Questa pubblicazione è stata sostenuta dalla Carolina del Sud Clinical &amp; Translational Research (SCTR) Institute, con una casa accademico presso la Medical University of South Carolina, NIH / NCATS Concessione Numero UL1 TR000062. Ulteriore supporto è stato fornito da premio di merito VA BX002327-01 a DRM. DJH è stato sostenuto da NIH / NCATS Concessione Numero TL1 TR000061 e dal NIH di Grant Numero T32 GM008716. SEA è stato sostenuto da NIH di Grant Numero T32 HL07260.

Tutte le procedure qui elencati sono stati approvati dalla cura e l&#39;uso degli animali Comitato Istituzionale presso la Medical University of South Carolina. Gli esperimenti qui descritti sono acute, esperimenti non di sopravvivenza. Come tale, non c&#39;è uso di unguento occhio e una suite operatoria sterile non è necessaria. L&#39;eutanasia si ottiene dissanguamento durante la raccolta del cuore. Preparazione 1. sperimentale <ol><li> Impostare sia una pressione costante o costante flusso Langendorff Perfusion Apparatus 4. </li><li> Preparare 4 L di Krebs modificato Henseleit Buffer (in mM: 112 NaCl, KCl 5, 1.2 MgSO4, 1 K 2 HPO 4, 1.25 CaCl 2, 25 NaHCO3, 11 D-glucosio, 0.2 acido ottanoico, pH = 7,4). <ol><li> Fai 10x azionari buffer. <ol><li> Sciogliere NaCl, KCl, MgSO 4, e K 2 HPO 4 a 3 litri di acqua ultrapura. </li><li> Sciogliere CaCl 2 in 500 ml di acqua ultrapura with 40 ml di 1 N HCl. </li><li> Aggiungere lentamente CaCl 2 soluzione al buffer realizzato in 1.2.1.1. Aggiungere acqua ultrapura abbastanza per un totale di 4 L. Consenti a mescolare per 1 ora prima di filtrare attraverso un filtro di 0,8 micron. Conservare a 4 ° C. </li><li> Sciogliere NaHCO 3 a 4 litri di acqua ultrapura. Lasciare a mescolare per 1 ora prima di filtrare attraverso a 0.8 micron filtro. Conservare a 4 ° C </li></ol></li><li> Unire 400 ml 10x tampone Krebs Henseleit con 400 ml 10x NaHCO 3 buffer. Aggiungere acqua ultrapura abbastanza per un totale di 4 L. Sciogliere glucosio e acido ottanoico mescolando. Filtrare 0,8 micron filtro. </li></ol></li><li> Sistema di Preparazione per il cuore. <ol><li> Aggiungere 2 L di tampone filtrata in una bottiglia 4 L 38 disposto sopra il rubinetto attaccato alla cannula aortica. Ciò fornirà una pressione di perfusione di 70-80 mmHg al cuore, che è la pressione di perfusione raccomandata 4. </li><li> Inserire gas dispersione gorgogliatore nel buffer nella bottiglia. </li><li> Eseguire il buffer attraverso il sistema, avendo cura di eliminare eventuali bolle dall&#39;interno del tubo. </li><li> Accendere gas (95% O 2/5% CO 2) almeno 30 minuti prima cuore è da collocare sul sistema. Utilizzare una sonda flusso di gas e analizzatore di gas del sangue per verificare la saturazione di ossigeno. </li></ol></li><li> Preparare scorte chirurgiche e pallone. <ol><li> Per la raccolta di cuore, preparare un paio di forbici chirurgiche 6,75 &quot;, un paio di forbici 4.5&quot;, due paia di hemostats, due set di pinze CaraMalto, due siringhe con 27 aghi G, 2 pezzi di 0-misuratore sutura di seta (su 15 cm di lunghezza), una cannula spinato 1/16 di pollice, e una cannula tracheale. </li><li> Per assemblare cannula tracheale, incollare un breve pezzo di tubo del diametro di plastica 1/16 di pollice a un connettore a Y. </li><li> Per assemblare apparato palloncino: inserire un ago sonda gastrica per un pezzo di tubo 1/16 di pollice, quindi collegare il tubo di alloggiamento trasduttore di pressione. Una piccola siringa collegata all&#39;altro lato della tr pressioneansducer alloggiamento consentirà l&#39;inflazione e la deflazione del palloncino. <ol><li> Tagliare un quadrato di plastica dell&#39;involucro e luogo punta di un ago sonda gastrica al centro. Avvolgere l&#39;involucro di plastica intorno all&#39;ago sonda gastrica. </li><li> Riempire palloncino con soluzione salina e legare con due punti di sutura. Gonfiare palloncino per assicurarsi che non ci siano perdite. Il volume di soluzione salina all&#39;interno del palloncino gonfiato dovrebbe essere circa 200 ml, ma può variare a seconda delle dimensioni del ratto. </li><li> In alternativa, utilizzare un lettino dito lattice al posto della pellicola trasparente. Posizionare gavage ago all&#39;interno dito culla, riempire con soluzione salina e legare. </li></ol></li></ol></li></ol> 2. Raccolta cuore <ol><li> Trattenere un ratto Sprague Dawley utilizzando un decapicone o altro sistema di vincolo manuale. Pesare topo. </li><li> Somministrare ketamina / miscela xylazina (0,85 mg / kg di ketamina e 0,15 mg / kg xilazina) tramite iniezione intraperitoneale per anestetizzare ratto. Questa dose di anestetico fornirà anestesia sufficienti per20-40 min, ma la procedura dovrebbe essere completata il più rapidamente possibile dopo anestesia è confermato. </li><li> Verificare il corretto livello di anestesia da test punta pizzico reflex. </li><li> Tracheotomia <ol><li> Rimuovere pelle da metà zampa a base di mascella, utilizzando hemostats per elevare Pelt e forbici per fare incisione. Mantenere la trippa elevata per separarlo dal muscolo sottostante, permettendo di tagliare lungo la trippa e rimuoverlo. </li><li> Utilizzando hemostats, sollevare il tessuto ghiandolare e fare una incisione orizzontale a fascia inferiore al tessuto ghiandolare. Utilizzare hemostats per diffondere incisione aperta, facendo attenzione a non intaccare le vene giugulari. </li><li> Utilizzando hemostats, pizzico muscolo sovrastanti la trachea, e con le forbici fare una incisione trasversale nel muscolo appena sotto la punta delle hemostats. Utilizzando hemostats, si sviluppa il tessuto oltre a rivelare trachea. </li><li> Inserire con cautela hemostats sotto la trachea, la compensazione fascia mentre procedendo delicatamente avanzamento e riavvolgimento emostatici. </li><li>Una volta hemostats sono collocati con successo dietro la trachea, un pizzico 0-0 sutura di seta con hemostats e hemisect la trachea con un piccolo paio di forbici affilate. </li><li> Inserire cannula tracheale nella trachea hemisected, tirare di sutura dietro la trachea, e legare la sutura intorno alla trachea