Este trabalho foi apoiado pela Key Project Medical Science and Technology Development Foundation, Nanjing Department of Health (No.YKK16098).

Os experimentos em animais foram aprovados pelo Comitê Institucional de Cuidados e Uso de Animais (IACUC) do Nanjing Drum Tower Hospital.
1. Preparação dos reagentes e animais
<ol><li>Dissolver o cloridrato de doxorubicina (Pfizer, EUA) em água esterilizada. Vórtice para obter uma solução Dox de 1 mg/mL e manter a 4 °C.</li>
	<li>Use camundongos C57BL/6 (8-10 semanas de idade; 25-30 g de peso). Para este estudo, os camundongos foram comprados no Centro de Pesquisa Animal Modelo da Universidade de Nanjing e mantidos na sala de animais do Nanjing Drum Tower Hospital.</li>
	<li>As gaiolas de camundongos livres de patógenos foram mantidas sob um ciclo claro/escuro de 12 h a uma temperatura constante de 23 °C. Todos os animais foram alimentados com uma dieta normal de comida e receberam comida e água ad libitum.</li>
</ol>2. Estabelecimento do modelo animal DCM
<ol><li>Randomize os camundongos em um grupo normal (n = 5) e dox (n = 5).</li>
	<li>Administre a solução Dox intraperitoneal a uma dose de 5 mg/kg usando uma seringa esterilizada de 1 mL 1x por semana para o grupo Dox. Trate os ratos de controle da mesma forma com a mesma quantidade de solução salina.</li>
	<li>Meça o peso corporal dos dois grupos semanalmente. Assim, ajuste a dose de injeção com base no peso corporal semanalmente por um total de 4 semanas, com uma dose acumulada de 20 mg/kg(Figura 1). NOTA: Um período de 4 semanas foi escolhido porque a ecocardiografia em 4 semanas mostrou diferença significativa na função cardíaca entre os dois grupos.</li>
</ol>3. Exame ecocardiográfico
<ol><li>No final da quarta semana, realize um exame ecocardiográfico dos camundongos.</li>
	<li>Anestesiar os camundongos com 2% de isoflurane intranasally. Se os camundongos não responderem a uma pitada da pele com uma pinça dentada ou estimular os dedos dos dedos e caudas, continue com o protocolo.</li>
	<li>Coloque o mouse em uma plataforma de manuseio de animais na posição supina. Para manter a anestesia, cubra o nariz e a boca do animal com um cone de nariz e entregue 2% de isoflurane. NOTA: Para anestesia IP, injete 4% de hidrato de cloro a uma dose de 0,2 mL/20 g.</li>
	<li>Remova a pele do peito cuidadosamente com uma máquina de barbear elétrica. Avalie a função cardíaca in vivo usando ecocardiografia transtorácica.</li>
	<li>Realize o ecocardiograma LV tanto nas visões parasternal de longo eixo quanto em eixo curto a uma taxa de quadros de 233 Hz. As dimensões sistólicas e terminais foram definidas como as fases correspondentes à onda ECG T e à onda R, respectivamente.</li>
	<li>Nos traçadores do modo M, meça o diâmetro médio lv end-sistólica (LVIDd), diâmetro diastólico final lv (LVIDs), espessura septal interventricular (IVS) e espessura posterior da parede LV (LVPW) de 3-5 batimentos cardíacos. Calcule também a fração de ejeção (EF) e o encurtamento de frações (FS) com base na ecocardiografia.</li>
</ol>4. Coloração histológica
<ol><li>Após a análise ecocardiográfica, sacrifique os camundongos por injeção intraventricular de 10% de cloreto de potássio.</li>
	<li>Perfunda o coração com cerca de 30 mL de soro fisiológico após dissecção até que o fígado e o pulmão fiquem pálidos.</li>
	<li>Extirpa o coração e lave-o completamente em solução tampão fosfato para extrusão sangue.</li>
	<li>Fixar o coração em 4% de formalina à temperatura ambiente por 24 h e tratar o tecido em uma caixa de parafina para que a cera de parafina esfrie e solidifique.</li>
	<li>Corte os corações em fatias de 5 μm de espessura para coloração patológica.</li>
	<li>De cera e seções de reidratação contendo músculo papilar.<ol>
<li>Incubar lâminas a 55 °C por 30 min. Em seguida, incubar em xileno 2x por 2 min cada; 100% etanol 2x por 2 min cada; 95% de etanol 2x por 2 min cada; 80% de etanol por 2 min; 75% de etanol por 2 min; e 50% de etanol por 2 min.</li>
	</ol>
</li>
	<li>Mancha usando hematoxilina e eosina (H&E), bem como a mancha de Masson.</li>
</ol>

