Agradecemos al Dr. Kelly Stevens para las pruebas iniciales del protocolo, el Dr. Michael Simons, Hauch Dr. Kip, y los miembros de sus dos laboratorios para la discusión general. Este trabajo es el apoyo de subvenciones del NIH HL087513 y HL094374 P01.

1. Los preparativos antes de iniciar <ol><li> Llenar cada lado del aparato de presión de perfusión con tampón Vasodilatador (4 mg / l papaverina + 1g / l adenosina en PBS) o 4% de paraformaldehído (PFA) en PBS, respectivamente. </li><li> Preparar una jeringa de insulina 1/2cc (con un 29G conectado permanentemente ½ &quot;aguja) llenándolo con 0,1 ml de heparina 1:100 (stock 5000U/ml) y doblar la aguja a un ángulo de 120 grados ~ con el bisel hacia arriba. Haga lo misma con una jeringa de 1 ml (con un 26G ½ &quot;aguja) llena con 0,3 ml de solución saturada de KCl. </li></ol> 2. Exponiendo el corazón y la canulación de la aorta <ol><li> Se anestesia el ratón usando el anestésico de elección. (Usamos una sobredosis de una mezcla de ketamina / xilazina. Inyección IP de 130 mg / kg de ketamina y 8,8 mg / kg de xilazina en solución salina) </li><li> Pin del ratón anestesiado en la bandeja de disección, hasta la parte ventral. Abrir la cavidad abdominal con una incisión central y retraer la pielpara exponer los órganos. Mover los intestinos a un lado para exponer una región de la vena cava posterior (PVC). </li><li> Inyectar solución de heparina en el PVC. Como se extrae la aguja, cubra el orificio de la aguja con un aplicador con punta de algodón para evitar fugas y mantenerla durante unos segundos hasta que los coágulos de PVC de pared y sellos. Esperar 2-3 minutos para heparina para dispersar a lo largo de la circulación del ratón. </li><li> Diseccionar el diafragma y la caja torácica por lo que se puede observar el corazón latiendo. Inyecte lentamente solución de KCl en el PVC hasta las detenciones del corazón. </li><li> Retire todos los órganos están debajo del diafragma y los impuestos especiales la parte posterior del ratón, dejando a la región anterior de la membrana intacta. Retire el diafragma, teniendo cuidado de cortar el PVC cerca del diafragma por lo que la porción proximal del corazón es fácil de localizar en los siguientes pasos. </li><li> Localice el extremo del corte de la aorta. Coloque un pedazo largo de 6-0 sutura de seda trenzada por debajo de la aorta de un lado a otro pocos milímetros anterioresm del extremo cortado de tal manera que la sutura se dobla hacia atrás sobre sí mismo. Cortar esta sutura ya a la mitad por lo que hay 2 piezas de sutura en la aorta. Insertar el angiocatéter en el extremo cortado de la aorta (Fig. 1A, B) y atar cada sutura con un nudo doble para sujetar el angiocatéter en su lugar y evitar cualquier contrapresión dentro de la aorta se escape. </li></ol> 3. La perfusión e inyección Microfil <ol><li> Conectar el angiocatéter al aparato de la presión de perfusión (Fig. 2) y comenzar la perfusión de los vasos con tampón vasodilatador (Fig. 1C) bombeando el aparato de perfusión a una presión de conducción de 100-110 mm Hg. Compruebe que el buffer se perfunde a través de las arterias coronarias, garantizando el líquido se sale de la PVC. Continuar a perfundir durante al menos 3 minutos o hasta que el fluido que sale del PVC es clara. (Continúe con los pasos a seguir, mientras que la perfusión). </li><li> Disección de las costillas y la espalda pin (o eliminar) la caja torácica para exponer el corazón. Una vez expuesto, ser careful no dejar que el corazón se secan apretando gotas de tampón en el corazón de una pieza tampón empapado de gasa. Despeja el timo para exponer el arco aórtico. Ligar las tres ramas principales de aorta con sutura de seda trenzada 6-0 para asegurar el líquido se desvía a través de las arterias coronarias, más que a través de estos vasos más grandes, de baja resistencia (Fig. 1D). </li><li> La perfusión del corazón con el fijador durante 15 minutos, luego enjuague con tampón vasodilatación por lo menos 2 minutos. Mientras tanto, ligar, tanto anterior vena cava para evitar que se escape Microfil del corazón después de la inyección (Fig. 1E). Coloque las suturas alrededor de la PVC y la aorta, pero no los apriete hasta después del llenado. </li><li> Preparar el Microfil (como se especifica en la tabla de reactivos) y cargarlo en una jeringa de 1 ml. Llenar la bandeja de disección con agua suficiente para cubrir el catéter (así como para prevenir la introducción de burbujas de aire cuando se cambia de un tubo de perfusión a la jeringa Microfil). Desconecte la perfusión APParatus del catéter y conectar la jeringa Microfil preparada. </li><li> Se inyecta la Microfil en la