Riconosciamo il sostegno offerto dai laboratori e dalla struttura animale dell&#39;Istituto di Neurofisiologia. Questo lavoro è stato sostenuto dalla Walter und Marga Boll-Stiftung, dalla Köln Fortune e dalla Deutsche Stiftung für Herzforschung.

La manipolazione degli animali deve essere conforme alle linee guida del comitato locale per la protezione degli animali e alla direttiva 2010/63 / UE del Parlamento europeo. 1. Preparare soluzioni <ol><li> Preparare la soluzione di Tyrode senza Ca 2+ (composizione in mM): NaCl 136, KCl 5,4, NaH 2 PO 4 0,33, MgCl2 1, glucosio 10, HEPES 5, 2,3-butanedione monoxime (BDM) 30. Regolare il pH a 7,4 Con NaOH a 4 ° C. </li><li> Preparare la soluzione di Tyrode con Ca 2+ (composizione in mM): NaCl 136, KCl 5,4, NaH 2 PO 4 0,33, MgCl2 1, glucosio 10, HEPES 5, BDM 30, CaCl2 0,9. Regolare il pH a 7,4 con NaOH a 4 ° C. </li><li> Preparare agarosio a basso contenuto di fusione al 4%: inserire 0,6 g di agarosio a bassa percentuale di fusione in soluzione di Tyrode da 15 ml senza Ca 2+ . Scaldare la miscela in un forno a microonde due volte a 750 W per 10-15 s finché l&#39;agarosio non si scioglie. Tenere la soluzione a una temperatura costante di 37 &amp;# 176; C e mescolare continuamente. </li><li> Mantenere il Dulbecco Modified Eagle Medium (DMEM) senza siero a 37 ° C in bolle con carbogeno (5% CO 2 , 95% O 2 ). </li></ol> 2. Preparare il Microtomo <ol><li> Accendere il microtomo. </li><li> Riempire la camera esterna del microtomo con ghiaccio. </li><li> Riempire la camera interna del microtomo con la soluzione di Tyrode ghiacciata senza Ca 2+ e continuamente ossigenare la soluzione con 100% O 2 . </li><li> Mettere una lamiera d&#39;acciaio nel porta-lama del microtomo. </li></ol> 3. Isolamento del cuore del mouse <ol><li> Iniettare 2,500 U di eparina sottocutanea. Attendere 15 min. </li><li> Sacrificare l&#39;animale per dislocazione cervicale. </li><li> Apri il petto con la sternotomia. </li><li> Separare con attenzione il pericardio usando piccole forbici e una pinza # 5. </li><li> Inserire una cannula nell&#39;arto ascendente e perfezionare le arterie coronarie in situ con Tyrode&#39;s ghiaccio freddoSoluzione senza Ca 2+ fino a rimuovere il sangue rimanente. </li><li> Rescondare delicatamente il cuore con una pinza e forbici e trasferire il cuore in soluzione fredda di Tyrode, guidata senza Ca 2+ . </li><li> Separare l&#39;atria dai ventricoli con un bisturi o forbici. </li></ol> 4. Incorporazione dei ventricoli in agarosio a basso contenuto di fusione del 4% <ol><li> Posizionare i ventricoli con l&#39;apice rivolto verso l&#39;alto nello stampo agarosio ( Figura 1 ). Posizionare il perno in mezzo dello stampo nella camera ventricolare sinistra. </li><li> Riempire lo stampo con agarosio a basso contenuto di fusione al 4% a 37 ° C, finché il cuore non sia completamente coperto. </li><li> Posizionare lo stampo sul ghiaccio per una tempra più rapida dell&#39;agarosio che impedisce il galleggiamento del tessuto. </li><li> Rimuovere il blocco di agarosio contenente i ventricoli dallo stampo con un bisturi. </li><li> Girare il blocco capovolto e riempire le camere ventricolari e il divario sul retro del blocco agarosio, cheS lasciata dal perno del molt, con agarosio a basso contenuto di fuso al 4% utilizzando una siringa con un ago da 20 G. </li><li> Tagliare il blocco di agarosio con uno scalpello per ottenere un fondo piatto del blocco e in posizione verticale dell&#39;apice cardiaco. </li></ol> 5. Sezione del tessuto ventricolare <ol><li> Fissare il blocco sul supporto del campione del microtomo con una goccia di colla cianoacrilato. Affrontare l&#39;apice cardiaco verso l&#39;alto. </li><li> Posizionare il supporto del campione nella camera del campione interno del microtomo, che è riempito di Tyrode senza ghiaccio senza Ca 2+ . Completamente coprire il blocco di agarosio con la soluzione di Tyrode. </li><li> Preparare le fette di asse corto a uno spessore di 150-400 μm, a seconda dell&#39;ulteriore applicazione (per registrazioni di elettrodo nitido 150-200 μm), mantenere la frequenza di vibrazione della lama a 60-70 Hz e spostare la lama in avanti in modo più lento possibile . </li><li> Utilizzare una spazzola fine per rimuovere attentamente l&#39;agarosio residuo dalle fette. </li><li> Trasferisce delicatamenteR sezioni con una pipetta Pasteur nella soluzione Tyrode con 0,9 mM Ca 2+ aerata con 100% O 2 e conservarle per almeno 30 minuti in ghiaccio per recuperare dalla procedura di affettatura. </li><li> Successivamente, tenere le fette per 30 minuti in DMEM a 37 ° C, aerata con carbogeno, per lavare BDM prima dell&#39;uso successivo. </li></ol> 6. Preparazione dell&#39;installazione dell&#39;elettrodo Sharp <ol><li> Per il pre-riscaldamento, accendere tutti i dispositivi elettrici 30 minuti prima che le registrazioni inizieranno. </li><li> Mettere un piatto di coltura da 3 cm sulla piastra riscaldante posta sul microscopio invertito. </li><li> Posizionare l&#39;elettrodo ad anello su misura ( figura 2 ) nel piatto e collegare i fili di messa a terra del preamplificatore e dell&#39;elettrodo di stimolazione. </li><li> Collegare i tubi flessibili del sistema di perfusione al piatto. </li><li> Riempire il serbatoio del sistema di perfusione con DMEM, aerato con carbogeno. </li><li> Accendere la pompa di perfusione e impostare la perfusioneTe a 2-3 ml / min. </li><li> Regolare la temperatura del DMEM nel piatto a 37 ° C regolando il riscaldatore di flusso e la piastra riscaldante. </li></ol> 7. Potenziali registrazioni di azione <ol><li> Mettere una fetta ventricolare nel piatto riempito con DMEM. </li><li> Controllare l&#39;integrità strutturale e la vitalità (sulla base della funzione contrattile) del tessuto con il microscopio invertito. </li><li> Riempire un elettrodo di vetro di registrazione con 3 M KCL. </li><li> Riempire un elettrodo di stimolazione con DMEM. </li><li> Posizionare l&#39;elettrodo di registrazione e l&#39;elettrodo di stimolazione sui supporti degli elettrodi. </li><li> Posizionare con cura l&#39;elettrodo di stimolazione sulla fetta e accendere lo stimolatore elettrico. Inizia con una frequenza di stimolazione di 1-2 Hz. </li><li> Spostare l&#39;elettrodo di registrazione con il micromanipolatore sulla posizione di registrazione desiderata. </li><li> Abbassare lentamente l&#39;elettrodo di registrazione fino a quando la punta tocca il tessuto. </li><li> Applicare un breve impulso elettrico rettangolare attraverso laL&#39;elettrodo di registrazione per penetrare nella membrana cellulare. </li><li> Riposizionare con cautela l&#39;elettrodo di registrazione fino a garantire un segnale stabile. </li><li> Avviare la registrazione dei potenziali di azione. </li></ol>

