Questo lavoro è stato finanziato dal US Army Grant (W81XWH-08-1-0701) e una borsa di studio da Carle Foundation Hospital.

La procedura è in piena compatibilità con numero di protocollo IACUC 13302 ed è stato approvato dalla divisione delle risorse animali. 1. Strumento Preparazione <ol><li> Autoclavare tutti gli strumenti non monouso utilizzati prima della procedura chirurgica per garantire la sterilità. </li><li> Sterilizzare gli strumenti utilizzati più volte in una sessione con una sterilizzatrice perle di vetro tra usi. </li></ol> 2. Hydrogel Preparazione <ol><li> Scongelare e le cellule staminali piatto almeno 24 ore prima della creazione del costrutto idrogel. </li><li> Preparate patch un giorno prima l&#39;intervento chirurgico utilizzando un apparato stereolitografia (o uno strumento simile) che è stato descritto in dettaglio in precedenti pubblicazioni 8,18. </li><li> Per creare l&#39;idrogel, prima di generare una progettazione assistita da computer (CAD) modello del patch desiderato ed esportare il design in formato stereolitografia (STL). </li><li> Creare una soluzione pre-gel per dissolving di poli (etilenglicole) dimetacrilato (PEGDMA) in PBS sterile 1x per ottenere 20% w / v soluzione. Contemporaneamente, sciogliere il fotoiniziatore 1- [4- (2-idrossi etossi) fenil] -2-idrossi-2-metil-1-propan-1-one in DMSO. Aggiungere il fotoiniziatore alla soluzione PEGDMA ad una finale w / v di 0,5%. Direttamente prima reticolazione, aggiungere 100 ml di densità di celle desiderato. Nota: Una densità delle cellule tra 2,0 x 10 5 -2.0 x 10 6 cellule / ml è raccomandato. </li><li> Pipettare la soluzione pre-gel in un piatto al centro della piattaforma SLA ed eseguire il software SLA con la STL formattato disegno già caricato. </li><li> Rimuovere le patch dall&#39;apparato incubare idrogel O / N a modificata mezzo di Eagle Dulbecco (DMEM) supplementato con siero 5% bovino fetale (FBS), 100 U / ml di penicillina, e 100 mg / ml di streptomicina per consentire cellule per regolare per l&#39;ambiente all&#39;interno della patch. </li></ol> 3. Preparazione degli animali e intubati oralesu <ol><li> Valutare ogni mouse prima della procedura per garantire che l&#39;animale è in buona salute, come determinato dalle loro abitudini di toelettatura e le interazioni con i loro compagni di gabbia. Nota: femmina 8-10 settimane di età C57BL / 6J sono stati usati per questa procedura, però, può essere utilizzato qualsiasi età mouse o tipo. </li><li> Inserire animali in una camera anestetico ed esporre al 5% isofluorano con un supporto 1 L / min O 2. Monitorare il livello di anestesia da pizzico piedi reflex. </li><li> Una volta che l&#39;animale sia adeguatamente anestetizzato, pesare e metterli sul supporto intubazione. </li><li> Dirigere una sorgente di luce verso cavità toracica dell&#39;animale e utilizzare pinze per ritrarre la superficie della laringe ed esporre le corde vocali. Guidare con attenzione un G angiocath 20 tra le corde vocali, e senza intoppi inserire nella trachea. Controllo corretto inserimento da un movimento meccanico della cavità toracica una volta che il catetere è collegato al piccolo ventilatore animali. </li><li> Regolare le impostazioni di ventilazione a the peso dell&#39;animale base alle linee guida del produttore. </li><li> Una volta intubato, mettere l&#39;animale in posizione supina su una piastra elettrica per evitare l&#39;ipotermia. Assicurarsi che il pad di riscaldamento non superi i 40 ° C. Se necessario, posizionare una barriera tra l&#39;animale e il pad di riscaldamento per ridurre al minimo il rischio di ustioni. </li><li> Rimuovere capelli dal sito chirurgico attraverso l&#39;uso di una crema depilatoria. Sterilizzare il sito da 3 scrub alternati di betadine e il 75% di etanolo per garantire una corretta sterilizzazione. Posizionare un drappo per esporre solo il sito chirurgico e posizionare veterinario pomata sugli occhi per prevenire la secchezza durante la procedura. </li></ol> 4. Procedura chirurgica <ol><li> La toracotomia viene eseguita sul lato sinistro dell&#39;animale, quindi, fare una incisione cutanea circa 1 cm a sinistra dello sterno, che corre lungo sterno. </li><li> Utilizzare pinze per separare lo strato muscolare della gabbia toracica sottostante in due fasi. Osservare una DelineLinea DELL&#39;INDICE DI che rappresenta gran pettorale muscoli dell&#39;animale. Sollevare questo muscolo un po &#39;, separarlo dal sottostante muscolo obliquo esterno e ritrarre mediale. Poi, liberare il muscolo obliquo esterno dalla gabbia toracica sottostante nello stesso modo, e retrarre