The study was supported by the Swiss National Foundation [SNF 310030-149986 to MNG], the University of Fribourg, and Fribourg Hospital.

Todos os animais receberam cuidados de acordo com a Convenção Europeia dos Animal Care. Os procedimentos cirúrgicos foram realizados em conformidade com a Lei de Protecção dos Animais Suíça depois de obter autorização do Serviço Veterinário do Estado, Fribourg, aprovado pelo Swiss Federal Veterinário, Suíça. Colheita 1. Coração NOTA: Todas as intervenções cirúrgicas foram realizadas sob anestesia isoflurano. Foram feitos esforços para diminuir o sofrimento animal. Em particular, todos os animais receberam uma injecção subcutânea de 0,1 mg / kg de buprenorfina pré-anestésica. O protocolo cirúrgico para indução de enfarte do miocárdio foi anteriormente descrito noutro local 13. <ol><li> Executar uma esternotomia sobre um animal myocardially infartada sob anestesia (animal entubado, 2,5% de isoflurano, a anestesia adequada confirmados pela pata pitada reflex). <ol><li> Abra o animal por o corte da pele e, em seguida, os músculoscom uma tesoura cirúrgica. Corte as costelas do lado esquerdo e direito, e em seguida, remover o peito. </li><li> Retirar as adesões restantes após a cirurgia anterior (LAD ligadura). Cortar a aorta e remover o coração. Colocar o tecido em KCl 1 M (em PBS), e em seguida lavar com PBS. </li></ol></li></ol> 2. Preparação do Tecido <ol><li> Colocar o coração infartado longitudinalmente em uma matriz de coração de rato acrílico. Mantê-lo a -20 ° C durante 1 h. </li><li> Cortar o coração diretamente na matriz usando uma lâmina de barbear com uma transversal. Certifique-se de cada fatia é de 2 mm de espessura. Cortar aproximadamente 5-7 diapositivos para cada coração (com base no nível de remodelação); isso é chamado de amostragem sistemática. </li><li> Neste passo, realizar a coloração com cloreto de 2,3,5-trifeniltetrazólio (TTC) é opcional (manter a orientação da base de fatias de coração-a-ápice). <ol><li> Incubar em 1% de TTC em PBS durante 50 min a 37 ° C. Incubar-lo em 4% de paraformaldeído (PFA) durante 20 min a 1 h. Coloque as seções between duas placas de vidro com espaçadores de 2 mm e tirar fotos com um microscópio estereológica acoplado com uma câmera de 15X de ampliação. CUIDADO! O paraformaldeído é tóxico. </li></ol></li><li> Deslize congelamento (1 opção st) <ol><li> Colocar cada lâmina em um molde de plástico (10 x 10 x 5 mm) com meio para cryotomy (temperatura de corte óptima, outubro) de montagem, manter a orientação (colocar o lado do vértice orientado da secção para baixo na parte inferior do molde). Congelar os blocos com vapores de 2-metilbutano em atmosfera de azoto líquido de arrefecimento para 10 - 15 min. Finalmente, armazenar o tecido a -80 ° C. </li></ol></li><li> Parafinização (2 opção nd) <ol><li> Colocar cada lâmina na incorporação de cassetes (manter a orientação, colocando o lado orientado para o vértice da secção para baixo na parte inferior da cassete) e, em seguida, em PFA a 4% durante 24 h. Coloque-o em uma noite máquina de processador de tecidos. <ol><li> Incubar-lo em etanol a 70% durante 2 h. Incubar em que etanol 95% durante 2 h. Incubar-lo em etanol a 100% durante 3 h. Incubar-lo em xilol durante 4 h. Incubar-lo em parafina (derretida a 60 ° C num forno), durante 5 h. Finalmente, fazer blocos, incorporando cada slide coração em parafina. </li></ol></li></ol></li></ol> 3. Masson-Goldner tricromo <ol><li> Fazer secções de tecido a partir de blocos de parafina com um micrótomo manual (espessura: 5 ^ M) ou a partir de blocos PTU com um criostato ajustado para -18 ° C (espessura: 7 mm). </li><li> Manchar um slide de cada peça do coração para cada rato (3 - 4 secções transversais por slide) com Masson-Goldner tricromo (ver anexo). NOTA: Iniciar a partir deste passo para as seções de parafina. <ol><li> Derreter as lâminas a 60 ° C num forno. De acordo com um exaustor, eles Desparafinar duas vezes em xilol durante 10 minutos cada. Rehidratar-los em 100% de etanol, 95% de etanol, 70% de etanol, e água destilada, durante 3 minutos cada. NOTA: Iniciar a partir deste passo para o criocortes. </li><li> Corrigi-los em Bouin solução durante a noite. Em seguida, lave-os em água corrente por 10 - 15 min. Lave-os em água destilada. Incubar-los em hematoxilina de Mayer por 3 min. </li><li> Retirar as lâminas e deixá-los em água destilada durante 5 minutos. Em seguida, incubam-los em ácido fucsina-Ponceau durante 5 min. Lave-os em ácido acético a 1% durante 1 min. </li><li> Em seguida, incubam-los em ácido fosfomolíbdico Orange G durante 1 min. Lave-os em ácido acético a 1% durante 1 min, incubar-las à luz corante verde durante 10 minutos, e lavá-los em ácido acético a 1% durante 1 min. </li><li> Desidratar-os em 70% de etanol (30 seg), 95% de etanol (30 seg), e etanol a 100% (5 min). Coloque uma gota de um meio de montagem resinoso nas secções de tecido, cobri-los com lamelas, e deixe-os secar. </li></ol></li></ol> Análise Tamanho 4. Infarto <ol><li> Adquirir uma imagem de cada slide em uma lupa estereoscópica (15X de ampliação) acoplado com uma câmera. Fotografar uma régua com a mesma configuraçãos. </li><li> Use software de análise de imagem para medir a espessura da cicatriz no meio do enfarte, a espessura do septo, área da cavidade do ventrículo esquerdo (VE), a área de enfarte e área de tecido do LV utilizando. <ol><li> Definir a escala com a imagem régua (Figura 1A). <ol><li> Clique em Medidas, em seguida, selecione Definir Fator de Conversão. Desenhar uma linha na barra de calibração. Botão direito do mouse sobre a imagem e clique sobre a calibração End. Observe o valor bar, e, em seguida, clique em OK. </li></ol></li><li> Escolha do LV a partir da imagem do coração coradas (Figura 1B). Use a ferramenta segmento múltipla. Selecione o ponto-a-ponto LV, e clique depois à direita Copiar / colar em qualquer lugar. </li><li> Medir a espessura da cicatriz e do septo usando a ferramenta de medição único segmento (Figura 1C </strong&gt;). </li><li> Detectar automaticamente o LV cavidade, enfarte, e áreas LV utilizando a ferramenta de medição de área automático no modo RGB (Figura 1D). <ol><li> Clique na ferramenta. Ative Só Fronteiras e Contígua no modo RGB. Finalmente, clique dentro da área de interesse para detectá-lo. Se necessário, use ferramentas de precisão. </li></ol></li></ol></li><li> Calcular o tamanho do enfarte como a relação da área de enfarte e a área de LV. </li><li> Calcular o índice de expansão da seguinte forma: [área da cavidade LV / toda a área LV] / [espessura enfarte / espessura do septo], com toda a área de LV incluindo tanto a cavidade do VE e áreas de tecido do LV. </li><li> Finalmente, calcule uma &quot;média&quot; para cada coração como segue: [média de índice de expansão das lâminas infartados] * [Número de lâminas infartados / número total de slides]. </li></ol>

