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.

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			<td height="21" style="height:21px;width:216px;">Zeiss Surgical Microscope</td>
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			<td>305122</td>
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			<td height="21" style="height:21px;">3 mL Syringe</td>
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			<td style="width:119px;">T8154</td>
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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 ...

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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

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Transplante ortotópico de aorta: um modelo de rato para estudar o desenvolvimento de vasculopatia crônica

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Medicina

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Um método para isolamento de ilhotas murino e transplante renal subcapsular

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

Desenvolvimento de bronquiolite obliterante em um modelo murino de transplante de pulmão ortotópico

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

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 ...

Cardiac Catheterization in Mice to Measure the Pressure Volume Relationship: Investigating the Bowditch Effect

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these strategies is to examine their effects on heart function. There are several techniques to measure in vivo cardiac mechanics (e.g., echocardiography, pressure/volume relations, etc.). Compared to echocardiography, real-time left ventricular (LV) pressure/volume analysis via catheterization is more precise and insightful in assessing LV function. Additionally, LV pressure/volume analysis provides the ability to instantaneously record changes during manipulations of contractility (e.g., &#946;-adrenergic stimulation) and pathological insults (e.g., ischemia/reperfusion injury). In addition to the maximum (+dP/dt) and minimum (-dP/dt) rate of pressure change in the LV, an accurate assessment of LV function via several load-independent indexes (e.g., end systolic pressure volume relationship and preload recruitable stroke work) can be attained. Heart rate has a significant effect on LV contractility such that an increase in the heart rate is the primary mechanism to increase cardiac output (i.e., Bowditch effect). Thus, when comparing hemodynamics between experimental groups, it is necessary to have similar heart rates. Furthermore, a hallmark of many cardiomyopathy models is a decrease in contractile reserve (i.e., decreased Bowditch effect). Consequently, vital information can be obtained by determining the effects of increasing heart rate on contractility. Our and others data has demonstrated that the neuronal nitric oxide synthase (NOS1) knockout mouse has decreased contractility. Here we describe the procedure of measuring LV pressure/volume with increasing heart rates using the NOS1 knockout mouse model.

This article describes the measurement of murine left ventricular function via pressure/volume analysis at different heart rates.

O cateterismo cardíaco em camundongos para medir o volume Pressão do relacionamento: Investigando o efeito Bowditch

Animal models that mimic human cardiac disorders have been created to test potential therapeutic strategies. A key component to evaluating these ...

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 ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Desenvolvimento de um modelo experimental não invasivo, assistido a laser de perda de células endoteliais córneas

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

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

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

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

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

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

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

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

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

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

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

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

Transplante intramial de hidrogéis injetáveis de carregamento de MSC após infarto do miocárdio em um modelo murino

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...