TWP was funded by the Irish Cardiac Society Brian McGovern Travelling Fellowship. WTP was funded by R01 HL095712 and an AHA Established Investigator Award.

Tutte le operazioni descritte sono state eseguite secondo protocolli approvati dalla cura e l&#39;uso di animali Comitato Istituzionale di Hospital di Boston per bambini. 1 Preparazione <ol><li> Eseguire basale cardiaca valutazione anatomica e funzionale da ecocardiografia prima di iniziare la procedura di iniezione, come il trasduttore di posizione fissa ottimale per l&#39;iniezione intramiocardico non può cedere le viste standard ottimali per delineare l&#39;anatomia e la valutazione della funzione. </li><li> La messa a punto di iniezione è mostrata nelle figure 1A-1C. Inserire una siringa vuota con un ago inguainato, conici orientato verso l&#39;alto, nel morsetto della siringa (vedere Figura 1C) e fissare la sonda ad ultrasuoni trasduttore nel morsetto scan-testa (vedi Figura 1B). Allentare la scansione testa di bloccaggio della sfera serraggio del giunto (Figura 1B) e modificare l&#39;orientamento del trasduttore in modo che sia allineato parallelamente all&#39;asse dell&#39;ago. Fissare il scan-posizione della testa serrando la palla blocco pinza giunto scan-testa. NOTA: Per l&#39;iniezione in topi adulti, un ago da 30 G con un 1 in / 2,5 cm di lunghezza è ottimale. Una siringa da 1 ml può essere utilizzato per volumi maggiori, mentre una siringa a tenuta di gas può essere utilizzato per un controllo più preciso della quantità minori (5-10 microlitri). </li><li> Applicare il gel per ultrasuoni liberamente alla punta del trasduttore con una spatola per coprire la testa lungo la sua interezza. Sfoderare con attenzione l&#39;ago e utilizzare l&#39;ago di montaggio controlli per far avanzare l&#39;ago direttamente sotto il trasduttore e all&#39;interno del gel per ultrasuoni per la visualizzazione. Fare piccoli aggiustamenti uso del supporto dell&#39;ago in modo che l&#39;ago è visualizzato chiaramente lungo la sua lunghezza l&#39;immagine ad ultrasuoni su. Se il trasduttore è stato correttamente allineato parallelamente l&#39;ago al punto 1.4, quindi l&#39;ago deve rimanere all&#39;interno del piano di imaging come è avanzata e ritirata con la manopola di comando dell&#39;iniezione (Figura 1C). </li><li> In fasi successive, non disturbare la necessitàle / trasduttore allineamento orizzontale mediante il movimento lungo l&#39;asse orizzontale. Piuttosto, indirizzare specifiche aree del cuore per iniezione modificando l&#39;(asse y) posizione verticale dell&#39;ago montare e spostando la piattaforma animale. </li><li> Spostare il trasduttore superiormente dalla piattaforma animale con il controllo dell&#39;altezza di scansione (Figura 1B) per consentire la successiva collocazione del topo anestetizzato sulla piattaforma animali. Questo non perturbare l&#39;allineamento asse x del trasduttore all&#39;asse lungo dell&#39;ago. </li><li> Rimuovere la siringa utilizzata per l&#39;allineamento dal morsetto della siringa e scartare con cura. Caricare il nuovo ago e siringa con l&#39;iniettato al volume di destinazione finale, consentendo spazio morto nella punta della siringa. Fate attenzione a rimuovere le bolle d&#39;aria. Posizionare la siringa nel morsetto della siringa senza regolare l&#39;allineamento asse x. Rientrare completamente la siringa con il controllo dell&#39;iniezione. NOTA: Ai fini della formazione iniziale, l&#39;uso di blu di Evancolorante (1%), Trypan macchia blu (0,4%) o una sospensione