Cette publication a été soutenue par la recherche clinique et translationnelle (SCTR) Institut Caroline du Sud, avec un foyer universitaire à l&#39;Université médicale de Caroline du Sud, NIH / RNTAC Grant Nombre UL1 TR000062. Un soutien supplémentaire a été fourni par VA prix du mérite BX002327-01 de DRM. DJH a été soutenue par le NIH / RNTAC Grant Nombre TL1 TR000061 et par le NIH Grant Nombre T32 GM008716. SEA a été soutenu par des subventions du NIH Nombre T32 HL07260.

Toutes les procédures énumérés ici ont été approuvés par le Comité de protection des animaux et l&#39;utilisation institutionnelle à l&#39;Université médicale de Caroline du Sud. Les expériences décrites ici sont, des expériences non-survie aigus. En tant que tel, il n&#39;y a aucune utilisation de pommade ophtalmique et un bloc opératoire stérile est pas nécessaire. L&#39;euthanasie est obtenu par saignée pendant la récolte du cœur. 1. Préparation expérimentale <ol><li> Mettre en place soit une pression constante ou un débit constant perfusion de Langendorff Appareil 4. </li><li> Préparer 4 litres de tampon de Krebs-Henseleit modifiée (en mM: NaCl 112, KCl 5, 1,2 MgSO 4, 1 K 2 HPO 4, CaCl2 1,25, NaHCO 3 25, 11 D-glucose, l&#39;acide octanoïque 0,2, pH = 7,4). <ol><li> Assurez 10x actions tampons. <ol><li> Dissoudre NaCl, KCl, MgSO 4 et K 2 HPO 4 dans 3 L d&#39;eau ultrapure. </li><li> Dissoudre CaCl2 dans 500 ml d&#39;eau ultra pure wie 40 ml de HCl 1 N. </li><li> Ajouter lentement solution de CaCl 2 à la mémoire tampon réalisés dans 1.2.1.1. Ajouter de l&#39;eau ultra pure assez pour totalisera 4 L. permettent de remuer pendant 1 heure avant de filtrer à travers un filtre de 0,8 um. Conserver à 4 ° C. </li><li> Dissoudre NaHCO 3 à 4 L d&#39;eau ultra-pure. Laisser agiter pendant 1 heure avant de filtrer à travers à 0,8 um filtre. Conserver à 4 ° C </li></ol></li><li> Mélanger 400 ml 10x tampon de Krebs Henseleit avec 400 ml 10x tampon NaHCO 3. Ajouter suffisamment d&#39;eau ultra-pure à totalisera 4 L. Dissoudre le glucose et l&#39;acide octanoïque, tout en agitant. Filtrer à travers 0,8 um filtre. </li></ol></li><li> Préparez-vous pour le système cardiaque. <ol><li> Ajouter 2 L de tampon filtré à un 4 L bouteille placé 38 en dessus du robinet fixé à la canule aortique. Cela donnera une pression de perfusion de 70-80 mmHg au cœur, qui est la pression de perfusion recommandé 4. </li><li> Insérez dispersion barboteur de gaz dans la mémoire tampon dans la bouteille. </li><li> Exécutez le tampon à travers le système, en étant sûr d&#39;éliminer les bulles à l&#39;intérieur du tube. </li><li> Activer le gaz (95% O 2/5% CO 2) d&#39;au moins 30 min avant de cœur doit être placé sur le système. Utilisation d&#39;une sonde d&#39;écoulement de gaz et l&#39;analyseur de gaz de sang pour vérifier la saturation en oxygène. </li></ol></li><li> Préparer fournitures chirurgicales et ballon. <ol><li> Pour la récolte de cœur, de préparer une paire de 6,75 &quot;ciseaux chirurgicaux, une paire de 4.5&quot; ciseaux, deux paires de pinces hémostatiques, deux ensembles de pince caramalt, deux seringues avec 27 g d&#39;aiguilles, 2 morceaux de 0-jauge suture de soie (environ 15 cm de longueur), une canule à barbillons 1/16 de pouce, et une canule trachéale. </li><li> Pour assembler canule trachéale, coller un court morceau de tuyau en plastique de diamètre 1/16 pouces à un connecteur en Y. </li><li> Pour assembler un appareil de ballon: fixer une aiguille de gavage à un morceau de tuyau 1/16 de pouce, puis fixer la tuyauterie à un logement de capteur de pression. Une petite seringue fixée à l&#39;autre côté de la pression de transducer logements permettra à l&#39;inflation et la déflation du ballon. <ol><li> Couper un carré de la pointe de pellicule plastique et le lieu d&#39;une aiguille de gavage dans le centre. Enveloppez la pellicule de plastique autour de l&#39;aiguille de gavage. </li><li> Remplissez ballon avec une solution saline et attacher avec deux points de suture. Gonflez ballon pour assurer qu&#39;il n&#39;y a pas de fuites. Le volume de solution saline à l&#39;intérieur du ballonnet gonflé doit être approximativement de 200 pi, mais peut varier en fonction de la taille du rat. </li><li> Sinon, utilisez