<ol>
	<li>Amsterdam, E. 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=AHA/ACC+Guideline+for+the+Management+of+Patients+with+Non-ST-Elevation+Acute+Coronary+Syndromes:+a+report+of+the+American+College+of+Cardiology/American+Heart+Association+Task+Force+on+Practice+Guidelines.">AHA/ACC Guideline for the Management of Patients with Non-ST-Elevation Acute Coronary Syndromes: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines.</a> J Am Coll Cardiol. 64, e139-e228 (2014).</li><li>Konstam, M. A., Kramer, D. G., Patel, A. R., Maron, M. S., Udelson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+remodeling+in+heart+failure:+current+concepts+in+clinical+significance+and+assessment.">Left ventricular remodeling in heart failure: current concepts in clinical significance and assessment.</a> JACC Cardiovasc Imaging. 4, 98-108 (2011).</li><li>Frobert, 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=Prognostic+Value+of+Troponin+I+for+Infarct+Size+to+Improve+Preclinical+Myocardial+Infarction+Small+Animal+Models.">Prognostic Value of Troponin I for Infarct Size to Improve Preclinical Myocardial Infarction Small Animal Models.</a> Front Physiol. 6, 353 (2015).</li><li>Redfors, B., Shao, Y., Omerovic, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+infarct+size+and+area+at+risk+assessment+in+mice.">Myocardial infarct size and area at risk assessment in mice.</a> Exp Clin Cardiol. 17, 268-272 (2012).</li><li>Guex, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma-functionalized+electrospun+matrix+for+biograft+development+and+cardiac+function+stabilization.">Plasma-functionalized electrospun matrix for biograft development and cardiac function stabilization.</a> Acta Biomater. 10, 2996-3006 (2014).</li><li>Landa, 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=Effect+of+injectable+alginate+implant+on+cardiac+remodeling+and+function+after+recent+and+old+infarcts+in+rat.">Effect of injectable alginate implant on cardiac remodeling and function after recent and old infarcts in rat.</a> Circulation. 117, 1388-1396 (2008).</li><li>Valente, 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=Optimized+heart+sampling+and+systematic+evaluation+of+cardiac+therapies+inmouse+models+of+ischemic+injury:+Assessment+of+cardiacremodeling+and+semi-automated+quantification+of+myocardial+infarctsize.">Optimized heart sampling and systematic evaluation of cardiac therapies inmouse models of ischemic injury: Assessment of cardiacremodeling and semi-automated quantification of myocardial infarctsize.</a> Curr. Protoc. Mouse Biol. 5, 359-391 (2015).</li><li>Csonka, C., 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=Measurement+of+myocardial+infarct+size+in+preclinical+studies.">Measurement of myocardial infarct size in preclinical studies.</a> J. Pharmacol. Toxicol. Methods. 61, 163-170 (2010).</li><li>Zornoff, L. A., Paiva, S. A., Minicucci, M. F., Spadaro, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+myocardium+infarction+in+rats:+Analysis+of+the+model.">Experimental myocardium infarction in rats: Analysis of the model.</a> Arq. Bras Cardiol. 93, 434-440 (2009).</li><li>Lichtenauer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+infarct+size+measurement+using+geometric+angle+calculation.">Myocardial infarct size measurement using geometric angle calculation.</a> Eur J Clin Invest. 44, 160-167 (2014).</li><li>Lutgens, E., 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=Chronic+myocardial+infarction+in+the+mouse:+cardiac+structural+and+functional+changes.">Chronic myocardial infarction in the mouse: cardiac structural and functional changes.</a> Cardiovasc Res. 41, 586-593 (1999).</li><li>Frobert, A., Valentin, J., Cook, S., Lopes-Vicente, L., Giraud, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+therapy+for+heart+failure+in+rat:+double+thoracotomy+for+myocardial+infarction+and+epicardial+implantation+of+cells+and+biomatrix.">Cell-based therapy for heart failure in rat: double thoracotomy for myocardial infarction and epicardial implantation of cells and biomatrix.</a> J Vis Exp. , e51390 (2014).</li><li>Takagawa, J., 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=Myocardial+infarct+size+measurement+in+the+mouse+chronic+infarction+model:+comparison+of+area-+and+length-based+approaches.">Myocardial infarct size measurement in the mouse chronic infarction model: comparison of area- and length-based approaches.</a> J Appl Physiol (1985). 102, 2104-2111 (2007).</li><li>Fishbein, M. C., 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=Early+phase+acute+myocardial+infarct+size+quantification:+validation+of+the+triphenyl+tetrazolium+chloride+tissue+enzyme+staining+technique.">Early phase acute myocardial infarct size quantification: validation of the triphenyl tetrazolium chloride tissue enzyme staining technique.</a> Am Heart J. 101, 593-600 (1981).</li><li>Weisman, H. F., Bush, D. E., Mannisi, J. A., Weisfeldt, M. L., Healy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+myocardial+infarct+expansion.">Cellular mechanisms of myocardial infarct expansion.</a> Circulation. 78, 186-201 (1988).</li><li>Mannisi, J. A., Weisman, H. F., Bush, D. E., Dudeck, P., Healy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+administration+after+myocardial+infarction+promotes+early+infarct+expansion.+A+study+in+the+rat.">Steroid administration after myocardial infarction promotes early infarct expansion. A study in the rat.</a> J Clin Invest. 79, 1431-1439 (1987).</li></ol>

