Ce travail a été financé par Grant US Army (W81XWH-08-1-0701) et une bourse de recherche de l&#39;Hôpital Carle Fondation.

La procédure est en pleine compatibilité avec le numéro de protocole IACUC 13302 et a été approuvé par la division des ressources animales. 1. Instrument Préparation <ol><li> Autoclave tous les instruments non jetables utilisés avant l&#39;intervention chirurgicale pour assurer la stérilité. </li><li> Stériliser les instruments utilisés plusieurs fois dans une session avec un stérilisateur à billes de verre entre les utilisations. </li></ol> 2. Préparation d&#39;hydrogel <ol><li> Décongeler les cellules souches et de la plaque d&#39;au moins 24 heures avant la création de la construction d&#39;hydrogel. </li><li> Préparer les correctifs un jour avant l&#39;intervention chirurgicale en utilisant un appareil de stéréolithographie (ou un instrument similaire) qui a été décrit en détail dans les publications précédentes 8,18. </li><li> Pour créer l&#39;hydrogel, d&#39;abord générer un design (CAD) modèle du patch désiré assistée par ordinateur et la conception exporter au format de stéréolithographie (STL). </li><li> Créer une solution de pré-gel par dissolving de poly (éthylène glycol) diméthacrylate (PEGDMA) dans du PBS 1x stérile pour obtenir une solution à 20% p / v. En même temps, dissoudre le photoinitiateur 1- [4- (éthoxy-hydroxy-2) phényl] -2-hydroxy-2-méthyl-1-propane-1-one dans du DMSO. Ajouter le photo-initiateur à la solution finale à une PEGDMA en poids / volume de 0,5%. Directement avant réticulation, ajouter 100 ml de la densité souhaitée de cellules. Remarque: Une densité de cellules entre 2,0 x 10 5 -2,0 x 10 6 cellules / ml est recommandée. </li><li> Introduire à la pipette la solution de pré-gel dans un plat au centre de la plate-forme SLA et exécuter le logiciel SLA avec la conception de la STL formaté précédemment téléchargée. </li><li> Retirer les plaques de l&#39;appareil et incuber les hydrogels O / N, dans Eagle modifié du milieu de Dulbecco (DMEM) supplémenté avec du sérum de 5% de fœtus bovin (FBS), 100 U / ml de pénicilline et 100 mg / ml de streptomycine afin de permettre pour les cellules à ajuster à l&#39;environnement dans le patch. </li></ol> 3. Préparation des animaux et intubati oralessur <ol><li> Évaluer chaque souris avant la procédure pour assurer que l&#39;animal est en bonne santé, tel que déterminé à partir de leurs habitudes de toilettage et les interactions avec leurs compagnons de cage. Note: 8-10 semaines vieilles souris C57BL / 6J femelles ont été utilisés pour cette procédure, cependant, tout âge de la souris ou de type peuvent être utilisés. </li><li> Placez les animaux dans une chambre d&#39;anesthésie et exposer à 5% isofluorane avec un 1 L / min 2 O soutien. Surveiller le niveau de l&#39;anesthésie par pincement de l&#39;orteil réflexe. </li><li> Une fois que l&#39;animal est correctement anesthésié, peser et de les placer sur le stand de l&#39;intubation. </li><li> Diriger une source de lumière vers la cavité thoracique de l&#39;animal et utiliser une pince pour retirer la surface du larynx et d&#39;exposer les cordes vocales. Guidez soigneusement un G angiocath 20 entre les cordes vocales, et insérer en douceur dans la trachée. Détecter l&#39;insertion correcte par un mouvement mécanique de la cavité thoracique, une fois que le cathéter est raccordé au petit ventilateur animal. </li><li> Réglez les paramètres de ventilation à THpoids e de l&#39;animal sur la base des directives du fabricant. </li><li> Une fois intubé, placer l&#39;animal dans la position couchée sur un coussin chauffant pour prévenir l&#39;hypothermie. Assurez-vous que le coussin chauffant ne dépasse pas 40 ° C. Si nécessaire, placer une barrière entre l&#39;animal et le tampon de chauffage pour minimiser les risques de brûlures. </li><li> Enlever les poils à partir du site chirurgical à travers l&#39;utilisation d&#39;une crème dépilatoire. Stériliser le site en 3 gommages alternées de bétadine et 75% d&#39;éthanol pour assurer la stérilisation approprié. Placez un drap pour exposer tout le site chirurgical, et placez vétérinaire pommade sur les yeux pour prévenir la sécheresse au cours de la procédure. </li></ol> 4. Procédure chirurgicale <ol><li> La thoracotomie est réalisée sur le côté gauche de l&#39;animal, par conséquent, faire une incision de la peau d&#39;environ 1 cm à gauche du sternum, qui court le long du sternum. </li><li> Utiliser des pinces pour séparer la couche musculaire sous-jacent à partir de la cage thoracique en deux étapes. Observer un delineligne d&#39;exploita- qui représente les principaux muscles pectoraux de l&#39;animal. Soulevez légèrement ce muscle, le séparer du muscle oblique externe sous-jacente et se rétracter en dedans. Ensuite, libérer le muscle oblique externe de la cage thoracique sous-jacente de la même manière, et rétracter