di microsfere fluorescenti come l&#39;iniettato può aiutare l&#39;operatore a confermare la competenza e il successo di iniezione mirati. </li></ol> 2 Iniezione <ol><li> Accendere la più calda integrato della piattaforma riscaldamento e set per 37 ° C. Posizionare la piattaforma animale 180 ° dal solito orientamento delle immagini, con la fascetta anestesia e testa dell&#39;animale più vicino all&#39;operatore. Questo permette al cuore (nella parte sinistra del torace) sia ipsilaterale al morsetto della siringa e l&#39;ago. Nota: minor senso orario ulteriori aggiustamenti rotazione alla piattaforma animale possono essere necessari per orientare il cuore correttamente per il piano parasternale asse corto di imaging che verrà utilizzato per l&#39;iniezione (Figura 2A). </li><li> Preparare topi per ecocardiografia come riportato in precedenza 9. Anestetizzare il mouse in una camera di induzione con il 2% isoflurano. Rimuovere peli del torace con i capelli crema di rimozione di unD Applicare lubrificante gel per entrambi gli occhi per evitare l&#39;essiccazione della sclera. </li><li> Sollevare il trasduttore mediante la scansione di controllo altezza della testa (Figura 1B). Posizionare il mouse supina anestetizzato in cima alla piattaforma animale riscaldato con il muso di un cono consegna 1-3% isoflurano (Figure 2A-2B). Inserire delicatamente una sonda rettale e nastro le quattro zampe agli elettrodi ECG, applicazione di gel per elettrodi per contatto elettrico. NOTA: un livello adeguato di anestesia deve essere garantita per il trattamento umano degli animali. Non ci dovrebbe essere alcun cambiamento nella frequenza cardiaca e nessuna risposta per il posizionamento dell&#39;ago attraverso la parete toracica. Controlli di temperatura omeostatici integrati della piattaforma degli animali dovrebbero essere utilizzati per mantenere la normotermia (37 ± 0,5 ° C), come ipotermia si tradurrà in bradicardia relativa, dilatazione ventricolare, ed eventuale disagio. </li><li> Una volta che il mouse è sicuro sulla piattaforma animali, abbassare il trasduttore sul depilated torace utilizzando la scansione di controllo altezza della testa (Figura 1B). La configurazione ottimale ultrasuoni per l&#39;iniezione è per il cuore di essere visualizzata con l&#39;orientamento asse corto parasternale, secondo la tecnica ecocardiografica standard. Ruotare la piattaforma animale 20-30 ° in senso orario per ottenere la finestra acustica ottimale per l&#39;iniezione nel piano di imaging asse corto (figure 2A-2B). Nota: Alternativamente, l&#39;iniezione può essere eseguita da un orientamento asse lungo parasternale dalla rotazione in senso antiorario della piattaforma animale (Figura 2C). </li><li> Utilizzare i comandi di regolazione della piattaforma di animali per regolare il campo di vista e di indirizzare ogni sito di iniezione desiderato nel miocardio ventricolare sinistro. Pan avanti e indietro dall&#39;apice alla base del cuore per indirizzare la posizione di iniezione desiderata nel ventricolo sinistro del miocardio (Figure 3A-3C). Nota: La papilla media parasternale asse corto (figura 3A) offre reproduciblE punti di riferimento che permettono followup l&#39;imaging del sito di iniezione. Nota: In alternativa, la vista lunga parasternale asse può essere utilizzato per indirizzare un sito di iniezione predefinito (Figura 3D). </li><li> Partendo con la siringa nel morsetto della siringa completamente retratto, far avanzare lentamente la siringa verso il petto