<ol>
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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=In+vitro+chronotropic+effects+of+Erythrina+senegalensis+DC+(Fabaceae)+aqueous+extract+on+mouse+heart+slice+and+pluripotent+stem+cell-derived+cardiomyocytes.">In vitro chronotropic effects of Erythrina senegalensis DC (Fabaceae) aqueous extract on mouse heart slice and pluripotent stem cell-derived cardiomyocytes.</a> J Ethnopharmacol. 165, 163-172 (2015).</li><li>Wang, 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=Cardiac+tissue+slices:+preparation,+handling,+and+successful+optical+mapping.">Cardiac tissue slices: preparation, handling, and successful optical mapping.</a> Am J Physiol Heart Circ Physiol. 308, H1112-H1125 (2015).</li><li>Bussek, A., 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=Tissue+slices+from+adult+mammalian+hearts+as+a+model+for+pharmacological+drug+testing.">Tissue slices from adult mammalian hearts as a model for pharmacological drug testing.</a> Cell Physiol Biochem. 24, 527-536 (2009).</li><li>Burnashev, N. A., Edwards, F. A., Verkhratsky, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamp+recordings+on+rat+cardiac+muscle+slices.">Patch-clamp recordings on rat cardiac muscle slices.</a> Pflugers Arch. 417, 123-125 (1990).</li><li>Pillekamp, F., 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=Establishment+and+characterization+of+a+mouse+embryonic+heart+slice+preparation.">Establishment and characterization of a mouse embryonic heart slice preparation.</a> Cell Physiol Biochem. 16, 127-132 (2005).</li><li>Pillekamp, F., 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=Neonatal+murine+heart+slices.+A+robust+model+to+study+ventricular+isometric+contractions.">Neonatal murine heart slices. 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Gli autori non hanno nulla da dichiarare.

