Questo lavoro è stato supportato da Key Project Medical Science and Technology Development Foundation, Nanjing Department of Health (No.YKK16098).

Gli esperimenti sugli animali sono stati approvati dall'Institutional Animal Care and Use Committee (IACUC) del Nanjing Drum Tower Hospital.
1. Preparazione dei reagenti e degli animali
<ol><li>Sciogliere la doxorubicina cloridrato (Pfizer, USA) in acqua sterilizzata. Vortice per ottenere una soluzione di Dox di 1 mg/mL e mantenere a 4 °C.</li>
	<li>Utilizzare topi C57BL/6 (8-10 settimane di età; 25-30 g di peso). Per questo studio, i topi sono stati acquistati dal Model Animal Research Center dell'Università di Nanchino e tenuti nella stanza degli animali del Nanjing Drum Tower Hospital.</li>
	<li>Le gabbie per topi prive di agenti patogeni sono state mantenute sotto un ciclo luce/buio di 12 ore a una temperatura costante di 23 °C. Tutti gli animali sono stati nutriti con una normale dieta chow e hanno ricevuto cibo e acqua ad libitum.</li>
</ol>2. Istituzione del modello animale DCM
<ol><li>Randomizzare i topi in un gruppo normale (n = 5) e dox (n = 5).</li>
	<li>Somministrare la soluzione di Dox per via intraperitoneale alla dose di 5 mg/kg utilizzando una siringa sterilizzata da 1 mL 1 volta a settimana per il gruppo Dox. Trattare i topi di controllo allo stesso modo con la stessa quantità di soluzione salina.</li>
	<li>Misurare il peso corporeo dei due gruppi settimanalmente. Di conseguenza, aggiustare la dose di iniezione in base al peso corporeo settimanalmente per un totale di 4 settimane, con una dose cumulativa di 20 mg/kg (Figura 1). NOTA: È stato scelto un periodo di tempo di 4 settimane perché l'ecocardiografia a 4 settimane ha mostrato una differenza significativa nella funzione cardiaca tra i due gruppi.</li>
</ol>3. Esame ecocardiografico
<ol><li>Alla fine della quarta settimana, condurre un esame ecocardiografico dei topi.</li>
	<li>Anestetizzare i topi con il 2% di isoflurano per via intranasale. Se i topi non rispondono a un pizzico di pelle con una pinzetta dentata o stimolando le dita dei piedi e la coda, continuare con il protocollo.</li>
	<li>Posizionare il mouse su una piattaforma di manipolazione degli animali in posizione supina. Per mantenere l'anestesia, coprire il naso e la bocca dell'animale con un cono nasale e fornire il 2% di isoflurano. NOTA: Per l'anestesia IP, iniettare il 4% di idrato di cloralio alla dose di 0,2 ml/20 g.</li>
	<li>Rimuovere accuratamente la pelliccia del torace con un rasoio elettrico. Valutare la funzione cardiaca in vivo utilizzando l'ecocardiografia transtoracica.</li>
	<li>Eseguire l'ecocardiogramma LV sia nell'asse lungo parasternale che nell'asse corto ad un frame rate di 233 Hz. Le dimensioni sistolica e diastolica finale sono state definite come le fasi corrispondenti rispettivamente all'onda T ECG e all'onda R.</li>
	<li>Sui tracciati in modalità M, misurare il diametro medio sistolico finale LV (LVIDd), il diametro diastolico terminale LV (LVID), lo spessore del setto interventricolare (IVS) e lo spessore della parete posteriore LV (LVPW) da 3-5 battiti cardiaci. Calcola anche la frazione di eiezione (EF) e l'accorciamento della frazione (FS) in base all'ecocardiografia.</li>
</ol>4. Colorazione istologica
<ol><li>Dopo l'analisi ecocardiografica, sacrificare i topi con l'iniezione intraventricolare di cloruro di potassio al 10%.</li>
	<li>Perfondere il cuore con circa 30 ml di soluzione salina dopo la dissezione fino a quando il fegato e il polmone diventano pallidi.</li>
	<li>Asportare il cuore e lavarlo accuratamente in soluzione tampone fosfato per estrudere il sangue.</li>
	<li>Fissare il cuore in formalina al 4% a temperatura ambiente per 24 ore e trattare il tessuto in una scatola di paraffina in modo che la cera di paraffina si raffreddi e si solidifichi.</li>
	<li>Tagliare i cuori in fette spesse 5 μm per la colorazione patologica.</li>
	<li>Decerazione e sezioni reidratate contenenti muscolo papillare.<ol>
<li>Incubare i vetrini a 55 °C per 30 min. Quindi, incubare in xilene 2 volte per 2 minuti ciascuno; 100% etanolo 2x per 2 minuti ciascuno; 95% di etanolo 2x per 2 minuti ciascuno; 80% di etanolo per 2 min; 75% di etanolo per 2 min; e 50% di etanolo per 2 min.</li>
	</ol>
</li>
	<li>Macchiare con ematossilina ed eosina (H & E) e la macchia di Masson.</li>
</ol>