cannulato, ancorando la sutura alla Y-inguine della cannula con un nodo chirurghi. </li><li> Utilizzando un ago 27 G, iniettare 1.000 U / kg di eparina nella vena giugulare e consentire a circolare per 30 sec. </li></ol></li><li> Toracotomia <ol><li> Rimuovere pelle da metà addome per regione medio-zampe anteriori da pizzicare con hemostats e il taglio con le forbici. </li><li> Fare un ¾ di pollice trasversale un&#39;incisione nella parete muscolare addominale, appena sotto il diaframma. </li><li> Effettuare una incisione verticale sul muro della parete toracica e addominale, taglio lungo lo sterno. </li><li> Tagliare il diaframma posteriore bilateralmente, poi diffuso gabbia toracica per rivelare il cuore, morsetto gabbia toracica con hemostats per assicurare rimane diffusione. </li&gt; <li> Utilizzando hemostats, rimuovere delicatamente il timo, esponendo l&#39;aorta ascendente. </li><li> Tease hemostats attraverso cappio aortica, avanzamento e riavvolgimento hemostats e delicatamente diffusione fascia per consentire punta di hemostats attraverso. </li><li> Utilizzare hemostats per tirare 0-0 sutura di seta dietro dell&#39;aorta ascendente, cravatta di sutura in una mezza piazza nodo allentato. </li><li> Girare rubinetto per avviare il flusso di tampone di perfusione, hemisect aorta e inserire la cannula nel lume aortico immediatamente. Rapidamente cinch un nodo mezzo quadrato e completare il nodo, stringendo a fondo. </li><li> Usare le forbici per sezionare cuore lontano dai grandi vasi, togliere il cuore dal petto e allegare alla colonna di perfusione. </li><li> Troncare tessuto polmonare in eccesso e permette cuore equilibrare su colonna per circa 15 minuti prima dell&#39;inserimento del palloncino. Utilizzare una portata normale di tampone attraverso il cuore di 10-20 ml / min. </li></ol></li></ol> 3. Langendorff perfusione e ischemia riperfusione <ol><li> Posizionamento di LV pressure balloon <ol><li> Sgonfiare pallone durante il rotolamento a forma di cono e girare rubinetto per assicurare pallone rimane sgonfio. </li><li> Excise lasciato atrio con un piccolo paio di forbici per esporre l&#39;accesso alla valvola mitrale. </li><li> Inserire palloncino attraverso atrio sinistro e la valvola mitrale in ventricolo sinistro. </li><li> Avviare il software di controllo della pressione (LabChart Pro). </li><li> Gonfiare pallone aprendo rubinetto e delicatamente crescente quantità di soluzione salina in mongolfiera fino diastolica lettura da micromanometro supera zero. </li><li> Per controllare la risposta lunghezza-tensione, gonfiare palloncino lentamente ad una pressione diastolica di 75 mmHg, sgonfiare lentamente, ripetere una volta. </li><li> Impostare la pressione diastolica di 10 mmHg modulando la quantità di soluzione salina nel pallone. Girare rubinetto per sigillare palloncino a questo livello di inflazione, e di mantenere il sistema di pressione chiuso tra LV palloncino e trasduttore di pressione. </li><li> Lower cuore in riscaldato da camera e coperchio della camera con la plastica. </li></ol></li><li> Annuncioministero della Ischemia riperfusione <ol><li> Inserire il fondo della camera riscaldata e lasciare da camera di riempire con tampone Effused. </li><li> Quando il buffer è sommerso cuore, girare rubinetto per arrestare il flusso di tampone nel cuore, inducendo ischemia globale. Ischemia tempo può variare a seconda degli obiettivi dell&#39;esperimento. </li><li> Dopo 30 min di ischemia, girate rubinetto per ripristinare il flusso di tampone in cuore, iniziando riperfusione. </li><li> Consentire riperfusione del cuore per 1 ora prima di terminare l&#39;esperimento. Utilizzare tempi riperfusione variabili, a seconda degli obiettivi dell&#39;esperimento. </li></ol></li><li> Cessazione Esperimento e raccolta di tessuto <ol><li> Utilizzando babordo di rubinetto, somministrare 10 ml di tampone fosfato salino a 4 ° C nel cuore ad una velocità di 10 ml / min per arrestare cuore. </li><li> Rimuovere cuore dalla colonna perfusione, tagliare via rimanente tessuto atriale. </li><li> Mettere il cuore in acciaio inox per affettare matrice di tessuto, coprire con Parafilm, e posto a -80 ° C per 8 minuti, o fino a quando il cuore ha una consistenza marshmallow-like. </li><li> Rimuovere blocco dal congelatore e inserire lamette in affettare matrice per tagliare cuore in 2 fette mm. Blocchi tessuto possono essere acquistati anche con i formati fetta altri di 2 mm. </li><li> Rimuovere fette uno alla volta e rilasciare in azoto liquido a lampeggiare congelare per studi biochimici. Conservare la fetta terzo ventricolo dall&#39;apice per infarto colorazione. </li><li> Rimuovere il tessuto da azoto liquido e conservare a -80 ° C fino al momento dell&#39;uso in esperimenti biochimici. </li></ol></li><li> Infarto colorazione <ol><li> Incubare la terza porzione ventricolare in 10 ml di 1% w / v di cloruro trifeniltetrazolio (TTC) a 37 ° C per 15 min. Nota: L&#39;incubazione in TTC può essere eseguita per 15 o 30 minuti con nessun cambiamento nell&#39;efficacia 15. </li><li> Trasferire fetta tessuto dalla TTC al 10% formalina tamponata e lasciar incubare a temperatura ambiente per una notte. Per ottenere risultati ottimali, sentirets devono essere tolti di formalina e ripreso entro 24 ore 4. </li><li> Fotografia macchiato fetta e l&#39;utilizzo di software come ImageJ per stimare le dimensioni dell&#39;infarto secondo il protocollo del produttore. Fotografare il più presto possibile dopo la fissazione, il colorante sbiadiscono nel tempo. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Isolato Il cuore perfuso di ratto può essere utilizzato con successo per studiare l&#39;effetto di un intervento farmacologico in precondizionamento cardiaco in ischemia riperfusione 9. Tuttavia, ci sono alcuni passaggi essenziali alla procedura che deve essere standardizzato per garantire risultati riproducibili. Il mantenimento di una temperatura di 37,4 ° C all&#39;interno del sistema è critico, in quanto anche lieve ipotermia e ipertermia può causare precondizionamento cardiaco 10,11. Il te...

