Este trabajo fue financiado por los EEUU Grant Ejército (W81XWH-08-1-0701) y una beca del Hospital Carle Fundación.

Procedimiento está en plena compatibilidad con número de protocolo IACUC 13.302 y ha sido aprobado por la división de los recursos animales. 1. Preparación de instrumentos <ol><li> Autoclave todos los instrumentos no desechables utilizados antes del procedimiento quirúrgico para garantizar la esterilidad. </li><li> Esterilizar los instrumentos utilizados varias veces en una sesión con un esterilizador de cuentas de cristal entre usos. </li></ol> 2. Preparación de hidrogel <ol><li> Descongele y células madre placa al menos 24 horas antes de la creación de la construcción de hidrogel. </li><li> Preparar parches un día antes de la intervención quirúrgica utilizando un aparato de estereolitografía (o instrumento similar) que se ha descrito con todo detalle en publicaciones anteriores 8,18. </li><li> Para crear el hidrogel, primero generar un modelo de diseño asistido por ordenador (CAD) del parche deseado y exportar el diseño a formato estereolitográfico (STL). </li><li> Crear una solución de pre-gel mediante dissolving poli (etilenglicol) dimetacrilato (PEGDMA) en 1X PBS estéril para obtener un 20% w solución / v. Al mismo tiempo, disolver el fotoiniciador 1- [4- (2-hidroxi-etoxi) -fenil] -2-hidroxi-2-metil-1-propano-1-ona en DMSO. Añadir el fotoiniciador a la solución PEGDMA en una final w / v de 0,5%. Directamente antes de la reticulación, añadir 100 ml de la densidad deseada de las células. Nota: Una densidad celular entre 2,0 x 10 5 -2,0 x 10 6 células se recomienda / ml. </li><li> Pipetear la solución de pre-gel en un plato en el centro de la plataforma de SLA y ejecutar el software SLA con el diseño STL formateado previamente cargado. </li><li> Retire los parches desde el aparato e incubar los hidrogeles O / N en medio de Dulbecco modificado por Eagle (DMEM) suplementado con 5% de suero bovino fetal (FBS), 100 U / ml de penicilina, y 100 mg / ml de estreptomicina para permitir células para ajustar al medio ambiente dentro del parche. </li></ol> 3. Animal Preparación y intubati Oralen <ol><li> Evaluar cada ratón antes del procedimiento para asegurarse de que el animal se encuentra en buen estado de salud, según se determina a partir de sus hábitos de aseo e interacciones con sus compañeros de jaula. Nota: Mujer 8-10 semanas de edad C57BL / 6J se utilizaron para este procedimiento, sin embargo, cualquier edad ratón o tipo pueden ser utilizados. </li><li> Coloque los animales en una cámara anestésica y exponer a 5% isofluorano con un soporte 1 L / min O 2. Vigilar el nivel de anestesia por reflejo pizca dedo del pie. </li><li> Una vez que el animal está anestesiado adecuadamente, pesar y colocarlos en el stand de la intubación. </li><li> Dirigir una fuente de luz hacia la cavidad del pecho del animal y el uso de fórceps para retraer la superficie de laringe y exponer las cuerdas vocales. Guiar con cuidado un G angiocath 20 entre las cuerdas vocales, y suavemente inserte en la tráquea. Detectar la inserción apropiada por un movimiento mecánico de la cavidad del pecho una vez que el catéter