This work was supported by grant 13IRG14560023 from the American Heart Association and NIH grants AI078972 and HL108054 to CJO. Support for AOB was from the NIH National Center for Advancing Translational Sciences NIH ULTR001120, and F31 A110417701.

NOTA: Todas as experiências com camundongos foram revisadas e aprovadas pelo Animal Care Institucional e Comissões de uso na University of Texas Health Science Center em San Antonio (protocolo nº 13032-34-01C). Cuidados com os animais e os protocolos experimentais aderiram ao Direito Público 89-544 (Animal Welfare Act) e suas alterações, as orientações serviços de saúde pública, e no Guia para o Cuidado e Uso de Animais de Laboratório (Departamento de Saúde e Serviços Humanos dos EUA). 1. Infecção <ol><li> Obter ratinhos BALB / c de ambos os sexos com idades entre 10-12 semanas de idade. NOTA: S. pneumoniae sorotipo 4 estirpe TIGR4 é um isolado clínico virulenta que foi seqüenciado 16. </li><li> Cresça TIGR4 em caldo de Todd-Hewitt a 37 ° C em 5% de CO2 até atingirem uma densidade óptica (DO) 620 de 0,5 (correspondendo a 1,0 x 10 8 unidades formadoras de colónias [] CFU / ml). Agregar a cultura de pneumococos por centrifugação durante 10 min a 3, 500 x g e eliminar o sobrenadante usando uma linha de vácuo. Suspender as bactérias e diluir com solução salina tamponada com fosfato estéril (PBS) a uma concentração final de 1,0 x 10 4 CFU / mL. </li><li> Utilizando uma câmara de indução, anestesiar ratos com 2,5% de isoflurano vaporizado em oxigênio. Confirme estado de anestesia por pitada toe suave com uma pinça sem corte. <ol><li> Segurando o rato anestesiado pela sua nuca e na vertical com uma mão, injetar cada rato por via intraperitoneal (ip) com 100 mL da S. pneumoniae suspensão utilizando uma seringa com uma agulha de calibre 27-30 (correspondente a uma dose de desafio de 1,0 x 10 3 CFU). Coloque a parte de trás do mouse em sua gaiola. Ratos normalmente despertar 30-40 seg após a injeção. </li></ol></li><li> Permitir que a infecção para continuar por pelo menos 24 (antecipado) ou 30 (avançado) hr. <ol><li> Eutanásia dos ratinhos por asfixia com CO 2. Execute deslocamento cervical para garantir rato tem falecido. A eutanásia é ainda confirmada pela remoçãodo coração no passo 1.6. </li></ol></li><li> Desinfetar a cauda com um algodão embebido em álcool e cortar uma seção de 2-3 mm. Recolhe 2 ul de sangue em série e diluir o sangue de 10 vezes em PBS contendo heparina de sódio 1 U / ml de cinco vezes. Diluições em série de placa sobre uma placa de agar tríptico de soja sangue e incubar durante a noite a 37 ° C em 5% de CO 2. O próximo dia de extrapolar colónia conta o nível de bacteremia. </li><li> Imobilizar o mouse em decúbito dorsal sobre uma plataforma cirúrgica. Pulverizar o peito com etanol 70% e seque. Usando uma tesoura cirúrgica e uma pinça abrir a cavidade torácica, retire a caixa torácica, e transecto o diafragma para expor o coração e os pulmões. Usando uma tesoura cortar os vasos sanguíneos ligados ao coração e extirpar suavemente o coração com cuidado para evitar contusões com uma pinça. </li><li> Lavar o coração com PBS e, em seguida, colocar em cassetes de colheita de amostras de tecidos que tais secções coronais seria obtido durante o corte do tecido. Para inclusão em parafina, place as cassetes em 10% solução tamponada com formalina e no dia seguinte enviar para inclusão em parafina. <ol><li> Alternativamente congelamento flash usando outubro Composto dentro de um cryomold que foi colocado em gelo seco. </li></ol></li></ol> 2. A visualização das lesões cardíacas e pneumococos dentro das lesões <ol><li> Reduzir secções de coração embebidos em parafina de tal forma que os quatro câmaras do coração são visíveis na secção de cada tecido. Cortar as lâminas com uma espessura de 5 mm. </li><li> Desparafinar e mancha slides com hematoxilina e eosina (H &amp; E) usando métodos padrão 17. </li><li> Ver H &amp; E manchada seções cardíacos usando um microscópio de luz em baixa (100X, 200X) e alta (imersão em óleo: 400X, 1000X) ampliações. Caracterizar os corações para a presença de microlesões no miocárdio. Aqui, adquirir