aorta hasta buen llenado de las arterias coronarias es evidente (Figuras 1F, 3A): las arterias se llenan primero, y luego el Microfil se &quot;derrame&quot; en los capilares como los sofocos tejido con el color de la Microfil. Una vez en el lado venoso, la naturaleza hidrófoba de la Microfil es para que aparezca inicialmente como esferas independientes como se desprende de los vasos más pequeños. Continuar para inyectar el Microfil hasta una columna continua llena las venas. Llenado completo será evidente cuando el Microfil es continua dentro de los vasos, y se sale a través del PVC. </li><li> Después de llenar es completa, rápidamente apretar las suturas que se colocaron previamente alrededor del PVC y la aorta para prevenir la naturaleza elástica del tejido cardíaco de apretar el Microfil fuera de los vasos. </li><li> Cubra el corazón con una gasa húmeda (empapado con agua de la bandeja de disección) Para evitar su desecación, y dejar que repose durante aproximadamente 1 hora a temperatura ambiente hasta que el Microfil ha polimerizado. Evitar cualquier tipo de presiones externas sobre el corazón durante el proceso de polimerización, como levantar o girar el corazón en un intento de obtener una visión temprana de los vasos llenos en la parte posterior del corazón. Esto puede exprimir Microfil de algunos buques en una zona más elástica del corazón, causando interrupciones en la Microfil. </li><li> Quitar el corazón y post-fijar en PFA al 4% durante toda la noche a 4 ° C. A continuación, almacenar en el 70% de etanol a 4 ° C. La vasculatura del corazón ya está listo para obtener imágenes de μCT. </li></ol> 4. Los resultados representativos Los buques que estén efectivamente perfundidos por Microfil tendrá Microfil continua, ininterrumpida a lo largo de los vasos (Fig. 3). El grado de llenado de los vasos coronarios se puede juzgar por ojo; venas se encuentra epicárdica 18, y puede ser fácilmente observado (Fig. 3A, punta de flecha); arterias,que son más intramiocárdica 18, también son visibles a través de la superficie del corazón (Fig. 3A, flecha). Llenado capilar es también evidente, como tejido cardíaco tiene una densidad muy alta de los capilares, y por lo tanto, cuando el relleno capilares, el tejido cardíaco se lave con el color de la Microfil (Fig. 3A, en estrella). Así, cualquier red vascular que fallaron en llenar será notable debido a la falta de Microfil (Fig. 3B, C). Las discontinuidades en el Microfil (asteriscos en la Fig. 3B) aparecen a menudo debido a la naturaleza hidrofóbica de la Microfil hará que se contraiga en sí mismo y hacer que &quot;rompe&quot; dentro de los vasos llenos. Estos &quot;descansos&quot; se puede reducir si la presión dentro de los vasos se mantiene a través adecuada como tie-offs de los puntos de salida vasculares del corazón. Otras discontinuidades pueden ser causados ​​por las burbujas de aire dentro de la Microfil. Para evitar la introducción de aire, asegúrese de que el angiocatéter está totalmente sumergido en el agua cuando se pasa de la perfusión AP<html> Paratus a la jeringa Microfil. Si una burbuja de aire se introduce, a menudo puede ser eliminado simplemente al continuar la perfusión Microfil hasta que la burbuja se ha empujado a través y fuera de los vasos coronarios. Redes vasculares no puede llenar completamente si una porción del lecho vascular está bloqueado (Fig. 3B, flecha). Mientras que la heparina inhibe la formación de coágulos de sangre, los bloqueos ocasionales pueden producirse debido a la perfusión de heparina incompleta antes de comenzar el procedimiento, o debido a otros factores desconocidos. Si ocurre un bloqueo, no hay, hasta donde sabemos, ningún método para desalojar el bloqueo para completar el relleno vascular. Llenado incompleto también puede resultar si la presión se usa demasiado poco durante el llenado, como el Microfil no se verán obligados a todos los lechos vasculares y redes capilares (Fig. 3C). Por el contrario, la presión excesiva puede provocar que los capilares se rompen y extravasación Microfil en el tejido circundante (Fig. 3D). files/ftp_upload/3740/3740fig1.jpg &quot;alt =&quot; Figura 1 &quot;/&gt; Figura 1. Descripción general del sistema de perfusión Microfil. (A) La aorta y el PVC se cortan aproximadamente al nivel del diafragma. (B) La aorta ascendente se canula con un angiocatéter. (C) La vasodilatación de amortiguación se perfunde a través de los vasos, impulsados ​​por el aparato de presión de perfusión (no se muestra), mientras que (D) las tres ramas principales fuera del arco aórtico se ligan. (S) 4% PFA se perfunde a través de las arterias coronarias, mientras tanto anterior venas