The authors have nothing to disclose.

Passos críticos dentro do Protocolo tecido fibrótico pode ser avaliada com precisão em um modelo de rato MI crónica utilizando amostragem sistemática do coração e imagem colhida análises dos cortes histológicos tricromáticas-manchado obtidos a partir da base para o ápice. Dois passos são particularmente importantes para implementação do protocolo de sucesso. Em primeiro lugar, o uso de KCl para a colheita coração permite que o músculo cardíaco a ser mantido...

O infarto do miocárdio (MI) é uma das principais causas de morte e incapacidade em todo o mundo. A doença cardíaca coronária é a causa principal; MI resulta de isquemia consecutiva a eventos coronarianos, como a oclusão. Quando reperfusão não é realizada dentro do primeiro 6 horas, isquemia induz necrose miocárdica irreversível. Em pacientes, o caracterização de MI conta com diferentes ferramentas de diagnóstico, incluindo sinais clínicos, eletrocardiograma, avaliação dos níveis plasmáticos de biomarcadores, ecocardiograma, imagiologia de ressonância magnética, e histológica 1. MI aguda e crónica são classificados como duas fases diferentes da lesão de acordo com a temporização da necrose do miocárdio em relação ao momento da oclusão coronária. A fase aguda, ocorrendo durante os primeiros 7 dias, está associada com a perda de cardiomiócitos, inflamação extensiva, e o recrutamento de fibroblastos. A fase sub-aguda, caracterizada por a cura do tecido cardíaco e a formação de uma cicatriz, ocorreentre 1 e 4 - 6 semanas. Expansão do infarto, afinamento da parede do ventrículo, e dilatação do ventrículo caracterizar a fase crônica. Remodelação extensa do ventrículo esquerdo resulta progressivamente de insuficiência cardíaca grave 2. MI induzida pelo permanente anterior esquerda artéria descendente (LAD) ligadura representa o modelo de roedor padrão de infarto crônico do miocárdio. Os imita ligadura coronária oclusão coronária. O tamanho do enfarte depende do local da ligadura. Caracterização de lesão isquémica do miocárdio em um modelo de roedor é classicamente realizada utilizando os níveis plasmáticos de biomarcadores, como a troponina I e T 3, ecocardiograma, ressonância magnética, e histologia 4,5. níveis de biomarcadores são correlacionados com a extensão da morte de cardiomiócitos. Ecocardiografia avalia a esquerda comprometimento da função ventricular resultante de anormalidades de movimento de parede regional. Além disso, técnicas de imagem não invasivos, como a ressonância magnética ou de alta resoluçãoecocardiografia, permitir a avaliação da redução do movimento da parede, o volume da área da cicatriz com diminuição da perfusão e miocárdio viável, eo afinamento da parede. dimensões do VE permitir a avaliação precisa do tamanho do infarto. Finalmente, a quantificação do miocárdio viável e mortos podem ser post-mortem realizados usando manchas específicas de secções histológicas de corações colhidas e permite a verificação do tamanho do enfarte (IS). Outra característica importante é a avaliação do índice de expansão do enfarte (EI) 6. A EI é associado com o infarto transmural e começa nos primeiros 3 dias. A EI é caracterizada por uma redução progressiva da espessura da parede, um aumento no tamanho da cavidade do VE, e consequentes alterações na forma do LV. A fim de avaliar a eficácia terapêutica de novos tratamentos - em particular, as estratégias baseadas em células regenerativas, matrizes, e o gene de avaliação de entrega precisas de MI em roedores é de importância primordial.Quando medido em uma única seção transversal obtida ao nível dos músculos papilares, o tamanho é pode ser tendenciosa, devido à grande variabilidade que existe no desenvolvimento do infarto seguinte ligadura LAD; o enfarte apex pode ser, em seguida, ocultado. Importante, as metodologias mais precisas para o tamanho determinado MI têm sido descritos para ratinhos ou ratos 7-9 10. No entanto, é insuficiente para quantificar com precisão remodelação LV ou reduções induzidas terapeuticamente (ou prevenções) da remodelação. Com efeito, este modo é geralmente expressa como uma percentagem do volume total do VE avaliada em secções transversais do coração. Embora este método é válido para infarto agudo do miocárdio, o afinamento da parede do LV que ocorre durante a remodelação permanece sub-avaliada 11,12. A quantificação morfométrica completa do tamanho do infarto e mudanças estruturais devem quantificar vários parâmetros, tais como comprimentos do endocárdio e epicárdio e diâmetros, bem como enfarte e áreas saudáveis. Nós descrevemos um aprox metodológicaoach para avaliar com precisão MI e remodelação em um modelo de rato crônica.