un lit latex de doigt à la place de la pellicule de plastique. Placez aiguille de gavage dans les doigtier, remplir avec une solution saline et attacher. </li></ol></li></ol></li></ol> 2. Récolte cœur <ol><li> Retenez un rat Sprague Dawley utilisant un decapicone ou un autre dispositif de retenue manuel. Peser rat. </li><li> Administrer la kétamine / xylazine mélange (0,85 mg / kg de kétamine et 0,15 mg / kg de xylazine) par injection intrapéritonéale à anesthésier rat. Cette dose d&#39;anesthésique fournira anesthésie suffisantes pour20-40 min, mais la procédure devrait être achevée aussi rapidement que possible après l&#39;anesthésie est confirmée. </li><li> Confirmez bon degré de l&#39;anesthésie par des tests pincement de l&#39;orteil réflexe. </li><li> Trachéotomie <ol><li> Retirer peau à partir de mi-patte à la base de la mâchoire, en utilisant des produits hémostatiques pour élever peau et ciseaux pour faire incision. Gardez la peau élevée à séparer du muscle sous-jacent, permettant de couper le long de la fourrure et le retirer. </li><li> Utilisation de pinces hémostatiques, soulevez le tissu glandulaire et de faire une incision horizontale au fascia inférieur au tissu glandulaire. Utilisez des pinces hémostatiques pour répandre incision ouverte, en prenant soin de ne pas entailler les veines jugulaires. </li><li> Utilisation de pinces hémostatiques, pincée muscle recouvrant la trachée et l&#39;aide de ciseaux font une incision transversale dans le muscle juste en dessous de la pointe des pinces hémostatiques. Utilisation de pinces hémostatiques, répartis tissu pour révéler la trachée. </li><li> Insérez soigneusement hémostatiques dans la trachée, de compensation fascia tout en progressant par doucement avancer et reculer hémostatiques. </li><li>Une fois hémostatiques sont placés avec succès derrière la trachée, pincez 0-0 suture de soie avec des pinces hémostatiques et hemisect la trachée avec une petite paire, de ciseaux. </li><li> Insérez canule trachéale dans la trachée hemisected, tirez suture derrière la trachée, et attacher la suture autour de la trachée canule, l&#39;ancrage de la suture à la Y-aine de la canule avec un noeud de chirurgiens. </li><li> En utilisant une aiguille 27 G, injecter 1 000 U / kg d&#39;héparine dans la veine jugulaire et laisser circuler pour 30 sec. </li></ol></li><li> Thoracotomie <ol><li> Retirer fourrure provenant milieu de l&#39;abdomen à la région mi-patte en pinçant avec pinces hémostatiques et couper avec des ciseaux. </li><li> Faire un ¾ de pouce transversale incision dans la paroi musculaire abdominale, juste en dessous du diaphragme. </li><li> Faire une incision verticale sur le mur de la paroi abdominale et la poitrine, coupe le long du sternum. </li><li> Coupez le diaphragme retour bilatéralement, puis étaler la cage thoracique pour révéler coeur, pince cage thoracique avec des pinces hémostatiques pour assurer qu&#39;il demeure propagation. </li&gt; <li> Utilisation de pinces hémostatiques, retirez délicatement le thymus, exposer l&#39;aorte ascendante. </li><li> Taquiner hémostatiques dans la boucle de l&#39;aorte, avancer et reculer hémostatiques et doucement propagation fascia pour permettre pointe de pinces hémostatiques travers. </li><li> Utilisez des pinces hémostatiques pour tirer 0-0 soie suture derrière l&#39;aorte ascendante, ligaturer dans un demi-carré noeud lâche. </li><li> Tournez le robinet pour commencer flux de tampon de perfusion, hemisect aorte et l&#39;insérer immédiatement la canule dans la lumière de l&#39;aorte. Rapidement sangler un nœud de demi-carré et compléter le noeud, serrer à fond. </li><li> Utilisez des ciseaux à disséquer coeur loin des gros vaisseaux, retirez le cœur de la poitrine et l&#39;attacher à la colonne de perfusion. </li><li> Coupez le tissu pulmonaire excès et permettre cœur à équilibrer sur la colonne pendant environ 15 minutes avant l&#39;insertion de ballon. Utiliser un débit normal de tampon à travers le cœur de 10-20 ml / min. </li></ol></li></ol> 3. perfusion de Langendorff et l&#39;ischémie-reperfusion <ol><li> Placement de LV préssure ballon <ol><li> Dégonfler ballon tout en roulant en forme de cône et tourner le robinet pour assurer