Cette recherche est soutenue par le Programme de recherche en sciences fondamentales par l’intermédiaire de la National Research Foundation of Korea (NRF) financée par le ministère de l’Éducation (NRF-2018R1D1A1A02049346)

Toutes les procédures de recherche sur les animaux ont été fournies conformément à la Loi sur le bien-être des animaux de laboratoire, au Guide pour le soin et l’utilisation des animaux de laboratoire et aux Lignes directrices et politiques pour les expériences de rongeurs fournies par le Comité institutionnel de soins et d’utilisation des animaux (IACUC) de l’École de médecine de l’Université catholique de Corée.
1. Préparation des MSC et des hydrogels injectables de gélatine
<ol><li>Culture MSCs dans un plat de culture de 100 mm à 37 °C et 5% CO2. Lorsque la croissance des MSC atteint 80% de confluence, laver le plat deux fois avec du DPBS et ajouter 1 mL de trypsine substitut à 37 °C pendant 3 min. REMARQUE : Les MSC ont été isolés de la moelle osseuse murine suivant lesprocédures conventionnelles 16, cultivés dans le milieu modifié de l’aigle de Dulbecco (DMEM) contenant 10% de sérum bovin fœtal (FBS) et 1% de solution antibiotique−antimycotique, et utilisés entre le passage 7\u20129 pour cette étude.</li>
	<li>Ajouter 9 mL de culture moyenne et centrifugeuse à 500 x g pendant 3 min. Ensuite, jetez le supernatant qui en résulte, resuspendez les cellules en 1 mL de PBS et maintenez la suspension cellulaire sur la glace.</li>
	<li>Diluer 10 μL de suspension cellulaire avec 10 μL de bleu Trypan et obtenir la concentration cellulaire à l’aide d’un compteur cellulaire automatisé.</li>
	<li>Resuspendez et transférez les MSC dans un tube de 1 mL à une densité de 1 x 107 cellules/mL.</li>
	<li>Préparez une solution conjuguée GH de 6,25 % dans PBS et séparez-la en 2 flacons. Ensuite, mélangez les solutions GH avec 6 μg/mL de HRP (gh solution A) ou 0,07 wt% de H2O2 (GH solution B). NOTE : Préparer l’acide propionique gélatine-hydroxylphylphyle (GH) conjugué selon les protocolesédités 12,15.<ol>
<li>Gardez un rapport volumétrique de 9:1 de gh solution conjuguée à HRP (GH solution A) et GH solution conjuguée à H2O2 (GH solution B), respectivement.</li>
	</ol>
</li>
	<li>Avant de mélanger les MSC avec la solution GH A, centrifugez brièvement la suspension cellulaire à 1 000 x g et aspirez soigneusement le supernatant qui en résulte. Par la suite, mélanger la pastille contenant des MSC avec la solution GH A.</li>
</ol>2. Chargement in situ du MSC et culture in vitro tridimensionnelle
<ol><li>Chargez la solution GH A (contenant des MSCs) et la solution GH B de chaque côté d’une double seringue. Plaque 300 μL des solutions GH combinées avec les MSC à une densité finale de 5 x 106 cellules/mL sur une glissière de chambre de huit puits.</li>
	<li>Après la formation in situ d’hydrogel et l’encapsulation suivante de MSC par l’intermédiaire de la liaison croisée enzymatique, ajoutez 700 μL de DMEM contenant 10% de FBS et 1% de solution antibiotique−antimycotique.</li>
	<li>Incuber la diapositive à 37 °C et 5 % de CO2 et remplacer le milieu de culture tous les 2\u20123 jours.</li>
</ol>3. Confirmation de la prolifération in vitro et de la survie des SMC dans les hydrogels GH
<ol><li>Pour déterminer la viabilité des SMC cultivés en 3D dans les hydrogels GH, utilisez un test de coloration des cellules vivantes ou mortes après le temps d’incubation prédéterminé.</li>
	<li>Après incubation des MSC encapsulés dans les hydrogels GH pendant 3, 5, 7 ou 14 jours, aspirez le milieu et lavez le puits deux fois avec du PBS.</li>
	<li>Préparer une solution de coloration contenant 5 μL de calcéine AM et 20 μL d’éthidium homodimer-1 (EthD-1) dans 10 mL de DPBS.</li>
	<li>Ajouter 200 μL de la solution de coloration au puits et incuber pendant 30 min dans l’obscurité à température ambiante.</li>
	<li>Aspirer la solution de coloration et laver le puits deux fois avec PBS.</li>
	<li>Séparez soigneusement la chambre de la glissière et placez un coverslip complet sur les hydrogels GH. Utilisez une microscopie confocale pour visualiser le degré de prolifération et les changements morphologiques des CSM encapsulés. REMARQUE : Les images fluorescentes ont été acquises sous grossissement de 200 x et photographiées aux longueurs d’onde excitation/émission de 470/540 nm pour la calcéine et de 516/607 nm pour EthD-1.</li>
</ol>4. Induction de l’infarctus du myocarde chez la souris
<ol><li>Anesthésier les souris C57BL/6 mâles de 7 semaines (20\u201222 g) par injection intraperitoneal d’un mélange de Zoletil (30 mg/kg) et rompun (10 mg/kg) en solution saline.</li>
	<li>Avant la chirurgie, dépolar le coffre de la souris à l’aide de crème d’épilation et stériliser la peau avec de l’iode.</li>