<table border="0" cellpadding="0" cellspacing="0" width="775">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acrylic rat heart matrix 2mm</td>
			<td style="width:119px;">72-5015</td>
			<td style="width:136px;">Harvard Appartus</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">INSPIRA ADVANCED VOLUME CONTROLLED VENTILATOR</td>
			<td style="width:119px;">HARVARD APPARATUS</td>
			<td style="width:136px;">557058</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CATHETER INSYTE 14G</td>
			<td style="width:119px;">BD</td>
			<td style="width:136px;">381267</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">O.C.T</td>
			<td style="width:119px;">BDHA361603E</td>
			<td style="width:136px;">VWR</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">TTC</td>
			<td style="width:119px;">T8877-10G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mayer hematoxylin</td>
			<td style="width:119px;">MHS32-1L</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Acid Fuchsin 
			CI 42685</td>
			<td style="width:119px;">F8129-50G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ponceau Xylidin 
			CI 16150</td>
			<td style="width:119px;">P2395-25G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Orange G 
			CI 16230</td>
			<td style="width:119px;">O3756-100G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Light green 
			CI 42095</td>
			<td style="width:119px;">L5382-25G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">KCl</td>
			<td style="width:119px;">P9333-500G</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Xylol</td>
			<td style="width:119px;">10315083</td>
			<td style="width:136px;">HoneyWell</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ethanol absolute</td>
			<td style="width:119px;">10303990</td>
			<td style="width:136px;">HoneyWell</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">2-methylbutane</td>
			<td style="width:119px;">M32631-1L</td>
			<td style="width:136px;">Sigma Aldrich</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Stereogical microscope</td>
			<td style="width:119px;">SM2800</td>
			<td style="width:136px;">Nikon</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Formaldehyde</td>
			<td style="width:119px;">99340</td>
			<td style="width:136px;">Reactolab</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Embedding cassette</td>
			<td style="width:119px;">K113.1</td>
			<td style="width:136px;">Carl Roth</td>
			<td style="width:273px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:247px;">Bersoft Image measurement Software</td>
			<td style="width:119px;"></td>
			<td style="width:136px;">Bersoft.com</td>
			<td style="width:273px;">Licensed software</td>
		</tr>
	</tbody>
</table>