cavas se ligan. (F) Utilizando una jeringa, Microfil se perfunde a través de las coronarias hasta que se observa que sale del PVC. <img src="/files/ftp_upload/3740/3740fig2.jpg" alt="Figura 2"/> Figura 2. Aparato de perfusión. Dos matraces Erlenmeyer, cada uno lleno con tampón Vasodilatación cualquiera o 4% PFA, se unen y se presuriza a través de tubos conectados a sus brazos laterales. El sistema está presurizado a través de bombeo manual delbombilla, y un manómetro de presión está conectado a uno de los matraces para permitir la supervisión y mantenimiento de la presión. Pequeños tubos se extienden a través de tapones de caucho y hacia abajo en el líquido en cada matraz. La presión que entra desde las bombas de armas blancas el líquido de cada matraz a cabo estos tubos más pequeños. Los tubos entonces se fusionan en una llave de paso que sólo permite que el líquido fluya desde un matraz al mismo tiempo. <img src="/files/ftp_upload/3740/3740fig3.jpg" alt="Figura 3"/> Figura 3. Muestra corazones microrrellenos. (A) Los buques que se llenan y se tienen pocos (si alguno) se rompe en el Microfil, y el tejido del corazón se teñirá del color de la Microfil debido a la llena de capilares (estrella, y comparar con C). Ambas arterias (flecha - Izquierda arteria descendente anterior) y venas (punta de flecha - vena coronaria izquierda) son visibles a través de la superficie del corazón. (B) Un corazón con cortes en el Microfil (asteriscos), así como bloqueos en algunos barcos que impedía micrófono completaRofil penetración. Los vasos bloqueados permanecen rojo (flecha), como la sangre no se barrió a cabo durante el proceso de perfusión. (C) Un corazón con las embarcaciones que se llenaron completamente. Tenga en cuenta que el tejido no se ha tomado el color amarillo de la Microfil, indicando el Microfil no penetran en los capilares. (D) Un corazón donde la rotura de capilares, durante el llenado, haciendo que el Microfil a la fuga en el tejido circundante (flecha).

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubbles+Targeted+to+Intercellular+Adhesion+Molecule-1+Bind+to+Activated+Coronary+Artery+Endothelial+Cells.">Microbubbles Targeted to Intercellular Adhesion Molecule-1 Bind to Activated Coronary Artery Endothelial Cells.</a> Circulation. 98, 1-5 (1998).</li><li>Wei, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Myocardial+Blood+Flow+With+Ultrasound-Induced+Destruction+of+Microbubbles+Administered+as+a+Constant+Venous+Infusion.">Quantification of Myocardial Blood Flow With Ultrasound-Induced Destruction of Microbubbles Administered as a Constant Venous Infusion.</a> Circulation. 97, 473-483 (1998).</li><li>Beckmann, N., Stirnimann, R., Bochelen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Magnetic+Resonance+Angiography+of+the+Mouse+Brain:+Application+to+Murine+Focal+Cerebral+Ischemia+Models.">High-Resolution Magnetic Resonance Angiography of the Mouse Brain: Application to Murine Focal Cerebral Ischemia Models.</a> Journal of Magnetic Resonance. 140, 442-450 (1999).</li><li>Kobayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+MR+angiography+of+intratumoral+vasculature+using+a+novel+macromolecular+MR+contrast+agent.">3D MR angiography of intratumoral vasculature using a novel macromolecular MR contrast agent.</a> Magnetic Resonance in Medicine. 46, 579-585 (2001).</li><li>Nezafat, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B1-insensitive+T2+preparation+for+improved+coronary+magnetic+resonance+angiography+at+3+T.">B1-insensitive T2 preparation for improved coronary magnetic resonance angiography at 3 T.</a> Magn. Reson. Med. 55, 858-864 (2006).</li><li>Wagner, S., Helisch, A., Ziegelhoeffer, T., Bachmann, G., Schaper, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+angiography+of+collateral+vessels+in+a+murine+femoral+artery+ligation+model.">Magnetic resonance angiography of collateral vessels in a murine femoral artery ligation model.</a> NMR in Biomedicine. 17, 21-27 (2004).</li><li>Cochet, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MR+angiography+and+velocity+measurement+in+mice+coronary+arteries+at+9.4+T:+assessment+of+coronary+flow+velocity+reserve.">In vivo MR angiography and velocity measurement in mice coronary arteries at 9.4 T: assessment of coronary flow velocity reserve.</a> Radiology. , 254-441 (2010).</li></ol>

Los ratones han sido manipulados con métodos aprobados por el Cuidado de Animales institucional y el empleo de la Universidad de Washington y de acuerdo con la Guía para el Cuidado y Uso de Animales de Laboratorio publicada por los EE.UU. Institutos Nacionales de Salud (NIH Publication No. 85-23, revisada en 1996).