dell&#39;animale ruotando il comando in senso orario iniezione (Figura 4A). Per consentire la visualizzazione chiara ecografia sia del cuore e la punta dell&#39;ago mentre si avvicina al petto, utilizzare un sacco di gel per ultrasuoni sul lato sinistro del petto e ottimizzare la finestra acustica impostando un ampio campo di vista sui controlli ecocardiografia. Impostare il punto / zona focale presso il sito di destinazione per l&#39;iniezione. Piccoli aggiustamenti al montaggio ago controlli possono ottimizzare l&#39;immagine dell&#39;ago lungo la sua lunghezza. NOTA: Alcune macchine ad ultrasuoni hanno una funzione software ago guida per estendere digitalmente una linea lungo l&#39;asse dell&#39;ago attraverso al miocardio bersaglio(Figura 4B). Tale software può essere utile ma non è indispensabile. </li><li> Con l&#39;animale opportunamente sedato (1-3% isoflurano miscelato con 0,5-0,8 L / min 100% di ossigeno), avanzare l&#39;ago attraverso la parete toracica del mouse e nel miocardio, osservando attentamente la posizione della punta dell&#39;ago smussato affatto volte. Smettere di avanzare quando la punta dell&#39;ago è all&#39;interno del miocardio bersaglio (Figura 4C). Tutta la punta smussata deve essere saldamente all&#39;interno del miocardio, per evitare perdite iniettato nello spazio pericardico. </li><li> Quando la punta è nella posizione desiderata, consegnare il iniettato spingendo lo stantuffo della siringa. Consegnare l&#39;iniettato lentamente, nell&#39;arco di 5-30 secondi (a seconda del volume di essere consegnati). Fino a 50 ml di dell&#39;iniettato possono essere forniti senza compromettere la funzione ventricolare. Un aspetto echobright transitoria alla regione miocardica iniettata può essere evidente dopo l&#39;iniezione di successo. A breve (secondi) periodo di relativa bradycardia è occasionalmente osservato con iniezione nel miocardio e rapidamente si risolve. </li><li> Una volta che l&#39;iniettato è stato somministrato, prontamente ritirare l&#39;ago con rotazione in senso antiorario della manopola iniezione. Il mouse deve essere tenuto sotto anestesia per parecchi minuti di osservazione ecocardiografico per confermare la funzione ventricolare preservata e senza complicazioni iatrogeni. Se indicato, più regioni di miocardio possono essere serialmente iniettati riposizionando dell&#39;angolo dell&#39;ago di approccio mediante regolazione della piattaforma animale. Dopo l&#39;iniezione intramiocardico, il mouse viene posizionato in una gabbia da sola e ha permesso di recuperare da anestesia sotto osservazione. </li><li> Non lasciare un animale incustodito fino a quando non ha ripreso conoscenza sufficiente per mantenere decubito sternale. Non restituire un animale che ha subito l&#39;iniezione intramiocardico alla compagnia di altri animali fino alla completa guarigione. La gabbia deve essere collocato su un blocco termoregolato con ridisposizione ady di acqua e mouse dieta. Disagio iatrogeni non è previsto e il fallimento di riprendere in modo appropriato comportamento normale poco dopo l&#39;intervento suggerisce una potenziale complicazione (vedi discussione). </li><li> Eliminare attentamente l&#39;ago immediatamente dopo il completamento dell&#39;iniezione intramiocardico per ridurre al minimo il rischio di lesioni diesis all&#39;operatore o agli astanti. Riutilizzo di un ago può provocare ottundimento della punta che rende più difficile per perforare il miocardio e risultante in un più alto rischio di complicanze. </li></ol>