<ol>
	<li>Weintraub, R. G., Semsarian, C., MacDonald, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dilated+cardiomyopathy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+of+oxidative+stress+and+cardiac+dysfunction+by+bisoprolol+in+an+animal+model+of+dilated+cardiomyopathy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+the+heart+mitochondrial+proteome+in+a+desmin+null+heart+failure+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coxsackievirus-induced+myocarditis+in+mice:+a+model+of+autoimmune+disease+for+studying+immunotoxicity.">Coxsackievirus-induced myocarditis in mice: a model of autoimmune disease for studying immunotoxicity.</a> Methods. 41 (1), 118-122 (2007).</li><li>Wang, Z. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin-induced+chronic+dilated+cardiomyopathy-the+apoptosis+hypothesis+revisited.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-140-5p+aggravates+doxorubicin-induced+cardiotoxicity+by+promoting+myocardial+oxidative+stress+via+targeting+Nrf2+and+Sirt2.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+and+long-term+models+of+doxorubicin-induced+cardiomyopathy+in+rats:+A+comparison+of+functional+and+histopathological+changes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CTRP3+protected+against+doxorubicin-induced+cardiac+dysfunction,+inflammation+and+cell+death+via+activation+of+Sirt1.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TGF-beta+pathway+mediates+doxorubicin+effects+on+cardiac+endothelial+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthracyclines:+molecular+advances+and+pharmacologic+developments+in+antitumor+activity+and+cardiotoxicity.">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. 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Nenhum conflito de interesses declarado.

Dox é um medicamento de quimioterapia antitumor periódico inespecífico comumente utilizado na clínica12. Seu principal efeito colateral é a cardiotoxicidade, caracterizada por cardiomiopatia e subsequente insuficiência cardíaca13. O mecanismo subjacente inclui danos da peroxidação lipídica miocárdica, inibição do reticulum sarcoplasmático miocárdio Ca2+-ATIVIDADE ATPase, e ativação do sistema local de renin-angiotensina miocárdica, resultando em...

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Uma das causas mais comuns de insuficiência cardíaca, o DCM é caracterizado pela dilatação ventricular e diminuição da função cardíaca e é a razão mais comum para transplante de coração em todo o mundo1. Para investigar melhor sua patogênese e encontrar tratamentos eficazes, o acesso a modelos animais maduros é especialmente importante. O objetivo dos experimentos descritos é estabelecer um modelo de rato estável de DCM que se assemelha ao DCM humano.
Devido à complexa patogênese do DCM, existem muitos métodos diferentes para fazer modelos animais correspondentes. Os modelos espontâneos de DCM2 são relativamente estáveis, mas são caros e não são facilmente disponíveis. Os modelos animais geneticamente modificados3 não são bem estabelecidos e requerem mais uso experimental. Modelos animais DCM induzidos por infecção viral4 ou defeitos autoimunes5 são fáceis de obter, mas não são totalmente representativos do DCM. Os modelos associados à toxicidade do miocárdio incluem modelos dcm induzidos pelo álcool e modelos animais DCM induzidos pelo Dox.
O modelo de cardiomiopatia induzido pelo Dox é obtido por injeção intraperitoneal de Dox6. O modelo explora o efeito colateral crônico mais grave do Dox: Após a exposição ao Dox, os pacientes desenvolvem sintomas de DCM de início tardio com uniformidade clínica7. O estresse oxidativo induzido pelo dox8 e o dano mitocondrial9, que levam à apoptose cardiomiocese, são sintomas na patogênese do DCM. Existem modelos de tratamento dox agudos e crônicos: uma única dose alta de Dox (15 mg/kg) induz um modelo de curto prazo para cardiomiopatia10, enquanto as injeções repetitivas de dox de baixa dose (seis semanais, 3 mg/kg) induzem um modelo de longo prazo para cardiomiopatia11. Com base no estudo apresentado, camundongos do tipo selvagem injetaram uma vez por semana durante um mês em uma dose de 5 mg/kg de morfologia e histologia do coração consistentes com as características do DCM ao final do tratamento, fornecendo uma maneira ideal de estabelecer um modelo de 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

A cardiomiopatia dilatada (DCM) refere-se a um espectro de distúrbios heterogêneos do miocárdio caracterizado por dilatação ventricular e desempenho cardíaco deprimido na ausência de hipertensão arterial, valvular, congênita ou doenças cardíacas isquêmicas, e que podem estar relacionadas a infecções, anormalidades autoimunes ou metabólicas ou herança familiar. Pode evoluir para insuficiência cardíaca congestiva com um prognóstico ruim. A doxorubicina (Dox) é amplamente empregada como uma droga quimioterápica, mas seu uso é limitado porque causa alterações semelhantes ao DCM do miocárdio. Sua toxicidade miocárdio é atribuída ao estresse oxidativo, inflamação crônica e apoptose cardiomiocócica. Um modelo de DCM explorando esses sintomas de DCM induzidos pelo Dox não foi estabelecido.