H &amp; E imagens usando um microscópio Zeiss Axioskop 2 equipado com um 100X 1,3 numérica objetivo abertura Plan-Neofluar. NOTA: Em fases iniciais l cardíacaesions são discriminados pela sua aparência colorida diferencial e, em alguns casos, a presença de células imunes (monócitos e granulócitos) que parecem ser monócitos. Nas lesões avançadas, particularmente aqueles observados em 30 horas, as células imunológicas são lesões vacuole-como ausentes e grandes são observados em seu lugar. É importante notar que, enquanto o influxo de células imunitárias durante as fases iniciais do desenvolvimento da lesão cardíaca pode ocorrer não parece ser um requisito. </li></ol> 3. Immunofluorescent Microscopia para pneumococos dentro Microle- <ol><li> Seção coração em cryomolds com microtome a uma espessura de 5 seções um e colocar em lâminas de vidro com carga positiva. Permitir secções cardíacas congelados para descongelar e ar seco. Fix secções durante 10 minutos em 10% de formalina tamponada neutra (pH 6,8-7,2 a 25 ° C). </li><li> Lavagem das lâminas (3x) em PBS durante 5 min, para permeabilizar 15 min em 0,2% de Triton X-100 em PBS, em seguida, lavar novamente em PBS. </li><li> Secti tecido Bloco<html> em em lâminas com 10% de soro de cabra em PBS durante 1 hora. </li><li> Lavar as lâminas com PBS, e incubar tampa com secções cardíacos de coelho anti-pneumococos do serotipo 4 anti-soro a 1: 1000 em PBS com 10% de soro de cabra durante 2 horas a 37 ° C. NOTA: Para os controles negativos investigadores devem usar ingênuo anti-soro de coelho </li><li> Após lavagem com PBS, a tampa e incubar secções com 10% de soro de cabra-PBS com cabra anti-coelho FITC anticorpo conjugado a 1: 2000 durante 30 min a 37 ° C. </li><li> Seguindo as instruções do fabricante, usar DAPI (4 &#39;, 6- diamidino-2-fenilindolo) em 5 mg / ml para a visualização de núcleos eucarióticos. Lave e montar secções de tecido com FluorSave. </li><li> Adquirir imagens fluorescentes utilizando um sistema de microscópio confocal tanto a baixa como alta ampliação. Ajustar a abertura numérica em conformidade para se obter resultados óptimos. Aqui, adquirir imagens fluorescentes usando um sistema confocal com um objetivo abertura numérica 60X 1,42. </li></ol>&quot;&gt; 4. Salvamento Antibiótico de fossa séptica Mice <ol><li> Comece com ratinhos infectados ip com 1,0 x 10 3 CFU de TIGR4 como anteriormente indicado na etapa 1. </li><li> Começando às 30 horas pós-infecção, administrar farmacêutica ampicilina grau ip (80 mg / kg de peso corporal) em 100 ul de solução salina a cada 12 h durante 36 h. Recolha corações no ponto de tempo desejado e processo tal como descrito no passo 1.6 para inclusão em parafina e tecido, o seccionamento. </li></ol> 5. Collagen Detection Deposition <ol><li> Usando xileno e lavagens álcool graduado com água, Desparafinar e tecidos hidrato seções deionizada usando métodos padrão 17. Tratar secções com ácido fosfomolíbdico aquosa a 0,2% durante 5 minutos e lavar com água desionizada durante 5 minutos. </li><li> Secções mancha com 0,1% de vermelho Sirius em ácido pícrico de 2 h à temperatura ambiente. </li><li> Lave os cortes em ácido clorídrico 0,01 N durante 3 min e enxaguar utilizando etanol a 70% durante 1 min. </li><li> Desidrataçãote as seções usando o idêntico, mas a fim de lavagens ao indicado na 5.1 reverter, desidratar seções cardíacas no aumento e classificados concentrações de etanol, em seguida, xileno. </li><li> Cortes de tecido Mount com uma solução de montagem para avaliação microscópica. </li><li> Para identificar as regiões de deposição de colágeno, examinar secções de tecido manchado por áreas de coloração leitura profunda com baixa e alta ampliação usando um microscópio de luz. Qualquer área dentro dos ventrículos do coração, que é o vermelho é uma área onde o colágeno tem sido depositado. Áreas sem essa mancha vermelha são locais de tecido saudável falta deposição de colágeno. Aqui, adquirir Picro Sirius Red imagens coradas utilizando um microscópio equipado com um objetivo abertura numérica 20X 0,5. NOTA: As manchas vermelhas no átrio tecidos saudáveis. </li></ol>