<ol>
	<li>Kochanek, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deaths:+final+data+for+2009.">Deaths: final data for 2009.</a> Natl Vital Stat Rep. 60 (3), 1-117 .</li><li>Strauer, B. E., Steinhoff, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=10+years+of+intracoronary+and+intramyocardial+bone+marrow+stem+cell+therapy+of+the+heart:+from+the+methodological+origin+to+clinical+practice.">10 years of intracoronary and intramyocardial bone marrow stem cell therapy of the heart: from the methodological origin to clinical practice.</a> J Am Coll Cardiol. 58, 1095-1104 (2011).</li><li>Cheng, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramyocardial+autologous+cell+engraftment+in+patients+with+ischaemic+heart+failure:+a+meta-analysis+of+randomised+controlled+trials.">Intramyocardial autologous cell engraftment in patients with ischaemic heart failure: a meta-analysis of randomised controlled trials.</a> Heart Lung Circ. 22 (11), 887-894 (2013).</li><li>Fischer-Rasokat, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pilot+trial+to+assess+potential+effects+of+selective+intracoronary+bone+marrow-derived+progenitor+cell+infusion+in+patients+with+nonischemic+dilated+cardiomyopathy:+final+1-year+results+of+the+transplantation+of+progenitor+cells+and+functional+regeneration+enhancement+pilot+trial+in+patients+with+nonischemic+dilated+cardiomyopathy.">A pilot trial to assess potential effects of selective intracoronary bone marrow-derived progenitor cell infusion in patients with nonischemic dilated cardiomyopathy: final 1-year results of the transplantation of progenitor cells and functional regeneration enhancement pilot trial in patients with nonischemic dilated cardiomyopathy.</a> Circ Heart Fail. 2, 417-423 (2009).</li><li>Seth, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Percutaneous+intracoronary+cellular+cardiomyoplasty+for+nonischemic+cardiomyopathy:+clinical+and+histopathological+results:+the+first-in-man+ABCD+(autologous+bone+marrow+cells+in+dilated+cardiomyopathy)+trial.">Percutaneous intracoronary cellular cardiomyoplasty for nonischemic cardiomyopathy: clinical and histopathological results: the first-in-man ABCD (autologous bone marrow cells in dilated cardiomyopathy) trial.</a> J Am Coll Cardiol. 48, 2350-2351 (2006).</li><li>Ladage, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Percutaneous+methods+of+vector+delivery+in+preclinical+models.">Percutaneous methods of vector delivery in preclinical models.</a> Gene Ther. 19, 637-641 (2012).</li><li>Zangi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+mRNA+directs+the+fate+of+heart+progenitor+cells+and+induces+vascular+regeneration+after+myocardial+infarction.">Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction.</a> Nat Biotechnol. 31, 898-907 (2013).</li><li>Laakmann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimally+invasive+closed-chest+ultrasound-guided+substance+delivery+into+the+pericardial+space+in+mice.">Minimally invasive closed-chest ultrasound-guided substance delivery into the pericardial space in mice.</a> Naunyn Schmiedebergs Arch Pharmacol. 386 (3), 227-238 (2013).</li><li>Respress, J. L., Wehrens, X. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transthoracic+echocardiography+in+mice.">Transthoracic echocardiography in mice.</a> J Vis Exp. (39), e1738 (2010).</li><li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cis-regulating+elements+and+trans-activating+factors+of+the+rat+cardiac+troponin+T+gene.">Characterization of cis-regulating elements and trans-activating factors of the rat cardiac troponin T gene.</a> J Biol Chem. 269, 30595-30603 (1994).</li><li>Shimshek, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Codon-improved+Cre+recombinase+(iCre)+expression+in+the+mouse.">Codon-improved Cre recombinase (iCre) expression in the mouse.</a> Genesis. 32, 19-26 (2002).</li><li>Lichtenauer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+and+intramyocardial+injection+of+apoptotic+white+blood+cell+suspensions+prevents+ventricular+remodelling+by+increasing+elastin+expression+in+cardiac+scar+tissue+after+myocardial+infarction.">Intravenous and intramyocardial injection of apoptotic white blood cell suspensions prevents ventricular remodelling by increasing elastin expression in cardiac scar tissue after myocardial infarction.</a> Basic Res Cardiol. 106 (4), 645-655 (2011).</li><li>Herrmann, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postinfarct+intramyocardial+injection+of+mesenchymal+stem+cells+pretreated+with+TGF-alpha+improves+acute+myocardial+function.">Postinfarct intramyocardial injection of mesenchymal stem cells pretreated with TGF-alpha improves acute myocardial function.</a> Am J Physiol Regul Integr Comp Physiol. 299 (1), 371-378 (2010).</li><li>Zhou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+injection+of+autologous+mesenchymal+stromal+cells+improves+myocardial+function.">Direct injection of autologous mesenchymal stromal cells improves myocardial function.</a> Biochem Biophys Res Commun. 390 (3), 902-907 (2009).</li><li>Campbell, N. G., Suzuki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+delivery+routes+for+stem+cell+therapy+to+the+heart:+current+and+future+approaches.">Cell delivery routes for stem cell therapy to the heart: current and future approaches.</a> J Cardiovasc Transl Res. 5 (5), 713-726 (2012).</li><li>Hou, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiolabeled+cell+distribution+after+intramyocardial,+intracoronary,+and+interstitial+retrograde+coronary+venous+delivery:+implications+for+current+clinical+trials.">Radiolabeled cell distribution after intramyocardial, intracoronary, and interstitial retrograde coronary venous delivery: implications for current clinical trials.</a> Circulation. 112, 150-156 (2005).</li><li>Dib, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+successful+training+on+methods+of+delivery+of+biologics+for+cardiac+regeneration:+a+report+of+the+International+Society+for+Cardiovascular+Translational+Research).">Recommendations for successful training on methods of delivery of biologics for cardiac regeneration: a report of the International Society for Cardiovascular Translational Research).</a> JACC Cardiovasc Interv. 3 (3), 265-275 (2010).</li><li>Dib, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+therapy+for+cardiovascular+disease:+a+comparison+of+methods+of+delivery.">Cell therapy for cardiovascular disease: a comparison of methods of delivery.</a> J Cardiovasc Transl Res. 4 (2), 177-181 (2011).</li></ol>