se conecta a la pequeña ventilador animal. </li><li> Ajustar la configuración de ventilación a the peso del animal en base a las instrucciones del fabricante. </li><li> Una vez intubado, coloque el animal en posición supina sobre una almohadilla térmica para evitar la hipotermia. Asegúrese de que la almohadilla térmica no exceda los 40 ° C. Si es necesario, colocar una barrera entre el animal y la almohadilla de calefacción para minimizar la posibilidad de quemaduras. </li><li> Retire el cabello del sitio quirúrgico a través del uso de una crema depilatoria. Esterilizar el sitio por 3 matorrales alternas de betadine y etanol al 75% para asegurar una correcta esterilización. Coloque un paño para exponer sólo el lugar de la cirugía, y coloque un ungüento veterinario en los ojos para evitar la sequedad durante el procedimiento. </li></ol> 4. Procedimiento Quirúrgico <ol><li> La toracotomía se realiza en el lado izquierdo del animal, por lo tanto, hacer una incisión en la piel de aproximadamente 1 cm a la izquierda del esternón, que corre a lo largo del esternón. </li><li> El uso de fórceps para separar la capa muscular de la caja torácica subyacente en dos pasos. Observar un delineCIONES línea que representa el pectoral mayor músculos del animal. Levante este músculo ligeramente, separarla del músculo oblicuo externo subyacente y retraer medialmente. A continuación, liberar el músculo oblicuo externo de la caja torácica subyacente de la misma manera, y retraer lateralmente, proporcionando una vista despejada de las segunda, tercera y cuarta costillas. </li><li> Realice la toracotomía entre las tercera y cuarta costillas. Levantar suavemente la cuarta costilla y utilizar un cauterizador para abrir la cavidad torácica entre las costillas tercera y cuarta. Coloque retractores para abrir aún más la cavidad y exponer el corazón. </li><li> El uso de fórceps para romper la delgada pericardio del corazón. </li><li> Ligar la arteria coronaria izquierda con una sutura de nylon monofilamento 8-0. Coloque la sutura aproximadamente 4 mm desde el vértice del corazón, directamente debajo de la punta inferior de la aurícula izquierda. Determinar la colocación de sutura adecuada por un blanqueo del miocardio ventricular y un aumento en el tamaño de la aurícula izquierda después de la suture se ha atado. </li></ol> 5. Colocación de parches y pegamento <ol><li> Mantenga los parches a 37 ° C y 5% de CO 2 condiciones hasta su uso. </li><li> Levante suavemente el parche usando una espátula de extremo plano y suavemente colocarlo en la superficie del corazón, en el sitio de miocardio. Nota: Para evitar que se salga el ventrículo izquierdo, el parche puede ser mantenido en su lugar por el mantenimiento de la ligera contacto utilizando la espátula o la punta de un par de pinzas. </li><li> Preparar el pegamento mezclando una proporción de 4: 1 de fibrinógeno a trombina. Mezclar la solución mediante pipeteo repetido hasta que empiece a espesar, generalmente dentro de 1 min después del comienzo de la preparación. </li><li> Una vez que la viscosidad de la solución alcanza el nivel deseado, transferir rápidamente aproximadamente 10 ml a