Função cardíaca A cardiomiopatia dilatada é caracterizada pela dilatação ventricular progressiva e disfunção contratil. A Figura 2 mostra imagens ecocardiográficas representativas dos dois grupos. Os camundongos tratados com dox apresentaram uma fração de ejeção ventricular esquerda significativamente reduzida e encurtamento fracionado ventricular esquerdo(Figura 3A,B). O diâmetro da LV também aumentou nas fase...

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

Descrito é um protocolo para estabelecer um modelo de cardiomiopatia dilatada induzida por Doxorubicina (DCM) em camundongos através de injeção intraperitoneal de doxorubicina a longo prazo.

Um modelo de murina induzida por doxorubicina de cardiomiopatia dilatada em 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

Research

JoVE Journal

Medicine

4.4K visualizações.  Clinical College of Nanjing Medical University. Descrito é um protocolo para estabelecer um modelo de cardiomiopatia dilatada induzida por Doxorubicina (DCM) em camundongos através de injeção intraperitoneal de doxorubicina a longo prazo.

Vídeo: 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.

Análise de deslocamento da Deformação Mecânica Miocárdica (DIAMANTE) revela heterogeneidade segmental da função cardíaca em zebrafish embrionário

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

Biologia do Desenvolvimento

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.

A ligação permanente da artéria coronária descendente anterior esquerda em Ratos: Um Modelo de Remodelação pós-infarto do miocárdio e insuficiência cardíaca

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.

Avaliação de Eletrofisiologia Cardíaca Pré-Plínica por Mapeamento Óptico de Dupla Tensão e Cálcio de Fatias Cardíacas Organotípicas Humanas

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

Bioengenharia

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.

Injeção de contraste percutânea guiada por ecocardiografia Intramiocárdica e entrega de célula em uma grande maquete pré-clínicos

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 induzida por taquicardia como um modelo de insuficiência cardíaca crônica em suínos

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.

Um modelo de cardiomiopatia induzida por doxorrubicina no 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.

Modelo de infarto do miocárdio murino usando ligadura permanente da artéria coronária descendente anterior esquerda

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.

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.

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.

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.

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

<meta charset="utf8"></head><body>Modelo Murino para Cardiomiopatia Diabética por Dieta Rica em Gordura e Injeção de STZ

Modeling and Evaluation of Murine Diabetic Cardiomyopathy Model

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus (T2DM), remain poorly understood. The myocardial toxicity associated with T2DM is attributed to factors such as lipotoxicity, glucotoxicity, oxidative stress, reduced cardiac efficiency, and lipoapoptosis. Compared to rats, mice offer greater accessibility, cost-effectiveness, and broader applicability for animal experiments. Insulin resistance and impaired insulin secretion are crucial factors in the pathophysiology of T2DM. We introduce a novel nongenetic murine model that replicates the progression of human DbCM induced by a combination of high-fat diet (HFD) feeding and streptozotocin (STZ) injection. In this study, we used wild-type C57BL/6J mice, administering an HFD regimen for 12 weeks, followed by intraperitoneal injections of STZ for an additional 12 weeks to induce characteristic manifestations of T2DM. We conducted oral glucose tolerance tests and measured serum insulin concentrations to confirm the development of insulin resistance and insufficient insulin secretion. Cardiac structure and function were rigorously assessed through noninvasive transthoracic echocardiography. Pathological characteristics were evaluated through Masson's trichrome staining and wheat germ agglutinin (WGA) staining, revealing pathological features related to DbCM. Therefore, we provide a robust and versatile method for establishing a nongenetic murine model of DbCM.

<meta charset="utf8"></head><body>Modelagem e Avaliação do Modelo de Cardiomiopatia Diabética Murina

This protocol outlines a method for inducing diabetic cardiomyopathy through a combination of high-fat diet feeding and streptozotocin injection. This approach aims to provide a reliable framework for scientific investigation into diabetic cardiomyopathy and to explore potential avenues for clinical treatment applications.

Modelagem e Avaliação do Modelo de Cardiomiopatia Diabética Murina

The underlying pathophysiological mechanisms of diabetic cardiomyopathy (DbCM), a leading cause of mortality among patients with type 2 diabetes mellitus ...