<ol><li>Weintraub, R. G., Semsarian, C., MacDonald, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dilated+cardiomyopathy%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Dilated cardiomyopathy.</a> Lancet. 390 (10092), 400-414 (2017).</li>
<li>Ichihara, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Attenuation+of+oxidative+stress+and+cardiac+dysfunction+by+bisoprolol+in+an+animal+model+of+dilated+cardiomyopathy%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">Attenuation of oxidative stress and cardiac dysfunction by bisoprolol in an animal model of dilated cardiomyopathy.</a> Biochemical and Biophysical Research Communications. 350 (1), 105-113 (2006).</li>
<li>Fountoulakis, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alterations+in+the+heart+mitochondrial+proteome+in+a+desmin+null+heart+failure+model%5BTitle%5D)+AND+%22Journal+of+Molecular+and+Cellular+Cardiology%22%5BJournal%5D)">Alterations in the heart mitochondrial proteome in a desmin null heart failure model.</a> Journal of Molecular and Cellular Cardiology. 38 (3), 461-474 (2005).</li>
<li>Fairweather, D., Rose, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coxsackievirus-induced+myocarditis+in+mice%3A+a+model+of+autoimmune+disease+for+studying+immunotoxicity%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Coxsackievirus-induced myocarditis in mice: a model of autoimmune disease for studying immunotoxicity.</a> Methods. 41 (1), 118-122 (2007).</li>
<li>Wang, Z. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+therapeutic+anti-CD4+monoclonal+antibody+inhibits+T+cell+receptor+signal+transduction+in+mouse+autoimmune+cardiomyopathy%5BTitle%5D)+AND+%22Chinese+Medical+Journal%22%5BJournal%5D)">A therapeutic anti-CD4 monoclonal antibody inhibits T cell receptor signal transduction in mouse autoimmune cardiomyopathy.</a> Chinese Medical Journal. 120 (15), 1319-1325 (2007).</li>
<li>Riad, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toll-like+receptor-4+deficiency+attenuates+doxorubicin-induced+cardiomyopathy+in+mice%5BTitle%5D)+AND+%22European+Journal+of+Heart+Failure%22%5BJournal%5D)">Toll-like receptor-4 deficiency attenuates doxorubicin-induced cardiomyopathy in mice.</a> European Journal of Heart Failure. 10 (3), 233-243 (2008).</li>
<li>Kankeu, C., Clarke, K., Passante, E., Huber, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Doxorubicin-induced+chronic+dilated+cardiomyopathy-the+apoptosis+hypothesis+revisited%5BTitle%5D)+AND+%22Journal+of+Molecular+Medicine%22%5BJournal%5D)">Doxorubicin-induced chronic dilated cardiomyopathy-the apoptosis hypothesis revisited.</a> Journal of Molecular Medicine. 95 (3), 239-248 (2017).</li>