The authors have nothing to disclose.

Biologics possono essere consegnati al miocardio mediante iniezione diretta intramiocardico, iniezione intrapericardica, o l&#39;amministrazione indiretta attraverso il flusso sanguigno. Cellulari basato recenti sperimentazioni di terapia in modelli con infarto miocardico hanno descritto un approccio toracotomia aperta per la fornitura di iniettato 12-14. Un fattore importante per il successo di un intervento terapeutico infarto dipende dalla scelta del percorso di consegna. La dose massima locale del biologi...

Le malattie cardiache sono la principale causa di morte per uomini e donne negli Stati Uniti, che rappresentano 600.000 decessi ogni anno 1. Modelli murini di malattie cardiovascolari sono di fondamentale importanza per lo studio dei meccanismi fisiopatologici e per esplorare potenziali terapie. Consegna miocardico di vettori di terapia genica, cellule staminali, RNA modificati, e altri agenti terapeutici permessi indagine del loro potenziale terapeutico per le malattie cardiache 2-7. Attualmente, ci sono opzioni limitate per la consegna del miocardio di agenti terapeutici in modelli murini 6. Iniezione intramiocardico sotto visualizzazione diretta è comunemente usato, ma richiede una sternotomia o toracotomia ed è limitata alla regione esposta del cuore. Mentre conveniente quando eseguita al momento di un&#39;altra manipolazione sperimentale come LAD legatura, la necessità di una procedura invasiva consegna intramiocardico limita potenziali disegni sperimentali e introduce uneffetti SUPPLEMENTARI dalla procedura (ad esempio, la fibrosi a causa di toracotomia). Consegna pericardico percutanea di vettori virali è stato segnalato, ma il sito e la distribuzione di agente terapeutico non è omogeneo ed è difficile da controllare 8. Percutanea risultati iniezione coronarici in distribuzione più omogenea del materiale iniettato, ma la consegna coronarica efficiente e riproducibile impugna in modelli murini. Qui, descriviamo una cassa tecnica di iniezione intramiocardico chiuso che permette minimamente invasiva, operatore controllato il targeting di agenti terapeutici sotto guida ecografica. La tecnica è facile da imparare, evita la necessità di toracotomia o sternotomia e le loro concomitanti complicazioni sperimentali, e offre una maggiore flessibilità sui tempi e siti di iniezione intramiocardico. Così, l&#39;iniezione intramiocardico ecocardiografia assistita rappresenta un metodo tecnicamente semplice e molto efficace di manipolare ilmiocardio in modelli sperimentali murini.