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The authors declare no conflict of interest in this study.

Neste relatório, um método altamente reprodutível para induzir S. pneumoniae mediada microlesões cardíacos em ratos durante IPD e as técnicas para a sua visualização é demonstrada. Os ratinhos são infectados com uma dose de bactérias que ultrapassa o limiar do sistema imunitário para a depuração, que conduz a bacteremia de alto grau, semelhante ao que ocorre durante a sepsia humano, e leva a uma eventual translocação bacteriana no miocárdio. Por razões ainda desconhecidas, S. pneumoniae</e...

Internação de adultos para pneumonia adquirida na comunidade (PAC) carrega um risco documentado para eventos cardíacos adversos que contribui para a mortalidade 1-4. Em um estudo recente realizado por Corrales-Medina et al. Complicações cardíacas foram encontrados para ser associado com e / ou responsáveis ​​por 27% dos óbitos associados à pneumonia 3. Streptococcus pneumoniae (pneumococo), a causa mais comum de PAC e sepse 5, foi diretamente associada a eventos cardíacos adversos em tantos como 19% dos pacientes adultos internados 6. Eventos cardíacos adversos associados com pneumonia incluem falha nova ou agravada cardíaca congestiva, arritmias e infarto do miocárdio em ordem decrescente de freqüência de 6. Num artigo recente PLoS Pathogens por Brown et al., S. pneumoniae foi encontrado para ser capaz de translocação através do endotélio vascular cardíaca, a entrada no myocardium, e formação de lesões microscópicas não purulentos discretos (microlesões) cheios de bactérias nos ventrículos durante a doença pneumocócica invasiva (DPI) 7. Evidência de formação microlesion cardíaca foi observada em amostras cardíacas a partir de primatas não humanos e os indivíduos que tinham sucumbido à infecção pneumocócica. Da mesma forma, camundongos infectados experimentalmente reproducibly desenvolvido microlesões cardíacos. Em camundongos, o tamanho microlesion e número foi positivamente correlacionada com a duração e gravidade de bacteremia, os níveis de troponina cardíaca em soros e eletrofisiologia cardíaca aberrante. A translocação bacteriana para o coração foi observado que ocorre através dos mesmos mecanismos responsáveis ​​pela translocação do pneumococo através da barreira sangue-cérebro e o desenvolvimento de meningite pneumocócica, ou seja, a proteína de ligação a colina A invasão mediada por células endoteliais vasculares em um receptor de laminina e Plaquetas receptor do fator de -activating forma dependente 8. Microlesformação de iões também exigiu a pneumocócica toxina pneumolisina de formação de poros que foi encontrado para matar cardiomiócitos 7. Microlesões cardíacos pneumocócicas são distintas do soft-tecido purulenta e abcessos cardíacos que são causadas por outras bactérias Gram-positivas, incluindo Staphylococcus aureus. Estes são caracterizados por um único focos das bactérias rodeadas por neutrófilos e a deposição de fibrina 9,10. Microle- formado por S. pneumoniae são menores em tamanho, distribuídos por todo o coração afetado, e carecem de infiltrados de células imunes. Durante as fases iniciais da infecção, microlesões causada por S. pneumoniae aparecem como áreas de tecido danificado ou inflamado reminiscente dos sinais patológicos de cardiomiopatia. Alguns monócitos podem ser observados durante este tempo, no entanto a sua presença é de curta duração e lesões rapidamente tornam-se necróticas na aparência e cheia de bactérias ao mesmo tempo continuar a GRow em tamanho até a morte do animal ou intervenção antimicrobiana. É importante salientar, no prazo de 3 dias de intervenção antibiótico, infiltração de células imunes profusa é observada no antigo local da lesão e isso é acompanhado por deposição de colágeno robusto. Remodelação cardíaca semelhante foi relatado para ocorrer após o infarto, juntamente com consequências duradouras na função cardíaca 11-15. Assim, microlesões são uma possível explicação para os eventos cardíacos adversos que ocorrem durante o IPD e, possivelmente, o aumento da incidência de mortalidade cardíaca relacionada com em indivíduos convalescentes que sobreviveram o episódio da doença. Nisto, a instrução é fornecido no modelo experimental de rato IPD e a formação da lesão cardíaca e visualização de microlesões cardíacas em estágios precoces e tardios da infecção. O protocolo para detecção de deposição de colágeno nos animais que foram salvos pela intervenção antimicrobiana é demonstrada. O objetivo deste artigo é o de facilitate a pesquisa de outros pesquisadores sobre esta patologia pneumocócica importante e romance.