	<li>Placez la souris sur une table d’opération et intubez en insérant un cathéter dans la trachée pour fournir de l’oxygène supplémentaire par ventilation mécanique.</li>
	<li>Coupez doucement la peau à l’aide de ciseaux chirurgicaux, puis pénètrez les muscles intercostals par micro ciseaux. Séparez les 2e et 3e côtes gauches à l’aide d’une suture en soie 5-0 pour maintenir une cavité thoracique ouverte.</li>
	<li>Ligate soigneusement l’artère coronaire descendante antérieure gauche (LAD) utilisant un support d’aiguille avec un 8-0 suture en polypropylène et couper la suture à l’aide d’électrocautery.</li>
	<li>Observez un changement de couleur immédiat dans la paroi ventriculaire gauche antérieure.</li>
</ol>5. Transplantation intramyocardique d’hydrogels GH à chargement par MSC
<ol><li>Après avoir induisé l’infarctus du myocarde par ligature LAD, injectez 10 μL de solutions GH à chargement MSC en deux points différents à la zone frontalière infarctus (total : 2 x 105 MSCs/20 μL) à l’aide d’une seringue double munie d’une aiguille 26G.<ol>
<li>Suivant la même procédure décrite à l’étape 1, préparez et transférez les solutions GH à chargement par MSC dans une double seringue. REMARQUE : Pour évaluer l’engraftment des hydrogels GH à chargement par MSC dans la zone infarctusée, les SMC et les conjugués gh ont été pré-étiquetés avec phk26 et isothiocyanate de fluorescéine (FITC), respectivement.</li>
	</ol>
</li>
	<li>Restaurer la cavité thoracique ouverte et fermer les muscles et la peau à l’aide de sutures 5-0. REMARQUE : Avant la fermeture de la poitrine, retirez l’air à l’aide d’une seringue cathéter.</li>
	<li>Retirez le tube trachéal et placez la souris dans une cage sous une lampe infrarouge pendant la récupération.</li>
	<li>Pour l’analgésie postopératoire, administrer des injections sous-cutanées de kétoprofène (5 mg/kg par jour) pour un minimum de 72 h. Toutes les souris devraient être étroitement surveillées pendant un moment approprié pour assurer le rétablissement approprié après des procédures chirurgicales aussi bien que le traitement adéquat de douleur.</li>
</ol>6. Échocardiographie
<ol><li>Quatre semaines après la transplantation, anesthésier d’abord la souris avec 5% d’isoflurane, puis ajuster la concentration d’isoflurane à 1%.</li>
	<li>Dépeler la poitrine à l’aide de crème d’épilation et placer la souris sur un coussin chauffant. Appliquer le gel transducteur à ultrasons sur la poitrine.</li>
	<li>Acquérir des vues parasternales à axe court bidimensionnel et enregistrer des tracés en mode M au niveau du muscle papillaire. REMARQUE : Placez un transducteur linéaire (7\u201215 MHz) dans la ligne parasternale gauche et visualisez les structures anatomiques.</li>
	<li>Mesurez les lignes correspondantes pour LVAW, LVID et LVPW pour obtenir l’épaisseur de la paroi cardiaque, la dimension de la chambre et le raccourcissement fractionnel. REMARQUE : Comparez la fonction cardiaque comprenant la fraction d’éjection (EF), le raccourcissement fractionnel (FS), et le volume systolique final (ESV) au niveau du muscle papillaire pour assurer l’évaluation appropriée au même endroit anatomique.</li>
</ol>7. Évaluation histologique
<ol><li>Au moment prédéterminé après la transplantation des hydrogels GH msc-chargement dans le coeur infarctus, euthanasier la souris dans une chambre de CO2 et de recueillir le cœur pour l’analyse histologique15.</li>
	<li>Pour l’hématoxyline et l’éosine (H&E) et la coloration trichrome (MT) de Masson, fixez les tissus cardiaques disséqués en paraformaldéhyde (PFA) à 4 % et intégris à la paraffine. Ensuite, coupez les blocs cardiaques intégrés à la paraffine en sections de série de 4 μm à l’aide d’une microtome et tachez les sections avec de la tache MT selon les protocoles standard17.</li>
	<li>Obtenez des images sur un scanner de diapositives à 20x grossissement et calculez la taille infarctus des groupes de traitement. Taille infarctus (%) = circonférence infarctus totale / circonférence totale du LV x 100</li>
	<li>Calculez les deux circonférences par mesure de la longueur médiane. Pour les circonférences intermédiaires LV, mesurez les longueurs de la ligne centrale entre les surfaces endocardiques et épicardiales. Pour les circonférences infarctus de la ligne médiane, mesurer les longueurs de l’infarctus, y compris plus de 50 % de toute l’épaisseur du myocarde18. REMARQUE : Toutes les analyses d’images ont été effectuées à l’aide du logiciel ImageJ.</li>
	<li>Mesurer l’épaisseur de la paroi de la cicatrice aux niveaux musculaires papillaires.</li>
	<li>Calculer la fraction de la zone de collagène. Zone de collagène (%) = superficie totale de fibrose interstitielle/zone myocyte x 100</li>
</ol>