lateralmente, fornendo una vista accessibile del secondo, terzo, e quarto costole. </li><li> Eseguire la toracotomia tra la terza e la quarta costola. Sollevare delicatamente la quarta costola e utilizzare un cauterizer per aprire la cavità toracica tra la terza e quarta costola. Posizionare divaricatori per aprire ulteriormente la cavità ed esporre il cuore. </li><li> Utilizzare pinze per rompere il sottile pericardio del cuore. </li><li> Legare l&#39;arteria coronaria sinistra con una sutura in nylon 8-0 monofilamento. Posizionare la sutura circa 4 mm dalla punta del cuore, direttamente sotto la punta inferiore dell&#39;atrio sinistro. Determinare il corretto posizionamento di sutura da un pallore del miocardio ventricolare ed un aumento delle dimensioni dell&#39;atrio sinistro dopo la Suture è stato legato. </li></ol> 5. Patch Placement e colla <ol><li> Mantenere patch a 37 ° C e 5% CO 2 condizioni fino al momento dell&#39;uso. </li><li> Sollevare delicatamente la patch con una spatola ended piana e posizionare delicatamente sulla superficie del cuore, nel sito di infarto. Nota: per evitare di scivolare fuori dal ventricolo sinistro, la patch può essere tenuto in posizione da mantenere leggermente contatto utilizzando la spatola o la punta di un paio di pinze. </li><li> Preparare la colla mescolando un rapporto 4: 1 di fibrinogeno in trombina. Miscelare la soluzione pipettando ripetutamente finché comincia ad addensarsi, di solito entro 1 minuto dopo l&#39;inizio della preparazione. </li><li> Una volta che la viscosità della soluzione raggiunge il livello desiderato, trasferire rapidamente circa 10 ml alla superficie patch. Nota: Il tempo di coagulazione della colla di fibrina è rapido, fornendo una piccola finestra di opportunità per un efficiente trasferimento della colla. </li></ol> 6. sutura <ol><li&gt; eseguire tutti i punti di sutura di chiusura con una sutura in nylon 6-0 monofilamento. </li><li> Chiudere lo strato costola con 3-4 singoli punti staccati. </li><li> Prima chiusura completa dello strato intercostali, inserire una cannula PE-10 nell&#39;incisione per evacuare la cavità toracica dopo la chiusura è completa e ristabilire la corretta pressione intrapleurica. </li><li> Chiudere i muscoli pettorali con 3-4 singoli punti staccati. </li><li> Sigillare lo strato di pelle con una sutura continua. </li><li> Dopo completa chiusura, collegare una siringa 1,0 ml al termine della cannula e utilizzarlo per evacuare la cavità toracica. </li><li> Applicare un tessuto adesivo al sito di incisione per rinforzare il sito di sutura. </li></ol> Trattamento 7. post-chirurgico <ol><li> Dare topi un&#39;iniezione di buprenorfina (0,05-1,0 mg / kg) e carprofen (2,2 mg / kg) per via sottocutanea immediatamente dopo la legatura coronarica e almeno 20 minuti prima del risveglio. Somministrare entrambi i farmaci 6-8 ore dopo l&#39;intervento chirurgico, E poi dare due volte al giorno per un massimo di tre giorni per controllare il dolore e angoscia. </li><li> Monitorare costantemente gli animali fino consapevole e ogni ora, per la prima dopo l&#39;intervento di 4 ore. Eseguire un secondo controllo la sera della chirurgia (2-4 ore più tardi) per somministrare una seconda dose di analgesico. </li><li> Monitorare gli animali ogni giorno dopo il secondo giorno dopo la procedura, fino a quando gli animali appaiono stabili. </li></ol> 8. Analisi di funzionamento del cuore e Istologia <ol><li> Eseguire ecocardiografi sui topi 4 settimane dopo la procedura per determinare l&#39;entità del danno a seguito del MI. </li><li> Anestetizzare topi con 4-5% isoflurano in una camera di anestesia per l&#39;induzione di anestesia per inalazione, e poi 2% isoflurano via maschera per l&#39;anestesia di mantenimento. </li><li> Eliminare l&#39;area di esposizione con una crema depilatoria e utilizzare un piccolo animale ultrasuoni per ottenere un 2D M-mode dell&#39;ecocardiografia scansione del cuore pulsante e registrare i parametri funzionali associato con esso. </li><li> Euthanize i topi in una camera di anidride carbonica, quindi raccogliere i cuori dagli animali e posto in una soluzione di formalina al 10% per 24 ore. Dopo 24 ore, trasferire i cuori al 70% di etanolo e conservare a 4 ° C fino al trattamento dei tessuti. </li><li> Elaborare i cuori e li incorpora in blocchi di paraffina. </li><li> Tagliare 5 millimetri sezioni attraverso la regione ventricolare del cuore, partendo all&#39;apice e terminando l&#39;atrio. </li><li> Stain fette con entrambi tricromica di Masson e ematossilina e eosina utilizzando protocolli standard sezionato. </li></ol>

