research+in+reconstructive+transplantation.">Animal models for basic and translational research in reconstructive transplantation.</a> Birth Defects Research. Part C, Embryo Today. 96 (1), 39-50 (2012).</li><li>Fleissig, Y., 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=Modified+heterotopic+hindlimb+osteomyocutaneous+flap+model+in+the+rat+for+translational+vascularized+composite+allotransplantation+research.">Modified heterotopic hindlimb osteomyocutaneous flap model in the rat for translational vascularized composite allotransplantation research.</a> Journal of Visualized Experiments: JoVE. (146), e59458 (2019).</li><li>Ulusal, A. E., Ulusal, B. G., Hung, L. M., Wei, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+hindlimb+allotransplantation+in+rats:+an+alternative+model+for+immunological+research+in+composite-tissue+allotransplantation.">Heterotopic hindlimb allotransplantation in rats: an alternative model for immunological research in composite-tissue allotransplantation.</a> Microsurgery. 25 (5), 410-414 (2005).</li><li>Jang, Y., Park, Y. E., Yun, C. W., Kim, D. H., Chung, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vest-collar+as+a+rodent+collar+to+prevent+licking+and+scratching+during+experiments.">The vest-collar as a rodent collar to prevent licking and scratching during experiments.</a> Lab Anim. 50 (4), 296-304 (2016).</li><li>Kern, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+rodent+orthotopic+forelimb+transplantation+model+that+allows+for+reliable+assessment+of+functional+recovery+resulting+from+nerve+regeneration.">A novel rodent orthotopic forelimb transplantation model that allows for reliable assessment of functional recovery resulting from nerve regeneration.</a> American Journal of Transplantation. 17 (3), 622-634 (2017).</li><li>Perez-Abadia, G., 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=Low-dose+immunosuppression+in+a+rat+hind-limb+transplantation+model.">Low-dose immunosuppression in a rat hind-limb transplantation model.</a> Transplant International. 16 (12), 835-842 (2003).</li><li>Sucher, R., 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=Orthotopic+hind-limb+transplantation+in+rats.">Orthotopic hind-limb transplantation in rats.</a> Journal of Visualized Experiments. (41), e2022 (2010).</li><li>Fleissig, Y. Y., Beare, J. E., LeBlanc, A. J., Kaufman, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+rat+hind+limb+transplant+as+an+experimental+model+of+vascularized+composite+allotransplantation:+Approaches+and+advantages.">Evolution of the rat hind limb transplant as an experimental model of vascularized composite allotransplantation: Approaches and advantages.</a> SAGE Open Medicine. 8, 2050312120968721 (2020).</li><li>Lindboe, C. F., Presthus, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+denervation,+immobilization+and+cachexia+on+fibre+size+in+the+anterior+tibial+muscle+of+the+rat.">Effects of denervation, immobilization and cachexia on fibre size in the anterior tibial muscle of the rat.</a> Acta Neuropathologica. 66 (1), 42-51 (1985).</li><li>Nazzal, J. A., Johnson, T. S., Gordon, C. R., Randolph, M. A., Lee, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+limb+allotransplantation+model+to+study+skin+rejection+in+the+rat.">Heterotopic limb allotransplantation model to study skin rejection in the rat.</a> Microsurgery. 24 (6), 448-453 (2004).</li></ol>

Modelos ortotópicos de transplante de membros em roedores foram descritos na literatura15,16,17; no entanto, eles requerem uma reparação nervosa, recolocação muscular, e uma osteossíntese perfeita do fêmur, o que pode ser um passo muito difícil. Esses modelos também estão associados a uma maior taxa de morbidade e mortalidade em roedores14, especialmente no seguimento de curto prazo, pois a recu...