ballon reste dégonflé. </li><li> Accise oreillette gauche avec une petite paire de ciseaux pour exposer l&#39;accès à la valve mitrale. </li><li> Insérez ballon à travers l&#39;oreillette gauche et la valve mitrale dans le ventricule gauche. </li><li> Lancer le logiciel de surveillance de la pression (LabChart Pro). </li><li> Gonflez ballon en ouvrant le robinet et doucement une quantité croissante de solution saline en ballon jusqu&#39;à la pression diastolique de micromanomètre dépasse zéro. </li><li> Pour vérifier la réponse longueur-tension, gonfler ballon lentement à une pression diastolique de 75 mmHg, dégonfler lentement, répéter une fois. </li><li> Régler la pression diastolique de 10 mmHg en modulant la quantité de solution saline dans le ballon. Tournez le robinet pour sceller ballon à ce niveau de l&#39;inflation, et de maintenir le système de pression fermée entre LV ballon et capteur de pression. </li><li> Basse coeur dans la chambre de la chambre et le couvercle chauffé avec du plastique. </li></ol></li><li> Annonceministère de l&#39;ischémie-reperfusion <ol><li> Branchez le fond de la chambre chauffée et permet de remplir la chambre avec un tampon épanché. </li><li> Lorsque le tampon a submergé coeur, tourner le robinet pour arrêter l&#39;écoulement de tampon dans le cœur, induisant une ischémie globale. temps d&#39;ischémie peut varier en fonction des objectifs de l&#39;expérience. </li><li> Après 30 min d&#39;ischémie, tourner le robinet pour restaurer le flux de tampon dans le cœur, initiant reperfusion. </li><li> Autoriser la reperfusion du coeur pendant 1 heure avant de terminer l&#39;expérience. Utilisez des temps de reperfusion variables, en fonction des objectifs de l&#39;expérience. </li></ol></li><li> Résiliation de l&#39;expérience et de récolte de tissus <ol><li> Utilisation du port de côté du robinet, administrer 10 ml de solution saline tamponnée phosphate à 4 ° C dans le cœur à un taux de 10 ml / min pour arrêter le coeur. </li><li> Retirer le cœur de la colonne de perfusion, rogner tissu auriculaire restant. </li><li> Placez coeur dans une matrice de découpage de tissu en acier inoxydable, couvrir avec Parafilm, et le lieu à -80 ° C pendant 8 minutes, ou jusqu&#39;à ce que le coeur a une consistance de guimauve-like. </li><li> Retirer le bloc du congélateur et insérer des lames de rasoir dans le tranchage matrice pour découper coeur en tranches de 2 mm. blocs de tissus peuvent également être achetés avec des tranches dimensions autres que 2 mm. </li><li> Retirer les tranches une à la fois et déposez-les dans de l&#39;azote liquide à clignoter geler pour les études biochimiques. Conserver la troisième tranche ventriculaire du sommet pour l&#39;infarctus coloration. </li><li> Retirer le tissu de l&#39;azote liquide et conserver à -80 ° C jusqu&#39;à utilisation dans des expériences biochimiques. </li></ol></li><li> Infarctus coloration <ol><li> Incuber la troisième tranche ventriculaire dans 10 ml de 1% p / v de chlorure de triphényltétrazolium (TTC) à 37 ° C pendant 15 min. Note: Incubation dans TTC peut être réalisée pour 15 ou 30 minutes, sans modification de 15 l&#39;efficacité. </li><li> Transfert tranche de tissu à partir de TTC à 10% de formaline tamponnée et laisser incuber à la température ambiante pendant une nuit. Pour des résultats optimaux, entendrets doivent être prises hors de formol et imagés dans les 24 heures 4. </li><li> Taché logiciel de tranche et de l&#39;utilisation de Droits tel que ImageJ pour estimer la taille de l&#39;infarctus selon le protocole du fabricant. Prenez des photos dès que possible après la fixation, comme le colorant va disparaître au fil du temps. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Le coeur de rat isolé perfusé peut être utilisé avec succès pour étudier l&#39;effet de l&#39;intervention pharmacologique sur préconditionnement cardiaque dans l&#39;ischémie reperfusion 9. Cependant, il ya certaines étapes essentielles de la procédure qui doit être normalisée afin d&#39;assurer des résultats reproductibles. Le maintien d&#39;une température de 37,4 ° C dans le système est critique, car même une légère hypothermie et l&#39;hyperthermie peuvent provoquer préconditionnement...

