Questa ricerca è supportata dal Basic Science Research Program attraverso la National Research Foundation of Korea (NRF) finanziata dal Ministero dell'Istruzione (NRF-2018R1D1A1A02049346)

Tutte le procedure di ricerca sugli animali sono state fornite in conformità con il Laboratory Animals Welfare Act, la Guida per la cura e l'uso degli animali da laboratorio e le linee guida e le politiche per gli esperimenti sui roditori fornite dall'Institutional Animal Care and Use Committee (IACUC) nella School of Medicine dell'Università Cattolica della Corea.
1. Preparazione di CSC e idrogel di gelatina iniettabili
<ol><li>CCI della cultura in un piatto da coltura da 100 mm a 37 °C e 5% CO2. Quando la crescita degli MSC raggiunge l'80% di confluenza, lavare il piatto due volte con DPBS e aggiungere 1 mL di sostituto della tripsiderina a 37 °C per 3 min. NOTA: I CSC sono stati isolati dal midollo osseo murino seguendole procedure convenzionali 16, coltivati nel Mezzo dell'Aquila Modificata (DMEM) di Dulbecco contenenti il 10% di siero bovino fetale (FBS) e l'1% di soluzione antibiotico-antimicotica, e utilizzati tra il passaggio 7\u20129 per questo studio.</li>
	<li>Aggiungere 9 mL di mezzo di coltura e centrifuga a 500 x g per 3 min. Successivamente, scartare il supernatante risultante, rimescolare le cellule in 1 mL di PBS e mantenere la sospensione cellulare sul ghiaccio.</li>
	<li>Diluire 10 μL di sospensione cellulare con 10 μL di blu Trypan e ottenere la concentrazione cellulare utilizzando un contatore di celle automatizzato.</li>
	<li>Resuspend e trasferire i MBC su un tubo da 1 ml ad una densità di 1 x 107 celle/ml.</li>
	<li>Preparare un 6,25 wt% di soluzione coniugata GH in PBS e separare in 2 flaconcini. Quindi, mescolare le soluzioni GH con 6 μg/mL di HRP (soluzione GH A) o 0,07 wt% di H2O2 (soluzione GH B). NOTA: Preparare coniugati gelatina-idrossifenil propionico (GH) secondo i protocollipubblicati 12,15.<ol>
<li>Mantenere un rapporto volumetrico 9:1 di soluzione coniugata GH a soluzione coniugata HRP (GH soluzione A) e GH coniugata rispettivamente a H2O2 (soluzione GH B).</li>
	</ol>
</li>
	<li>Prima di miscelare i CBC con la soluzione GH A, centrifugare brevemente la sospensione cellulare a 1.000 x g e aspirare con cura il supernatante risultante. Successivamente, mescolare il pellet contenente MSC con la soluzione GH A.</li>
</ol>2. In situ MSC-loading e coltura tridimensionale in vitro
<ol><li>Caricare gh soluzione A (contenente MSC) e GH soluzione B su entrambi i lati di una doppia siringa. Piastra 300 μL delle soluzioni GH combinate con MBC ad una densità finale di 5 x 106 celle/mL su uno scivolo a camera a otto poi.</li>
	<li>Dopo la formazione di idrogel in situ e la successiva incapsulamento MSC tramite cross-linking enzimatico, aggiungere 700 μL di DMEM contenente il 10% di FBS e l'1% di soluzione antibiotico-antimicotica.</li>
	<li>Incubare lo scivolo a 37 °C e 5% di CO2 e sostituire il mezzo di coltura ogni 2\u20123 giorni.</li>
</ol>3. Conferma della proliferazione in vitro e della sopravvivenza dei CG MSC all'interno degli idrogel gh
<ol><li>Per determinare la fattibilità dei CBC coltivati in 3D all'interno degli idrogel GH, utilizzare un saggio di colorazione delle cellule vive / morte dopo il tempo di incubazione predeterminato.</li>
	<li>Dopo l'incubazione dei CBC incapsulati negli idrogel GH per 3, 5, 7 o 14 giorni, aspirare il mezzo e lavare il pozzo due volte con PBS.</li>
	<li>Preparare una soluzione di colorazione contenente 5 μL di calceina AM e 20 μL di omodimero di etidio-1 (EthD-1) in 10 mL di DPBS.</li>
	<li>Aggiungere 200 μL della soluzione di colorazione al pozzo e incubare per 30 minuti al buio a temperatura ambiente.</li>
	<li>Aspirare la soluzione di colorazione e lavare il pozzo due volte con PBS.</li>
	<li>Separare accuratamente la camera dallo scivolo e posizionare un coverslip completo sugli idrogel GH. Utilizzare una microscopia confocale per visualizzare il grado di proliferazione e i cambiamenti morfologici degli MSC incapsulati. NOTA: Le immagini fluorescenti sono state acquisite con ingrandimento inferiore a 200x e immagini alle lunghezze d'onda di eccitazione/emissione di 470/540 nm per la calceina e 516/607 nm per EthD-1.</li>
</ol>4. Induzione dell'infarto del miocardio nei topi
<ol><li>Anestetizzare topi maschi C57BL/6 di 7 settimane (20\u201222 g) con iniezione intraperitoneale di una miscela di Zoletil (30 mg/kg) e Rompun (10 mg/kg) in soluzione salina.</li>
	<li>Prima dell'intervento chirurgico, depilate il torace del topo usando la crema per la depilazione e sterilizzare la pelle con iodio.</li>