<ol>
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Les auteurs n’ont aucun conflit d’intérêts à déclarer avec cet ouvrage.

Hydrogels GH injectables ont un grand potentiel pour les applications in vivo en raison de leur capacité à incorporer homogènement divers agents thérapeutiques in situ. En outre, leurs propriétés physiques et biochimiques peuvent être facilement manipulées en fonction des besoins dépendants de la maladie. À cet égard, des hydrogels injectables ont été proposés pour répondre aux principales limitations de la thérapie actuelle par cellules souches cardiaques entravées par une mauvaise survie et la rétenti...

La thérapie cardiaque de cellules souches est apparue comme approche potentielle pour la réparation et la régénération myocardiques1,2. Malgré les résultats positifs récents dans les modèles animaux et les essais cliniques, l’application de la thérapie à base de cellules souches pour la réparation du myocarde est limitée en raison de la faible rétention et la mauvaise survie des cellules injectées dans les tissus cardiaques infarctus3,4. En conséquence, l’utilisation de l’ingénierie tissulaire à base de cellules, y compris les biomatériaux injectables5, les patchscardiaques 6, et les feuillescellulaires 7, a été intensivement étudié pour améliorer la rétention cellulaire et l’intégration dans le myocarde hôte.
Parmi les diverses approches potentielles de la réparation des tissus cardiaques bioingénieurs, les hydrogels injectables combinés à des types de cellules appropriés, tels que les cellules souches mésenchymiques (CSM), les cellules souches embryonnaires (ESC) et les cellules souches pluripotentes induites (IPSC), sont une option attrayante pour livrer efficacement les cellules dans les régions myocardiques8,9. La gélatine, un polymère naturel bien connu, peut être utilisée comme matrice injectable en raison de sa grande biocompatibilité, de sa biodégradabilité considérable et de sa réduction de l’immunogénicité par rapport à un large éventail de biomatériaux utilisés dans les applications biomédicales. Bien que les plates-formes injectables à base de gélatine aient un grand potentiel, leur applicabilité in vivo reste limitée en raison de leur faible rigidité mécanique et de leur dégradabilité facile dans l’environnement physiologique.
Pour surmonter ces limitations, une conception nouvelle et simple d’hydrogels à base de gélatine composé d’acide propionique hydroxyphényle a été proposée pour des applications in vivo. Les conjugués à l’acide propionique gélatine-hydroxyphényle (GH) peuvent être reliés in situ en présence d’une enzyme, la peroxidase de raifort (HRP), et par la suite encapsuler divers médicaments, biomolécules ou cellules dans l’hydrogel, suggérant un grand potentiel dans les applications d’ingénierietissulaire 10,11,12,13,14. En outre, nous avons récemment étudié les effets thérapeutiques des hydrogels de GH contenant des MSCs encapsulés et avons démontré leur utilisation dans la réparation et la régénération cardiaques réussies après MI dans un modèle murin15. Dans ce protocole, nous décrivons une technique simple pour l’encapsulation et la prolifération tridimensionnelle in vitro (3D) des MSCs dans les hydrogels de GH. Nous introduisons également une procédure chirurgicale conçue pour produire un modèle murin d’MI par ligature d’artère coronaire et transplantation intramyocardique des hydrogels de GH MSC-chargement dans le coeur infarctus.

