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: Todos los experimentos con ratones fueron revisados ​​y aprobados por los Comités Use y Cuidado de Animales Institucional de la Universidad de Texas Health Science Center en San Antonio (protocolo # 13032-34-01C). Cuidado de los animales y los protocolos experimentales se adhirieron a la Ley Pública 89-544 (Ley de Protección de los Animales) y sus modificaciones, directrices servicios de salud pública, y la Guía para el Cuidado y Uso de Animales de Laboratorio (Departamento de Salud y Servicios Humanos de Estados Unidos). 1. Infección <ol><li> Obtener ratones BALB / c de ambos sexos entre las edades de 10 a 12 semanas de edad. NOTA: S. pneumoniae serotipo 4 cepa TIGR4 es un aislado clínico virulento que ha sido secuenciado 16. </li><li> Crecer TIGR4 en caldo de Todd-Hewitt a 37 ° C en 5% de CO 2 hasta alcanzar una densidad óptica (OD) 620 de 0,5 (correspondiente a 1,0 x 10 8 unidades formadoras de colonias [CFU / ml)]. Sedimentar la cultura neumocócica por centrifugación durante 10 min a 3, 500 xg y eliminar el sobrenadante usando una línea de vacío. Suspender y bacterias con solución salina tamponada con fosfato estéril (PBS) se diluye hasta una concentración final de 1,0 x 10 4 CFU / ml. </li><li> El uso de una cámara de inducción, anestesiar a los ratones con 2,5% de isoflurano vaporizado en oxígeno. Confirme estado anestesiado por pizca dedo del pie suave con pinzas romas. <ol><li> Sosteniendo el ratón anestesiado por su pescuezo y en posición vertical con una mano, se inyectan cada ratón por vía intraperitoneal (ip) con 100 l de la S. pneumoniae suspensión con una jeringa con una aguja de calibre 27-30 (correspondiente a una dosis de prueba de 1,0 x 10 3 CFU). Coloque la parte posterior del ratón en su jaula. Los ratones normalmente despiertan 30-40 segundos después de la inyección. </li></ol></li><li> Deje que la infección de proceder por lo menos 24 (anticipada) o 30 (avanzado) hr. <ol><li> La eutanasia a los ratones por asfixia de CO 2. Realizar dislocación cervical para garantizar ratón ha fallecido. La eutanasia se ve confirmado por la eliminacióndel corazón en el paso 1.6. </li></ol></li><li> Desinfectar la cola con un algodón con alcohol y cortar una sección de 2.3 mm. Recoger 2 l de sangre en serie y diluir la sangre de 10 veces en PBS que contenía heparina sódica 1 U / ml cinco veces. Placa de diluciones en serie en una placa de agar sangre de soja tríptico y se incuba durante la noche a 37 ° C en 5% de CO 2. El siguiente extrapolar días de la colonia cuenta el nivel de bacteriemia. </li><li> Inmovilizar el ratón en una posición supina sobre una plataforma quirúrgica. Rocíe el pecho con etanol al 70% y secar. Con unas tijeras quirúrgicas y pinzas abren la cavidad torácica, retire la caja torácica, y seccionar el diafragma para exponer el corazón y los pulmones. Con unas tijeras cortan los vasos sanguíneos conectados al corazón y los impuestos especiales suavemente el corazón con cuidado para evitar hematomas con fórceps. </li><li> Enjuague el corazón con PBS y luego se coloca en casetes de recogida de muestras de tejido tal que las secciones coronales se obtendrían durante el corte del tejido. Para la inclusión en parafina, place los cassettes en 10% de solución amortiguadora de formalina y al día siguiente para enviar inclusión en parafina. <ol><li> Alternativamente congelación ultrarrápida usando Compuesto OCT dentro de un criomolde que se ha colocado en hielo seco. </li></ol></li></ol> 2. La visualización de las lesiones cardiacas y neumococos dentro de las lesiones <ol><li> Reduzca secciones cardíacos incluidos en parafina de tal manera que las 4 cámaras del corazón son visibles en cada sección de tejido. Cortar las diapositivas en espesor de 5 micras. </li><li> Desparafinar y mancha diapositivas con hematoxilina y eosina (H &amp; E) utilizando métodos estándar 17. </li><li> Ver H &amp; E manchadas secciones cardiacas utilizando un microscopio de luz a baja (100X, 200X) y alta (aceite de inmersión: 400X, 1.000X) aumentos. Caracterizar corazones la presencia de microlesiones en el miocardio. En este caso, la adquisición de H &amp; E imágenes utilizando un microscopio Zeiss Axioskop 2 equipado con un 100X 1.3 numérica