	<li>Posizionare il mouse su un tavolo operatorio e intubare inserendo un catetere nella trachea per fornire ossigeno supplementare attraverso la ventilazione meccanica.</li>
	<li>Tagliare delicatamente la pelle usando forbici chirurgiche e quindi penetrare nei muscoli intercostali con micro forbici. Separare la seconda e la terza costola sinistra usando una sutura di seta 5-0 per mantenere una cavità toracica aperta.</li>
	<li>Legare con cura l'arteria coronarica discendente anteriore sinistro (LAD) utilizzando un portaaghi con un 8-0 sutura in polipropilene e tagliare la sutura usando l'elettrocauteria.</li>
	<li>Osservare un cambiamento di colore immediato nella parete ventricolare sinistra anteriore.</li>
</ol>5. Trapianto intramiocardico di idrogel GH a carico di MSC
<ol><li>Dopo aver indotto l'infarto del miocardio mediante legatura LAD, iniettare 10 μL di soluzioni GH a caricamento MSC in due punti diversi nella zona di confine infarto (totale: 2 x 105 MSC/20 μL) utilizzando una doppia siringa dotata di un ago 26G.<ol>
<li>Seguendo la stessa procedura descritta nella fase 1, preparare e trasferire le soluzioni GH a caricamento MSC in una doppia siringa. NOTA: Per valutare l'innesto degli idrogel GH a carico di MSC all'interno dell'area infarcted, gli MSC e i coniugati GH sono stati preetichetti rispettivamente con PHK26 e isotiocianato di fluoresceina (FITC).</li>
	</ol>
</li>
	<li>Ripristinare la cavità toracica aperta e chiudere i muscoli e la pelle usando 5-0 suture. NOTA: Prima della chiusura del torace, rimuovere l'aria usando una siringa catetere.</li>
	<li>Rimuovere il tubo tracheale e posizionare il mouse in una gabbia sotto una lampada a infrarossi durante il recupero.</li>
	<li>Per l'analgesia postoperatoria, somministrare iniezioni sottocutanee di Chetoprofene (5 mg/kg al giorno) per un minimo di 72 ore. Tutti i topi devono essere attentamente monitorati per un tempo appropriato per garantire un corretto recupero dopo le procedure chirurgiche e un adeguato trattamento del dolore.</li>
</ol>6. Ecocardiografia
<ol><li>Quattro settimane dopo il trapianto, inizialmente anestetizza il topo con il 5% di isoflurane e quindi regola la concentrazione di isoflurane all'1%.</li>
	<li>Depilate il petto usando la crema per la depilazione e posizionare il mouse su una pastiglia riscaldante. Applicare il gel trasduttore ad ultrasuoni sul petto.</li>
	<li>Acquisire viste bidimensionali parasternali a basso asse e registrare tracciati in modalità M a livello del muscolo papillare. NOTA: Posizionare un trasduttore di array lineare (7\u201215 MHz) nella linea parasternale sinistra e visualizzare le strutture anatomiche.</li>
	<li>Misurare le linee corrispondenti per LVAW, LVID e LVPW per ottenere lo spessore della parete cardiaca, la dimensione della camera e l'accorciamento frazionato. NOTA: Confrontare la funzione cardiaca tra cui la frazione di espulsione (EF), l'accorciamento frazionato (FS) e il volume estolico finale (ESV) a livello del muscolo papillare per garantire una corretta valutazione nella stessa posizione anatomica.</li>
</ol>7. Valutazione istologica
<ol><li>Al momento predeterminato dopo il trapianto di idrogel GH a carico di MSC nel cuore infarto, eutanasiare il topo in una camera di CO2 e raccogliere il cuore per l'analisi istologica15.</li>
	<li>Per la colorazione tricromatica ed eosina (H&E) e tricromo (MT) di Masson, fissare i tessuti cardiaci sezionati in paraformaldeide (PFA) al 4% e incorporare nella paraffina. Successivamente, tagliare i blocchi cardiaci incorporati nella paraffina in sezioni seriali da 4 μm utilizzando un microtomo e macchiare le sezioni con macchia MT secondo i protocolli standard17.</li>
	<li>Acquisire immagini su uno scanner di diapositive con ingrandimento 20x e calcolare la dimensione infaritta dei gruppi di trattamento. Dimensioni infarct (%) = circonferenza totale infarct / circonferenza totale LV x 100</li>
	<li>Calcolare entrambe le circonferenze in base alla misurazione della lunghezza della linea mediana. Per le circonferenze della linea mediana LV, misurare le lunghezze dell'linea centrale tra le superfici endocardiale ed epicardiale. Per le circonferenze infariche della linea mediana, misurare le lunghezze di infarto, compreso più del 50 % dello spessore intero del miocardio18. NOTA: Tutte le analisi delle immagini sono state eseguite utilizzando il software ImageJ.</li>
	<li>Misurare lo spessore della parete della cicatrice ai livelli muscolari papillari.</li>
	<li>Calcola la frazione dell'area del collagene. Area di collagene (%) = superficie totale della fibrosi interstiziale/area del miocita x 100</li>
</ol>