<table>
	<tbody>
		<tr>
			<td>4 % paraformaldehyde (PFA)</td>
			<td>Intron</td>
			<td>IBS-BP031-2</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 silk suture</td>
			<td>AILEE</td>
			<td>SK534</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 polypropylene suture</td>
			<td>ETHICON</td>
			<td>M8732H</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chamber slide</td>
			<td>Nunc LAB-TEK</td>
			<td>154534</td>
			<td></td>
		</tr>
		<tr>
			<td>Angiocath Plus (22GA) catheter</td>
			<td>BD Angiocath Plus</td>
			<td>REF382423</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimyocotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>GYROGEN</td>
			<td>1582MGR</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slipe</td>
			<td>MARIENFELD</td>
			<td>101242</td>
			<td></td>
		</tr>
		<tr>
			<td>Deluxe High Temperature Cautery kit</td>
			<td>Bovie</td>
			<td>QTY1</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EOSIN</td>
			<td>SIGMA-ALDRICH</td>
			<td>HT110116</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>EMSURE</td>
			<td>K49350783 739</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fechtner conjunctiva forceps titanium</td>
			<td>WORLD PRECISISON INSTRUMENTS</td>
			<td>WP1820</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate isomer I (FITC)</td>
			<td>SIGMA-ALDRICH</td>
			<td>F7250</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>HEBU</td>
			<td>HB0458</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair removal cream</td>
			<td>Ildong Pharmaceutical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Stoelting</td>
			<td>50300</td>
			<td>Homeothermic Blanket System</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>50301</td>
			<td>Replacement Heating Pad for 50300 (10 X 12.5cm)</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>SIGMA-ALDRICH</td>
			<td>HHS80</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxide (HRP; 250-330 U/mg)</td>
			<td>SIGMA-ALDRICH</td>
			<td>P8375</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2; 30 wt % in H2O)</td>
			<td>SIGMA-ALDRICH</td>
			<td>216763</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Green Pharmaceutical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD cell staining kit</td>
			<td>Thermo Fisher</td>
			<td>R37601</td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical ventilator</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifuge</td>
			<td>HANIL</td>
			<td>Micro 12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holder</td>
			<td>KASCO</td>
			<td>37-1452</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissor</td>
			<td>HEBU</td>
			<td>HB7381</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>OLYMPUS</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>MT staining kit</td>
			<td>SIGMA-ALDRICH</td>
			<td>HT1079-1SET</td>
			<td>Weigert&#8217;s iron hematoxylin solution</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>HT15-1KT</td>
			<td>Trichrome Stain (Masson) Kit</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>LK LABKOREA</td>
			<td>H06-660-107</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>PHK26 staining kit</td>
			<td>SIGMA-ALDRICH</td>
			<td>MINI26</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide scanner</td>
			<td>Leica</td>
			<td>SCN400</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>HEBU</td>
			<td>HB7454</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td>3M micopore</td>
			<td>1530-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue cassette</td>
			<td>Scilab Korea</td>
			<td>Cas3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Transducer gel</td>
			<td>SUNGHEUNG</td>
			<td>SH102</td>
			<td></td>
		</tr>
		<tr>
			<td>Trout-Barraquer needle holder curved</td>
			<td>KASCO</td>
			<td>50-3710c</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound system</td>
			<td>Philips</td>
			<td>Affiniti 50</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>JUNSEI</td>
			<td>25175-0430</td>
			<td></td>
		</tr>
	</tbody>
</table>

intramyocardial transplantation of msc-loading injectable hydrogels after myocardial infarction in a murine model