latéralement, fournissant une vue dégagée de la deuxième, troisième, et quatrième nervures. </li><li> Effectuez la thoracotomie entre les troisième et quatrième côtes. Soulevez doucement la quatrième côte et utiliser un cauterizer pour ouvrir la cavité thoracique entre les troisième et quatrième côtes. Placez rétracteurs à ouvrir davantage la cavité et d&#39;exposer le coeur. </li><li> Utilisez une pince à rompre le mince péricarde du cœur. </li><li> Ligaturer l&#39;artère coronaire gauche avec une suture en nylon monofilament 8-0. Placer la suture d&#39;environ 4 mm de la pointe du cœur, directement au-dessous de l&#39;extrémité inférieure de l&#39;oreillette gauche. Déterminer le placement de suture appropriée par un blanchiment du myocarde ventriculaire et une augmentation de la taille de l&#39;oreillette gauche après la suture a été liée. </li></ol> 5. Patch placement et de colle <ol><li> Gardez correctifs à 37 ° C et 5% de CO 2 conditions jusqu&#39;à utilisation. </li><li> Soulevez doucement le patch en utilisant une spatule indéterminée plat et placer doucement sur la surface du cœur, sur le site de l&#39;infarctus. Remarque: Pour éviter de glisser hors du ventricule gauche, le patch peut être maintenu en place par le maintien de la légère contact en utilisant soit la spatule ou la pointe d&#39;une paire de pinces. </li><li> Préparer la colle en mélangeant un ratio de 4: 1 du fibrinogène à la thrombine. Mélanger la solution par pipetage répété jusqu&#39;à ce qu&#39;il commence à épaissir, habituellement dans 1 min après le début de la préparation. </li><li> Une fois que la viscosité de la solution atteigne le niveau désiré, transférer rapidement d&#39;environ 10 ml à la surface du patch. Remarque: Le temps de coagulation de la colle de fibrine est rapide, fournissant une petite fenêtre d&#39;opportunité pour un transfert efficace de la colle. </li></ol> 6. Suture <ol><li&gt; Effectuer toutes les sutures de fermeture en utilisant une suture en nylon 6-0 monofilament. </li><li> Fermez la couche de nervure trois à quatre points de suture séparés individuels. </li><li> Avant la fermeture complète de la couche intercostal, insérer une canule PE-10 dans l&#39;incision à évacuer la cavité de la poitrine après la fermeture est complète et rétablir la pression intrapleurale correspondant. </li><li> Fermez les muscles pectoraux trois à quatre points de suture séparés individuels. </li><li> Sceller la couche de la peau avec une suture continue. </li><li> Suite à la fermeture complète, fixer une seringue de 1,0 ml à l&#39;extrémité de la canule et l&#39;utiliser pour évacuer la cavité thoracique. </li><li> Appliquer un adhésif tissulaire au site d&#39;incision à renforcer le site de suture. </li></ol> 7. Traitement post-opératoire <ol><li> Envoyer des souris une injection de buprénorphine (0,05-1,0 mg / kg) et le carprofène (2,2 mg / kg) par voie sous- cutanée immédiatement après la ligature coronaire et moins 20 min avant le réveil. Administrer deux médicaments 6-8 h après la chirurgie, Et ensuite donner deux fois par jour pour un maximum de trois jours pour contrôler la douleur et la détresse. </li><li> Surveiller en permanence les animaux jusqu&#39;à conscient et toutes les heures pour la première 4 heures après la chirurgie. Effectuer un deuxième bilan de la nuit de la chirurgie (2-4 heures plus tard) d&#39;administrer une deuxième dose d&#39;analgésique. </li><li> Surveiller les animaux tous les jours après le deuxième jour suivant la procédure, jusqu&#39;à ce que les animaux semblent stables. </li></ol> 8. Analyse de la fonction cardiaque et histologie <ol><li> Effectuer Échocardiographes sur les souris 4 semaines après la procédure afin de déterminer l&#39;étendue des dommages suite à la MI. </li><li> Anesthésier la souris avec 4-5% d&#39;isoflurane dans une chambre pour l&#39;induction de l&#39;anesthésie anesthésie par inhalation, puis 2% d&#39;isoflurane au masque pour entretien de l&#39;anesthésie. </li><li> Dégagez la zone d&#39;imagerie avec une crème dépilatoire et utiliser un petit échographie animale pour obtenir un balayage de l&#39;échocardiographie 2D M-mode du coeur battant et enregistrer des paramètres asso fonctionnelleciées avec elle. </li><li> Euthanasier les souris dans une chambre de dioxyde de carbone, puis recueillir les coeurs des animaux et les placer dans une solution de formol à 10% pendant 24 heures. Après 24 heures, transférer les cœurs à 70% d&#39;éthanol et de garder à 4 ° C jusqu&#39;à ce que le traitement des tissus. </li><li> Traiter les cœurs et les intégrer dans des blocs de paraffine. </li><li> Coupez 5 mm articles à travers la région ventriculaire du coeur, à partir de la pointe et se terminant à l&#39;atrium. </li><li> Stain tranches avec deux trichrome et hématoxyline Masson et aux taches de éosine utilisant des protocoles standards sectionné. </li></ol>

