Este trabalho foi financiado pelo US Army Grant (W81XWH-08-1-0701) e uma bolsa de Carle Hospital Foundation.

Procedimento está em total compatibilidade com o número de protocolo do IACUC 13302 e foi aprovado pela divisão de recursos animais. 1. Instrumento Preparação <ol><li> Autoclave todos os instrumentos não descartáveis ​​usados ​​antes do procedimento cirúrgico para garantir a esterilidade. </li><li> Esterilizar instrumentos utilizados várias vezes em uma sessão com um esterilizador talão de vidro entre os usos. </li></ol> 2. Preparação Hidrogel <ol><li> Descongelar e células-tronco placa pelo menos 24 horas antes da criação do constructo hidrogel. </li><li> Prepare remendos um dia antes do procedimento cirúrgico usando um aparelho de estereolitografia (ou instrumento similar), que foi descrito em detalhes em publicações anteriores 8,18. </li><li> Para criar o hidrogel, gerar primeiro um design (CAD) modelo assistido por computador do patch desejado e exportar o projeto para o formato stereolithographic (STL). </li><li> Criar uma solução pré-gel por dissolving de poli (etilenoglicol) dimetacrilato (PEGDMA) em 1x PBS estéril para obter uma solução a 20% w / v. Ao mesmo tempo, dissolve-se o fotoiniciador 1- [4- (2-hidroxi-etoxi) -fenil] -2-hidroxi-2-metil-1-propano-1-ona em DMSO. Adicionar o fotoiniciador para a solução final de PEGDMA com um w / v de 0,5%. Directamente antes da reticulação, adicionar 100 ml de a densidade de células desejada. Nota: A densidade de células entre 2,0 x 10 5 -2,0 x 10 6 células / ml é recomendada. </li><li> Pipetar a solução de pré-gel em um prato no centro da plataforma de SLA e executar o software SLA com o desenho STL formatada previamente carregado. </li><li> Remover os remendos do aparelho e incubar os hidrogéis O / N em meio de Eagle modificado por Dulbecco (DMEM) suplementado com soro a 5% de bovino fetal (FBS), 100 U / ml de penicilina, e 100 mg / ml de estreptomicina para permitir células para ajustar para o ambiente dentro do patch. </li></ol> 3. Preparação Animal e intubati Oralem <ol><li> Avalie cada rato antes do procedimento para garantir que o animal está em boas condições de saúde, como determinado a partir de seus hábitos de higiene e interações com seus companheiros de gaiola. Nota: fêmeas de 8-10 semanas de idade C57BL / 6J foram usadas para este procedimento, no entanto, qualquer tipo ou idade do rato pode ser utilizado. </li><li> Inserir os animais numa câmara de anestesia e expor a 5% de isofluorano com de 1 L / min de O2 suporte. Monitorar o nível de anestesia por toe pitada reflexo. </li><li> Uma vez que o animal está devidamente anestesiados, pesar e colocá-los no stand intubação. </li><li> Dirigir uma fonte de luz em direcção cavidade torácica do animal e utilizar uma pinça para retrair a superfície da laringe e expor as cordas vocais. Passe cuidadosamente um G angiocath 20 entre as cordas vocais, e suavemente inserir na traquéia. Detectar uma inserção correcta por um movimento mecânico da cavidade torácica uma vez que o cateter está ligado ao ventilador pequeno de animais. </li><li> Ajustar as definições de ventilação para poe peso do animal com base nas orientações do fabricante. </li><li> Uma vez entubado, colocar o animal em decúbito dorsal sobre uma almofada de aquecimento para evitar a hipotermia. Certifique-se de que a almofada de aquecimento não deve exceder 40 ° C. Se necessário, colocar uma barreira entre o animal e a almofada de aquecimento para minimizar a possibilidade de queimaduras. </li><li> Remover o cabelo do local cirúrgico através da utilização de um creme depilatório. Esterilizar o site por 3 esfrega alternadas de betadine e etanol 75% para garantir a esterilização adequada. Inserir uma cortina para expor apenas o local cirúrgico, e colocar veterinário unguento para os olhos para evitar a secura durante o procedimento. </li></ol> 4. Procedimento Cirúrgico <ol><li> A toracotomia é realizada no lado esquerdo do animal, portanto, fazer uma incisão na pele aproximadamente 1 cm para a esquerda do esterno, que atravessa o comprimento do esterno. </li><li> Use uma pinça para separar a camada muscular subjacente da caixa torácica em dois passos. Observar uma Delineating linha que representa os músculos peitorais maiores do animal. Levante esta muscular ligeiramente, separá-lo do músculo oblíquo externo subjacente e retrair medial. Em seguida, liberar o músculo oblíquo externo da caixa torácica subjacente da mesma maneira, e retrair