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Vevo 2100 ultrasound imaging system&#160;</td>
			<td style="width:144px;">Visualsonics</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Vevo Integrated Rail System III</td>
			<td style="width:144px;">Visualsonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Microscan MS400 transducer</td>
			<td style="width:144px;">Visualsonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Microscan MS550D transducer</td>
			<td style="width:144px;">Visualsonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">PrecisionGlide needles</td>
			<td style="width:144px;">BD</td>
			<td style="width:119px;">305128</td>
			<td>30G x 1 inch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">1cc slip tip syringe</td>
			<td style="width:144px;">Exel International&#160;</td>
			<td style="width:119px;">26048</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Gastight 50ul glass syringes</td>
			<td style="width:144px;">Hamilton&#160;</td>
			<td style="width:119px;">1705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Trypan blue stain (0.4%)</td>
			<td style="width:144px;">Gibco&#160;</td>
			<td style="width:119px;">15250</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Isoflurane</td>
			<td style="width:144px;">Baxter</td>
			<td style="width:119px;">AHN3640</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Aquasonic 100</td>
			<td style="width:144px;">Parker Laboratories</td>
			<td style="width:119px;">(01-08)</td>
			<td>or equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:313px;">Polystyrene microspheres (red fluorescent)</td>
			<td style="width:144px;">Life Technologies</td>
			<td style="width:119px;">F-8842</td>
			<td>or equivalent</td>
		</tr>
	</tbody>
</table>

ultrasound-guided transthoracic intramyocardial injection in mice

Modelli murini di malattie cardiovascolari sono importanti per lo studio dei meccanismi fisiopatologici e di esplorare potenziali terapie rigenerative. Gli esperimenti che coinvolgono l&#39;iniezione infarto sono attualmente svolte da accesso chirurgico diretto attraverso una toracotomia. Mentre conveniente quando eseguita al momento di un&#39;altra manipolazione sperimentale come legatura dell&#39;arteria coronaria, la necessità di una procedura invasiva consegna intramiocardico limita potenziali disegni sperimentali. Con la sempre migliore risoluzione ultrasuoni e modalità di imaging non invasive avanzate, è ora fattibile per eseguire di routine, iniezione intramiocardico percutanea ecoguidata. Questa modalità efficiente e affidabile fornisce agenti ad una zona mirata di miocardio. I vantaggi di questa tecnica includono la prevenzione di morbilità chirurgica, la possibilità di indirizzare le regioni del miocardio selettivo sotto guida ecografica, e la possibilità di esprimere iniettato al myocardium in più intervalli di tempo predeterminati. Con la tecnica praticata, complicanze da iniezione intramiocardico sono rari, e topi tornare rapidamente alla normale attività sul recupero da anestetico. Seguendo la procedura descritta in questo protocollo, l&#39;operatore con esperienza di base ecocardiografia può rapidamente diventare competente in questa versatile tecnica, mini-invasiva.

Murino iniezione intramiocardico di blu tipo e microsfere fluorescenti Iniezione di colorante blu di Evan è utile per scopi di formazione. Subito dopo l&#39;iniezione, eutanasia il mouse e rimuovere il cuore per visualizzare la posizione del colorante blu iniettato. Figura 5 mostra un esempio di una iniezione di successo, con colorante blu infiltrante il miocardio a livello muscolare metà papillari (Figura 5A, regione racchiusa dalla linea tratteggiata). Evan...

Watch this Scientific Journal Video about Ultrasound-guided Transthoracic Intramyocardial Injection in Mice at JoVE.com

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Iniezione percutanea intramiocardico ecocardiografia-guida rappresenta una modalità efficiente, affidabile, e il targeting per la fornitura di agenti di trasferimento di geni o cellule nel cuore murino. Seguendo la procedura descritta in questo protocollo, l&#39;operatore può rapidamente diventare competente in questa versatile tecnica, mini-invasiva.

Ultrasound-guided transtoracica intramiocardico iniezione nei topi

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

ultrasound-guided-transthoracic-intramyocardial-injection-in-mice

Research

JoVE Journal

Medicine

14.7K Views.  Boston Children's Hospital.  Iniezione percutanea intramiocardico ecocardiografia-guida rappresenta una modalità efficiente, affidabile, e il targeting per la fornitura di agenti di trasferimento di geni o cellule nel cuore murino. Seguendo la procedura descritta in questo protocollo, l'operatore può rapidamente diventare competente in questa versatile tecnica, mini-invasiva. 

