Diese Veröffentlichung wurde von der South Carolina Clinical &amp; Translational Research (SCTR) Institute unterstützt, mit einem akademischen Heimat an der Medizinischen Universität von South Carolina, NIH / NCATS Statt Anzahl UL1 TR000062. Weitere Unterstützung wurde von VA Merit Award BX002327-01 DRM zur Verfügung gestellt. DJH wurde von NIH / NCATS Statt Anzahl TL1 TR000061 von NIH Zuschusses Anzahl T32 GM008716 unterstützt. SEA wurde vom NIH Zuschusses Anzahl T32 HL07260 unterstützt.

Alle hier aufgeführten Verfahren wurden von der Institutional Animal Care und Verwenden Committee an der Medizinischen Universität von South Carolina genehmigt. Die hier beschriebenen Experimente sind akute, nicht-Überlebensexperimente. Als solche gibt es keine Verwendung von Augensalbe und eine sterile OP-Bereich nicht erforderlich ist. Sterbehilfe wird durch Ausbluten bei der Ernte des Herzens erreicht. 1. Versuchsvorbereitung <ol><li> Bis entweder einen konstanten Druck oder konstanten Fluss Langendorff Perfusion 4 Stellen. </li><li> Herstellung von 4 L modifizierte Krebs-Henseleit-Puffer (in mM: 112 NaCl, 5 KCl, 1,2 MgSO 4, 1 K 2 HPO 4, 1,25 CaCl 2, 25 NaHCO 3, 11 D-Glucose, 0,2 Octansäure, pH = 7,4). <ol><li> Machen 10x stock Puffer. <ol><li> Aufzulösen NaCl, KCl, MgSO 4 und K 2 HPO 4 in 3 L Reinstwasser. </li><li> Löse CaCl 2 in 500 ml Reinstwasser with 40 ml 1 N HCl. </li><li> Langsam CaCl2-Lösung in den Puffer in 1.2.1.1 vorgenommen. Fügen Sie genug hochreinem Wasser auf insgesamt 4 L. Lassen Sie für 1 Stunde vor dem Filtrieren durch ein 0,8 um Filter zu rühren. Lagerung bei 4 ° C. </li><li> Löse NaHCO 3 in 4 L Reinstwasser. Man lässt 1 h, bevor es durch einen Filter an 0,8 um-Filter rühren. Lagerung bei 4 ° C </li></ol></li><li> Kombinieren Sie 400 ml 10x Krebs Henseleit-Puffer mit 400 ml 10x NaHCO3-Puffer. Fügen Sie genug hochreinem Wasser auf insgesamt 4 L. auflösen Glukose und Oktansäure unter Rühren. Man filtriert durch 0,8-um-Filter. </li></ol></li><li> Bereiten System für Herz. <ol><li> In 2 l gefiltert Puffer zu einem 4-l-Flasche 38 in platziert über dem Hahn an die Aortenkanüle angebracht. Dadurch wird ein Perfusionsdruck von 70-80 mmHg in das Herz, die die empfohlene Perfusionsdruck 4 zu liefern. </li><li> Legen Gasdispersions bubbler in den Puffer in der Flasche. </li><li> Führen Sie den Puffer durch das System, dass auf jeden Fall alle Blasen aus dem Inneren der Rohrleitung zu beseitigen. </li><li> Einzuschalten Gas (95% O 2/5% CO 2) mindestens 30 min vor Herz ist auf dem System platziert werden. Verwenden Sie ein Gasstrom-Sonde und Blutgasanalysegerät, um die Sauerstoffsättigung zu überprüfen. </li></ol></li><li> Bereiten chirurgisches Material und Ballon. <ol><li> Für die Ernte von Herz, bereiten ein Paar von 6,75 &quot;chirurgische Schere, ein Paar von 4.5&quot; Schere, zwei Paare von Hämostatika, zwei Sätze von Karamellenmalz Zange zwei Spritzen mit 27 G Nadeln, 2 Stück von 0-Gauge-Seidenfaden (ca. 15 cm in der Länge), einer 1/16 inch Stacheldraht Kanüle und eine Trachealkanüle. </li><li> Um Trachealkanüle montieren, kleben Sie ein kurzes Stück von 1/16 Zoll Durchmesser Kunststoffschlauch mit einem Y-Verbinder. </li><li> Um Ballongerät montieren: fügen Sie eine Sondennadel an ein Stück 1/16 Zoll Schlauch, dann bringen Sie Schlauch an Druckwandlergehäuse. Eine kleine Spritze zu der anderen Seite des Druck tr befestigtansducer Gehäuse wird Aufblasen und Entleeren des Ballons zu ermöglichen. <ol><li> Schneiden Sie ein Quadrat der Plastikverpackung und Platz Spitze einer Magensonde Nadel in der Mitte. Wickeln Sie die Plastikfolie um die Sondennadel. </li><li> Füllen Ballon mit Kochsalzlösung und binden mit zwei Nähten. Blasen Sie Ballon, um sicherzustellen, keine Lecks vorhanden sind. Das Volumen der Salzlösung innerhalb des aufgeblasenen Ballons sollte ungefähr 200 &amp; mgr; l, kann aber abhängig von der Größe der Ratte variieren. </li><li> Alternativ können Sie einen Latex