objetivo abertura Plan-Neofluar. NOTA: en las primeras etapas l cardiacaesions son discriminados por su apariencia de color diferencial y en algunos casos la presencia de células inmunes (monocitos y granulocitos) lo que parecen ser los monocitos. En lesiones avanzadas, en particular los que se observan en 30 h, las células inmunes son lesiones vacuolas como ausentes y grandes se observan en su lugar. Es importante observar que mientras que puede ocurrir la afluencia de células inmune durante las primeras etapas del desarrollo de la lesión cardiaca no parece ser un requisito. </li></ol> 3. inmunofluorescencia microscopía para neumococos dentro microlesiones <ol><li> Sección corazón en criomoldes con micrótomo hasta un espesor de 5 micras y secciones lugar en portaobjetos de vidrio cargados positivamente. Permitir secciones cardiacas congelados se descongelen y secar al aire. Fijar secciones durante 10 min en 10% de formalina tamponada neutra (pH 6.8 a 7.2 a 25 ° C). </li><li> Lavado de los portaobjetos (3x) en PBS durante 5 min, permeabilizar durante 15 min en 0,2% de Triton X-100 en PBS, luego se lava de nuevo en PBS. </li><li> Secti tejido Bloquear<html> en el portaobjetos con 10% de suero de cabra en PBS durante 1 hora. </li><li> Se lavan los portas con PBS, tapa e incubar las secciones cardiacas con conejo anti-serotipo 4 neumococo antisuero a escala 1: 1000 en PBS con 10% de suero de cabra durante 2 horas a 37 ° C. NOTA: Para los controles negativos investigadores deben utilizar antisuero de conejo ingenuo </li><li> Después de lavar con PBS, la cubierta e incubar las secciones con 10% de suero de cabra-PBS con cabra anti-conejo FITC anticuerpo conjugado a 1: 2000 durante 30 minutos a 37 ° C. </li><li> Siguiendo las instrucciones del fabricante, utilice DAPI (4 &#39;, 6- diamidino-2-fenilindol) a 5 mg / ml para la visualización de los núcleos eucariotas. Lave y montar las secciones de tejido con Fluorsave. </li><li> Adquirir imágenes fluorescentes utilizando un sistema de microscopía confocal, tanto en baja y alta magnificación. Ajuste la apertura numérica en consecuencia para obtener resultados óptimos. Aquí, adquirir imágenes fluorescentes utilizando un sistema confocal con un objetivo 60X apertura numérica 1,42. </li></ol>&quot;&gt; 4. Antibiótico Rescate de ratones séptico <ol><li> Comience con ratones infectados ip con 1,0 x 10 3 UFC de TIGR4 como se indica anteriormente en el paso 1. </li><li> A partir de las 30 horas después de la infección, administrar farmacéutica ampicilina grado ip (80 mg / kg de peso corporal) en 100 l de solución salina cada 12 horas durante 36 horas. Recoge los corazones en el punto de tiempo deseado y el proceso como se describe en el paso 1.6 para la inclusión en parafina y el tejido, el corte. </li></ol> 5. El colágeno Detección Deposición <ol><li> El uso de xileno y lavados de alcohol graduadas con agua, Desparafinar y tejidos hidrato secciones desionizada utilizando métodos estándar 17. Tratar secciones con ácido acuoso 0,2% fosfomolıbdico durante 5 minutos y enjuagar con agua desionizada durante 5 min. </li><li> Secciones mancha con 0,1% rojo sirio en ácido pícrico durante 2 horas a temperatura ambiente. </li><li> Lavar las secciones en ácido clorhídrico 0,01 N durante 3 min y enjuagar el uso de etanol al 70% durante 1 min. </li><li> DeshidrataciónTA El secciones usando el idéntico pero el orden de lavados al descrito en 5.1 revertir, deshidratar secciones cardiacas en el aumento y la clasificación concentraciones de etanol y después xileno. </li><li> Secciones de tejido monte con una solución de montaje para la evaluación microscópica. </li><li> Para identificar las regiones del depósito de colágeno, examinar secciones de tejido teñido de áreas de tinción de lectura profunda en condiciones de baja y alta magnificación utilizando un microscopio de luz. Cualquier área dentro de los ventrículos del corazón, que es de color rojo es un área donde el colágeno se ha depositado. Áreas sin esta mancha roja son sitios de tejido sano que carecen de la deposición de colágeno. Aquí, adquirir Picro Sirius Red imágenes teñidas utilizando un microscopio equipado con un objetivo 20X apertura numérica 0.5. NOTA: Las manchas rojas atrio en los tejidos sanos. </li></ol>