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The authors declare that they have no competing financial interests.

Con questo approccio al modello MI murino, abbiamo sviluppato un sistema che riduce al minimo il danno alle zone non miocardio che sono associati con altre tecniche MI murine. Queste aree includono danni causati da tracheostomia, il taglio dello strato muscolare, e la rottura delle nervature per esporre la cavità toracica. Crediamo che questi cambiamenti migliorare le generali chirurgici risultato grazie alla cura di mantenere il più delle grandi strutture, compr...

Un infarto del miocardio (MI) è definito come l&#39;interruzione di sangue ad una regione del cuore causata dalla occlusione di un&#39;arteria coronaria. Il danno derivante da un MI è dovuto al rimodellamento del tessuto cardiaco vitale in tessuto cicatriziale non funzionale, che diminuisce la capacità del cuore o, più specificamente, il ventricolo sinistro, per battere correttamente. Ciò si traduce in una diminuzione del volume di sangue che può essere consegnato al corpo ad ogni battito cardiaco, chiamata volume ictus, e la percentuale di sangue che viene pompato dal cuore ad ogni battito cardiaco, noto come frazione di eiezione 6. Questi, insieme ad altre funzioni diminuita, aumenta la tensione sul resto del cuore per mantenere la funzione adeguata. Spesso, questo aumento ceppo può diventare così grave che provoca un secondo attacco cardiaco, un fenomeno visto in circa il 10% degli individui 7. Mentre le pratiche mediche si sono evoluti per trattaresubito dopo un infarto miocardico, nessuna tecnica è stata sviluppata per fermare, rallentare o invertire gli effetti collaterali negativi di rimodellamento tissutale. Terapie con cellule staminali sono emersi come una possibile via per un tale trattamento, tuttavia, nonostante il loro potenziale promettente, le cellule staminali non hanno dimostrato con successo in ambito clinico. Una teoria le loro carenze è l&#39;incapacità di garantire le cellule benefiche rimangono nella sede dell&#39;infarto abbastanza lungo per generare risultati favorevoli 5. È stato dimostrato che non più del 24% di cellule che sono semplicemente iniettato nel sito di infarto sopravvissuto, rimanendo al primo danneggiata sito: 1 giorno post-parto 2. Una possibile prospettiva per affrontare la questione della conservazione delle cellule è quello di sviluppare sistemi di idrogel biocompatibile che incapsulano sia cellule o terapeutica, che possono essere consegnati al sito danneggiato. L&#39;idrogel di scelta in questo protocollo è un poli (glicole etilenico) dimetacrilato grazie al suo impiego precedente incapsulamento della pila procedures, tuttavia, qualsiasi idrogel grado di incapsulamento può essere utilizzato 8. La consegna della patch direttamente al sito di lesione assicura il contatto cellula-tessuto per un periodo prolungato di tempo, aumentando la lunghezza di tempo le cellule possono fornire fattori positivi al miocardio sottostante. Un collo di bottiglia per l&#39;approccio patch è la difficoltà di aderire il cerotto alla superficie del cuore. Molti gruppi hanno superato questo attraverso una varietà di tecniche, i più diffusi essendo una semplice sutura per legare il costrutto alla superficie cardiaca 9,10. Questo si è dimostrato efficace in un numero di casi in cui il costrutto è fatto di un materiale più rigido, ma non riesce quando tentato su un sistema idrogel, a causa della concentrazione di acqua ad alta e delicata natura del costrutto patch. Per ovviare a questo, abbiamo utilizzato una colla di fibrina sistema adesivo esterno che imita la chimica di formazione del coagulo. Colla di fibrina è stata utilizzata in numerosi studi medici, incLuding lacrime Dura, fistole bronchiali, e trapianto di cornea, mettendo in evidenza la biocompatibilità del prodotto come sigillante ferita 11-13. Inoltre, la fibrina è stato utilizzato per una varietà di scopi cardiache, compreso il trattamento chirurgico delle fratture del ventricolo sinistro e coronarici chirurgia di bypass, tuttavia, il suo uso come una colla adesione per una patch cardiaca non è comunemente usato 14-17. Una semplice formulazione di trombina e fibrinogeno risultati in una colla biocompatibile che può essere posizionato direttamente sulla parte esterna di un cerotto cardiaco esterno, fornendo un sistema di aderenza sostenibili in modo cerotto all&#39;interazione cuore.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Harvard Model 687 Mouse Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>55-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Inintech Biosciences LLC Dry Glass Bead Sterilizer</td>
			<td>Fisher Scientific</td>
			<td>NC9531961</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica MZ6 surgical microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cautery Kit</td>
			<td>Gemini</td>
			<td>GEM 5917</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate Forceps - 0.4mm Tips Angled</td>
			<td>Fine Science Tools</td>
			<td>11063-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Agricola Retractor - 3.5cm Spread</td>
			<td>Fine Science Tools</td>
			<td>17005-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring Scissors - 2.5mm Blades Straight</td>
			<td>Fine Science Tools</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Needle Holder - w/Lock Tungsten Carbide 14cm</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Scissors - Delicate Straight 10.5 cm</td>
			<td>Fine Science Tools</td>
			<td>14060-10</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 monofilament suture</td>
			<td>Ethicon</td>
			<td>8730P</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 Silk suture</td>
			<td>Ethicon</td>
			<td>639G</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T7009</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Sigma</td>
			<td>F3879</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetbond Tissue Adhesive</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
	</tbody>
</table>