Élucidation des événements sous-jacents de la réponse cardiaque à la fois à l&#39;ischémie et la reperfusion sont essentiels pour améliorer le traitement des infarctus 1 et cardiaques procédures chirurgicales qui nécessitent du myocarde clampage aortique 2. Alors que les modèles in vivo d&#39;une lésion de reperfusion d&#39;ischémie permettent une analyse de point de terminaison très utiles, ils ne sont pas aussi efficaces pour étudier les effets fonctionnels de la lésion de reperfusion d&#39;ischémie aiguë en temps réel. En outre, dans les modèles d&#39;ischémie reperfusion vivo produisent généralement la variabilité significative de la taille de l&#39;infarctus, et la livraison directe de médicaments au cœur au moment de la reperfusion est difficile. L&#39;utilisation d&#39;un système de cœur isolé de Langendorff pour l&#39;étude des lésions d&#39;ischémie de reperfusion permet une évaluation fonctionnelle en temps réel des traitements pharmacologiques, zone uniforme de tissu infarci et livraison instantanée du médicament directement dans le myocarde. D&#39;abord décrit by Langendorff Oscar en 1895 3, au coeur de Langendorff isolé est un modèle robuste pour étudier les lésions de reperfusion d&#39;ischémie, après avoir été utilisé dans la recherche de reperfusion d&#39;ischémie pendant les 40 dernières années 4,5. Ici, quelques modifications sont apportées pour optimiser le coeur isolé pour l&#39;analyse fonctionnelle. Dans cathétérisme situ de l&#39;aorte alors que le cœur bat assure que le cœur ne connaît pas le préconditionnement ischémique, qui pourrait altérer les résultats des essais ischémie reperfusion 6. Pour faciliter cela, une trachéotomie est réalisée, ce qui permet la ventilation et assurer la stabilité physiologique du rat au cours de la chirurgie. Le cœur est ensuite fixé à une colonne à chemise d&#39;eau en spirale à travers laquelle le verre tampon Krebs Henseleit est envoyé par perfusion rétrograde directement dans l&#39;aorte. Un ballon rempli de solution saline est introduite dans le ventricule gauche et fixé à un transducteur de pression, ce qui permet la mesure du temps réel de la pression de l&#39;intérieur de l&#39;un ventriculed calcul des paramètres fonctionnels multiples. À la fin de l&#39;expérience, le cœur est rincée avec une solution saline froide pour arrêter la contraction et le flash congelés dans l&#39;azote liquide pour permettre une analyse en aval du taux d&#39;ADN, ARN et de protéines. Ainsi modifié, le cœur de Langendorff perfusé sert d&#39;un système efficace pour la surveillance directe de l&#39;effet physiologique des interventions pharmacologiques à tout moment aiguë au cours de la lésion d&#39;ischémie reperfusion.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma Aldrich</td>
			<td align="right">203726</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Sigma Aldrich</td>
			<td>RES20765-A7</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Octanoic Acid</td>
			<td>Sigma Aldrich</td>
			<td>C2875</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3,5-triphenyltetrazolium chloride</td>
			<td>Sigma Aldrich</td>
			<td>T8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical Pressure Transducer</td>
			<td>MEMSCAP</td>
			<td>SP844</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Peristaltic Pump</td>
			<td>Cole Parmer</td>
			<td>EW-07521-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Easy Load Pump Head</td>
			<td>Cole Parmer</td>
			<td>EW-07518-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated circulating water bath</td>
			<td>Lauda</td>
			<td>M3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing Flow Module</td>
			<td>Transonic</td>
			<td>TS410</td>
			<td></td>
		</tr>
		<tr>
			<td>Modular Research Console</td>
			<td>Transonic</td>
			<td>T402</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline flow sensor</td>
			<td>Transonic</td>
			<td>ME2PXN</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab Data Acquisition Device</td>
			<td>AD Instruments</td>
			<td>PL3508</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart data acquisition software</td>
			<td>AD Instruments</td>
			<td>MLU60/8</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and assessment of ischemia-reperfusion injury in langendorff-perfused rat hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