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The authors declare no conflict of interest in this study.

En este informe, un método altamente reproducible para inducir S. pneumoniae mediada microlesiones cardíacas en ratones durante IPD y las técnicas para su visualización se demuestra. Los ratones se infectan con una dosis de bacterias que supera el umbral del sistema inmune para el despacho, dando lugar a bacteremia de alto grado, similar a la que ocurre durante la sepsis humana, y conduce a la translocación bacteriana eventual en el miocardio. Por razones aún desconocidas, S. pneumoniae en el cor...

La hospitalización de adultos para la neumonía adquirida en la comunidad (NAC) conlleva un riesgo documentado de eventos cardiacos adversos que contribuyen a la mortalidad 1-4. En un estudio reciente de Corrales-Medina et al. Se encontraron complicaciones cardíacas que se asocia con y / o responsable del 27% de las muertes por neumonía asociada a 3. Streptococcus pneumoniae (neumococo), la causa más común de la PAC y la sepsis 5, se ha asociado directamente con eventos cardíacos adversos en hasta el 19% de los pacientes adultos ingresados ​​6. Eventos cardíacos adversos asociados con la neumonía son la insuficiencia nuevo o empeorado cardíaca congestiva, arritmias, infarto de miocardio y en orden decreciente de frecuencia 6. En un reciente artículo de PLoS Pathogens por Brown et al., S. pneumoniae se encontró que era capaz de traslocación a través del endotelio vascular cardíaco, la entrada en el myocardium, y la formación de lesiones microscópicas no purulentas discretas (microlesiones) llenos de bacterias en los ventrículos durante la enfermedad neumocócica invasiva (ENI) 7. Se observó evidencia de formación microlesión cardiaca en muestras cardíacas de los primates no humanos y personas que habían sucumbido a la infección neumocócica. Asimismo, los ratones infectados experimentalmente desarrollaron de manera reproducible microlesiones cardíacas. En los ratones, el tamaño y el número microlesión se correlacionó positivamente con la duración y la gravedad de la bacteriemia, los niveles de troponina cardíaca en el suero, y la electrofisiología cardiaca aberrante. La translocación bacteriana en el corazón se encontró que se produzca a través de los mismos mecanismos responsables de la translocación del neumococo través de la barrera sangre-cerebro y el desarrollo de la meningitis neumocócica, es decir, la proteína de unión a colina-A mediada por invasión de células endoteliales vasculares en un receptor de laminina y de plaquetas receptor del factor de -activating manera dependiente de 8. Microlesformación de iones también requiere la neumolisina neumocócica toxina formadora de poros que se encontró para matar a los cardiomiocitos 7. Microlesiones cardíacos neumocócicas son distintos del purulenta de partes blandas y abscesos cardíacas que son causadas por otras bacterias Gram-positivas, incluido el Staphylococcus aureus. Estos se caracterizan por una sola focos de bacterias rodeadas por los neutrófilos y 9,10 deposición de fibrina. Microlesiones formado por S. pneumoniae son de menor tamaño, distribuidos a lo largo de un corazón afectado, y carecen de infiltrados de células inmunes. Durante las primeras etapas de la infección, microlesiones causadas por S. pneumoniae aparecen como áreas de tejido dañado o inflamado evocador de las muestras patológicas de la cardiomiopatía. Algunos monocitos se pueden observar durante este tiempo, sin embargo, su presencia es de corta duración y las lesiones se convierten rápidamente necrótico en apariencia y llena de bacterias, mientras que al mismo tiempo continuar a GRujo de tamaño hasta la muerte del animal o la intervención antimicrobiana. Es importante destacar que, en el plazo de 3 días de la intervención a los antibióticos, profusa infiltración de células inmunes se observa en el antiguo sitio de la lesión y esto va acompañado de robusto depósito de colágeno. La remodelación cardiaca similar se ha reportado que ocurre después de un infarto junto con consecuencias duraderas sobre la función cardíaca 11-15. Por lo tanto, microlesiones son una posible explicación para los eventos cardíacos adversos que ocurren durante el IPD y, posiblemente, el aumento de la incidencia de la mortalidad relacionada con el corazón en personas convalecientes que han sobrevivido al episodio de la enfermedad. En este documento, se proporciona la instrucción en el modelo experimental de ratón de IPD y formación de la lesión cardiaca y la visualización de microlesiones cardíacos en las primeras y últimas etapas de la infección. El protocolo para la detección de la deposición de colágeno en los animales que han sido salvados por la intervención antimicrobiana se demuestra. El objetivo de este artículo es facilitar la investigación de otros investigadores sobre este importante y novedoso patología neumocócica.

