Este projeto foi financiado pela Associação Alemã de Pesquisa (DFG, HE 4578/3-1).

A. Cartilagem Biópsia (Room Cirurgia; passos 1-5 na sala de preparação não-estéril) <ol><li> Realizar um controlo de peso final do coelho (coelho branco da Nova Zelândia, fêmea, 3,5-4,0 kg de peso corporal, 6 meses de idade) a fim de ser capaz de drogas de dose adequada e para monitorar o peso após a cirurgia. </li><li> Induzir a anestesia ao coelho por uma injecção intravenosa de 10 mg / kg de propofol. </li><li> Após entubação, manter a anestesia com 1,5 mg / kg / min de propofol e de 0,05 mg / kg por via intravenosa de fentanil. Monitorar anestesia usando capnografia, oximetria de pulso e freqüência cardíaca. </li><li> Raspar o joelho a ser operado com um cortador elétrico e vácuo da pele. </li><li> Desinfetar o joelho completamente raspada e cobrir o resto do coelho com uma compressa esterilizada. </li><li> Palpar a patela e realizar uma incisão medial da patela. </li><li> Abrir a articulação do joelho por uma artrotomia parapatelar média sob condições estéreis. Tente evitar qualquer corte pequeno suvasos sanguíneos perficial. </li><li> Deslocar a patela lateralmente. </li><li> Inspecione a articulação do joelho para eventuais anomalias e lesões de cartilagem macroscópicos. </li><li> Retire cuidadosamente pequenos pedaços de cartilagem (2-3 mm) para fora da tróclea femoral ossis com um bisturi estéril (Figura 1). </li><li> Colocar imediatamente estas biópsias da cartilagem num tubo de 50 ml com meio completo (DMEM + 10% de soro fetal de vitelo (FCS) + 1% de penicilina / estreptomicina (pen / estrep)) para continuar com o direito em cultura in vitro, após a operação. </li><li> Limpe os defeitos e lavá-los com soro fisiológico estéril. </li><li> Mude a posição da patela dentro do sulco da tróclea. </li><li> Fechamento da ferida em camadas com suturas um único botão (4-0 Vicryl) e uma sutura cutânea contínua (4-0 Monocryl). Use material de sutura absorvível. </li><li> Finalmente, selar a ferida com um spray de limpeza permeável ao vapor de água. </li></ol> B. In vitro Cultura <ol><li> Processar a biópsia cpeças artilage o mais rapidamente possível após a operação em um fluxo laminar tecido capa de cultura estéril. </li><li> Lava-se a estéril colhida cartilagem 2x em PBS com subsequente centrifugação a 500 xg durante 3 minutos, à TA. </li><li> Cortar cartilagem em pedaços de 1 mm, 3, a transferência para um Falcon de 50 ml e digerem num agitador durante 30 min com 10 ml de tripsina-EDTA (0,25%) e 10 ml de PBS durante 30 min </li><li> Após 30 minutos, parar a tripsinização adicionando meio completo. Então, agite as peças de cartilagem para o outro para 12 horas com colagenase A (0,21 U / mg) em meio livre de soro (DMEM + 1% Pen / Strep). </li><li> Centrifugar a solução resultante a 170 xg durante 3 minutos à temperatura ambiente. </li><li> Ressuspender sedimentos celulares em 5 ml de meio completo. </li><li> Semente os condrócitos isolados sobre a 25 cm 2 frascos de cultura de tecidos em 5 ml de meio completo e manter numa incubadora de cultura de tecidos humidificada a 37 ° C, 5% de CO 2. </li><li> Cresça condrócitos a uma densidade de 80%. </li><li> Solte a células ffrascos rom por lavagem em PBS 2x antes de expor a 0,05% de tripsina-EDTA por 3 minutos. Uma vez que os condrócitos separar, parar de tripsinização pela adição de 10 mL médio completo. </li><li> Centrifugar a suspensão a 300 xg (3 minutos) e ressuspender o pellet em meio completo. </li><li> Células de sementes em frascos de cultura de tecidos (75 cm 2) e por partes a uma razão de 1:3 (~ 5.000 células / cm 2) a cada 5 ° dia ou ao 80% de confluência. </li><li> Determinar o número de células e viabilidade de tripan coloração azul. </li></ol> C. Cell-semeadura da Matriz <ol><li> Antes da implantação de lavagem e as células de libertação, tal como descrito acima. Efectuar a contagem de células e transferir 5 x 10 4 células para um tubo de microcentrífuga de 1,5 ml. Depois centrifugar a 500 xg durante 5 min à TA. </li><li> Ressuspender o sedimento em 15 mL meio completo para uma completa saturação da matriz. </li><li> Corte a bicamada colágeno andaime para as dimensões exatas do defeito trochlear por uma biópsia estérilsoco. </li><li> Matriz lugar num prato de cultura de tecido de tamanho adequado (por exemplo, placas de 24 poços). </li><li> Células de semente para o lado poroso, por células da matriz adesiva. Isto impede a migração de células para longe da matriz. </li><li> Adicione matrizes de células semeadas, sem mais meio de cultura de células dentro de uma incubadora durante 1 hora para permitir a aderência completa dos condrócitos. </li><li> Em seguida, coloque célula-matriz-construções em recipientes de cultura (por exemplo, placas de 24 poços) com meio completo fresco. Tome cuidado para não lavar as células fora da matriz, mesmo que forte adesão é esperado. </li><li> Agora, célula-matriz-construções podem ser levados para a sala de cirurgia para re-implantação. </li></ol> D. Matrix-assistida Transplante autólogo de condrócitos <ol><li> Realizar artrotomia parapatelar ao joelho contralateral como acima descrito (A. 1-8). </li><li> Criar dois defeitos condrais isoladas no sulco troclear com um operatin ar estérilg furadeira (3,6 mm de diâmetro). </li><li> Limpar o defeito e lavar com solução salina estéril. </li><li> Deve ser dada atenção para não abrir a cavidade da medula, a qualquer momento. Se necessário, vedar a parte inferior dos defeitos com cauterização eléctrica contra a hemorragia. </li><li> Implante as matrizes semeados com os condrócitos de cultura com o lado poroso virados para baixo (Figura 2). Em seguida, pressione-se encaixam nos furos e lave a superfície circundante. </li><li> Vedar as matrizes implantados com um pouco de cola de fibrina (Tissucol Duo S) para garantir a fixação segura. </li><li> Após a coagulação, mudar a patela no sulco troclear e aplicar gama completa de movimento para a articulação do joelho algumas vezes. </li><li> Deslocar a patela lateralmente mais uma vez e verifique se há algum evento ou sinal de fixação instável. As matrizes devem ser realizadas ainda no local. </li><li> Substitua a patela novamente e terminar a operação com o fechamento da ferida em camadas e spray de vestir, como descrito acima. </li></ol>