<li>Zhao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNA-140-5p+aggravates+doxorubicin-induced+cardiotoxicity+by+promoting+myocardial+oxidative+stress+via+targeting+Nrf2+and+Sirt2%5BTitle%5D)+AND+%22Redox+Biology%22%5BJournal%5D)">MicroRNA-140-5p aggravates doxorubicin-induced cardiotoxicity by promoting myocardial oxidative stress via targeting Nrf2 and Sirt2.</a> Redox Biology. 15, 284-296 (2018).</li>
<li>O'Connell, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Short-term+and+long-term+models+of+doxorubicin-induced+cardiomyopathy+in+rats%3A+A+comparison+of+functional+and+histopathological+changes%5BTitle%5D)+AND+%22Experimental+and+Toxicologic+Pathology%22%5BJournal%5D)">Short-term and long-term models of doxorubicin-induced cardiomyopathy in rats: A comparison of functional and histopathological changes.</a> Experimental and Toxicologic Pathology. 69 (4), 213-219 (2017).</li>
<li>Yuan, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CTRP3+protected+against+doxorubicin-induced+cardiac+dysfunction%2C+inflammation+and+cell+death+via+activation+of+Sirt1%5BTitle%5D)+AND+%22Journal+of+Molecular+and+Cellular+Cardiology%22%5BJournal%5D)">CTRP3 protected against doxorubicin-induced cardiac dysfunction, inflammation and cell death via activation of Sirt1.</a> Journal of Molecular and Cellular Cardiology. 114, 38-47 (2018).</li>
<li>Sun, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+TGF-beta+pathway+mediates+doxorubicin+effects+on+cardiac+endothelial+cells%5BTitle%5D)+AND+%22Journal+of+Molecular+and+Cellular+Cardiology%22%5BJournal%5D)">The TGF-beta pathway mediates doxorubicin effects on cardiac endothelial cells.</a> Journal of Molecular and Cellular Cardiology. 90, 129-138 (2016).</li>
<li>Minotti, G., Menna, P., Salvatorelli, E., Cairo, G., Gianni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anthracyclines%3A+molecular+advances+and+pharmacologic+developments+in+antitumor+activity+and+cardiotoxicity%5BTitle%5D)+AND+%22Pharmacological+Reviews%22%5BJournal%5D)">Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity.</a> Pharmacological Reviews. 56 (2), 185-229 (2004).</li>
<li>Vejpongsa, P., Yeh, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevention+of+anthracycline-induced+cardiotoxicity%3A+challenges+and+opportunities%5BTitle%5D)+AND+%22Journal+of+The+American+College+of+Cardiology%22%5BJournal%5D)">Prevention of anthracycline-induced cardiotoxicity: challenges and opportunities.</a> Journal of The American College of Cardiology. 64 (9), 938-945 (2014).</li>
<li>Renu, K., V, G. A., P, B. T., Arunachalam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+mechanism+of+doxorubicin-induced+cardiomyopathy+-+An+update%5BTitle%5D)+AND+%22European+Journal+of+Pharmacology%22%5BJournal%5D)">Molecular mechanism of doxorubicin-induced cardiomyopathy - An update.</a> European Journal of Pharmacology. 818, 241-253 (2018).</li>
</ol>