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			<td height="21" style="height:21px;width:308px;">Name of Reagent/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:308px;">Todd-Hewitt broth&#160;</td>
			<td style="width:175px;">Neogen</td>
			<td>7161A</td>
			<td></td>
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			<td height="42" style="height:42px;width:308px;">O.C.T Compound&#160;</td>
			<td style="width:175px;">Tissue-Tek (Sakura Finetek)&#160;</td>
			<td style="width:119px;">4583</td>
			<td></td>
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			<td style="width:175px;">Fisher Scientific (Acros)</td>
			<td>9002-93-1</td>
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			<td height="21" style="height:21px;width:308px;">Normal Goat Serum</td>
			<td style="width:175px;">Abcam</td>
			<td style="width:119px;">ab7481</td>
			<td></td>
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			<td height="21" style="height:21px;">anti-serotype 4 pneumococcus antiserum&#160;</td>
			<td>Statens Serum Institut</td>
			<td style="width:119px;">16747</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">goat anti-rabbit FITC conjugated antibody&#160;</td>
			<td style="width:175px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">111-096-144</td>
			<td></td>
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			<td height="21" style="height:21px;width:308px;">DAPI</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">D1306</td>
			<td></td>
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			<td height="21" style="height:21px;width:308px;">Fluorsave</td>
			<td style="width:175px;">Millipore</td>
			<td>345789</td>
			<td></td>
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			<td height="21" style="height:21px;width:308px;">Permount</td>
			<td>Fisher Scientific</td>
			<td>S70104</td>
			<td></td>
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			<td height="21" style="height:21px;width:308px;">Ampicillin</td>
			<td style="width:175px;">Sigma</td>
			<td>A9393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">phosphomolybdic acid 0.2%&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>26357-01</td>
			<td></td>
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			<td height="21" style="height:21px;width:308px;">0.1% Sirius Red in picric acid&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>26357-02</td>
			<td></td>
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			<td height="21" style="height:21px;">0.01 N hydrochloric acid&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>26357-03</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Tissue Tack Microscope Slides</td>
			<td style="width:175px;">Polysciences, Inc</td>
			<td style="width:119px;">24216</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Paralube Vet Ointment</td>
			<td style="width:175px;">Dechra</td>
			<td>12920060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Hematoxylin and Eosin Staining Kit</td>
			<td style="width:175px;">unspeciified&#160;</td>
			<td style="width:119px;"></td>
			<td>Standard</td>
		</tr>
	</tbody>
</table>

visualization of streptococcus pneumoniae within cardiac microlesions and subsequent cardiac remodeling

Durante a bacteremia Streptococcus pneumoniae pode translocar através do endotélio vascular para o miocárdio e formar bactérias cheios de lesões microscópicas discretas (microlesões) que são notáveis ​​devido à ausência de infiltrado de células imunitárias. Devido a sua liberação de produtos cardiotóxicos, S. pneumoniae dentro microlesões são pensados ​​para contribuir para a insuficiência cardíaca que é freqüentemente observado durante a doença pneumocócica invasiva fulminate em adultos. Nisto é demonstrado um protocolo para a infecção experimental do rato que leva à formação reprodutíveis microlesion cardíaca dentro de 30 horas. Instrução é fornecida na identificação microlesion em hematoxilina &amp; eosina seções coração manchado e as distinções morfológicas entre microlesões precoces e tardias são realçados. Instrução é fornecida em um protocolo de verificação de S. pneumoniae dentro microlesões usando anticorpos contra polissacarídeo capsular e pneumocócicaA microscopia de imunofluorescência. Por último, um protocolo para a intervenção antibiótico que resgata ratinhos infectados e para a detecção e avaliação da formação de cicatriz no coração dos ratos convalescentes é fornecido. Juntos, esses protocolos irá facilitar a investigação dos mecanismos moleculares subjacentes invasão cardíaca pneumocócica, a morte de cardiomiócitos, remodelação cardíaca como resultado da exposição a S. pneumoniae, e a resposta imune para o pneumococos no coração.