la superficie del parche. Nota: El tiempo de coagulación de la cola de fibrina es rápida, proporcionando una pequeña ventana de oportunidad para la transferencia eficiente de la cola. </li></ol> 6. La sutura <ol><li&gt; Realizar todas las suturas de cierre utilizando una sutura de nylon monofilamento 6-0. </li><li> Cierre la capa de costilla con tres y cincuenta y siete suturas interrumpidas individuales. </li><li> Antes del cierre completo de la capa intercostal, insertar una cánula PE-10 en la incisión para evacuar la cavidad del pecho después del cierre es completo y restablecer la presión intrapleural adecuada. </li><li> Cierre los músculos pectorales con tres y cincuenta y siete suturas interrumpidas individuales. </li><li> Sellar la capa de la piel con una sutura continua. </li><li> Tras el cierre completo, adjunte una jeringa 1,0 ml hasta el final de la cánula y utilizarla para evacuar la cavidad torácica. </li><li> Aplicar un adhesivo de tejido para el sitio de la incisión para reforzar el sitio de sutura. </li></ol> Tratamiento 7. Post-quirúrgica <ol><li> Dé ratones una inyección de buprenorfina (0,05-1,0 mg / kg) y carprofeno (2,2 mg / kg) por vía subcutánea inmediatamente después de la ligadura coronaria y menos 20 min antes de la reactivación. Administrar ambos fármacos 6-8 hr después de la cirugía, Y luego dar dos veces al día durante un máximo de tres días para controlar el dolor y la angustia. </li><li> Continuamente monitorear los animales hasta consciente y cada hora durante la primera después de la cirugía de 4 horas. Realizar un segundo chequeo de la noche de la cirugía (2-4 horas más tarde) para administrar una segunda dosis de analgésico. </li><li> Monitorear los animales todos los días después de que el segundo día después del procedimiento, hasta que los animales parecen estables. </li></ol> 8. Análisis de la función del corazón y de Histología <ol><li> Realizar ecocardiografías en los ratones 4 semanas siguiendo el procedimiento para determinar la extensión del daño después de la MI. </li><li> Anestesiar ratones con 4-5% isoflurano dentro de una cámara de anestesia para la inducción de la anestesia de inhalación, y luego 2% de isoflurano a través de mascarilla para el mantenimiento de la anestesia. </li><li> Despeje el área de imagen con una crema depilatoria y utilizar un pequeño animal de ultrasonido para obtener una exploración ecocardiografía en modo M 2D del corazón que late y registrar los parámetros asso funcionalciados con él. </li><li> La eutanasia a los ratones en una cámara de dióxido de carbono, a continuación, recoger los corazones de los animales y el lugar en una solución de formalina al 10% durante 24 horas. Después de 24 horas, la transferencia de los corazones de etanol al 70% y mantener a 4 ° C hasta el procesamiento de tejidos. </li><li> Procesar los corazones y les incrustar en bloques de parafina. </li><li> Cortar 5 mm secciones a través de la región ventricular del corazón, comenzando en el vértice y que termina en el atrio. </li><li> Manchas seccionó rebanadas con tanto tricrómico de Masson y hematoxilina y eosina utilizando protocolos estándar. </li></ol>