Nas últimas duas décadas, o VCA evoluiu como um tratamento revolucionário para pacientes que sofrem desfiguração grave, incluindo face1, amputações de membros superiores2,peniano3e outros defeitos complexos do tecido4,5. No entanto, as consequências da imunossupressão ao longo da vida ainda dificultam uma aplicação mais ampla dessas complexas cirurgias reconstrutivas. Pesquisas básicas são cruciais para melhorar as estratégias anti-rejeição. O aumento do tempo de preservação do VCA também é essencial para melhorar a logística de transplante e aumentar o pool de doadores (já que os doadores de VCA devem preencher mais critérios do que doadores de órgãos sólidos, incluindo tom de pele, tamanho anatômico, gênero). Nesse contexto, os transplantes de membros de ratos são amplamente utilizados em estudos sobre a rejeição imune de atores6,7, novos protocolos de indução de tolerância8e estudos de preservação9,10,11. Assim, esses modelos VCA são um elemento-chave para dominar a pesquisa translacional VCA.
Retalhos osteomyocutâneos têm sido descritos na literatura como modelos confiáveis para estudar VCA em ratos8,12,13,14. Embora os transplantes ortotópicos de membros inteiros permitam uma avaliação a longo prazo da função do enxerto, é um procedimento demorado associado a maiores taxas de morbidade e mortalidade pós-operatórias14. Em contraste, os modelos heterotópicos de transplante de membros não são funcionais, mas permitem estudos reprodutíveis sobre VCA. Os resultados pós-operatórios podem ser antecipados de forma confiável antes do início de um estudo de transplante de VCA de ratos. Este estudo relata um modelo de transplante de retalho osteomyocutâneo heterotópico no rato que inclui desfechos e complicações possíveis frequentes que podem surgir intra-operatóriamente e no pós-operatório durante um período de seguimento de três semanas.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24 GA angiocatheter</td>
			<td>BD Insyte Autoguard</td>
			<td>381412</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 suture Black monofilament non absorbable suture</td>
			<td>Ethicon</td>
			<td>1667</td>
			<td>Used to suture the E-collar to the back of the neck</td>
		</tr>
		<tr>
			<td>4-0 suture Coated Vicryl Plus Antibacterial</td>
			<td>Ethicon</td>
			<td>VCP496</td>
			<td></td>
		</tr>
		<tr>
			<td>Adson Tissue Forceps, 11 cm, 1 x 2 Teeth with Tying Platform</td>
			<td>ASSI</td>
			<td>ASSI.ATK26426</td>
			<td></td>
		</tr>
		<tr>
			<td>Bipolar cords</td>
			<td>ASSI</td>
			<td>228000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Black Polyamide Monofilament USP 10-0, 4 mm 3/8c</td>
			<td>AROSurgical</td>
			<td>T04A10N07-13</td>
			<td>Used to perform the microvascular anastomoses</td>
		</tr>
		<tr>
			<td>Buprenorphine HCl</td>
			<td>Pharmaceutical, Inc</td>
			<td>42023-179-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dilating Forceps</td>
			<td>Fine science tools (FST)</td>
			<td>18131-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scissors 15 cm, Round Handle 8 mm diameter, Straight Slender Tapered Blade 7 mm, Lipshultz Pattern</td>
			<td>ASSI</td>
			<td>ASSI.SAS15RVL</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Micro Clamps 5.5 x 1.5 mm</td>
			<td>Fine science tools (FST)</td>
			<td>18040-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Elizabethan collar</td>
			<td>Braintree Scientific</td>
			<td>EC-R1</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps 13.5 cm long, Flat Handle, 9 mm wide Straight Tips 0.1 mm diameter (x2)</td>
			<td>ASSI</td>
			<td>ASSI.JFL31</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey Micro Needle Holder</td>
			<td>Fine science tools (FST)</td>
			<td>12500-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Lock Flush Solution, USP, 100 units/mL</td>
			<td>BD PosiFlush</td>
			<td>306424</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary</td>
			<td>14043-704-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewelers Bipolar Forceps Non Stick 11 cm, straight pointed tip, 0.25 mm tip diameter</td>
			<td>ASSI</td>
			<td>ASSI.BPNS11223</td>
			<td></td>
		</tr>
		<tr>
			<td>Lone Star Elastic Stays</td>
			<td>CooperSurgical</td>
			<td>3314-8G</td>
			<td>Used to retract the inguinal ligament for femoral vessels dissection</td>
		</tr>
		<tr>
			<td>Lone Star Self-Retaining Retractors</td>
			<td>CooperSurgical</td>
			<td>3301G</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Mosquito Hemostats</td>
			<td>Fine science tools (FST)</td>
			<td>13010-12</td>
			<td>Used to retract the inguinal fat pad distally</td>
		</tr>
		<tr>
			<td>Needle Holder, 15 cm Round Handle, 8 mm diameter, Superfine Curved Jaw 0.2 mm tip diameter, without lock</td>
			<td>ASSI</td>
			<td>ASSI.B1582</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Suture Black Monolfilament 8-0, 6.5 mm 3/8c</td>
			<td>Ethilon</td>
			<td>2808G</td>
			<td>Used to ligate collateral branches on the femoral vessels</td>
		</tr>
		<tr>
			<td>Offset Bone Nippers</td>
			<td>Fine science tools (FST)</td>
			<td>16101-10</td>
			<td></td>
		</tr>
		<tr>
			<td>S&#38;T Vascular Clamps 5.5 x 1.5 mm</td>
			<td>Fine science tools (FST)</td>
			<td>00398-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Walton scissors</td>
			<td>Fine science tools (FST)</td>
			<td>14077-09</td>
			<td></td>
		</tr>
	</tbody>
</table>