Video: Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Transthoracic Echocardiography in Mice

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene targeting methods can be used to generate mice with altered cardiac size and function,1-3 and as a result, in vivo techniques are needed to evaluate their cardiac phenotype. Transthoracic echocardiography, pulse wave Doppler (PWD), and tissue Doppler imaging (TDI) can be used to provide dimensional measurements of the mouse heart and to quantify the degree of cardiac systolic and diastolic performance. Two-dimensional imaging is used to detect abnormal anatomy or movements of the left ventricle, whereas M-mode echo is used for quantification of cardiac dimensions and contractility.4,5 In addition, PWD is used to quantify localized velocity of turbulent flow,6 whereas TDI is used to measure the velocity of myocardial motion.7 Thus, transthoracic echocardiography offers a comprehensive method for the noninvasive evaluation of cardiac function in mice.

Transthoracic echocardiography offers a noninvasive method for the evaluation of cardiac function in mice. A combination of ultrasound and Doppler imaging modalities can be used to obtain dimensional measurements of the heart and intracardiac blood flow, which together provide an assessment of cardiac systolic and diastolic performance.

L&#39;ecocardiografia transtoracica in topi

In recent years, murine models have become the primary avenue for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene ...

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 ...

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

Intramiocardico cellulare di consegna: Osservazioni in murini Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

Substernal Thyroid Biopsy Using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration

Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1. There is a wide variation in the published incidence rates due to the lack of a standardized definition for STG. Biopsy is often required to differentiate benign from malignant lesions. Unlike cervical thyroid, the overlying sternum precludes ultrasound-guided percutaneous fine needle aspiration of STG. Consequently, surgical mediastinoscopy is performed in the majority of cases, causing significant procedure related morbidity and cost to healthcare. Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA) is a frequently used procedure for diagnosis and staging of non-small cell lung cancer (NSCLC). Minimally invasive needle biopsy for lesions adjacent to the airways can be performed under real-time ultrasound guidance using EBUS. Its safety and efficacy is well established with over 90% sensitivity and specificity. The ability to perform EBUS as an outpatient procedure with same-day discharges offers distinct morbidity and financial advantages over surgery. As physicians performing EBUS gained procedural expertise, they have attempted to diversify its role in the diagnosis of non-lymph node thoracic pathologies. We propose here a role for EBUS-TBNA in the diagnosis of substernal thyroid lesions, along with a step-by-step protocol for the procedure.

Substernal thyroid lesions are common, and need to be differentiated from malignancy. Obtaining percutaneous fine needle biopsy is not possible due to its retrosternal location. This article proposes a protocol for biopsy of substernal thyroid lesions using Endobronchial Ultrasound-guided Transbronchial Needle Aspiration (EBUS-TBNA).

Retrosternale Tiroide biopsia endobronchiale Utilizzando ecoguidata transbronchiale Needle Aspiration

Substernal thyroid goiter (STG) represents about 5.8% of all mediastinal lesions1. There is a wide variation in the published incidence rates due to the ...

Ultrasound-guided Botulinum Toxin-A Injections: A Method of Treating Sialorrhea

Neurological diseases can be complicated by sialorrhea, an excessive flow of saliva. Patients suffering from moderate to severe sialorrhea have an impaired quality of life, often worsened by correlated complications such as aspiration pneumonia, oral infections, dental caries, and maceration of the skin. Diverse therapeutic approaches have been proposed for the treatment of sialorrhea, including surgery and the use of anticholinergic agents, with limited results and the possible occurrence of serious adverse events. Recently, botulinum toxin (BoNT) injection within the major salivary glands has been proposed in patients refractory to anticholinergic therapy, with the aim of inhibiting local acetylcholine release and gland activity. 
In order to obtain a better outcome in terms of reduction of saliva production, efficacy, duration, and avoidance of major adverse events, we developed an ultrasound-guided BoNT-type A injection technique accurately described in the text. Here we present a method of treating sialorrhea with bilateral parotid and submandibular gland BoNT-type A injections under ultrasound guidance. Four quadrants of the parotid gland and two quadrants of the submandibular gland are visualized and injected using two accesses and one access, respectively. 
The ultrasound-guided procedure provides a simple, non-invasive, real-time visualization of the muscular and glandular tissues and their surrounding structures, optimizing treatment efficacy and safety.