Fingerling anstelle der Plastikfolie. Platzieren Sie eine Magensonde Nadel innerhalb Fingerling, füllen mit Kochsalzlösung und Krawatte ab. </li></ol></li></ol></li></ol> 2. Ernte das Herz <ol><li> Restrain eine Sprague Dawley-Ratte mit einem decapicone oder andere manuelle Rückhaltevorrichtung. Wiegen Ratte. </li><li> Verabreichen Ketamin / Xylazin-Mischung (0,85 mg / kg Ketamin und 0,15 mg / kg Xylazin) durch intraperitoneale Injektion an Ratten anästhesiert. Diese Dosis des Anästhetikums ausreichend für Anästhesie liefern20-40 min, aber das Verfahren sollte möglichst schnell nach der Narkose wird bestätigt, durchgeführt werden. </li><li> Bestätigen richtigen Grad der Anästhesie durch Tests toe Prise Reflex. </li><li> Tracheotomie <ol><li> Entfernen pelt von Mitte Vorderpfote auf Basis des Kiefers, mit Gefäßklemmen, um Fell und Schere zu erheben, um Einschnitt zu machen. Halten Sie das Fell angehoben, um es von der darunter liegenden Muskeln zu trennen, so dass man auf dem Fell schneiden und entfernen. </li><li> Verwendung hemostats, heben Drüsengewebe und machen einen horizontalen Schnitt in Faszie schlechter als das Drüsengewebe. Verwenden hemostats zu Einschnitt spreizt, kümmert sich nicht um nick die Halsvenen. </li><li> Verwendung Hämostaten pinch Muskeldarüberliegenden Luftröhre, und mit einer Schere einen Quereinschnitt im Muskel genau unter der Spitze der Hämostatika. Verwendung hemostats, verbreiten Gewebe auseinander, um die Luftröhre zu offenbaren. </li><li> Gefäßklemmen vorsichtig unter Luftröhre, Clearing Faszie, während voran durch leichtes Vor- und Zurück hemostats. </li><li>Sobald hemostats erfolgreich hinter der Luftröhre gelegt, kneifen 0-0 Seidennaht mit Gefäßklemmen und hemisect die Luftröhre mit einem kleinen, scharfen Schere. </li><li> Legen Trachealkanüle in die Trachea hemiseziert, ziehen Naht hinter der Luftröhre, und binden Sie den Faden um die Kanüle eingeführt Luftröhre, die Verankerung des Nahtmaterials zu der Y-Leiste der Kanüle mit einem Chirurgen Knoten. </li><li> Unter Verwendung einer 27 G Nadel injizieren 1.000 U / kg Heparin in die Halsschlagader und lassen Sie sie für 30 Sekunden zu zirkulieren. </li></ol></li><li> Thorakotomie <ol><li> Entfernen pelt von Mitte Bauch bis Mitte Vorderpfote Region durch Kneifen mit Gefäßklemmen und Schneiden mit einer Schere. </li><li> Machen Sie eine ¾-Zoll-Quereinschnitt in die Bauchmuskelwand, direkt unterhalb des Zwerchfells. </li><li> Machen Sie eine vertikale Inzision bis die Bauchdecke und Brustwand, Schneiden entlang dem Brustbein. </li><li> Schneiden Sie die Blende wieder auf bilateraler Ebene, dann verbreiten Brustkorb zu Herzen, Klemmbrustkorb mit Gefäßklemmen, um sicherzustellen, bleibt es verbreitet zu offenbaren. </li&gt; <li> Verwendung hemostats, entfernen Sie vorsichtig Thymus, Aussetzen der aufsteigenden Aorta. </li><li> Necken hemostats durch Aorten-Schleife, Vorschieben und Zurückziehen hemostats und sanft Verbreitung Faszie zu Spitze Gefäßklemmen ermöglichen durch. </li><li> Verwenden hemostats zu 0-0 Seidennaht hinten aufsteigenden Aorta zu ziehen, binden Naht zu einem lockeren Halb quadratischen Knoten. </li><li> Schalten Hahn, um den Fluss von Perfusionspuffer, hemisect Aorta beginnen und sofort legen Kanüle in Aortenlumen. Schnell cinch eine Halbkreuzknoten und füllen Sie die Knoten, Anziehen gründlich. </li><li> Mit einer Schere Herzen weg von den großen Gefäßen zu sezieren, Herz aus der Brust zu entfernen und um die Perfusion Spalte. </li><li> Abschneiden von überschüssigem Lungengewebe und lassen Herz auf der Säule für etwa 15 min vor dem Einsetzen des Ballons äquilibrieren. Verwenden Sie einen normalen Durchflussrate von Puffer durch das Herz von 10-20 ml / min. </li></ol></li></ol> 3. Langendorff-Perfusion und Ischämie-Reperfusionsverletzung <ol><li> Platzierung der LV vorssure Ballon <ol><li> Lassen Sie die Luft Ballon beim Rollen in eine Kegelform und schalten Hahn, um sicherzustellen, Ballon