partial heterotopic hindlimb transplantation model in rats

As alotransplantações compostas vascularizadas (VCA) representam a opção de reconstrução mais avançada para pacientes sem possibilidades cirúrgicas autólogas após um defeito complexo tecidual. Os transplantes de rosto e mão mudaram a vida dos pacientes desfigurados, dando-lhes um novo órgão social estético e funcional. Apesar dos resultados promissores, o VCA ainda é pouco executado devido a comorbidades de imunossupressão ao longo da vida e complicações infecciosas. O rato é um modelo animal ideal para estudos in vivo que investigam vias imunológicas e mecanismos de rejeição de enxertos. Ratos também são amplamente utilizados em novas técnicas de preservação de enxerto de tecido composto, incluindo estudos de perfusão e criopreservação. Os modelos utilizados para VCA em ratos devem ser reprodutíveis, confiáveis e eficientes com baixa morbidade e mortalidade pós-operatória. Os procedimentos heterotópicos de transplante de membros preenchem esses critérios e são mais fáceis de realizar do que transplantes ortotópicos de membros. Dominar modelos microcirúrgicos de roedores requer experiência sólida em microcirurgia e cuidados com animais. Aqui é relatado um modelo confiável e reprodutível de transplante de retalhos heterotópicos parciais em ratos, os desfechos pós-operatórios e os meios de prevenção de possíveis complicações.

Neste estudo de operador único, foram realizados 30 transplantes de membros parciais heterotópicos sintéricos. O sucesso foi definido no pós-operatório 21 como a ausência de falha do VCA ou complicações que requerem eutanásia. A evolução normal do enxerto está representada na Figura 3. A duração média para aquisição parcial de membros e inset de enxerto no receptor foi de 35 e 105 min, respectivamente; a média de isquemia foi de 105 min. Durante o acompanhamento, ocorreram ...

Watch this Scientific Journal Video about Partial Heterotopic Hindlimb Transplantation Model in Rats at JoVE.com

Partial Heterotopic Hindlimb Transplantation Model in Rats

Este artigo apresenta um protocolo de transplante de retalho osteomyocutâneo heterotópico em ratos e seus resultados potenciais no acompanhamento a médio prazo.

Modelo de transplante de hindlimb heterotópico parcial em ratos

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities ...