a hydrogel construct and fibrin-based glue approach to deliver therapeutics in a murine myocardial infarction model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

Durante la procedura chirurgica, legatura dell&#39;arteria coronarica sinistra può essere identificato da un marcato blanching valle dell&#39;arteria occlusa. Come test prima di sposarsi, la sutura può essere stretto per breve tempo per verificare se è in un luogo adeguato. Inoltre, poiché occlusione delle arterie risultati in diminuzione quasi istantanea nella capacità dell&#39;atrio sinistro per battere correttamente, l&#39;atrio sinistro si allargherà in risposta ad un riflusso di sangue nel sistema. <p cla...

Watch this Scientific Journal Video about A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model. at JoVE.com

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

Un idrogel Costruire e fibrina-based Glue approccio per fornire Therapeutics in una murino Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

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JoVE Journal

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8.6K Views.  University of Illinois at Urbana-Champaign. This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

Video: A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

Infarto acuto del miocardio in topi

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

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Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Legatura dell&#39;arteria coronarica e di iniezione intramiocardico in un modello murino di infarto

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

A Murine chiuso del torace modello di ischemia miocardica e riperfusione

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Infarto miocardico e funzionale Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

La valutazione dell&#39;outcome primario in un modello suino di infarto miocardico acuto

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Infarto del miocardio in topi neonati, un modello di rigenerazione cardiaca

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Modello di infarto miocardico Murine utilizzando la legatura permanente dell'arteria coronaria discendente anteriore sinistra

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

Un modello di criolesionismo per studiare l'infarto miocardico nel mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Establishing a Swine Model of Post-myocardial Infarction Heart Failure for Stem Cell Treatment

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major causes of mortality and morbidity around the world. Cell-based therapies for cardiac repair and improvement of left ventricular function after MI have attracted considerable attention. Accordingly, the safety and efficacy of these cell transplantations should be tested in a preclinical large animal model of HF prior to clinical use. Pigs are widely used for cardiovascular disease research due to their similarity to humans in terms of heart size and coronary anatomy. Therefore, we sought to present an effective protocol for the establishment of a porcine chronic HF model using closed-chest coronary balloon occlusion of the left circumflex artery (LCX), followed by rapid ventricular pacing induced with pacemaker implantation. Eight weeks later, the stem cells were administered by intramyocardial injection in the peri-infarct area. Then the infarct size, cell survival, and left ventricular function (including echocardiography, hemodynamic parameters, and electrophysiology) were evaluated. This study helps establish a stable preclinical large animal HF model for stem cell treatment.

We sought to establish a swine model of heart failure induced by left circumflex artery blockage and rapid pacing to test the effect and safety of intramyocardial administration of stem cells for cell-based therapies.

Istituzione di un modello suina di infarto post-miocardico per il trattamento delle cellule staminali

Although advances have been achieved in the treatment of heart failure (HF) following myocardial infarction (MI), HF following MI remains one of the major ...

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

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

Trapianto intramiocardico di idrogel iniettabili a carico MSC dopo infarto del miocardio in un modello murino

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