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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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Todd-Hewitt broth&#160;</td>
			<td style="width:175px;">Neogen</td>
			<td>7161A</td>
			<td></td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100&#160;</td>
			<td style="width:175px;">Fisher Scientific (Acros)</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">DAPI</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Fluorsave</td>
			<td style="width:175px;">Millipore</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:308px;">Permount</td>
			<td>Fisher Scientific</td>
			<td>S70104</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.01 N hydrochloric acid&#160;</td>
			<td>Electron Microscopy Sciences</td>
			<td>26357-03</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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 bacteriemia Streptococcus pneumoniae puede trasladar a través del endotelio vascular en el miocardio y formar bacterias llenos discretas lesiones microscópicas (microlesiones) que son notable debido a la ausencia de la infiltración de células inmunes. Debido a su liberación de productos cardiotóxicos, S. pneumoniae dentro microlesiones se cree que contribuyen a la insuficiencia cardíaca que se observa con frecuencia durante la enfermedad neumocócica invasiva fulminante en adultos. En esto se demuestra un protocolo para la infección experimental de ratón que lleva a la formación reproducible microlesión cardíaco dentro de 30 horas. La instrucción es proporcionada en la identificación microlesión en hematoxilina y eosina secciones de corazón de colores y las distinciones morfológicas entre microlesiones principios y finales se destacan. La instrucción es proporcionada en un protocolo para la verificación de S. pneumoniae dentro microlesiones utilizando anticuerpos contra el polisacárido capsular de neumococo ymicroscopía de inmunofluorescencia. Por último, se proporciona un protocolo de intervención antibiótico que rescata los ratones infectados y para la detección y evaluación de la formación de cicatrices en los corazones de los ratones convalecientes. Juntos, estos protocolos facilitar la investigación de los mecanismos moleculares que subyacen a la invasión cardiaca neumocócica, muerte de los cardiomiocitos, la remodelación cardiaca como resultado de la exposición a S. pneumoniae, y la respuesta inmune a los neumococos en el corazón.

Visualización de microlesiones por H &amp; E Se observaron microlesiones cardíacos en los ventrículos del H &amp; E manchadas secciones cardíacas de ratones con IPD tras su desafío ip con TIGR4. La Figura 1 muestra el aumento en el tamaño de estas lesiones entre 24 y 30 horas después de la infección (hpi), con un mayor número de hematoxilina teñidas (es decir, púrpura-azul) neumococos visible dentro de la lesión. Microlesiones se caracterizaron ...

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.

Visualización de<em&gt; Streptococcus pneumoniae</em&gt; Dentro cardíacos microlesiones y la remodelación cardiaca posterior

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 vistas.  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. 

Video: 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.

Prueba de esfuerzo cardíaco inducida por dobutamina y supervisado por cateterismo cardíaco en ratones

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

Investigación

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.

Monitoreo de la Presión de Oclusión predice con precisión las venas pulmonares durante la ablación 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.

Un modelo de remodelamiento cardiaco A través de la constricción de la aorta abdominal en ratas

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.

Preparación y<em&gt; In Vitro</em&gt; Caracterización de Magnetized miR-modificado células endoteliales

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.

Resonancia Magnética Cardíaca para la Evaluación del Trombo Cardíaco Sospechoso: Técnicas Convencionales y 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.

Aislamiento y Purificación de Pericitas 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.

Un modelo de roedor isquémico de pericardio 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.

Pruebas robotizadas de las posiciones de la cámara para determinar la configuración ideal para la visualización estéreo en 3D de la cirugía a corazón abierto

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 animal grande de donación después de la muerte circulatoria y perfusión regional norterámica para la evaluación 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.

Análisis de disfunción contráctil cardíaca y transitorios de Ca2+ en miocitos de roedores

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