bleibt entleerten. </li><li> Excise linken Vorhof mit einer kleinen Schere, um den Zugang zu Mitralklappe aussetzen. </li><li> Legen Ballon durch linken Vorhof und Mitralklappe in den linken Ventrikel. </li><li> Startdruck-Monitoring-Software (LabChart Pro). </li><li> Blasen Sie Ballon durch Öffnen Absperrhahn und sanft zunehmende Menge an Kochsalzlösung in Ballon, bis der diastolische Druck Lesung aus Mikromanometer Null übersteigt. </li><li> Auf Länge-Spannung Antwort überprüfen, aufblasen Ballon langsam zu einer diastolischen Druck von 75 mmHg, entleeren langsam, einmal zu wiederholen. </li><li> Eingestellt diastolische Druck 10 mmHg durch Modulieren der Menge von Salzlösung in den Ballon. Schalten Hahn zu Ballon auf diesem Niveau der Inflation zu versiegeln und zu pflegen geschlossenen Drucksystem zwischen LV Ballon und Druckwandler. </li><li> Unteren Herzkammer in die erhitzt und Abdeckung mit Kunststoffkammer. </li></ol></li><li> AnzeigeDienst der Ischämie-Reperfusionsverletzung <ol><li> Stecker die Unterseite der erwärmten Kammer und erlauben Kammer mit ergossenen Puffer zu füllen. </li><li> Als Puffer Herzen unter Wasser, drehen Hahn, den Fluss von Puffer in das Herz zu stoppen, zu induzieren globale Ischämie. Ischämie Zeit kann je nach den Zielen des Experiments variieren. </li><li> Nach 30 Minuten der Ischämie, schalten Hahn, um die Strömung des Puffers in das Herz wiederherzustellen, Initiierung der Reperfusion. </li><li> Lassen Sie Reperfusion des Herzens für 1 h vor Beendigung des Experiments. Verwenden variable Reperfusion Zeiten, abhängig von den Zielen des Experiments. </li></ol></li><li> Kündigung Experiment und Ernten von Tissue <ol><li> Verwendung Seitenöffnung des Absperrhahns verabreichen 10 ml phosphatgepufferter Salzlösung bei 4 ° C in das Herz mit einer Rate von 10 ml / min, um das Herz anzuhalten. </li><li> Herz entfernen Perfusion Spalte wegschneiden restlichen Vorhofgewebe. </li><li> Zeigen Herz in einen Edelstahlgewebe Schneidematrix, Deckel mit Parafilm und Stelle bei -80 ° C für 8 Minuten, oder bis das Herz hat einen Marshmallow artige Konsistenz. </li><li> Entfernen Block vom Gefrierschrank und legen Sie in Rasierklingen schneiden Matrix zu Herzen in 2 mm dicke Scheiben schneiden. Gewebeblöcke können auch mit anderen als 2 mm Scheibe Größen erworben werden. </li><li> Scheiben entfernen ein zu einer Zeit und fallen in flüssigem Stickstoff auf Blitzeis für biochemische Untersuchungen. Bewahren Sie den dritten Ventrikel Scheibe von der Spitze zur Infarkt Färbung. </li><li> Entfernen Gewebe aus flüssigem Stickstoff und bei -80 ° C bis zur Verwendung in biochemischen Experimenten. </li></ol></li><li> Infarkt-Färbung <ol><li> Inkubieren des dritten ventrikulären Scheibe in 10 ml 1% w / v Triphenyltetrazoliumchlorid (TTC) bei 37 ° C für 15 min. Anmerkung: Die Inkubation in TTC kann für 15 oder 30 Minuten bei unveränderter Wirksamkeit 15 durchgeführt werden. </li><li> Übertragen Gewebeschnitt von TTC zu 10% Formalin und lassen Sie sie bei Raumtemperatur über Nacht inkubiert. Für optimale Ergebnisse zu hörents sollte von Formalin aufgenommen und innerhalb von 24 Stunden 4 abgebildet werden. </li><li> Fotografie Buntscheibe und Verwendung Software wie ImageJ zu Infarktgröße schätzen, nach dem Protokoll des Herstellers. Fotografieren so bald wie möglich nach der Fixierung, da der Farbstoff im Laufe der Zeit verblassen. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Isoliert perfundierten Rattenherzen erfolgreich eingesetzt werden kann, um die Wirkung von pharmakologischen Intervention Herz Präkonditionierung Reperfusionsverletzung 9 untersuchen. Es gibt jedoch einige wesentliche Schritte der Prozedur, die um reproduzierbare Ergebnisse zu gewährleisten standardisiert werden müssen. Aufrechterhalten einer Temperatur von 37,4 ° C innerhalb des Systems ist entscheidend, denn auch milder Hypothermie und Hyperthermie kann Herz Präkonditionierung 10,11 führen....