histological quantification of chronic myocardial infarct in rats

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ischemic injury. Following the initial acute phase, chronic remodeling of the left ventricle (LV) modifies the structure and function of the heart. Permanent coronary ligation in small animals has been widely used as a reference model for a chronic model of MI. Thinning of the infarcted wall progressively develops to transmural fibrosis. Histological assessment of infarct size is commonly performed; nevertheless, a standardization of the methods for quantification is missing. Indeed, important methodological aspects, such as the number of sections analyzed and the sampling and quantification methods, are usually not described and therefore preclude comparison across investigations. Too often, quantification is performed on a single section obtained at the level of the papillary muscles. Because novel strategies aimed at reducing infarct expansion and remodeling are under investigation, there is an important need for the standardization of accurate heart sampling protocols. We describe an accurate method to quantify the infarct size using a systematic sampling of harvested rat heart and image analyses of trichromatic stained histological sections obtained from base to apex. We also provide evidence that calculating the expansion index (EI) allowed for infarct size assessment, taking into account changes of the left ventricle throughout the remodeling.

pós-LAD ligadura seis semanas, corações foram colhidas de ratos Lewis. secções de tecido de 2 mm foram obtidas a partir do vértice para a base. Um procedimento de coloração TTC foi realizada para visualizar a área de enfarte, que aparece em branco, eo miocárdio saudável, que aparece em vermelho (Figura 2). Dependendo do local de ligação da ADA, a dimensão do enfarte varia. Para grande MI, infartos transmurais foram observados a partir do vértice para a bas...

Watch this Scientific Journal Video about Histological Quantification of Chronic Myocardial Infarct in Rats at JoVE.com

Histological Quantification of Chronic Myocardial Infarct in Rats

The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections.

Quantificação histológico de miocárdio crônico infarto em ratos

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ...

histological-quantification-of-chronic-myocardial-infarct-in-rats

Research

JoVE Journal

Medicine

14.4K visualizações.  University of Fribourg. The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections. 

Vídeo: Histological Quantification of Chronic Myocardial Infarct in Rats

Acute Myocardial Infarction in Rats

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

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

Infarto Agudo do Miocárdio em Ratos

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

Medicina

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Aguda e crônica teste sensorial tátil após a lesão medular em ratos

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Constrição crônica do nervo ciático e Teste de Hipersensibilidade dor em ratos

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem 1. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema 1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images 1.
	The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery SALLI MRI in Rats

Contrast enhanced cardiac magnetic resonance (CMR) imaging allows comprehensive in vivo assessment of the heart in small animal models of cardiovascular disease. Here we detail the procedures to perform CMR imaging, reconstruction and analysis using Small Animal Lock-Locker Inversion Recovery (SALLI) in rats.

Avaliação da função cardíaca e Morfologia do miocárdio Usando Pequenos Animais Inversion Recovery Olha-locker (SALLI) MRI em ratos

Small animal magnetic resonance  imaging is an important tool to study cardiac function and changes in myocardial  tissue. The high heart rates of small ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Ressonância magnética e PET no rato modelos de Infarto do Miocárdio

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Ultrasonic Assessment of Myocardial Microstructure

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. Ultrasonic assessments of cardiac tissue can include analyses of backscatter signal intensity within a given region of interest. Previously established techniques have relied predominantly on the integrated or mean value of backscatter signal intensities, which may be susceptible to variability from aliased data from low frame rates and time delays for algorithms based on cyclic variation. Herein, we describe an ultrasound-based imaging algorithm that extends from previous methods, can be applied to a single image frame&#160;and accounts for the full distribution of signal intensity values derived from a given myocardial sample. When applied to representative mouse and human imaging data, the algorithm distinguishes between subjects with and without exposure to chronic afterload resistance. The algorithm offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Echocardiography is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. We describe an ultrasound-based imaging algorithm that offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Avaliação ultrassônica da microestrutura miocárdio

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Respostas Bexiga Distensão Evocado Visceromotor como um modelo para Bexiga dor em camundongos

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

Exame ecocardiográfico e histológico da morfologia cardíaca no Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Avaliação da reserva de fluxo coronariano após reperfusão de isquemia miocárdica em ratos

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

Método invasivo semi-mínimo para induzir infarto do miocárdio em ratos e a avaliação da função cardíaca por um sistema cardíaco de trabalho isolado

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...