L’un des principaux problèmes auxquels sont confrontées les thérapies actuelles de cellules souches cardiaques pour prévenir l’insuffisance cardiaque postinfarcte est le faible taux de rétention et de survie des cellules transplantées dans le myocarde blessé, limitant leur efficacité thérapeutique. Récemment, l’utilisation de biomatériaux d’échafaudage a attiré l’attention pour améliorer et maximiser la thérapie par cellules souches. L’objectif de ce protocole est d’introduire une technique simple et simple pour transplanter les cellules souches mésenchymales dérivées de la moelle osseuse (MSCs) à l’aide d’hydrogels injectables d’acide propionique hydroxyphényle (GH); les hydrogels sont favorables en tant que plate-forme de livraison de cellules pour des applications d’ingénierie de tissu cardiaque en raison de leur capacité à être in situ inter-liée et à la biocompatibilité élevée. Nous présentons une méthode simple pour fabriquer des hydrogels GH à chargement par MSC (MSC/hydrogels) et évaluer leur survie et leur prolifération dans la culture in vitro tridimensionnelle (3D). En outre, nous démontrons une technique pour la transplantation intramyocardique de MSC/hydrogels chez les souris, décrivant une procédure chirurgicale pour induire l’infarctus du myocarde (MI) par ligature antérieure gauche d’artère coronaire (LAD) et transplantation suivante de MSC/hydrogels.

Pour livrer efficacement les CSM au myocarde infarctus, des hydrogels in situ à chargement in situ à liaison croisée décrits à la figure 1 ont été utilisés dans ce protocole. Avant la transplantation in vivo, la prolifération et la survie des SMC dans les hydrogels GH ont été confirmées par un test de coloration 3D in vitro des cellules vivantes/mortes (vivant : vert; mort : rouge). Comme le montre la figure 2, lesimages représentatives présentaien...

Watch this Scientific Journal Video about Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model at JoVE.com

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

La thérapie à base de cellules souches est apparue comme une stratégie efficace pour réparer les tissus cardiaques blessés après l’infarctus du myocarde. Nous fournissons une application in vivo optimale pour la transplantation de cellules souches utilisant des hydrogels de gélatine qui peuvent être enzymatiquement liés.

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 ...

intramyocardial-transplantation-msc-loading-injectable-hydrogels

Research

JoVE Journal

Medicine

4.3K vues.  The Catholic University of Korea. La thérapie à base de cellules souches est apparue comme une stratégie efficace pour réparer les tissus cardiaques blessés après l’infarctus du myocarde. Nous fournissons une application in vivo optimale pour la transplantation de cellules souches utilisant des hydrogels de gélatine qui peuvent être enzymatiquement liés.

Vidéo: Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine 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 ...

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 ...

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.

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 ...

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 Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by reduced blood supply to the heart, resulting in the loss of oxygen to and the ensuing necrosis of the heart muscle. The MI model has gained popularity for its use as a short-term ischemia-reperfusion model and a long-term permanent ligation model. Below, we describe a reliable method for the permanent ligation of the LAD. With mouse genetic engineering technology becoming more advanced, and with an increasing availability of quality murine surgical instruments, the mouse has become a popular model for MI surgeries. Our surgical model incorporates the use of an easily reversible anesthetic for the rapid recovery of the mouse; a minimally invasive endotracheal intubation without involving a tracheotomy; and a thoracentesis through the original thoracotomy site without creating an additional incision in the chest, as is done in some other methods, to effectively remove excess blood and air from the chest cavity. This method is comparatively less invasive than other methods, which dramatically reduces surgical and post-surgical complications and mortality and improves reproducibility.

Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

Murine gauche interventriculaire antérieure (LAD) Coronary Artery Ligature: un modèle amélioré et simplifié pour Myocardial Infarction

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by ...

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 ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

Retard de l’accouchement intramyocardique des cellules souches après une blessure à l’Ischémie Reperfusion dans un modèle murine

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...