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Os autores não têm nada a revelar.

O protocolo apresentado fornece uma técnica estabelecida 9,12,13 e facilmente reprodutível para isolar condrócitos autólogos para a proliferação e posterior re-implantação em defeitos da cartilagem criadas artificialmente em joelhos de coelho. A utilização de condrócitos autólogos para a remodelação e reparação de lesões da cartilagem articular já está em uso clínico proporcionar resultados satisfatórios a longo prazo 6. Principais problemas como, p...

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent/equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Biochrom AG</td>
			<td>F 0415</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td>10 103 586 001</td>
			<td>0.21 U/mg</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>PAN Biotech GmbH</td>
			<td>3702-P103009</td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Biochrom AG</td>
			<td>A 2213</td>
			<td>10,000 U/ml/10,000 &mu;g/ml</td>
		</tr>
		<tr>
			<td>PBS Dulbecco (1X)</td>
			<td>Biochrom AG</td>
			<td>L1815</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td>Merck KgaA</td>
			<td>410230</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25 %/0.02 %</td>
			<td>Biochrom AG</td>
			<td>L2163</td>
			<td>in PBS w/o Ca2+, Mg2+</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Delta Select GmBH</td>
			<td>1819340</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl solution (0.9%)</td>
			<td>Bbraun</td>
			<td>8333A193</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dishes 100 mm/150 mm</td>
			<td>TPP AG</td>
			<td>93100/93150</td>
			<td>Growth area 60.1 mm2/147.8 mm2</td>
		</tr>
		<tr>
			<td>Tissue culture flasks 25/75 mm2</td>
			<td>TPP AG</td>
			<td>90025/90075</td>
			<td>25 mm2, 75 mm2</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (50 ml)</td>
			<td>TPP AG</td>
			<td>91050</td>
			<td>Gamma-sterilized</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Brand GmbH+Co KG</td>
			<td>717810</td>
			<td>Neubauer</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td>Sigma-Aldrich</td>
			<td>L8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Spray dressing (OpSite)</td>
			<td>Smith&amp;Nephew</td>
			<td>66004978</td>
			<td>Permeable for water vapor</td>
		</tr>
		<tr>
			<td>Chondro-Gide&Ograve;</td>
			<td>Geistlich Pharma AG</td>
			<td>30915.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch</td>
			<td>pfm medical ag</td>
			<td>48351</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissucol Duo S</td>
			<td>Baxter</td>
			<td>3419627</td>
			<td>0.5 ml</td>
		</tr>
	</tbody>
</table>

matrix-assisted autologous chondrocyte transplantation for remodeling and repair of chondral defects in a rabbit model