Apresentamos um modelo cirúrgico confiável e reprodutível de transplante heterotópico de escalada em ratos. Esta técnica é ideal para estudos de curto prazo sobre preservação ou rejeição imunológica em VCA. A descrição passo a passo permitirá que os estagiários dominem rapidamente a cirurgia.Para começar, faça uma incisão circunferencial na pele acima do tornozelo no terço distal da perna. Esqueleto e cauterizar a artéria safena e o ramo terminal da artéria popliteal usando fórceps bipolares. Cauterize e corte os músculos gastrocnemius, soleus, tibialis anterior e bíceps femoris até que o osso tibial seja exposto.Faça uma incisão de 2,5 centímetros na linha inguinal direita e disseque a almofada de gordura inguinal. Use um retrátil de anzol para agarrar o ligamento inguinal. Segure a almofada de gordura inguinal distally com fórceps de fixação e retraia a almofada de gordura para expor os vasos femorais.Disseca os vasos femorais, individualize os ramos de Murphy e ligante com laços de nylon 8/0. Heparinize o rato doador injetando a heparina na veia dorsal peniana usando uma agulha calibre 27,5. Complete a incisão da pele ao redor do quadril.Use os fórceps bipolares para cauterizar os músculos bíceps femoris e glúteos superficialis, em seguida, cauterizar e cortar o nervo ciático no comprimento do fêmur médio. Exponha o fêmur proximalmente ao nível da crista femoral posterior e liga os vasos femorais com laços de nylon ao nível do ligamento inguinal. Realize uma arteriotomia na artéria femoral logo abaixo da ligadura e dilate para inserir o angiocateter de calibre 24.Cauterize e corte o músculo restante debaixo do pedículo para expor o lado anterior do fêmur. Use o cortador de osso para cortar a tíbia proximally e corte o fêmur o mais distally possível. Lave a retalho parcial com dois mililitros de soro fisiológico de heparina para obter uma saída venosa clara.Coloque a plataforma traseira no gelo em uma gaze estéril até a transferência microvascular. Disseque a almofada de gordura inguinal e recline a almofada de gordura inguinal distally para expor os vasos femorais. Use um gancho para retrair o ligamento inguinal e os fórceps de fixação para segurar a almofada de gordura inguinal distally.Disseque os vasos femorais, individualize os ramos de Murphy, e ligante com laços de nylon como demonstrado. Ligate ambas as naves acima dos vasos epigástricos usando laços de nylon. Coloque os grampos aproximadores proximicamente, dilate a lente do vaso e enxágue com soro fisiológico de heparina.Faça uma incisão no flanco esquerdo acima do quadril e crie um bolso subcutâneo com um túnel subcutâneo para o vinco inguinal. Coloque a parte proximal do membro parcial e a almofada de gordura inguinal através do túnel subcutâneo para transferência microvascular. Realize as anastomoses venosas e arteriais usando suturas de nylon.Remova os dois grampos aproximadores e observe a revascularização do membro. Realize um teste de ordenha em ambos os vasos para avaliar a patência de cada anastomose. Faça uma incisão longitudinal da pele no lado medial do membro transplantado e insira o enxerto.Remova o excesso de pele do enxerto e feche a ferida com suturas separadas e uma sutura em execução usando suturas absorventes 4/0. Sutura juntar as almofadas de gordura inguinal do membro transplantado e o receptor usando duas suturas absorvíveis separadas e fechar o vinco inguinal após o último check-up da anastomose microvascular. A evolução do modelo de retrocesso heterotópico foi monitorada até o final do estudo.O crescimento capilar foi observado durante a primeira semana pós-operatória, e a retração cutânea apareceu após duas semanas. Falha vascularizada de aotransplante composto pode ocorrer durante a primeira semana pós-operatória devido à trombose microvascular. A trombose venosa foi a causa da eutanásia precoce em 20% dos casos, todos ocorridos antes do quinto dia de pós-operatório.A pele parecia azul e ficava mais escura a cada dia. A automutilação é uma preocupação séria em enxertos não-sentidos e ocorre frequentemente entre o segundo e o sétimo dia. Se limitado a menos de um terço da superfície do enxerto e diz respeito apenas à pele, o debridamento cirúrgico e a sutura utilizando suturas não absorvíveis podem ser discutidos com o veterinário da equipe.A prevenção da automutilação depende do uso de uma coleira e costurada no pescoço até o sétimo dia de pós-operatório e limpeza de qualquer sangue ou crosta nas feridas cirúrgicas do animal. Autofagia grave de múltiplas camadas do enxerto leva à eutanásia do animal. Cistos dérmicos apareceram após o pós-operatório 14, às vezes com um centro necrosado cutâneo antes da fístula.É crucial garantir que a colocação do enxerto não impeça o animal de ser ambulatorial, e que o pedículo não esteja torcido ou apertado durante o inset do enxerto. Nossa equipe realizou estudos de preservação nos membros adquiridos antes do transplante. Os efeitos de diferentes durações do armazenamento estático a frio foram avaliados, bem como diferentes protocolos de perfusão de membros.