Botulinum toxin is injected into the salivary glands for the treatment of sialorrhea. The ultrasound-guided botulinum toxin injection in both the parotid and submandibular glands demonstrates a sustained efficacy and the absence of serious side effects.

Ecoguidata botulinica iniezioni di tossina-A: un metodo di trattamento Scialorrea

Neurological diseases can be complicated by sialorrhea, an excessive flow of saliva. Patients suffering from moderate to severe sialorrhea have an ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Ultrasuono-guida di iniezione intracardiaca di cellule staminali mesenchimali umane per aumentare Homing all'intestino per l'utilizzo in modelli murini di malattie intestinali infiammatorie sperimentali

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

L'iniezione intramiocardica ecocardiografia-guida percutanea contrasto e consegna delle cellule in un modello preclinico grande

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Esame ecocardiografico transtoracico nel modello del coniglio

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

Endoscopic Ultrasound-Guided Biliary Drainage: Endoscopic Ultrasound-Guided Hepaticogastrostomy in Malignant Biliary Obstruction

Patients with unresectable malignant biliary obstruction often require biliary drainage to decompress the biliary system. Endoscopic Retrograde Cholangiopancreatography (ERCP) is the primary biliary drainage method whenever possible. Percutaneous Transhepatic Biliary Drainage (PTBD) is used as a salvage method if ERCP fails. Endoscopic Ultrasound-Guided Biliary Drainage (EUS-BD) provides a feasible alternative biliary drainage method where one of the methods is EUS guided Hepaticogastrostomy (EUS-HGS). Here we describe the EUS-HGS technique in a case of unresectable malignant hilar biliary obstruction to achieve biliary drainage.
Presented here is the case of a 71-year-old female with painless jaundice and weight loss for 2 weeks. Computed Tomography (CT) imaging showed a 4 x 5 cm hilar tumor with lymphadenopathy and liver metastasis. EUS fine needle biopsy (FNB) of the lesion was consistent with cholangiocarcinoma. Her bilirubin levels were 212 &#181;mol/L (&lt;15) during presentation.
A linear echoendoscope was used to locate the left dilated intrahepatic ducts (IHD) of the liver. The segment 3 dilated IHD was identified and punctured using a 19 G needle. Contrast was used to opacify the IHDs under fluoroscopic guidance. The IHD was cannulated using a 0.025-inch guidewire. This was followed by the dilation of the fistula tract using a 6 Fr electrocautery dilator along with a 4 mm biliary balloon dilator. A partially covered metallic stent of 10 cm in length was deployed under fluoroscopic guidance. The distal part opens in the IHD and the proximal part was deployed within the working channel of the echoendoscope that subsequently released into the stomach. The patient was discharged three days after the procedure. Follow up performed in the second and fourth weeks showed that the bilirubin levels were 30 &#181;mol/L and 14 &#181;mol/L, respectively. This indicates that EUS-HGS is a safe method for biliary drainage in unresectable malignant biliary obstruction.

Endoscopic Ultrasound-Guided Biliary Drainage (EUS-BD) is an alternative method of biliary decompression in malignant biliary obstruction. Here we describe the technique of EUS guided-Hepaticogastrostomy (EUS-HGS) in a case of unresectable malignant hilar biliary obstruction.

Drenaggio biliare guidato da ultrasuoni endoscopici: epaticogastrostomia endoscopica guidata da ultrasuoni nell'ostruzione biliare maligna

Patients with unresectable malignant biliary obstruction often require biliary drainage to decompress the biliary system. Endoscopic Retrograde ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

Impianto di cardiomiociti derivati da cellule staminali pluripotenti indotte ecoguidate in topi infartuati miocardici

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...