Nessun conflitto di interessi dichiarato.

Dox è un farmaco chemioterapico antitumorale periodico non specifico comunemente usato nella pratica clinica12. Il suo principale effetto collaterale è la cardiotossicità, caratterizzata da cardiomiopatia e successiva insufficienza cardiaca13. Il meccanismo sottostante include il danno della perossidazione lipidica miocardica, l'inibizione del reticolo sarcoplasmatico miocardico Ca2+-ATPasi e l'attivazione del sistema renninico-angiotensina locale miocardico, con conseguente aumento della produzione di AT II e sovraccarico intracellulare di calcio14. A causa dell'effetto letale di una grande dose di iniezione intraperitoneale di Dox, in questo protocollo è stato utilizzato un modo alternativo per stabilire un modello affidabile.
Il modello DCM stabilito da questo metodo è molto simile alla patogenesi del DCM umano. Oltre all'insufficienza cardiaca, la complicanza più comune di Dox, l'ascite è stata osservata nei topi DCM. Pertanto, durante lo sviluppo del modello, si deve prestare attenzione al funzionamento sterile e alla sostituzione tempestiva delle siringhe. Inoltre, per la dose di Dox, sono stati confrontati due metodi di somministrazione: un lungo ciclo di trattamento con una piccola dose e un trattamento a breve termine con una dose elevata. I risultati mostrano che l'iniezione continua di Dox a 5 mg / kg per quattro settimane può portare a un modello accurato di DCM, ridurre la mortalità dei topi e un tempo più breve per la creazione del modello.
In sintesi, un modello animale DCM murino stabile, affidabile ed economico può essere stabilito con successo mediante iniezione intraperitoneale di Dox. Questa ricerca fornisce un modello ideale per lo studio del DCM. Contribuirà a rivelare la patogenesi della cardiomiopatia dilatativa o dell'insufficienza cardiaca cronica ed esplorare nuovi farmaci o trattamenti per future applicazioni cliniche.

An erratum was issued for:&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/t/61158">A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo</a>. A figure was updated.
Figure 1 was updated from:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61158/61158fig1v2.jpg" /> 
Figure 1: Schematic diagram of a Dox-induced dilated cardiomyopathy. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61158/61158fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61158/61158fig1v5.jpg" /> 
Figure 1: Schematic diagram of a Dox-induced dilated cardiomyopathy. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61158/61158fig1v5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www-jove-com.bdigitaluss.remotexs.co/t/61158">A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo</a>. A figure was updated.

Erratum: A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Una delle cause più comuni di insufficienza cardiaca, DCM è caratterizzata da dilatazione ventricolare e diminuzione della funzione cardiaca ed è la ragione più comune per il trapianto di cuore in tutto il mondo1. Al fine di indagare ulteriormente la sua patogenesi e trovare trattamenti efficaci, l'accesso a modelli animali maturi è particolarmente importante. Lo scopo degli esperimenti descritti è quello di stabilire un modello murino stabile di DCM che assomigli al DCM umano.
A causa della complessa patogenesi del DCM, ci sono molti metodi diversi per realizzare modelli animali corrispondenti. I modelli DCMspontanei 2 sono relativamente stabili, ma sono costosi e non facilmente disponibili. I modelli animali geneticamente modificati3 non sono ben consolidati e richiedono un uso più sperimentale. I modelli animali DCM indotti da infezione virale4 o difetti autoimmuni5 sono facili da ottenere, ma non sono del tutto rappresentativi del DCM. I modelli associati alla tossicità miocardica includono modelli DCM indotti da alcol e modelli animali DCM indotti da Dox.
Il modello di cardiomiopatia indotta da Dox è ottenuto mediante iniezione intraperitoneale di Dox6. Il modello sfrutta l'effetto collaterale cronico più grave di Dox: dopo l'esposizione a Dox, i pazienti sviluppano sintomi di DCM ad esordio tardivo con uniformità clinica7. Lo stress ossidativo indotto da Dox8 e il danno mitocondriale9, che portano all'apoptosi cardiomiocitaria, sono sintomi nella patogenesi del DCM. Esistono modelli di trattamento Dox acuti e cronici: una singola dose elevata di Dox (15 mg/kg) induce un modello a breve termine per la cardiomiopatia10,mentre iniezioni ripetitive di Dox a basso dosaggio (sei settimanali, 3 mg/kg) inducono un modello a lungo termine per la cardiomiopatia11. Sulla base dello studio presentato, i topi wild type iniettati per via intraperitoneale una volta alla settimana per un mese alla dose di 5 mg/kg mostrano morfologia e istologia del cuore coerenti con le caratteristiche del DCM entro la fine del trattamento, fornendo un modo ideale per stabilire un modello DCM.