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The authors declare that they have no competing financial interests.

Con este enfoque para el modelo murino MI, hemos desarrollado un sistema que minimiza el daño a las áreas no miocardio que se asocian con otras técnicas de MI murinos. Estas áreas incluyen los daños causados ​​por una traqueotomía, el corte de la capa muscular, y la rotura de costillas para exponer la cavidad torácica. Creemos que estos cambios mejoran el resultado global gracias quirúrgicos para el cuidado de mantener la mayor cantidad de las principal...

Un infarto de miocardio (MI) se define como la interrupción de la sangre a una región del corazón causada por la oclusión de una arteria coronaria principal. El daño resultante de un MI es debido a la remodelación del tejido cardiaco viable en el tejido de la cicatriz no funcional, lo que disminuye la capacidad del corazón o, más específicamente, el ventrículo izquierdo, a latir correctamente. Esto resulta en una disminución en el volumen de sangre que se puede entregar al cuerpo con cada latido del corazón, conocido como el volumen de carrera, y el porcentaje de sangre que se bombea fuera del corazón con cada latido del corazón, conocida como la fracción de eyección 6. Estos, junto con otras funciones disminuido, aumenta la tensión en el resto del corazón para mantener la función adecuada. A menudo, este aumento de la tensión puede llegar a ser tan grave que provoca un segundo ataque al corazón, un fenómeno visto en aproximadamente el 10% de los individuos 7. Mientras que las prácticas médicas han evolucionado para tratarInmediatamente después de un infarto de miocardio, ninguna técnica ha sido desarrollada para detener, lento o revertir los efectos secundarios negativos de la remodelación de tejidos. Terapias con células madre han surgido como una posible vía para un tratamiento de este tipo, sin embargo, a pesar de su prometedor potencial, las células madre no han demostrado su eficacia en el ámbito clínico. Una teoría para sus deficiencias es la incapacidad para asegurar las células beneficiosos permanecen en el sitio de miocardio tiempo suficiente para generar resultados favorables 5. Se ha demostrado que no más del 24% de las células que simplemente se inyecta en el lugar de miocardio sobrevivió y se mantuvo en el sitio 1 día después de la entrega dañado 2. Una posible perspectiva para abordar este problema de la retención de células es el desarrollo de sistemas de hidrogel biocompatible que encapsulan cualquiera de las células o la terapéutica, que pueden ser entregados al sitio dañado. El hidrogel de elección en este protocolo es un poli (etilenglicol) dimetacrilato debido a su uso anterior en pr encapsulación de célulasocedures, sin embargo, cualquier hidrogel capaz de encapsulación se puede utilizar 8. La entrega del parche directamente al sitio de la lesión asegura el contacto de célula a tejido durante un período prolongado de tiempo, el aumento de la longitud de tiempo, las células pueden proporcionar factores beneficiosos para el miocardio subyacente. Un cuello de botella para el enfoque de parche es la dificultad de adherir el parche a la superficie del corazón. Muchos grupos han superado este a través de una variedad de técnicas, la más prevalente ser una sutura simple de atar el constructo a la superficie del corazón 9,10. Esto ha demostrado ser exitosa en una serie de casos en los que el constructo está hecha de un material más rígido, pero falla cuando intento en un sistema de hidrogel, debido a la alta concentración de agua y delicada naturaleza de la construcción de parche. Para superar esto, hemos utilizado un sistema de adhesivo externa pegamento de fibrina que imita la química de la formación de coágulos. El pegamento de fibrina se ha utilizado en numerosas cirugías Medical, IncLuding lágrimas Dura, fístulas bronquiales, y trasplante de córnea, destacando la biocompatibilidad del producto como un sellador herida 11-13. Además, la fibrina se ha utilizado para una variedad de propósitos cardíacas, incluyendo el tratamiento quirúrgico de las rupturas del ventrículo izquierdo y cirugías de bypass de arteria coronaria, sin embargo, su uso como un pegamento de adherencia para un parche cardíaco no se usa comúnmente 14-17. Una formulación sencilla de trombina y fibrinógeno resultados en una cola biocompatible que se puede colocar directamente en el exterior de un parche cardíaco externo, proporcionando un sistema de adhesión viable para asegurar el parche a la interacción corazón.

<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 el procedimiento quirúrgico, la ligadura de la arteria coronaria izquierda se puede identificar por un marcado blanqueo de aguas abajo de la arteria ocluida. Como una prueba antes de atar el nudo, la sutura se puede apretar brevemente para comprobar si está en el lugar apropiado. Adicionalmente, puesto que la oclusión de los resultados de la arteria en disminución casi instantánea en la capacidad de la aurícula izquierda a latir correctamente, la aurícula izquierda se agrandará en respuesta a un reflujo ...

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.

Un hidrogel Construct y fibrina enfoque basado Pegamento para ofrecer Terapéutica en un infarto de miocardio 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 vistas.  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.

Video: 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 de miocardio en ratas

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

Investigación

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 de las arterias coronarias y la inyección intramiocárdica en un 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.

Un murino tórax cerrado modelo de isquemia y reperfusión miocárdica

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 de miocardio y la evaluación de resultados funcionales en los cerdos

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.

La evaluación de resultado primaria en un modelo porcino de infarto agudo de miocardio

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.

El infarto de miocardio en ratones recién nacidos, un modelo de regeneración cardiaca

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 de miocardio murino usando ligadura permanente de la arteria coronaria descendente anterior izquierda

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.

Un modelo de criolesiones para estudiar el infarto de miocardio en el ratón

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.

Establecer un modelo porcino de infarto de infarto post-miocardio insuficiencia cardíaca para el tratamiento de células madre

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.

Trasplante intramiocardio de hidrogeles inyectables msc-loading después de infarto de miocardio en un 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 ...