Aufklärung der Ereignisse des Herz Reaktion auf sowohl Ischämie und Reperfusion zugrunde liegen weiter zu verbessern die Behandlung von Herzinfarkt und Herz 1 operative Eingriffe, die Aorten-Querspann 2 erfordern. Während in vivo Modellen von Ischämie-Reperfusionsverletzung ermöglichen sehr nützlich Endpunktanalyse, sind sie nicht so wirksam für die Untersuchung der funktionellen Auswirkungen von Ischämie-Reperfusionsverletzung akut in Echtzeit. Zusätzlich kann in vivo Ischämie-Reperfusion-Modelle erzeugen im Allgemeinen zu signifikanten Schwankungen in der Infarktgröße und die direkte Abgabe von Arzneimittel an das Herz zum Zeitpunkt der Reperfusion ist eine Herausforderung. Die Verwendung einer Langendorff-Herzen isoliert System für die Untersuchung ischämischen Reperfusionsschäden ermöglicht Echtzeit-funktionelle Bewertung der pharmakologischen Behandlungen, gleichmäßige Flächen infarzierten Gewebe und momentane Abgabe von Arzneimittel direkt in den Herzmuskel. Zuerst beschrieben by Oscar Langendorff 1895 3 ist der Langendorff-Herz isoliert ein robustes Modell für die Untersuchung ischämischen Reperfusionsschäden, nachdem er in Ischämie-Reperfusion Forschung für den letzten 40 Jahren 4,5 verwendet. Hier werden einige Modifikationen vorgenommen, um das isolierte Herz zur Funktionsanalyse zu optimieren. In situ Kanülierung der Aorta, während das Herz schlägt, wird sichergestellt, dass das Herz nicht ischämischen Präkonditionierung, die die Ergebnisse der Ischämie-Reperfusion den Versuchen 6 verändern würden auftreten. Um dies zu erleichtern, wird ein Luftröhrenschnitt durchgeführt, so dass Belüftung und gewährleisten physiologische Stabilität der Ratte während der Operation. Das Herz wird dann auf eine Glaswassermantel spiral Säule, durch die Krebs-Henseleit-Puffer wird durch retrograde Perfusion direkt in die Aorta geliefert befestigt. Eine Kochsalzlösung gefüllten Ballon wird in den linken Ventrikel eingeführt und mit einem Druckwandler, der für die Echtzeit-Messung von Drücken von innerhalb der Herzkammer ein ermöglicht befestigtd Berechnung mehrerer funktioneller Parameter. Am Ende des Experiments wird das Herz mit kalter Kochsalzlösung gespült, um die Kontraktion und in flüssigem Stickstoff eingefroren, um Downstream-Analyse von DNA, RNA und Protein-Ebene ermöglichen verhaften. So modifizierte, dient der Langendorff-perfundierten Herzen als ein wirksames System für die direkte Überwachung der physiologischen Wirkung von pharmakologische Interventionen zu jeder Zeit während des akut ischämischen Reperfusionsschäden.

