The authors would like to thank the many individuals who have worked on and perfected this technique and have been responsible for the generation of many manuscripts both in this lab and others. This work was supported by National Institutes of Health Grant EY12707 (PMS) and an unrestricted grant from Research to Prevent Blindness to Department of Ophthalmology.

NOTA: Todos os animais utilizados neste procedimento são tratados de acordo com a Associação de Pesquisa em Visão e Oftalmologia declaração para o Uso de Animais em Oftálmica e Vision Research, bem como as diretrizes estabelecidas pelo animal comitê de supervisão em Saint Louis University. Nota: Todos os instrumentos cirúrgicos e soluções são esterilizadas antes da cirurgia para limitar a infecção microbiana do olho. Deve notar-se que, enquanto os animais experimentam alguma dor com este procedimento, não empregamos analgésicos. A razão para isto é porque todos são analgésicos e anti-inflamatórios vez que as respostas de transplante de córnea envolvem a inflamação, a utilização de drogas anti-inflamatórias iria comprometer a nossa capacidade para determinar quais os factores que estão envolvidos na falha de enxerto da córnea. 1. Anestesia <ol style="margin-left: 40px;"><li> Coloque ratos doadores e receptores sob anestesia geral por meio de injeções IP de cetamina (86.98 mg / kg) e xilazina (13,04 mg / kg). </li><li> Mantenha o mouse destinatário sob anestesia durante todo o processo, que normalmente leva 30 minutos a uma hora. Consequentemente, monitorar constantemente o mouse para detectar quaisquer sinais de recuperar a consciência. </li><li> Aplicar puralube pomada para o olho que não vai passar por cirurgia depois que o animal é anestesiado para prevenir o ressecamento. </li></ol> 2. Alongamento de córnea <ol style="margin-left: 40px;"><li> Obtenção do botão da córnea do doador. <ol><li> Uma vez que o animal é completamente anestesiados, atingir midríase por administração adequada de um par de gotas oculares de tropicamida a 1% e 2,5% de cloridrato de fenilefrina. </li><li> Posicione a cabeça do animal doador horizontalmente em uma placa colocada em um suporte móvel resistente. Fixar a cabeça com uma tira de fita adesiva em volta do pescoço para assegurar que o olho está em uma posição horizontal ao longo de toda a operação. </li><li> Usando um trephine 2 mm de diâmetro, cuja ponta foi tingida com azul de metileno, para foraforrar o local do enxerto da córnea central. </li><li> Com uma lâmina afiada, penetrar na córnea e injectar Healon dentro da câmara anterior para a aprofundar a reduzir a possibilidade de danos no endotélio dador e lente subjacente. </li><li> Extirpar o enxerto doador com Vannas tesoura e coloque em um prato contendo solução salina equilibrada de Hanks até o uso. </li><li> Após o enxerto de doadores tem sido removido, eutanásia do camundongo doador através de inalação de CO2. </li></ol></li><li> Preparando o leito do enxerto. <ol><li> Repita os mesmos passos como descrito em 2.1.1 através 2.1.2 para o destinatário. </li><li> Usando uma trefina de diâmetro 1,5 mm, delinear local do enxerto destinatário. </li><li> Com uma lâmina afiada, penetrar na córnea e injectar Healon dentro da câmara anterior para a aprofundar a reduzir a possibilidade de danos para a lente subjacente. </li><li> Remova o botão central da córnea delineado a partir do destinatário usando Vannas tesoura e descarte. </li></ol></li><li> Suturar oenxerto <ol><li> Coloque a córnea do doador sobre o enxerto cama na córnea do destinatário. Certifique-se de que Healon adequado está sob a córnea do doador para proteger as células endoteliais do doador de dano pelo contato direto com a lente. </li><li> Usando micropinças derrubadas super fino, coloque a primeira mordida de nylon 11-0 sutura no lado do doador, por meio do doador com 90% de profundidade de espessura total para o lado do destinatário, em seguida, amarrar. </li><li> Uma vez que a córnea está ancorada no lugar, realizar suturas interrompidas midcardinal de tal forma que a córnea tem de 8 a 10 pontos totais e da córnea do doador está firmemente alinhados com e anexados ao leito do enxerto corneano destinatário. </li></ol></li><li> O aprofundamento da câmara anterior <ol><li> Aprofundar a câmara anterior através da injecção de HBSS ou bolha de ar para dentro da câmara anterior e verificar cuidadosamente a integridade do enxerto da córnea para detecção de fugas com uma esponja de celulose. NOTA: Se a câmara anterior não pode ser reformado, então há uma alta probabilidade de cataract que fará com que as futuras avaliações da córnea transplantada muito difícil e também irá levar à disfunção endotélio corneano doador e falha assim enxerto. </li></ol></li><li> Avaliação final <ol><li> Observar o olho para determinar que a pupila é redonda e a profundidade da câmara anterior é normal. NOTA: Se o aluno não é redondo, isso indica que, durante a sutura da íris foi danificado e, assim, o enxerto