<table><tbody><tr><td>4% paraformaldehyde</td><td>servicebio</td><td>CAS30525-89-4</td><td></td></tr><tr><td>C57BL/6 mice</td><td>Model Animal Research Center of Nanjing University</td><td>\</td><td></td></tr><tr><td>Doxorubicin hydrochloride</td><td>Pfizer</td><td>CAS25316-40-9</td><td></td></tr><tr><td>echocardiography</td><td>Visualsonics</td><td>\</td><td></td></tr><tr><td>Hematoxylin and Eosin staining kit</td><td>Solarbio</td><td>G1120</td><td></td></tr><tr><td>Masson staining kit</td><td>Solarbio</td><td>G1343</td><td></td></tr><tr><td>phosphate buffer solution</td><td>Sigma</td><td>P5368</td><td></td></tr><tr><td>potassium chloride</td><td>Sigma</td><td>CAS7447-40-7</td><td></td></tr><tr><td>sterilized syringe</td><td>Millipore</td><td>SLGP033RB</td><td></td></tr></tbody></table>

a doxorubicin-induced murine model of dilated cardiomyopathy in vivo

La cardiomiopatia dilatativa (DCM) si riferisce a uno spettro di disturbi miocardici eterogenei caratterizzati da dilatazione ventricolare e prestazioni cardiache depresse in assenza di ipertensione, cardiopatie valvolari, congenite o ischemiche e che possono essere correlati a infezioni, anomalie autoimmuni o metaboliche o ereditarietà familiare. Può progredire in insufficienza cardiaca congestizia con una prognosi sfavorevole. La doxorubicina (Dox) è ampiamente impiegata come farmaco chemioterapico, ma il suo uso è limitato perché provoca cambiamenti simili a DCM del miocardio. La sua tossicità miocardica è attribuita allo stress ossidativo, all'infiammazione cronica e all'apoptosi cardiomiocitaria. Non è stato stabilito un modello di DCM che sfrutti questi sintomi di DCM indotti da Dox.

Funzione cardiaca La cardiomiopatia dilatativa è caratterizzata da progressiva dilatazione ventricolare e disfunzione contrattile. La Figura 2 mostra immagini ecocardiografiche rappresentative dei due gruppi. I topi trattati con Dox hanno mostrato una frazione di eiezione ventricolare sinistra marcatamente ridotta e un accorciamento frazionario ventricolare sinistro (Figura 3A,B). Il diametro LV è aumentato anche nella fase diastolica e sistolica (Figura 3C,D). Ciò ha rivelato che i topi trattati con Dox avevano una funzione cardiaca compromessa.
Colorazione istologica La colorazione patologica è stata eseguita per osservare i cambiamenti patologici dei topi trattati con soluzione salina e dei topi trattati con Dox. Le fibre miocardiche dei topi nel gruppo di controllo erano disposte in modo ordinato, senza infiltrarsi nei leucociti. Nel gruppo Dox, le miofibre miocardiche erano disordinate e rotte e i cardiomiociti erano più piccoli e più sottili (Figura 4A). La colorazione di Masson ha mostrato che i topi trattati con Dox avevano più fibrosi interstiziale (Figura 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61158/61158fig1v5.jpg"> Figura 1: Diagramma schematico di una cardiomiopatia dilatativa indotta da Dox. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61158/61158fig1v5large.jpg" target="_blank">Fare clic qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61158/61158fig2v2.jpg"> Figura 2: L'ecocardiografia dei due gruppi. (A) Gruppo di controllo; (B) Gruppo Dox. A sinistra è la sezione dell'asse lungo e a destra è la sezione dell'asse corto. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61158/61158fig2v2large.jpg" target="_blank">Fare clic qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61158/61158fig3v2.jpg"> Figura 3: Le variabili della funzione cardiaca tra i gruppi. a)EF; b)FS; (C) LVEDd; (D) LVED. *P < 0.05, T-test dellostudente. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61158/61158fig3v2large.jpg" target="_blank">Fare clic qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61158/61158fig4v2.jpg"> Figura 4: Colorazione istologica. (A) Colorazione H&E; (B) Colorazione di Masson. A sinistra è il gruppo di controllo e a destra è il gruppo Dox. La freccia superiore mostra che i cardiomiociti trattati con Dox erano più piccoli e più sottili e le miofibre erano disordinate e rotte. La freccia inferiore denota la presenza di fibrosi interstiziale. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61158/61158fig4v2large.jpg" target="_blank">Fare clic qui per visualizzare una versione più grande di questa figura.</a>
5

Watch this Scientific Journal Video about A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo at JoVE.com

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

È descritto un protocollo per stabilire un modello di cardiomiopatia dilatativa indotta da doxorubicina (DCM) nei topi tramite iniezione intraperitoneale a lungo termine di doxorubicina.