L&#39;appareil ventriculaire gauche ballon permet un suivi en temps réel de la pression développée par le pouvoir ventricule gauche (Figure 1). Comme décrit précédemment 7, cette trace de pression peut être utilisée pour calculer un grand nombre de paramètres de la fonction ventriculaire. Ces calculs peuvent être effectués dans la phase initiale, ainsi que la phase de reperfusion, moyennées sur plusieurs traces à l&#39;intérieur de chaque groupe, et on les compare afin de déter...

Watch this Scientific Journal Video about Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts at JoVE.com

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

Induction et de l&#39;évaluation de l&#39;ischémie-reperfusion dans coeurs de rat Langendorff perfusés

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

induction-assessment-ischemia-reperfusion-injury-langendorff-perfused

Research

JoVE Journal

Medicine

22.6K vues.  Medical University of South Carolina. The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

Vidéo: Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

En temps réel d&#39;imagerie numérique de leucocytes-endothélial interaction dans l&#39;ischémie-reperfusion (IRI) du muscle Cremaster Rat

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

Recherche

Médecine

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite tissue. Ischemia reperfusion injury (IRI) is a known cause of partial free flap failure and has no effective treatment. Establishing a laboratory model of this injury can prove costly both financially as larger mammals are conventionally used and in the expertise required by the technical difficulty of these procedures typically requires employing an experienced microsurgeon. This publication and video demonstrate the effective use of a model of IRI in rats which does not require microsurgical expertise. This procedure is an in situ model of a transverse abdominis myocutaneous (TRAM) flap where atraumatic clamps are utilized to reproduce the ischemia-reperfusion injury associated with this surgery. A laser Doppler Imaging (LDI) scanner is employed to assess flap perfusion and the image processing software, Image J to assess percentage area skin survival as a primary outcome measure of injury.

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.

In situ Transverse lambeau de grand droit: un modèle de rat de myocutané ischémie reperfusion

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischémie-reperfusion modèle de lésions rénales aiguës et post blessures fibrose chez la souris

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Modèle murin de Intestinal Ischémie-reperfusion

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Évaluation de la réserve de flux coronaire après la réperfusion d'ischémie myocardique chez les rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Un modèle préclinique de rat pour l'étude des dommages d'ischémie-réperfusion dans la microchirurgie reconstructive

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Optocardiographie et études d'électrophysiologie des coeurs perfusés d'Ex Vivo Langendorff

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

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, ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Lésion d’ischémie-reperfusion d’organe en simulant des changements hémodynamiques dans le modèle de greffe de foie de rat

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

Évaluation en temps réel de la microperfusion de la moelle épinière dans un modèle porcin d’ischémie/reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...