Visualização de microlesões por H &amp; E Microlesões cardíacas foram observadas nos ventrículos de H &amp; E coradas secções cardíacas de ratos com IPD seguintes seu desafio ip com TIGR4. A Figura 1 demonstra o aumento no tamanho das lesões entre 24 e 30 horas pós-infecção (hpi), com maior número de hematoxilina -stained (ie, azul-púrpura) pneumococos visível no interior da lesão. Microle- foram caracterizadas por uma morfologia semelhante...

Watch this Scientific Journal Video about Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling at JoVE.com

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals.

Visualização de<em&gt; Streptococcus pneumoniae</em&gt; Dentro Microle- cardíacas e remodelação cardíaca Subsequente

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled ...

visualization-streptococcus-pneumoniae-within-cardiac-microlesions

Research

JoVE Journal

Medicine

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

8.9K visualizações.  The University of Texas Health Science Center at San Antonio. Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals. 

Vídeo: Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Teste de estresse cardíaco induzido por dobutamina e Monitorado por cateterismo cardíaco em ratos

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

Medicina

Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation

Cryoballoon ablation (CBA) is an established therapy for atrial fibrillation (AF). Pulmonary vein (PV) occlusion is essential for achieving antral contact and PV isolation and is typically assessed by contrast injection. We present a novel method of direct pressure monitoring for assessment of PV occlusion.
Transcatheter pressure is monitored during balloon advancement to the PV antrum. Pressure is recorded via a single pressure transducer connected to the inner lumen of the cryoballoon. Pressure curve characteristics are used to assess occlusion in conjunction with fluoroscopic or intracardiac echocardiography (ICE) guidance. PV occlusion is confirmed when loss of typical left atrial (LA) pressure waveform is observed with recordings of PA pressure characteristics (no A wave and rapid V wave upstroke). Complete pulmonary vein occlusion as assessed with this technique has been confirmed with concurrent contrast utilization during the initial testing of the technique and has been shown to be highly accurate and readily reproducible.
We evaluated the efficacy of this novel technique in 35 patients. A total of 128 veins were assessed for occlusion with the cryoballoon utilizing the pressure monitoring technique; occlusive pressure was demonstrated in 113 veins with resultant successful pulmonary vein isolation in 111 veins (98.2%). Occlusion was confirmed with subsequent contrast injection during the initial ten procedures, after which contrast utilization was rapidly reduced or eliminated given the highly accurate identification of occlusive pressure waveform with limited initial training.
Verification of PV occlusive pressure during CBA is a novel approach to assessing effective PV occlusion and it accurately predicts electrical isolation. Utilization of this method results in significant decrease in fluoroscopy time and volume of contrast.

Effective pulmonary vein isolation utilizing a cryoballoon depends on complete pulmonary vein occlusion. The point of occlusion can be effectively predicted by direct analysis of pulmonary vein pressure waveform analysis during balloon inflation using a simple and reproducible technique.

Monitorização da Pressão direta é capaz de predizer oclusão da veia pulmonar durante ablação Cryoballoon

Cryoballoon  ablation (CBA) is an established therapy for atrial fibrillation (AF).  Pulmonary vein (PV) occlusion is essential for achieving antral ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

Um Modelo de Cardiologia remodela Através constrição da Aorta Abdominal em Ratos

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

preparação e<em&gt; In Vitro</em&gt; Caracterização de magnetizado miR-modificado Células Endoteliais

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

Ressonância magnética cardíaca para avaliação de suspeita de trombo cardíaco: técnicas convencionais e emergentes

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Isolamento e purificação de Pericytes cardíacos murinos

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

Um modelo de roedor isquêmico de pericárdio intacto

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...