Un modello murino indotto da doxorubicina di cardiomiopatia dilatativa in vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...

a-doxorubicin-induced-murine-model-of-dilated-cardiomyopathy-in-vivo

Universidad San Sebastian

Research

JoVE Journal

Medicine

4.5K Views.  Clinical College of Nanjing Medical University. È descritto un protocollo per stabilire un modello di cardiomiopatia dilatativa indotta da doxorubicina (DCM) nei topi tramite iniezione intraperitoneale a lungo termine di doxorubicina.

Video: A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

L'analisi dello spostamento della deformazione meccanica miocardiale (DIAMOND) rivela l'eterogeneità segmentale della funzione cardiaca nel pesce zebra embrionale

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

Ricerca

Biologia dello sviluppo

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Legatura permanente dell&#39;arteria discendente anteriore sinistra coronarica nei topi: un modello di post-infarto rimodellante miocardico e insufficienza cardiaca

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Medicina

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

Valutazione elettrofisiologia cardiaca preclinica mediante mappatura ottica a doppia tensione e calcio delle fette cardiache di organitypica umana

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Bioingegneria

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 ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

Cardiomiopatia tachicardia-indotta come un modello di insufficienza cardiaca cronica in maiali

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as cardiomyopathy. Because of its convenience and amenability to high throughput genetic manipulations, the generation of acquired cardiomyopathy models, such as the doxorubicin-induced cardiomyopathy (DIC) model in adult zebrafish, is opening the doors to new research avenues, including discovering cardiomyopathy modifiers via forward genetic screening. Different from the embryonic zebrafish DIC model, both initial acute and later chronic phases of cardiomyopathy can be determined in the adult zebrafish DIC model, enabling the study of stage-dependent signaling mechanisms and therapeutic strategies. However, variable results can be obtained with the current model, even in the hands of experienced investigators. To facilitate future implementation of the DIC model, we present a detailed protocol on how to generate this DIC model in adult zebrafish and describe two alternative ways of intraperitoneal (IP) injection. We further discuss options on how to reduce variations to obtain reliable results and provide suggestions on how to appropriately interpret the results.

A method to generate a doxorubicin-induced cardiomyopathy model in adult zebrafish (Danio rerio) is described here. Two alternative ways of intraperitoneal injection are presented and conditions to reduce variations among different experimental groups are discussed.

Un modello di cardiomiopatia indotta da doxorubicina in Zebrafish adulto

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

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

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

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

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

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

Revealing Subtle Changes in Cardiac Function using Transthoracic Dobutamine Stress Echocardiography in Mice

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic dobutamine stress echocardiography in humans, a reductionist investigation approach to unmask subtle changes in cardiac function has become possible. Here, we provide a protocol for using this technique in mice to facilitate expanded analysis of LV architecture and function in physiology and pathology enabling the observation of alterations in models of cardiac disease hidden in unstressed hearts. This investigation can be performed in one and the same animal and allows both, basal and pharmacologically stress-induced measurements. We outline detailed criteria for appropriate anesthesia, imaging-based LV analysis, consideration of intra- and interobserver variability, and obtaining positive inotrope response that can be attained in mice after intraperitoneal injection of dobutamine under near physiological conditions. To recapitulate the characteristics of human physiology and disease in small animal models, we highlight critical pitfalls in evaluation, e.g., a pronounced Bowditch effect in mice. To further meet translational objectives, we compare stress-induced effects in humans and mice. When used in translational studies, attention must be paid to physiological differences between mice and human. Experimental rigor dictates that some parameters assessed in patients can only be used with caution due to restrictions in spatial and temporal resolution in mouse models.