Delucidazione degli eventi di riferimento la risposta cardiaca sia ischemia e riperfusione sono essenziali per migliorare il trattamento di infarto 1 e cardiache procedure chirurgiche che richiedono miocardio clampaggio aortico 2. Mentre i modelli in vivo di ischemia riperfusione consentono analisi endpoint molto utili, essi non sono efficaci per studiare gli effetti funzionali di ischemia riperfusione acutamente in tempo reale. Inoltre, in modelli di ischemia riperfusione vivo producono generalmente significativa variabilità nelle dimensioni dell&#39;infarto e consegna diretta di farmaco al cuore al momento della riperfusione è impegnativo. L&#39;utilizzo di un sistema Langendorff cuore isolato per studiare il danno da ischemia riperfusione permette valutazione funzionale in tempo reale di trattamenti farmacologici, superficie uniforme tessuto infartuato e consegna istantanea di farmaco direttamente al miocardio. Prima descritto by Oscar Langendorff nel 1895 3, il cuore Langendorff isolato è un modello affidabile per lo studio di ischemia riperfusione, essendo stato utilizzato nella ricerca ischemia riperfusione per gli ultimi 40 anni di 4,5. Qui, alcune modifiche sono fatte per ottimizzare il cuore isolato per l&#39;analisi funzionale. In situ incannulamento dell&#39;aorta mentre il cuore batte assicura che il cuore non sperimenta precondizionamento ischemico, tali da modificare i risultati di studi ischemia riperfusione 6. Per facilitare ciò, viene eseguita una tracheotomia, consentendo la ventilazione e garantire la stabilità fisiologica del ratto durante l&#39;intervento chirurgico. Il cuore è poi collegato a un camicia d&#39;acqua colonna tortile vetro attraverso il quale tampone Krebs Henseleit avviene tramite perfusione retrograda direttamente nell&#39;aorta. Un palloncino soluzione salina viene inserita nel ventricolo sinistro e collegato ad un trasduttore di pressione, che consente la misurazione in tempo reale delle pressioni dall&#39;interno un ventricolod calcolo dei molteplici parametri funzionali. Al termine dell&#39;esperimento, cuore viene lavata con soluzione salina fredda per arrestare la contrazione e flash congelati in azoto liquido per consentire l&#39;analisi valle dei livelli di DNA, RNA e proteine. Così modificato, cuore perfuso Langendorff serve come un sistema efficace per il monitoraggio diretto degli effetti fisiologici degli interventi farmacologici in qualsiasi momento acutamente durante il danno da ischemia riperfusione.