Le fetture ventricolari consentono studi elettrofisiologici, farmacologici e meccanici con una struttura tissue preservata in vivo e accesso diretto della tecnologia di misura a tutte le regioni del cuore. Le proprietà del potenziale fisiologico di azione sono state dimostrate in fette embrionali, neonatali e adulti 9 , 10 , 11 . La vitalità delle fette, ad eccezione degli strati superficiali direttamente danneggiati...

Le fette di tessuto sottile sono state utilizzate frequentemente nella scienza di base poiché Yamamot e Mcllwain hanno mostrato nel 1966 che l&#39;attività elettrica delle fette del cervello è mantenuta in vitro 1 . Da allora, studi elettrofisiologici e farmacologici sono stati condotti su fette da cervello 2 , fegato 3 , polmone 4 e tessuto miocardico 5 , 6 , 7 . Le prime registrazioni di patch-clamp in fette ventricolari da cuori di ratto neonatale sono state descritte nel 1990 8 , ma questa tecnica è caduta nell&#39;oblio per un certo tempo. Più di un decennio più tardi, il nostro gruppo ha stabilito un nuovo metodo per preparare le embrioni murine 9 , le neonate 10 e le 11 fette di cuore adulte. Queste fette di tessuto vitale possono essere utilizzate per esperimenti acuti (fetta adultaS possono essere coltivati ​​per diverse ore) o esperimenti di coltura a breve termine (fette embrionali e neonatali possono essere coltivate per alcuni giorni). Le fette mostrano in vivo caratteristiche elettrofisiologiche simili e una diffusione di eccitazione omogenea valutata con potenzialità di azione dell&#39;elettrodo acuto e registrazioni a matrice di microelettrodi 11 . Grazie alla loro morfologia &quot;bidimensionale&quot;, consentono l&#39;accesso diretto degli elettrodi di registrazione a tutte le regioni del ventricolo, che li rende uno strumento interessante per le indagini elettrofisiologiche e solleva nuove opzioni sperimentali rispetto ai cuori interi perfezionati da Langendorff. La risposta del farmaco delle fette ai bloccanti del canale ionico come verapamil (bloccante canale L-tipo Ca 2+ ), lidocaina (bloccante Na + -cannel), 4-aminopiridina (bloccante K + -kanelare dipendente dalla tensione non selettiva) e linopirdina (KCNQ K + - blocco canale) 9 , 11 </suP&gt; corrispondevano a effetti noti sui cardiomiociti dissociati. Le misure di forza isometrica hanno rivelato una relazione positiva di frequenza di forza e suggerito fortemente la funzione contrattuale intatta 10 . Questi risultati hanno dimostrato che le fettine ventricolari murine sono adatte come un modello di tessuto in vitro per studi fisiologici e farmacologici. Inoltre, le fetture ventricolari di cuori riceventi in combinazione con registrazioni di elettrodo nitide hanno dimostrato di essere uno strumento molto utile per caratterizzare l&#39;integrazione elettrica e meccanica, nonché la maturazione dei cardiomiociti fetali 12 , 13 e 14 trapiantati. In sintesi, le fetture ventricolari sono un prezioso e ben consolidato modello di tessuto multicellulare e devono essere considerati complementari ai cardiomiociti dissociati e ai cuori perfezionati da LangendorffNella ricerca cardiovascolare, con il grande vantaggio di fornire una struttura tissutale in vivo (in contrasto con le cellule dissociate), nonché l&#39;accesso diretto delle tecnologie di misura come le registrazioni di elettrodo nitido a tutte le regioni del cuore (in contrasto con i preparati cardiaci interi).