Defeitos da cartilagem articular são consideradas um importante problema de saúde porque a cartilagem articular tem uma capacidade limitada de auto-regeneração 1. Lesões de cartilagem não tratadas levar a dor contínua, afetam negativamente a qualidade de vida e predispõem para a osteoartrose. Durante as últimas décadas, várias técnicas cirúrgicas têm sido desenvolvidos para o tratamento de tais lesões. No entanto, até agora não foi possível conseguir uma reparação completa em termos de cobertura com o defeito da cartilagem articular ou hialina de proporcionar recuperação satisfatória a longo prazo 2-4. Portanto, as lesões da cartilagem articular continuam a ser um alvo privilegiado para técnicas regenerativas, como Engenharia de Tecidos. Em contraste com outras técnicas cirúrgicas, que muitas vezes conduzem à formação de tecido fibroso ou fibrocartilaginoso, Tissue Engineering visa restaurar completamente a complexa estrutura e as propriedades da cartilagem articular original usando o potencial condrogénico das células transplantadas. Rdesenvolvimentos ecent abriu possibilidades promissoras para terapias de regeneração da cartilagem. A primeira abordagem baseada em células para o tratamento de cartilagem em toda a espessura ou lesões osteocondrais foi realizada em 1994 por Lars Peterson e Pad Brittberg pioneiro condrócitos autólogos clínica (ACI) 5. Hoje em dia, a técnica está bem estabelecido clinicamente para o tratamento de grandes defeitos na cartilagem hialina do joelho, mantendo bons resultados clínicos até 10 a 20 anos após a implantação 6. Nos últimos anos, o implante de condrócitos autólogos foram submetidos a uma rápida progressão. O uso de um tridimensional colagénio matriz artificial no qual as células são replantadas subsequentemente transformou-se cada vez mais popular 7-9. MACT composto por dois procedimentos cirúrgicos: Em primeiro lugar, a fim de recolher os condrócitos, uma biópsia da cartilagem precisa ser realizada a partir de uma área de cartilagem não peso-rolamento de tjoint ele joelho. Em seguida, os condrócitos a ser extraídos, purificados e expandiu-se para um número suficiente de células in vitro. Os condrócitos são então semeadas em uma matriz tridimensional e pode ser subsequentemente re-implantado. Ao preparar um implante de tecido de engenharia, a taxa de proliferação e capacidade de diferenciação são fundamentais para a regeneração de tecidos bem sucedida 10. A utilização de uma matriz tridimensional, como uma célula transportadora é pensado para apoiar estas características celulares 11. O protocolo a seguir vai resumir e demonstrar uma técnica para o isolamento de condrócitos a partir de biópsias de cartilagem, a sua proliferação in vitro e a sua sementeira em um 3D-matriz (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Suíça). Finalmente, a implantação das células-matriz-construções em defeitos condrais criadas artificialmente de articulação do joelho de um coelho vai ser descrito. Esta técnica pode ser usada como uma configuração experimentalpara novas experiências de reparação da cartilagem.

A técnica cirúrgica descrita permite uma implantação bem sucedida e isolamento de condrócitos autólogos para um defeito condral artificial. A configuração experimental resultou em uma integração bem sucedida do implante na cartilagem ao redor. Após 12 semanas in vivo, o defeito condral foi preenchido por tecido de reparação com uma superfície homogénea e intacta, o que reduziu tensão de cisalhamento e danos no implante (Figura 4). Além disso, nenhum...

Watch this Scientific Journal Video about Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model at JoVE.com

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Uma técnica experimental para o tratamento de lesões condrais no joelho do coelho é descrito. O implante de condrócitos autólogos semeadas numa matriz é um método bem aceite para a remodelação e reparação de lesões da cartilagem articular que fornecem resultados satisfatórios a longo prazo. Matrix-assistida transplante de condrócitos autólogos (MACT) oferece um método padronizado e implante clinicamente estabelecida.