<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;apparato ventricolo sinistro balloon permette il monitoraggio in tempo reale della pressione sviluppata dalle amministrazioni ventricolo sinistro (figura 1). Come descritto in precedenza 7, questa traccia di pressione può essere utilizzata per calcolare molti dei parametri della funzione ventricolare. Questi calcoli possono essere effettuati in fase iniziale nonché la fase di riperfusione, mediati su tracce multiple all&#39;interno di ciascun gruppo, e confrontati per determinare se l...

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.

Induzione e valutazione di ischemia-riperfusione Injury in Langendorff-perfusi Hearts Rat

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

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JoVE Journal

Medicine

22.6K Views.  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.

Video: 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.

In tempo reale Imaging digitale di leucociti-endotelio in ischemia-riperfusione (IRI) del muscolo Cremaster Rat

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

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Medicina

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 trasversale retto addominale miocutaneo Flap: un modello di ratto di miocutaneo ischemia riperfusione

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.

Ischemia-riperfusione Modello di danno renale acuta e post Infortunio fibrosi nei topi

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.

Modello murino di ischemia-riperfusione intestinale Injury

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.

Valutazione della riserva del flusso coronario dopo la miaocardia Ischemia Reperfusione nei ratti

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 modello di ratto preclinico per lo studio delle lesioni ischemia-reperfusione in microchirurgia ricostruttiva

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.

Optocardiografia ed elettrofisiologia Studi di Ex Vivo Langendorff-perfusi

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.

Consegna intramiocardica ritardata di cellule staminali dopo l'infortunio di ricottamento ischemia in un modello 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.

Lesione da ischemia-riperfusione dell'organo simulando cambiamenti emodinamici nel modello di trapianto di fegato di ratto

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.

Valutazione in tempo reale della microperfusione del midollo spinale in un modello suino di ischemia/riperfusione

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