<table border="0" cellpadding="0" cellspacing="0" width="895">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Leica VT 1000s</td>
			<td style="width:340px;">Leica Microsystems, Wetzlar, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;">Microtome with vibrating blade.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:248px;">Stainless Steel Blades</td>
			<td style="width:340px;">Campden Instruments, Loughborough, England</td>
			<td>7550-1-SS</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur pipettes&#160;</td>
			<td>Sigma-Aldrich, St. Louise, USA</td>
			<td>Z627992&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Fine brush, e.g. size 6 (4/32&#34;)</td>
			<td style="width:340px;">VWR, International, Radnor, USA</td>
			<td>149-2125</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Preparation table</td>
			<td style="width:340px;"></td>
			<td style="width:140px;"></td>
			<td style="width:167px;">self made</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Molt for embedding ventricles in agarose</td>
			<td style="width:340px;"></td>
			<td style="width:140px;"></td>
			<td style="width:167px;">self made</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:248px;">1 ml Syringe</td>
			<td style="width:340px;">Becton, Dickinson;&#160; Franklin Lakes, USA</td>
			<td>300013</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">27Gx3/4`` Needles</td>
			<td rowspan="2" style="width:340px;">Braun, Melsungen, Germany</td>
			<td>4657705</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">20G 11/2`` Needles</td>
			<td>4657519</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Small scissor</td>
			<td style="width:340px;">WPI, Sarasota, USA</td>
			<td>501263</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Tweezers #5, 0.1 x 0.06 mm tip</td>
			<td style="width:340px;">WPI, Sarasota, USA</td>
			<td>500342</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Oxygen gas (medical grade O2)</td>
			<td style="width:340px;">Linde, Munich, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Carbogen gas (95 % O2, 5 % CO2)&#160;</td>
			<td style="width:340px;">Linde, Munich, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">NaCL</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">7647-14-5</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">KCL</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">746436</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:248px;">CaCl2</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">746495</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:248px;">KH2PO4</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">NIST200B&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">HEPES</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">51558</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:248px;">NaHCO3</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">S5761&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">D(+)-Glucose</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">G8270&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:248px;">MgSO4</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">M7506&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">NaOH</td>
			<td style="width:340px;">Sigma-Aldrich, St. Louise, USA</td>
			<td style="width:140px;">S8045&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Cyanoacrylate glue&#160;</td>
			<td style="width:340px;">Henkel, D&#252;sseldorf, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Low-melt Agarose&#160;</td>
			<td style="width:340px;">Roth, Karlsruhe, Germany</td>
			<td style="width:140px;">6351.2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Heparin-sodium-25000 I.E./5mL</td>
			<td style="width:340px;">Ratiopharm, Ulm, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Dulbecco&#39;s Modified Eagle Medium&#160; (DMEM), high glucose, GlutaMAX</td>
			<td style="width:340px;">ThermoScientific, Waltham, USA</td>
			<td style="width:140px;">10566016</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">SEC-10LX Amplifier</td>
			<td style="width:340px;">npi electronic GmbH, Tamm, Germany</td>
			<td>SEC-10LX</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">EPC 9</td>
			<td style="width:340px;">HEKA Elektronik GmbH, Lambrecht, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Zeiss Axiovert 200</td>
			<td style="width:340px;">Zeiss, Oberkochen, Germany</td>
			<td style="width:140px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">&#160;Low magnification Micromanipulator</td>
			<td style="width:340px;">Narashige, Tokyo, Japan</td>
			<td style="width:140px;">Nm-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">High magnification, three-axis micromanipulator</td>
			<td style="width:340px;">Narashige, Tokyo, Japan</td>
			<td style="width:140px;">MHW-3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Peristaltic perfusion pump</td>
			<td style="width:340px;">Multi Channel Systems, Reutlingen, Germany</td>
			<td style="width:140px;">PPS2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">2-channel temperature controller</td>
			<td style="width:340px;">Multi Channel Systems, Reutlingen, Germany</td>
			<td style="width:140px;">TCO02</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:248px;">Square pulse stimulator</td>
			<td style="width:340px;">Natus Europe GmbH, Planegg, Germany</td>
			<td style="width:140px;">Grass SD9</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:248px;">Glass capillaries</td>
			<td style="width:340px;">WPI, Sarasota, USA</td>
			<td style="width:140px;">1B150F-1</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