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The authors declare that they have no competing financial interests.

Avec cette approche pour le modèle MI murin, nous avons développé un système qui minimise les dommages aux zones non-myocarde qui sont associés à d&#39;autres techniques MI murins. Ces domaines comprennent les dommages causés par une trachéotomie, la coupe de la couche musculaire et la rupture des nervures afin d&#39;exposer la cavité de la poitrine. Nous croyons que ces changements améliorent les résultats globaux chirurgicales grâce au soin apporté ?...

Un infarctus du myocarde (IM) est défini comme l&#39;interruption de sang à une région du cœur provoquée par l&#39;occlusion d&#39;une artère coronaire principale. Le dommage résultant d&#39;un infarctus du myocarde est dû au remodelage du tissu cardiaque viable dans du tissu cicatriciel non fonctionnel, ce qui diminue la capacité du cœur ou, plus précisément, du ventricule gauche, à frapper correctement. Il en résulte une diminution du volume de sang qui peut être délivrée au corps avec chaque battement de coeur, connu sous le volume d&#39;éjection systolique, et le pourcentage de sang qui est pompé hors du cœur avec chaque battement du coeur, dite fraction d&#39;éjection 6. Ceux-ci, ainsi que d&#39;autres fonctions diminué, augmente la pression sur le reste du cœur de maintenir une fonction adéquate. Souvent, cette souche accrue peut devenir si grave que cela provoque une deuxième crise cardiaque, un phénomène vu dans environ 10% des individus 7. Bien que les pratiques médicales ont évolué pour traiterimmédiatement après un infarctus du myocarde, aucune technique a été développée pour arrêter, lente, ou inverser les effets secondaires négatifs de remodelage tissulaire. Thérapies par cellules souches ont émergé comme une avenue possible pour un tel traitement, cependant, en dépit de leur potentiel prometteur, les cellules souches ont pas fait ses preuves dans le cadre clinique. Une théorie pour leurs lacunes est l&#39;incapacité à assurer que les cellules restent bénéfiques sur le site de l&#39;infarctus assez longtemps pour générer des résultats favorables 5. Il a été démontré que pas plus de 24% des cellules qui sont simplement injecté dans le site de l&#39;infarctus a survécu et est resté au endommagé le site un jour après l&#39;accouchement 2. Une perspective possible pour aborder cette question de la rétention cellulaire est de développer des systèmes d&#39;hydrogel biocompatibles qui encapsulent soit des cellules ou thérapeutiques, qui peuvent être livrés avec le site endommagé. L&#39;hydrogel de choix dans ce protocole est un poly (éthylène glycol) diméthacrylate raison de son utilisation précédente dans l&#39;encapsulation cellulaire procedures, cependant, toute hydrogel capable d&#39;encapsulation peut être utilisé 8. La livraison de la pièce directement sur le site de la lésion assure un contact de cellule à tissu sur une longue période de temps, ce qui augmente la durée pendant laquelle les cellules peuvent fournir des facteurs bénéfiques pour le myocarde sous-jacent. Un goulot d&#39;étranglement à l&#39;approche de patch est la difficulté d&#39;adhérer le patch à la surface du cœur. De nombreux groupes ont surmonté ce à travers une variété de techniques, la plus répandue étant une suture simple de lier la construction à la surface du cœur 9,10. Cela a fait ses preuves dans un certain nombre de cas dans lesquels le produit de construction est fabriqué en un matériau plus rigide, mais échoue lorsque la tentative sur un