lateralmente, proporcionando uma vista desafogada das segunda, terceira e quarta costelas. </li><li> Realizar a toracotomia entre a terceira ea quarta costelas. Suavemente levantar a quarta costela e usar um cauterizador para abrir a cavidade do peito entre as terceira e quarta costela. Coloque afastadores para abrir ainda mais a cavidade e expor o coração. </li><li> Use uma pinça para romper o pericárdio do coração fina. </li><li> Ligadura da artéria coronária esquerda com uma sutura de nylon 8-0 monofilamento. Coloque a sutura de aproximadamente 4 mm a partir do vértice do coração, directamente abaixo da extremidade inferior do átrio esquerdo. Determinar a colocação adequada de sutura por um branqueamento do miocárdio ventricular e um aumento no tamanho do átrio esquerdo, após o Suture foi amarrado. </li></ol> 5. remendo Colocação e Glue <ol><li> Manter manchas a 37 ° C e 5% de CO 2 condições até à sua utilização. </li><li> Suavemente levantar o remendo com uma espátula plana terminada e colocá-lo suavemente na superfície do coração, no local do enfarte. Nota: Para evitar que escorregue do ventrículo esquerdo, o patch pode ser mantido no lugar por levemente mantendo contato usando a espátula ou a ponta de um par de fórceps. </li><li> Preparar a cola por mistura de uma proporção de 4: 1 de fibrinogénio a trombina. Misture a solução por pipetagem repetida até que começa a engrossar, geralmente dentro de 1 min após o início da preparação. </li><li> Uma vez que a viscosidade da solução atinge o nível desejado, transferir rapidamente aproximadamente 10 ml a superfície do emplastro. Nota: O tempo de coagulação da cola de fibrina é rápido, proporcionando uma pequena janela de oportunidade para a transferência eficiente da cola. </li></ol> 6. Sutura <ol><li&gt; Executar todas as suturas de fechamento usando uma sutura de nylon 6-0 monofilamento. </li><li> Feche a camada de costela com 3-4 suturas interrompidas individuais. </li><li> Antes do fechamento completo da camada intercostal, inserir uma cânula de PE-10 para dentro da incisão para evacuar a cavidade torácica após o encerramento é completa e restabelecer a pressão intrapleural adequada. </li><li> Feche os músculos peitorais com 3-4 suturas interrompidas individuais. </li><li> Selar a camada de pele com sutura contínua. </li><li> Depois do encerramento completo, anexar uma seringa de 1,0 ml para a extremidade da cânula e usá-lo para evacuar a cavidade torácica. </li><li> Aplicar um adesivo de tecido para o local da incisão para reforçar o local da sutura. </li></ol> 7. O tratamento pós-operatório <ol><li> Submeter ratinhos uma injecção de buprenorfina (0,05-1,0 mg / kg) e carprofeno (2,2 mg / kg) por via subcutânea imediatamente após a ligação coronária e pelo menos 20 min antes da avivamento. Administrar as duas drogas 6-8 horas após a cirurgiaE, em seguida, dar duas vezes por dia por até três dias para controlar a dor e angústia. </li><li> Monitorar continuamente os animais até consciente e cada hora para a seguinte primeira cirurgia 4 horas. Realize um segundo exame a noite da cirurgia (2-4 horas mais tarde) para administrar uma segunda dose de analgésico. </li><li> Monitorar os animais diariamente após o segundo dia após o procedimento, até que os animais parecem estáveis. </li></ol> 8. Análise da função cardíaca e Histologia <ol><li> Realizar ecocardiógrafos nos ratos 4 semanas após o procedimento para determinar a extensão dos danos após a MI. </li><li> Anestesiar ratos com 4-5% de isoflurano dentro de uma câmara de anestesia para a indução da anestesia inalatória e, em seguida 2% de isoflurano através de máscara facial para manutenção da anestesia. </li><li> Limpe a área de imagem com um creme depilatório e usar um pequeno ultra-som animal para obter uma varredura ecocardiografia 2D M-mode do batimento cardíaco e gravar os parâmetros asso funcionalciados com ele. </li><li> Eutanásia os ratos numa câmara de dióxido de carbono, em seguida, recolher os corações dos animais e em lugar de uma solução de formalina a 10% durante 24 h. Após 24 horas, transferir os corações para 70% de etanol e manter a 4 ° C até o processamento de tecidos. </li><li> Processar os corações e incorporá-los em blocos de parafina. </li><li> Cortar 5 milímetros secções através da região ventricular do coração, começando no vértice e terminando no átrio. </li><li> Stain fatias com tanto tricrômico de Masson e hematoxilina e eosina utilizando protocolos padrão segmentado. </li></ol>