<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.

Die linke Kammerballon Vorrichtung ermöglicht eine Echtzeitüberwachung des Drucks durch den Auftraggeber linken Ventrikel (Abbildung 1) entwickelt. Wie zuvor 7 beschrieben, kann diese Druckspur verwendet, um viele der Parameter der Herzfunktion zu berechnen. Diese Berechnungen können in der Basisphase als auch der Reperfusionsphase gemacht werden, gemittelt über mehrere Spuren in jeder Gruppe, und verglichen, um zu bestimmen, ob die pharmakologische Intervention ergab Herz Vorbehandlung, ...

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.

Induktion und Beurteilung der Ischämie-Reperfusion Injury in Langendorff-perfundierten Rattenherzen

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 Aufrufe.  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.

Serie: 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.

Real-time Digital Imaging von Leukozyten-Endothel-Interaktion in der Ischämie-Reperfusionsschaden (IRI) der Ratte Cremastermuskel

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

Forschung

Medizin

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 Quer Bauchmuskel myokutane Flap: Eine Ratte Modell myokutane Ischämie 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 ...

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.

Ischämie-Reperfusion-Modell des akuten Nierenschädigung und nach der Verletzung Fibrose bei Mäusen

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.

Murine Modell der intestinalen Ischämie-Reperfusionsschaden

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.

Bewertung des koronaren Flussreservats nach Myokard-Ischämie-Reperfusion bei Ratten

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.

Ein präklinisches Rattenmodell zur Untersuchung von Ischämie-Reperfusionsverletzungen in der rekonstruktiven Mikrochirurgie

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.

Optokardiographie und Elektrophysiologie Studien an Ex Vivo Langendorff-perfundierten Herzen

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.

Verzögerte intramyokardiale Lieferung von Stammzellen nach Ischämie-Reperfusionsverletzung in einem Murine-Modell

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.

OrganIschemia-Reperfusion Verletzung durch Simulation hämodynamischer Veränderungen im Lebertransplantationsmodell der Ratte

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.

Echtzeit-Beurteilung der Rückenmarksmikroperfusion in einem Schweinemodell der Ischämie/Reperfusion

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