<meta charset="utf8"></head><body>Rivelazione di sottili cambiamenti nella funzione cardiaca utilizzando l'ecocardiografia da stress transtoracica con dobutamina nei topi

Left ventricular dysfunction constitutes the final common pathway for a host of cardiac disorders. We here provide a detailed protocol of transthoracic dobutamine stress echocardiography approach for comprehensive evaluation of the left ventricular function of mouse models of cardiac disease as well as cardiac phenotyping.

Rivelazione di sottili cambiamenti nella funzione cardiaca utilizzando l'ecocardiografia da stress transtoracica con dobutamina nei topi

Left ventricular (LV) dysfunction paves the final pathway for a multitude of cardiac disorders. With the non-invasive high-frequency transthoracic ...

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. This paper describes a protocol for studying the role of galectin 3 (Gal-3) in the temporal evolution of cardiac healing and remodeling in an experimental animal model of MI. 
The procedures described include an experimental model of MI with a permanent coronary ligature in male C57BL/6J (control) and Gal-3 knockout (KO) mice, an echocardiography procedure to study cardiac remodeling and systolic function in vivo, a histological evaluation of interstitial myocardial fibrosis with picrosirius red-stained and rhodamine-conjugated lectin-stained sections for studying myocyte hypertrophy by the cross-sectional area (MCSA), and the quantification of infarct size and cardiac remodeling (scar thinning, septum thickness, and expansion index) by planimetry in slices stained with Masson's trichrome and triphenyl tetrazolium chloride. Gal-3 KO mice with MI showed disrupted cardiac remodeling and an increase in the scar thinning ratio and the expansion index. At the onset of MI, myocardial function and cardiac remodeling were also severely affected. At 4 weeks post MI, the natural evolution of fibrosis in infarcted Gal-3 KO mice was also affected. 
In summary, the experimental model of MI is a suitable model for studying the temporal evolution of cardiac repair and remodeling in mice with the genetic deletion of Gal-3 and other animal models. The lack of Gal-3 affects the dynamics of cardiac repair and disrupts the evolution of cardiac remodeling and function after MI.

<meta charset="utf8"></head><body>Un modello sperimentale di infarto del miocardio per lo studio della riparazione e del rimodellamento cardiaco nei topi knockout

Here, we describe an experimental model of myocardial infarction, an echocardiography procedure to study cardiac remodeling and function, and procedures for quantifying fibrosis and hypertrophy in picrosirius red-stained and rhodamine-stained sections, as well as the infarct size and expansion index in slices stained with Masson's trichrome.

Un modello sperimentale di infarto del miocardio per lo studio della riparazione e del rimodellamento cardiaco nei topi knockout

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. ...

Un modello murino indotto da doxorubicina di cardiomiopatia dilatativa in vivo

Riepilogo

Esplora Altri Video

L'analisi dello spostamento della deformazione meccanica miocardiale (DIAMOND) rivela l'eterogeneità segmentale della funzione cardiaca nel pesce zebra embrionale

Legatura permanente dell'arteria discendente anteriore sinistra coronarica nei topi: un modello di post-infarto rimodellante miocardico e insufficienza cardiaca

Valutazione elettrofisiologia cardiaca preclinica mediante mappatura ottica a doppia tensione e calcio delle fette cardiache di organitypica umana

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

Cardiomiopatia tachicardia-indotta come un modello di insufficienza cardiaca cronica in maiali

Un modello di cardiomiopatia indotta da doxorubicina in Zebrafish adulto

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

Rivelazione di sottili cambiamenti nella funzione cardiaca utilizzando l'ecocardiografia da stress transtoracica con dobutamina nei topi

Rivelazione di sottili cambiamenti nella funzione cardiaca utilizzando l'ecocardiografia da stress transtoracica con dobutamina nei topi

Un modello sperimentale di infarto del miocardio per lo studio della riparazione e del rimodellamento cardiaco nei topi knockout