partial-heterotopic-hindlimb-transplantation-model-in-rats

Research

JoVE Journal

Immunology and Infection

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Modelo murino de CD40-ativação de células B

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Imunologia e Infecção

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Transplante Limb-Hind ortotópico em ratos

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

No método de imagem in vivo Distinguir inflamação aguda e crônica

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Crônica de Longo Prazo<em&gt; Pseudomonas aeruginosa</em&gt; Infecção das vias aéreas em ratos

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

In Vitro e In Vivo de avaliação da atividade supressiva, T, B e células mieloides e respostas Humoral de pacientes transplantados

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

A Recovery Cardiopulmonary Bypass Model Without Transfusion or Inotropic Agents in Rats

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ complications related to prolonged CPB still compromise the outcome of cardiovascular surgeries, and may worsen postoperative morbidity and mortality. Animal models recapitulating the clinical usage of CPB enable the clarification of the pathophysiological processes that occur during CPB, and facilitate pre-clinical studies to develop strategies protecting against these complications. Rat CPB models are advantageous because of their greater cost-effectiveness, convenient experimental processes, abundant testing methods at the genetic or protein levels, and genetic consistency. They can be used for investigating the immune system activation and synthesis of proinflammatory cytokines, compliment activation, and production of oxygen free radicals. The rat models have been refined and have gradually taken the place of large-animal models. Here, we describe a simple CPB model without transfusion and/or inotropic agents in a rat. This recovery model allows the study of the long-term multiple organ sequelae of CPB.

Here, we present a protocol to describe a simple recovery cardiopulmonary bypass model without transfusion or inotropic agents in a rat. This model allows the study of the long-term multiple organ sequelae of cardiopulmonary bypass.

Um modelo de recuperação de Bypass cardiopulmonar sem transfusão ou inotrópicos em ratos

Cardiopulmonary bypass (CPB) is indispensable in cardiovascular surgery. Despite the dramatic refinement of CPB technique and devices, multi-organ ...

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

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

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

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

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

Optimization of the Cuff Technique for Murine Heart Transplantation

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

We introduce an inner tube approach to the cuff technique for mouse cervical heterotopic heart transplantation to help evert the vessel over the cuff. We found that cooperation between two experienced surgeons markedly shortens the operation time.

Otimização da Técnica de Manguito para Transplante de Coração de Murine

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to ...

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

We describe three methods of bone marrow transplantation (BMT): BMT with total-body irradiation, BMT with shielded irradiation, and BMT method with no pre-conditioning (adoptive BMT) for the study of clonal hematopoiesis in mouse models.

Procedimentos de transplante de medula óssea em camundongos para estudar hematopoiese clonal

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Purificação e Expansão de Células T Assassinos Naturais Invariantes para Estudos In vitro e In vivo

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...