Robotized Testing of Camera Positions to Determine Ideal Configuration for Stereo 3D Visualization of Open-Heart Surgery

Stereo 3D video from surgical procedures can be highly valuable for medical education and improve clinical communication. But access to the operating room and the surgical field is restricted. It is a sterile environment, and the physical space is crowded with surgical staff and technical equipment. In this setting, unobscured capture and realistic reproduction of the surgical procedures are difficult. This paper presents a method for rapid and reliable data collection of stereoscopic 3D videos at different camera baseline distances and distances of convergence. To collect test data with minimum interference during surgery, with high precision and repeatability, the cameras were attached to each hand of a dual-arm robot. The robot was ceiling-mounted in the operating room. It was programmed to perform a timed sequence of synchronized camera movements stepping through a range of test positions with baseline distance between 50-240 mm at incremental steps of 10 mm, and at two convergence distances of 1100 mm and 1400 mm. Surgery was paused to allow 40 consecutive 5-s video samples. A total of 10 surgical scenarios were recorded.

The human depth perception of 3D stereo videos depends on the camera separation, point of convergence, distance to, and familiarity of the object. This paper presents a robotized method for rapid and reliable test data collection during live open-heart surgery to determine the ideal camera configuration.

Teste robotizado de posições da câmera para determinar configuração ideal para visualização estéreo 3D de cirurgia de coração aberto

Stereo 3D video from surgical procedures can be highly valuable for medical education and improve clinical communication. But access to the operating room ...

Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ donor pool. However, the DCD process is associated with the risk of cardiac tissue injury due to the inevitable period of warm ischemia. Normothermic regional perfusion (NRP) allows for an in situ organ assessment, allowing the procurement of hearts determined to be viable. Here, we describe a clinically relevant large-animal model of DCD followed by NRP. Circulatory death is established in anesthetized pigs by stopping mechanical ventilation. After a preset warm ischemia period, an extracorporeal membrane oxygenator (ECMO) is used for a NRP period lasting at least 30 min. During this reperfusion period, the model allows the collection of various myocardial biopsies and blood samples for initial cardiac evaluation. Once NRP is weaned, biochemical, hemodynamic, and echocardiographic assessments of cardiac function and metabolism can be performed before organ procurement. This protocol closely simulates the clinical scenario previously described for DCD and NRP in heart transplantation and has the potential to facilitate studies aimed at decreasing ischemia-reperfusion injury and enhance cardiac functional preservation and recovery.

The protocol describes a large-animal (porcine) model of donation after circulatory death, followed by thoracoabdominal normothermic regional perfusion that closely simulates the clinical scenario in heart transplantation, and has the potential to facilitate therapeutic studies and strategies.

Modelo de Doação em Animais de Grande Porte após Morte Circulatória e Perfusão Regional Normotérmica para Avaliação Cardíaca

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ ...

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury and/or remodeling. One approach for assessing these functional alterations utilizes unloaded shortening and Ca2+ transient analyses in primary adult cardiac myocytes. For this approach, adult myocytes are isolated by collagenase digestion, made Ca2+ tolerant, and then adhered to laminin-coated coverslips, followed by electrical pacing in serum-free media. The general protocol utilizes adult rat cardiac myocytes but can be readily adjusted for primary myocytes from other species. Functional alterations in myocytes from injured hearts can be compared to sham myocytes and/or to in vitro therapeutic treatments. The methodology includes the essential elements needed for myocyte pacing, along with the cell chamber and platform components. The detailed protocol for this approach incorporates the steps for measuring unloaded shortening by sarcomere length detection and cellular Ca2+ transients measured with the ratiometric indicator Fura-2 AM, as well as for raw data analysis.

Analysis of Cardiac Contractile Dysfunction and Ca2+ Transients in Rodent Myocytes

A set of protocols are presented that describe the measurement of contractile function via sarcomere length detection along with calcium (Ca2+) transient measurement in isolated rat myocytes. The application of this approach for studies in animal models of heart failure is also included.

Análise da disfunção contrátil cardíaca e dos transientes de Ca2+ em miócitos de roedores

Contractile dysfunction and Ca2+ transients are often analyzed at the cellular level as part of a comprehensive assessment of cardiac-induced injury ...