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The authors declare that they have no competing financial interests.

Com esta abordagem para o modelo murino MI, desenvolvemos um sistema que minimiza o dano para as zonas não miocárdio, que estão associados a outras técnicas de MI de murino. Estas áreas incluem danos causados ​​por uma traqueostomia, o corte da camada muscular, e à ruptura de nervuras para expor a cavidade torácica. Nós acreditamos que estas mudanças melhorar os resultados globais cirúrgicos graças ao cuidado para manter o máximo das principais estr...

Um enfarte do miocárdio (MI) é definida como a interrupção de sangue para uma região do coração causada por oclusão de uma artéria coronária principal. O dano resultante de um MI é devido à remodelação do tecido cardíaco viável em tecido cicatricial não-funcional, o que diminui a capacidade do coração ou, mais especificamente, no ventrículo esquerdo, para bater apropriadamente. Isto resulta numa diminuição no volume de sangue que pode ser entregue ao corpo com cada batimento cardíaco, conhecido como o volume de curso, e a percentagem de sangue que é bombeado para fora do coração com cada batimento cardíaco, conhecido como a fracção de ejecção 6. Estas, juntamente com outras funções diminuídas, aumenta a pressão sobre o resto do coração para manter a função adequada. Muitas vezes, esse aumento da pressão pode se tornar tão grave que faz com que um segundo ataque cardíaco, um fenômeno visto em aproximadamente 10% dos indivíduos 7. Enquanto práticas médicas têm evoluído para tratarrescaldo de um MI, nenhuma técnica foi desenvolvida para parar, reduzir ou reverter os efeitos colaterais negativos da remodelação do tecido. Terapias com células-tronco surgiram como uma via possível para um tal tratamento, no entanto, apesar do seu potencial promissor, as células-tronco não têm sido bem sucedidos no contexto clínico. Uma teoria para as suas deficiências é a incapacidade de garantir as células benéficos permanecer no local de enfarte longo o suficiente para gerar resultados favoráveis ​​5. Demonstrou-se que não mais do que 24% de células que são simplesmente injectada no local de enfarte sobreviveram e permaneceram no local danificado um dias pós-parto 2. A perspectiva possível para resolver este problema de retenção celular é desenvolver sistemas de hidrogel biocompatíveis que encapsulam células ou terapêuticas, que podem ser entregues com o sítio danificado. O hidrogel de escolha neste protocolo é um poli (etileno-glicol) dimetacrilato devido ao seu uso anterior em encapsulamento de células procedures, no entanto, qualquer hidrogel capaz de encapsulação pode ser utilizado 8. A entrega do remendo directamente para o local da lesão assegura o contacto célula-a-tecido durante um período prolongado de tempo, aumentando o período de tempo as células podem fornecer factores benéficos para o miocárdio subjacente. Um gargalo para o remendo abordagem é a dificuldade de fazer aderir o penso à superfície do coração. Muitos grupos superar este através de uma variedade de técnicas, sendo o mais prevalente de uma sutura simples amarrar a construção à superfície do coração de 9,10. Isto tem sido bem sucedida num certo número de casos em que a construção é feita de um material mais rígido, mas falha quando tentada em um sistema de hidrogel, devido à concentração elevada de água e delicada natureza da construção remendo. Para superar isto, nós utilizamos um sistema de adesivo externa cola de fibrina que imita a química da formação de coágulos. A cola de fibrina tem sido utilizada em vários gabinetes médicos, Including lágrimas Dura, fístulas brônquicas, e transplante da córnea, destacando a biocompatibilidade do produto na forma de um selante de ferida 11-13. Além disso, a fibrina tem sido usado para uma variedade de fins cardíacas, incluindo o tratamento cirúrgico das rupturas do ventrículo esquerdo e cirurgias de revascularização miocárdica, no entanto, a sua utilização como uma cola de aderência para uma correção cardíaca não é comumente usado 14-17. Uma formulação simples de trombina e fibrinogénio resulta em uma cola biocompatível que pode ser colocado directamente no lado de fora de um remendo cardíaca externa, proporcionando um sistema de adesão viável para garantir a interacção remendo coração.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Harvard Model 687 Mouse Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>55-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Inintech Biosciences LLC Dry Glass Bead Sterilizer</td>
			<td>Fisher Scientific</td>
			<td>NC9531961</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica MZ6 surgical microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cautery Kit</td>
			<td>Gemini</td>
			<td>GEM 5917</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate Forceps - 0.4mm Tips Angled</td>
			<td>Fine Science Tools</td>
			<td>11063-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Agricola Retractor - 3.5cm Spread</td>
			<td>Fine Science Tools</td>
			<td>17005-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors - 2.5mm Blades Straight</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Needle Holder - w/Lock Tungsten Carbide 14cm</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors - Delicate Straight 10.5 cm</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 monofilament suture</td>
			<td>Ethicon</td>
			<td>8730P</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 Silk suture</td>
			<td>Ethicon</td>
			<td>639G</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T7009</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Sigma</td>
			<td>F3879</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
	</tbody>
</table>