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Gli idrogel GH iniettabili hanno un grande potenziale per applicazioni in vivo grazie alla loro capacità di incorporare omogeneamente diversi agenti terapeutici in situ. Inoltre, le loro proprietà fisiche e biochimiche possono essere facilmente manipolate in base a requisiti dipendenti dalla malattia. A questo proposito, sono stati proposti idrogel iniettabili per affrontare le principali limitazioni dell'attuale terapia con cellule staminali cardiache ostacolate dalla scarsa sopravvivenza e ritenzione cellulare (cioè...

La terapia con cellule staminali cardiache è emersa come un potenziale approccio per la riparazione e la rigenerazione delmiocardio 1,2. Nonostante i recenti risultati positivi nei modelli animali e negli studi clinici, l'applicazione della terapia a base di cellule staminali per la riparazione del miocardio è limitata a causa della bassa ritenzione e della scarsa sopravvivenza delle cellule iniettate nei tessuti cardiaci infarti3,4. Di conseguenza, l'uso dell'ingegneria tissutale a base cellulare, compresi i biomateriali iniettabili5,le chiazze cardiache6e ifogli cellulari 7,è stato intensamente studiato per migliorare la ritenzione cellulare e l'integrazione all'interno del miocardio ospite.
Tra i vari approcci potenziali alla riparazione del tessuto cardiaco bioingegnerato, gli idrogel iniettabili combinati con tipi di cellule appropriati, come le cellule staminali mesenchimali (MCC), le cellule staminali embrionali (ESC) e le cellule staminali pluripotenti indotte (IPSC), sono un'opzione interessante per fornire efficacemente le cellule nelle regioni del miocardio8,9. La gelatina, un noto polimero naturale, può essere utilizzata come matrice iniettabile grazie alla sua grande biocompatibilità, alla notevole biodegradabilità e alla ridotta immunogenicità rispetto a una vasta gamma di biomateriali utilizzati in applicazioni biomediche. Sebbene le piattaforme iniettabili a base di gelatina abbiano un grande potenziale, la loro applicabilità in vivo rimane limitata in base alla loro bassa rigidità meccanica e alla facile degradabilità nell'ambiente fisiologico.
Per superare questi limiti, è stato proposto un nuovo e semplice design di idrogel a base di gelatina costituiti da acido propionico idrossifenile per applicazioni in vivo. I coniugati gelatino-idrossifenil propionico (GH) possono essere reticolati in situ in presenza di un enzima, la perossidasi di rafano (HRP), e successivamente incapsulare vari farmaci, biomolecole o cellule all'interno dell'idrogel, suggerendo un grande potenziale nelle applicazioni di ingegneriatissutale 10,11,12,13,14. Inoltre, abbiamo recentemente studiato gli effetti terapeutici degli idrogel gh contenenti MSC incapsulati e dimostrato il loro uso nella riparazione e rigenerazione cardiaca di successo dopo l'MI in un modello murino15. In questo protocollo, descriviamo una semplice tecnica per l'incapsulamento e la proliferazione tridimensionale in vitro (3D) dei CCI all'interno degli idrogel GH. Introduciamo anche una procedura chirurgica progettata per generare un modello MI murino attraverso la legatura dell'arteria coronarica e il trapianto intramiocardico di idrogel GH a caricamento MSC nel cuore infarto.