murine short axis ventricular heart slices for electrophysiological studies

Cardiomiociti murini sono stati ampiamente utilizzati per studi in vitro di fisiologia cardiaca e nuove strategie terapeutiche. Tuttavia, le preparazioni multicellulari di cardiomiociti dissociati non rappresentano la struttura complessa in vivo di cardiomiociti, non miociti e matrice extracellulare, che influenza sia le proprietà meccaniche che elettrofisiologiche del cuore. Qui descriviamo una tecnica per preparare fette ventricolari vitali di cuori adulti con un tessuto tissutale in vivo e dimostrare la loro idoneità alle registrazioni elettrofisiologiche. Dopo l&#39;escissione del cuore, i ventricoli vengono separati dall&#39;atria, perfezionati con soluzione Ca 2+ -libera contenente 2,3-butanedione monoxime e incorporati in un blocco agarosio a basso contenuto di fusione al 4%. Il blocco è posto su un microtomo con una lama vibrante, e fettine di tessuto con uno spessore di 150-400 μm sono preparate mantenendo la vibrazione freDella lama a 60-70 Hz e spostare la lama in avanti in modo più lento possibile. La spessore delle fette dipende dall&#39;ulteriore applicazione. Le fette vengono conservate in soluzione di Tyrode ghiacciata con 0,9 mM Ca 2+ e 2,3-butanedione monoxime (BDM) per 30 minuti. Successivamente, le fette vengono trasferite a 37 ° C di DMEM per 30 minuti per lavare il BDM. Le fette possono essere utilizzate per studi elettrofisiologici con elettrodi taglienti o matrici di microelettrodi, per misurare le forze per analizzare la funzione contrattile o per indagare l&#39;interazione tra i cardiomiociti derivanti dalle cellule staminali trapiantati e il tessuto ospite. Per registrazioni di elettrodo nitide, una fetta viene posta in un piatto di coltura da 3 cm sulla piastra riscaldante di un microscopio invertito. La fetta è stimolata con un elettrodo unipolare e potenziali d&#39;azione intracellulari di cardiomiociti all&#39;interno della fetta sono registrati con un elettrodo di vetro nitido.

L&#39;infarto miocardico porta ad una perdita praticamente irreversibile di cardiomiociti. La terapia sostitutiva cellulare che utilizza cardiomiociti derivanti da cellule staminali per la rigenerazione cardiaca esogena è un approccio terapeutico promettente. L&#39;integrazione elettrica e la maturazione delle cellule trapiantate sono fondamentali per la sicurezza e l&#39;efficienza della terapia sostitutiva delle cellule. Pe...

Watch this Scientific Journal Video about Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies at JoVE.com

Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

Qui descriviamo la preparazione di fette ventricolari vitali da topi adulti e il loro utilizzo per registrazioni potenziali di azione elettrodo. Queste preparazioni multicellulari forniscono una struttura tissutale conservata in vivo , che li rende un prezioso modello per studi elettrofisiologici e farmacologici in vitro .

Fette di cuore ventricolare dell&#39;asse corto dell&#39;urina per gli studi elettrofisiologici

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular ...

murine-short-axis-ventricular-heart-slices-for-electrophysiological

Research

JoVE Journal

Medicine

7.3K Views.  University of Cologne.  Qui descriviamo la preparazione di fette ventricolari vitali da topi adulti e il loro utilizzo per registrazioni potenziali di azione elettrodo. Queste preparazioni multicellulari forniscono una struttura tissutale conservata in vivo , che li rende un prezioso modello per studi elettrofisiologici e farmacologici in vitro . 

Video: Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Riduzione del ventricolo sinistro stress di parete e di miglioramento della funzione in mancanza di Cuori con Algisyl-LVR

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

Ricerca

Medicina

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Modello murino di guarigione delle ferite

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

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 ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

Murini eterotopica cuore Tecnica Trapianto

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Use of Two Intracorporeal Ventricular Assist Devices As a Total Artificial Heart

Mechanical circulatory support (MCS) has been introduced as a viable alternative to heart transplantation primarily through the use of intracorporeal ventricular assist devices (VADs) for support of the left ventricle. However, certain clinical scenarios warrant biventricular mechanical support. One strategy for some patients is the excision of both ventricles and the implantation of two VAD pumps as a total artificial heart (TAH). This has recently been made possible by the improvements in device design and the small profile of centrifugal devices. This TAH approach remains experimental with many important challenges such as the device settings to balance the right and left circulation, the orientation of the devices and the outflow graft with their influence on hemolysis and stability, and the outcome of chronic support using such an orientation. This protocol aims to provide a reproducible approach for total artificial heart replacement with two intracorporeal centrifugal VADs in a cow model.