a hydrogel construct and fibrin-based glue approach to deliver therapeutics in a murine myocardial infarction model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

Durante o procedimento cirúrgico, a ligação da artéria coronária esquerda pode ser identificado por um acentuado branqueamento a jusante da artéria oclusa. Como um teste antes de amarrar o nó, a sutura pode ser apertado brevemente para verificar se ele está no lugar apropriado. Além disso, uma vez que a oclusão da artéria os resultados na redução quase instantâneo na capacidade do átrio esquerdo para bater apropriadamente, átrio esquerdo irá aumentar em resposta a um refluxo de sangue no sistema. <p...

Watch this Scientific Journal Video about A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model. at JoVE.com

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

Um hidrogel Construir e fibrina à base de cola Approach to Deliver Therapeutics em um Infarto do Miocárdio modelo murino.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

a-hydrogel-construct-fibrin-based-glue-approach-to-deliver

Research

JoVE Journal

Medicine

8.6K visualizações.  University of Illinois at Urbana-Champaign. This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

Vídeo: A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

Infarto Agudo do Miocárdio em Ratos

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

Medicina

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Ligadura da artéria coronária e injeção intramiocárdica em um modelo murino de infarto

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Um modelo fechado de tórax murino de isquemia do miocárdio e reperfusão

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Infarto do Miocárdio e Avaliação de Resultados Funcional em porcos

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Avaliação do resultado principal em um modelo suíno de Infarto Agudo do Miocárdio

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Infarto do miocárdio em ratos neonatais, um modelo de regeneração cardíaca

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Modelo de infarto do miocárdio murino usando ligadura permanente da artéria coronária descendente anterior esquerda

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

Um modelo Crioferimento para estudar infarto do miocárdio no mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Estabelecendo um modelo suíno de insuficiência cardíaca pós-miocárdio para tratamento de células-tronco

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

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 ...