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

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

Uno dei principali problemi che devono affrontare le attuali terapie con cellule staminali cardiache per prevenire l'insufficienza cardiaca postinfarct è il basso tasso di ritenzione e sopravvivenza delle cellule trapiantate all'interno del miocardio ferito, limitandone l'efficacia terapeutica. Recentemente, l'uso di biomateriali impalcatura ha attirato l'attenzione per migliorare e massimizzare la terapia con cellule staminali. L'obiettivo di questo protocollo è introdurre una tecnica semplice e diretta per trapiantare le cellule staminali mesenchimali derivate dal midollo osseo (MSC) utilizzando idrogel di acido propionico idrossifenile iniettabile (GH); gli idrogel sono favorevoli come piattaforma di somministrazione cellulare per applicazioni di ingegneria dei tessuti cardiaci a causa della loro capacità di essere collegati in situ incrociati e dell'elevata biocompatibilità. Presentiamo un metodo semplice per fabbricare idrogel GH a caricamento MSC (MSC/idrogel) e valutarne la sopravvivenza e la proliferazione in coltura tridimensionale (3D) in vitro. Inoltre, dimostriamo una tecnica per il trapianto intramiocardico di MSC / idrogel nei topi, descrivendo una procedura chirurgica per indurre l'infarto miocardico (MI) tramite la legatura coronarica discendente anteriore sinistro (LAD) e il successivo trapianto MSC / idrogels.

Per fornire efficacemente i CFC al miocardio infartuato, in questo protocollo sono stati utilizzati idrogel collegabili incrociati in situ a caricamento MSC descritti nella figura 1. Prima del trapianto in vivo, la proliferazione e la sopravvivenza dei MBC negli idrogel gh sono stati confermati da un saggio di colorazione in cellule vive / morte in vitro 3D (vivo: verde; morto: rosso). Come mostrato nella figura 2, le immagini rappresentative mostravano una suff...

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

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

La terapia a base di cellule staminali è emersa come una strategia efficiente per riparare i tessuti cardiaci feriti dopo l'infarto del miocardio. Forniamo un'applicazione in vivo ottimale per il trapianto di cellule staminali utilizzando idrogel di gelatina che possono essere enzimaticamente incrociati.

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 ...

intramyocardial-transplantation-msc-loading-injectable-hydrogels

Research

JoVE Journal

Medicine

4.3K Views.  The Catholic University of Korea. La terapia a base di cellule staminali è emersa come una strategia efficiente per riparare i tessuti cardiaci feriti dopo l'infarto del miocardio. Forniamo un'applicazione in vivo ottimale per il trapianto di cellule staminali utilizzando idrogel di gelatina che possono essere enzimaticamente incrociati.

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

Acute Myocardial Infarction in Rats

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

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

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 ...

Ricerca

Medicina

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 ...

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 ...

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

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

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

Un 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 ...

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

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

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

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

Murino discendente anteriore sinistra (LAD) Coronary legatura dell&#39;arteria: una migliore e semplificata del modello di infarto miocardico

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

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

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

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

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 ...

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, ...