système d&#39;hydrogel, en raison de la concentration élevée en eau et de la nature fragile de la construction de patch. Pour surmonter cela, nous avons utilisé un système d&#39;adhésif de la colle de fibrine externe qui imite la chimie de la formation de caillot. La colle de fibrine a été utilisé dans de nombreux cabinets médicaux, incLUDING larmes dura, fistules bronchiques, et la greffe de cornée, soulignant la biocompatibilité du produit comme un produit d&#39;étanchéité de la plaie 11-13. En outre, la fibrine a été utilisé pour une variété de fins cardiaques, y compris le traitement chirurgical des ruptures ventriculaires gauche et pontages coronariens de l&#39;artère, cependant, son utilisation comme colle d&#39;adhérence pour un patch cardiaque n&#39;a pas été couramment utilisé 14 à 17. Un simple formulation de thrombine et de fibrinogène se traduit par une colle biocompatible qui peut être placé directement sur l&#39;extérieur d&#39;une pièce cardiaque externe, en fournissant un système d&#39;adhérence adéquate pour assurer patch pour interaction cardiaque.

<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.

Au cours de l&#39;intervention chirurgicale, la ligature de l&#39;artère coronaire gauche peut être identifié par un net blanchiment en aval de l&#39;artère occluse. Comme un test avant d&#39;attacher le noeud, la suture peut être serré brièvement pour vérifier si elle est à l&#39;endroit approprié. En outre, étant donné que l&#39;occlusion de l&#39;artère résultats dans presque instantanée diminution de la capacité de l&#39;oreillette gauche à battre correctement, l&#39;oreillette gauche va agrandir en...

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 hydrogel construire et de fibrine-colle à base son approche et proposer Therapeutics dans un infarctus du myocarde murin modèle.

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 vues.  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.

Vidéo: 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.

Infarctus aigu du myocarde chez le rat

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

Recherche

Médecine

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.

Ligature de l&#39;artère coronaire et injection intramyocardique dans un modèle murin d&#39;infarctus

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 murin à thorax fermé Modèle d&#39;ischémie myocardique et la reperfusion

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.

Infarctus du myocarde et d&#39;évaluation fonctionnelle de résultats chez le porc

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.

Évaluation des résultats primaires dans un modèle porcin de infarctus aigu du myocarde

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.

Infarctus du myocarde chez les souris néonatales, Un modèle de régénération cardiaque

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.

Modèle d'infarctus myocarde de Murine utilisant la ligation permanente de l'artère coronaire descendante antérieure gauche

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 modèle cryo-blessure pour étudier l'infarctus du myocarde chez la souris

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.

Établissement d’un modèle porcin d’insuffisance cardiaque d’infarctus post-myocardique pour le traitement des cellules souches

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.

Transplantation intramyocardique d’hydrogels injectables à chargement MSC après l’infarctus du myocarde dans un modèle murin

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