Matrix-assistida Transplante autólogo de condrócitos para remodelação e reparação de lesões condrais em um modelo de coelho

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

matrix-assisted-autologous-chondrocyte-transplantation-for-remodeling

Research

JoVE Journal

Medicine

14.3K visualizações.  Klinikum rechts der Isar der Technischen Universität München. Uma técnica experimental para o tratamento de lesões condrais no joelho do coelho é descrito. O implante de condrócitos autólogos semeadas numa matriz é um método bem aceite para a remodelação e reparação de lesões da cartilagem articular que fornecem resultados satisfatórios a longo prazo. Matrix-assistida transplante de condrócitos autólogos (MACT) oferece um método padronizado e implante clinicamente estabelecida.

Vídeo: Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

Um modelo do rato de No Utero Transplante

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Medicina

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 ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Um modelo de pele Rato Fetal de reparação de feridas Scarless

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

A Rat carótida Balão Injury modelo para testar Therapeutics remodela Anti-vasculares

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

Particulas de Lentivirus terapia gênica mediada por vetor de hepatócitos Ex Vivo para transplante autólogo em suínos

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Exame ecocardiográfico transtorácico no modelo coelho

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Transplante de Epitélio de Pigmento De Retina em um modelo de primata não humano para doenças degenerativas da retina

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.

Um modelo simplificado para transplante de válvula cardíaca heterotópica em roedores

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of ...

Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. However, lung transplantation is limited by a shortage of available donor organs. As such, there is high waitlist mortality. Ex situ lung perfusion (ESLP) has increased donor lung utilization rates in some centers by 15%-20%. ESLP has been applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs. Large animal (in vivo) transplantation models are required to validate ongoing in vitro research findings. Anatomic and physiologic differences between humans and pigs pose significant technical and anesthetic challenges. An easily reproducible transplant model would permit the&#160;in vivo&#160;validation of current ESLP strategies and the preclinical evaluation of various interventions designed to improve donor lung function. This protocol describes a porcine model of orthotopic left lung allotransplantation. This includes anesthetic and surgical techniques, a customized surgical checklist, troubleshooting, modifications, and the benefits and limitations of the approach.

This protocol describes a juvenile porcine model of orthotopic left lung allotransplantation designed for use with ESLP research. Focus is made on anesthetic and surgical techniques, as well as critical steps and troubleshooting.

Transplante ortotópico de pulmão esquerdo em modelo suíno juvenil para PESL

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. ...

Transcatheter Pulmonary Valve Replacement from Autologous Pericardium with a Self-Expandable Nitinol Stent in an Adult Sheep Model

Transcatheter pulmonary valve replacement has been established as a viable alternative approach for patients suffering from right ventricular outflow tract or bioprosthetic valve dysfunction, with excellent early and late clinical outcomes. However, clinical challenges such as stented heart valve deterioration, coronary occlusion, endocarditis, and other complications must be addressed for lifetime application, particularly in pediatric patients. To facilitate the development of a lifelong solution for patients, transcatheter autologous pulmonary valve replacement was performed in an adult sheep model. The autologous pericardium was harvested from the sheep via left anterolateral minithoracotomy under general anesthesia with ventilation. The pericardium was placed on a 3D shaping heart valve model for non-toxic cross-linking for 2 days and 21 h. Intracardiac echocardiography (ICE) and angiography were performed to assess the position, morphology, function, and dimensions of the native pulmonary valve (NPV). After trimming, the crosslinked pericardium was sewn onto a self-expandable Nitinol stent and crimped into a self-designed delivery system. The autologous pulmonary valve (APV) was implanted at the NPV position via left jugular vein catheterization. ICE and angiography were repeated to evaluate the position, morphology, function, and dimensions of the APV. An APV was successfully implanted in&#160;sheep J. In this paper, sheep J was selected to obtain representative results. A 30 mm APV with a Nitinol stent was accurately implanted at the NPV position without any significant hemodynamic change. There was no paravalvular leak, no new pulmonary valve insufficiency, or stented pulmonary valve migration. This study demonstrated the feasibility and safety, in a long-time follow-up, of developing an APV for implantation at the NPV position with a self-expandable Nitinol stent via jugular vein catheterization in an adult sheep model.

This study demonstrates the feasibility and safety of developing an autologous pulmonary valve for implantation at the native pulmonary valve position by using a self-expandable Nitinol stent in an adult sheep model. This is a step toward developing transcatheter pulmonary valve replacement for patients with right ventricular outflow tract dysfunction.

Substituição da válvula pulmonar transcateter de pericárdio autólogo com um Stent nitinol auto-expansível em um modelo de ovelha adulto

Transcatheter pulmonary valve replacement has been established as a viable alternative approach for patients suffering from right ventricular outflow ...