é considerada uma falha técnica. </li><li> Aplicar pomada antibiótica ao olho. Opcional: Feche a tampa do olho com uma sutura de seda 7-0. </li><li> Observe ratos até que estejam totalmente acordado e depois individualmente abrigá-los por um período mínimo de dois dias após a cirurgia. </li></ol></li></ol> Remoção 3. Sutura <ol style="margin-left: 40px;"><li> Naqueles casos em que a tampa de sutura é usado, anestesiar os ratos, tal como descrito acima e remover o fio de sutura na tampa 48 h. </li><li> Anestesie camundongos no dia 7 pós-operatório. Remover o sutures fixar o enxerto da córnea. Uma vez que as suturas são removidas eo animal está totalmente desperto, coloque-o na gaiola. </li></ol> 4. Avaliação Clínica <ol style="margin-left: 40px;"><li> Examinar o olho para indicações de complicações processuais que incluem, catarata (opacificação do cristalino), hifema (sangue na câmara anterior), uma câmara anterior, que não é da profundidade adequada, ou opacidade significativa da córnea. Considere aqueles que demonstram estas complicações como &quot;comprometida&quot; e sacrificá-los por inalação de CO2. <ol><li> Realizar todos os exames em ratos não anestesiados. Segure o mouse com restringindo um lado, portanto, o mouse para que o outro lado pode proptose do olho para permitir uma melhor visão do olho. Uma vez que as observações são concluídas, devolver o animal para sua gaiola. </li></ol></li><li> Já um observador familiarizado com os grupos de tratamento avaliam córneas transplantadas 2-3 vezes por semana para os sinais de transplante de córneaepisódios de rejeição ou falência do transplante. Use o microscópio cirúrgico ou um biomicroscópio de lâmpada de fenda horizontal para estas observações. <ol><li> Avaliar cada córnea para a opacidade utilizando uma escala de 0 a 5. A escala é definida como se segue: <ol><li> Atribuir uma pontuação de 0 a essas córneas que não têm sinais de opacidade. </li><li> Atribuir uma pontuação de 1 a essas córneas que mostram opacidade superficial mínima. </li><li> Atribuir uma pontuação de 2 a córneas que exibem opacidade leve e profundo, mas o aluno subjacente e íris são ainda perceptíveis. </li><li> Atribuir uma pontuação de 3 a córnea que são apresentados opacidade do estroma da íris, em que não pode ser visto em pormenor, com a excepção de as margens da pupila. </li><li> Atribuir uma pontuação de 4 a córnea que exibir densa opacidade estromal e se há estruturas subjacentes podem ser vistos. </li><li> Atribuir uma pontuação de 5 a córnea que exibir completa opacidade e edema estromal intensivo, com a pupila e íris totalmente obscurecido. </li></ol></li><li><html> Também avaliar cada córnea para o grau de infiltração de vasos sanguíneos (neovascularização), utilizando uma escala de classificação de 1 a 8. Para realizar isto, ver a córnea como consistindo em quatro quadrantes iguais e determinar a quantidade de vasos sanguíneos em cada um destes quadrantes, com um marcar essa gama vontade de 0 (sem vasos) a 2 de extensa vascularização do que quadrante. Adicione as pontuações individuais de cada quadrante para calcular a pontuação final neovascularização. </li><li> Classifique córneas como agudamente rejeitados se eles têm uma pontuação de 3 para duas observações consecutivas para o tempo aponta até 5 semanas. </li><li> Classifique camundongos cuja córneas foram claros em 5 semanas, mas desenvolver opacificação às vezes&gt; 45 dias após o enxerto, com uma pontuação de 3 para dois momentos consecutivos, como tendo sofrido rejeições de fim de prazo. Use curvas de sobrevida de Kaplan-Meier para analisar a sobrevida do enxerto. </li></ol></li></ol> 5. A manipulação do Modelo em-left: 40px; &quot;&gt; <li> Preparação das células individuais do baço. <ol><li> Para preparar células individuais, primeira eutanásia do rato doador. Em seguida, remover o baço. </li><li> Inserir o baço em um filtro de células e rompê-la com um êmbolo da seringa a partir de uma seringa de 3 ml. </li><li> Lavar as células e ressuspender em 10 ml de Solução Salina Equilibrada de Hanks. </li><li> Retirar 10 jul de suspensão de células e adicionar 10 ul de 0,4% de azul tripano e misturar. Acrescentar que a hemocitômetro e contar as células na grade central. O número de células na câmara de ar é a contagem de células de 10 x 4 x 2 (factor de diluição em azul tripano) x 10 (volume no tubo). </li></ol></li><li> Injecção para a câmara anterior. <ol><li> Anestesiar ratos, tal como descrito anteriormente. </li><li> Realizar injecções usando um microscópio de dissecação. Para cada injecção intracameral, usar 10 6 células do baço em um volume de 0.005 ml e uma seringa de microlitro 0,25 ml, equipado com uma agulha 33 G. NOTA: Outras manipulaçõesdo modelo pode ser realizada pelo tratamento dos animais com os reagentes que actuam como antagonistas ou agonistas para determinar o papel que um factor particular pode desempenhar após a cirurgia do enxerto corneano ortotópico. </li></ol></li></ol>