Tejido cardíaco tiene una demanda metabólica muy alta, y por tanto requiere un suministro constante de nutrientes y el oxígeno de la sangre entregada por la vasculatura coronaria. Las enfermedades de los vasos coronarios, los cuales disminuyen la función coronaria debido a la estenosis del vaso y el bloqueo, puede llevar a la hipoxia tisular y la isquemia, y poner a los pacientes afectados por el riesgo de infarto de miocardio y un daño irreparable al músculo del corazón. Una mejor comprensión del estado de enfe...

<html><table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> 1 ml jeringas </td><td> <a href="http://www.bd.com/" target="_blank">Becton Dickinson</a> </td><td> BD-309602 </td><td></td></tr><tr><td> 1/2cc jeringas para insulina con agujas 29G ½ conectado permanentemente &#39; </td><td> <a href="http://www.bd.com/" target="_blank">Becton Dickinson</a> </td><td> BD-309306 </td><td></td></tr><tr><td> 2 &quot;x 2&quot; Almohadillas de gasa </td><td> Med101store.com </td><td> SKU 2208 </td><td></td></tr><tr><td> 24G ¾ &quot;Angiocath IV del catéter </td><td> <a href="http://www.bd.com/" target="_blank">Becton Dickinson</a> </td><td> BD-381112 </td><td></td></tr><tr><td> 26G ½ &quot;agujas de calibre </td><td>lacio &quot;&gt; Becton Dickinson </td><td> BD-305111 </td><td></td></tr><tr><td> La adenosina </td><td> <a href="http://www.sigmaaldrich.com/" target="_blank">Sigma</a> </td><td> A9251 </td><td> 1 g / l en PBS durante Buffer vasodilatación (con papaverina) </td></tr><tr><td> Pinzas angulares Graefe </td><td> <a href="http://www.finescience.com/" target="_blank">Herramientas de Bellas Ciencia</a> </td><td> 11052-10 </td><td></td></tr><tr><td> De algodón con punta de aplicadores: 6 &quot;no estéril </td><td> <a href="http://www.cardinal.com/" target="_blank">Cardinal Health</a> </td><td> C15055-006 </td><td></td></tr><tr><td> Curvas Tijeras quirúrgicas </td><td> <a href="http://www.finescience.com/" target="_blank">Herramientas de Bellas Ciencia</a> </td><td> 14085-09 </td><td></td></tr><tr><td> Estereoscopio de disección y la fuente de luz </td><td> <a href="http://www.nikon.com/" target="_blank">Nikon</a> </td><td> NA </td><td> NA </td></tr><tr><td> Bandeja de disección, 11.5 x 7.5 pulgadas </td><td> <a href="http://www.coleparmer.com/" target="_blank">Cole-Parmer</a> </td><td> YO-10915-12 </td><td> Lleno de alquitrán para la fijación abajo el ratón </td></tr><tr><td> Bellas pinzas curvas </td><td> <a href="http://www.aesculapusa.com/" target="_blank">Aesculap</a> </td><td> FD281R </td><td> Necesitas dos </td></tr><tr><td> La heparina, 5.000 U / ml de caldo </td><td> <a href="http://www.apppharma.com/" target="_blank">APP Pharmaceuticals LLC</a> </td><td> NDC 63323-047-10 </td><td> 1:100 dilución en agua </td></tr><tr><td> KCl </td><td> <a href="http://www.fishersci.com/" target="_blank">Pescador</a> </td><td> P217 </td><td> Solución saturada en H2O </td></tr><tr><td> Ketamina (Ketaset), 100 mg / ml de stock </td><td> Fort Dodge, de Overland Park, KS, EE.UU. </td><td> NDC 0856-2013-01 </td><td> Mezclado como el peso corporal 130 mg / kg, con xilacina en s 0,9%aline </td></tr><tr><td> Microfil </td><td> <a href="http://www.flowtech-inc.com/" target="_blank">Flujo Tech</a> </td><td> MV-122 (amarillo). Otras opciones de color están también disponibles. </td><td> Mezclar 1:1 en peso, con 10% en volumen de agente de curado. Prepararse inmediatamente antes de la inyección, y agitar para asegurar que esté bien mezclado </td></tr><tr><td> No estériles de sutura: 6-0, trenzado de seda </td><td> <a href="http://www.harvardapparatus.com/" target="_blank">Aparato de Harvard</a> </td><td> 723287 </td><td></td></tr><tr><td> Papaverina </td><td> <a href="http://www.americanregent.com/" target="_blank">América Regent Inc.