Here, we present a protocol using two centrifugal pumps as a total artificial heart replacement.

Utilizzo di due dispositivi di assistenza ventricolare intracorporea come un cuore artificiale totale

Mechanical circulatory support (MCS) has been introduced as a viable alternative to heart transplantation primarily through the use of intracorporeal ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Valutazione elettrofisiologica di Atria murino con Mapping ottico ad alta risoluzione

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Optocardiografia ed elettrofisiologia Studi di Ex Vivo Langendorff-perfusi

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Murine Precision-Cut Liver Slices come un modello Ex Vivo di biologia del fegato

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Location, Dissection, and Analysis of the Murine Stellate Ganglion

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter of investigation in the pathophysiology of ventricular arrhythmias (VA). Neurons in the stellate ganglia (SG)&#160;&#8211;&#160;bilateral star-shaped structures of the sympathetic chain &#8211;&#160;are an important component of the sympathetic infrastructure. The SG are a recognized target for&#160;treatment via cardiac sympathetic denervation in patients with therapy-refractory VA. While neuronal remodeling and glial activation in the SG have been described in patients with VA, the underlying cellular and molecular processes that potentially precede the onset of arrhythmia are only insufficiently understood and should be elucidated to improve autonomic modulation. Mouse models allow us to study sympathetic neuronal remodeling, but identification of the murine SG is challenging for the inexperienced investigator. Thus, in-depth cellular and molecular biological studies of the murine SG are lacking for many common cardiac diseases. Here, we describe a basic repertoire for dissecting and studying the SG in adult mice for analyses at RNA level (RNA isolation for gene expression analyses, in situ hybridization), protein level (immunofluorescent whole mount staining), and cellular level (basic morphology, cell size measurement). We present potential solutions to overcome challenges in the preparation technique, and how to improve staining via quenching of autofluorescence. This allows for the visualization of neurons as well as glial cells via established markers in order to determine cell composition and remodeling processes. The methods presented here allow characterizing the SG to gain further information on autonomic dysfunction in mice prone to VA and can be complemented by additional techniques investigating neuronal and glial components of the autonomic nervous system in the heart.

Pathophysiological changes in the cardiac autonomic nervous system, especially in its sympathetic branch, contribute to the onset and maintenance of ventricular arrhythmias. In the present protocol, we show how to characterize murine stellate ganglia to improve the understanding of the underlying molecular and cellular processes.

Posizione, dissezione e analisi del Ganglio Stellato Murine

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

Use of a Percutaneous Ventricular Assist Device/Left Atrium to Femoral Artery Bypass System for Cardiogenic Shock

The left atrial to femoral artery bypass (LAFAB) system is a mechanical circulatory support (MCS) device used in cardiogenic shock (CS) that bypasses the left ventricle by draining blood from the left atrium (LA) and returning it to the systemic arterial circulation via the femoral artery. It can provide flows ranging from 2.5-5 L/min depending on the size of the cannula. Here, we discuss the mechanism of action of LAFAB, available clinical data, indications for its use in cardiogenic shock, steps of implantation, post-procedural care, and complications associated with the use of this device and their management.
We also provide a brief video of the procedural component of device therapy, including the pre-placement preparation, percutaneous placement of the device via transseptal puncture under echocardiographic guidance and the post-operative management of device parameters.

The following article describes the stepwise procedure for placement of a device (e.g., Tandemheart) in cardiogenic shock (CS) that is a percutaneous left ventricular assist device (pLVAD) and a left atrial to femoral artery bypass (LAFAB) system that bypasses and supports the left ventricle (LV) in CS.

Uso di un dispositivo di assistenza ventricolare percutanea / atrio sinistro al sistema di bypass dell'arteria femorale per lo shock cardiogeno

The left atrial to femoral artery bypass (LAFAB) system is a mechanical circulatory support (MCS) device used in cardiogenic shock (CS) that bypasses the ...