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Ophthalmol. 52, 375-396 (2007).</li><li>Patel, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft+survival+and+endothelial+outcomes+in+the+new+era+of+endothelial+keratoplasty.">Graft survival and endothelial outcomes in the new era of endothelial keratoplasty.</a> Exp. Eye Res. 95, 40-47 (2012).</li><li>Anshu, A., Price, M. O., Tan, D. T., Price, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+keratoplasty:+a+revolution+in+evolution.">Endothelial keratoplasty: a revolution in evolution.</a> Surv. Ophthalmol. 57, 236-252 (2013).</li></ol>

The authors have no competing financial interests.

O modelo murino de transplante de córnea aqui descrito permite ao investigador para estudar rejeições humano em um modelo que é preditiva de quais fatores são mais associada tanto rejeição 15,17,18,20, 26-30 e 21-25 aceitação de córnea aloenxertos. Ao contrário do transplante de córnea humana, no qual os pacientes recebem tratamento com esteróides ou tópica ou sistêmica, quer tratar ou prevenir a rejeição de 31, este modelo é normalmente usado para determinar os fatores...

O transplante de córnea é um dos tipos comuns e mais bem sucedidas do transplante realizados em seres humanos. As razões pelas quais esta cirurgia é realizada são resultado de uma lesão, uma doença infecciosa, ou outras formas de doença não infecciosa de córnea 2. Números da Associação Eye Bank of America indicam que mais de 46 mil foram realizados em 2011 (veja o site em: restoresight.org/eye_banks/eye_banks.html). Uma indicação do seu sucesso é que as taxas de falha de um ano por córneas alogênicos variam de 10 a 15% e em 5 anos o sucesso é superior a 70% 3-8. Como vários estudos têm demonstrado, o sucesso de aloenxertos da córnea está directamente relacionada com o facto de o olho é um local imunologicamente privilegiado. Os factores responsáveis ​​para o status de córneas como um sítio imunologicamente privilegiado incluem a falta de ambos os vasos sanguíneos e linfáticos na córnea, uma ausência relativa de células apresentadoras de antigénios, factores produzidos pela córnea que suppress Funtions efetoras imunes 9-15, baixa expressão de antigénios MHC 16, e a expressão de FasL 17-20. No entanto, apesar destes fatores predisponentes desses enxertos para o sucesso, eles passam por rejeição 3-7. Consequentemente, a compreensão desses mecanismos que mediam essa rejeição, assim como testar várias terapias para prevenir a rejeição é de fundamental importância. Para esse fim, nós descrevemos aqui um modelo murino de transplante de córnea, que tem sido usado por mais de 20 anos para estudar o transplante de córnea em um ambiente experimental controlado. Como as respostas de transplante envolve muitos fatores diferentes que trabalham em conjunto que irá final determinam se o tecido transplantado falhar ou for bem-sucedido, não é possível compreender a importância desses fatores em qualquer modelo in vitro. Consequentemente, estudos utilizando animais intactos são necessários para determinar quais fatores são importantes para o sucesso ou failure de tecido transplantado. Enquanto outras espécies de animais têm sido utilizados para estudar o transplante de córnea, o modelo murino tem diversas vantagens quando comparado com a utilização de outras espécies. A primeira é a existência de muitas cepas de camundongos que expressam certos transgenes ou foram gene-alvo que falta expressão de fatores imunológicos específicos, cuja função no transplante podem ser melhor estudados. Além disso, existem muitos factores de ambos os reagentes (recombinantes e anticorpos que neutralizam factores) que são específicos para os ratos, e que não existem para muitas outras espécies de animais. Por causa da existência desses fatores, este modelo tem sido amplamente utilizado para identificar os fatores relevantes envolvidos em respostas de córnea aguda de aloenxerto 15, 17,18,20 -29. Além disso, muitos dos factores envolvidos no transplante de córnea são também conhecidos para serem funcionais no transplante de outros tecidos.

<table><tbody><tr><td>Zeiss Surgical Microscope</td><td>Zeiss</td><td>Rebuilt</td><td></td></tr><tr><td>1 ml Syringe</td><td>BD</td><td>305122</td><td></td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309602</td><td></td></tr><tr><td>Vannus Scissors</td><td>Stortz</td><td>E-3387</td><td></td></tr><tr><td>11-0 Sutures</td><td>Alcon</td><td>717939M</td><td></td></tr><tr><td>Trephine 2.0 mm</td><td>Katena</td><td>K 2-7520</td><td></td></tr><tr><td>Trephine 1.5 mm</td><td>Katena</td><td>K 2-7510</td><td></td></tr><tr><td>Tricaine Hydrochloride 0.5%</td><td>Alcon</td><td>NDC 0065-0741-12</td><td></td></tr><tr><td>Healon</td><td>Abbott</td><td>Healon OVD</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>7-0 Sutures</td><td>Alcon</td><td>8065</td><td></td></tr><tr><td>2.5% Phenylephrine HCl</td><td>Alcon</td><td>NDC 61314-342-02</td><td></td></tr><tr><td>1% Tropicamide</td><td>Bausch &amp;amp; Lomb</td><td>NDC-24208-585-59</td><td></td></tr><tr><td>Hamilton Syringe</td><td>Hamilton</td><td>7654-01</td><td></td></tr><tr><td>33 gauge needle</td><td>Hamilton</td><td>90033</td><td></td></tr><tr><td>Cell Strainer (100 &amp;mu;m nylon)</td><td>BD Falcon</td><td>352360</td><td></td></tr><tr><td>Hemocytometer</td><td>Cardinal Health</td><td>B3175</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154</td><td></td></tr></tbody></table>

murine corneal transplantation: a model to study the most common form of solid organ transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

O modelo murino de transplante de córnea tem sido usado por mais de 20 anos com sucesso para caracterizar os mecanismos de rejeição do enxerto corneano tanto 19-23 e aceitação da córnea do enxerto 13, 15,16,18, 24-27. Este modelo foi utilizado para determinar a importância da expressão de FasL na aceitação do enxerto corneano, em que os animais que não possuem FasL não foram capazes de aceitar enxertos da córnea 15. Também tem sido usado para demonstrar que factor de cresci...

Watch this Scientific Journal Video about Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation at JoVE.com

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Murino transplante de córnea: Um modelo para estudar a forma mais comum de Transplante de Órgãos Sólidos

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

murine-corneal-transplantation-model-to-study-most-common-form-solid

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

9.6K visualizações.  Saint Louis University. Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Vídeo: Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rato Transplante Renal: Modelos de rejeição de enxertos

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Medicina

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

Rim murino técnica de transplante

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murino Transplante de pele de espessura total

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Imunologia e Infecção

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 ...

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 ...

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 ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

Murine Modelo de Transplante de Aorta Cervical usando uma técnica de manguito não sutura modificada

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ transplantation. While the heterotopic vascularized murine cardiac transplantation model was initially utilized in studies of graft rejection using combinations of mismatched inbred mouse strains, access to genetically modified strains and therapeutic modalities can provide powerful new preclinical insights. Fundamentally, the surgical methodology for this technique has not changed since its development, especially with respect to important factors such as aseptic technique, anesthesia, and analgesia, which make material impacts on postsurgical morbidity and mortality. Additionally, improvements in perioperative management are expected to provide improvements in both animal welfare and experimental outcomes. This paper reports upon a protocol developed in collaboration with a subject matter expert in veterinary anesthesia and describes the surgical technique with an emphasis on perioperative management. Additionally, we discuss the implications of these refinements and provide details on troubleshooting critical surgical steps for this procedure.

The present paper describes a modified technique for heterotopic vascularized cardiac transplantation with updated aseptic technique, analgesia, and anesthesia.

Protocolo de transplante cardíaco heterotópico murino modificado que atende aos padrões contemporâneos de técnica asséptica, anestesia e analgesia

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Um modelo de cultura de órgão endotelial corneano porcina usando Split córnea botões

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

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.