</a> </td><td> NDC 0517-4010-01 </td><td> 4 mg / l en PBS durante Buffer vasodilatación (con adenosina) </td></tr><tr><td> Paraformaldehído </td><td> <a href="http://www.sigmaaldrich.com/" target="_blank">Sigma</a> </td><td> P6148 </td><td> Preparado como solución al 4% </td></tr><tr><td> Aparato de perfusión </td><td></td><td></td><td> Ver figura 2 </td></tr><tr><td> Tijeras de primavera </td><td> <a href="http://www.finescience.com/" target="_blank">Herramientas de Bellas Ciencia</a> </td><td> 15018-10 </td><td></td></tr><tr><td> Xilazina (Anased), 20 mg / dL de valores </td><td> <a href="http://www.lloydlab.com/" target="_blank">Lloyd Labs</a> </td><td> NADA # 139-236 </td><td> Mezclado como el peso corporal 8,8 mg / kg, con ketamina en 0,9% de solución salina </td></tr></tbody></table>

retrograde perfusion and filling of mouse coronary vasculature as preparation for micro computed tomography imaging

La visualización de la vasculatura se está convirtiendo cada vez más importante para la comprensión de muchos estados patológicos diferentes. Si bien existen varias técnicas para obtener imágenes de la vasculatura, pocos son capaces de visualizar la red vascular en su conjunto, mientras que se extiende a una resolución que incluye el 1,2 vasos más pequeños. Además, muchas de las técnicas de colada vasculares destruir el tejido circundante, evitando su posterior análisis de la muestra 3-5. Un método que evita estos problemas es de micro-tomografía axial computerizada (μCT). μCT imagen puede escanear a resoluciones menores de 10 micras, es capaz de producir reconstrucciones en 3D de la red vascular, y deja el tejido intacto para su posterior análisis (por ejemplo, histología y morfometría) 11.06. Sin embargo, los vasos de imágenes ex vivo por métodos μCT requiere que los vasos se llena con un compuesto radiopaco. Como tal, la representación exacta de la vasculatura producido por μCT imagen dependellenado fiable y completa de los vasos. En este protocolo, se describe una técnica para el llenado de los vasos coronarios del ratón en la preparación de imágenes μCT. Dos técnicas que predominan existen para el llenado de los vasos coronarios: en vivo a través de la perfusión de la canalización y retrógrado de la aorta (o una rama del arco aórtico) 12-14, o ex vivo a través de un sistema de perfusión Langendorff 15-17. A continuación se describe un método in vivo canulación aórtica, que ha sido específicamente diseñado para garantizar el llenado de todos los buques. Nosotros utilizamos un compuesto viscosidad baja radiopaco llamado Microfil que puede perfundir a través de los vasos más pequeños para llenar todos los capilares, así como los dos lados arteriales y venosas de la red vascular. Los buques son perfundidos con buffer usando un sistema de perfusión a presión, y se rellena con Microfil. Para asegurarse de que Microfil llena los pequeños vasos de resistencia más altos, que ligan las ramas grandes emanating de la aorta, que desvía el Microfil en las coronarias. Una vez finalizado el llenado, para evitar que la naturaleza elástica del tejido cardíaco de exprimir Microfil cabo de algunos vasos, que ligar accesibles los puntos importantes de salida vasculares inmediatamente después del llenado. Por lo tanto, nuestra técnica está optimizado para el llenado completo y la retención máxima del agente de carga, lo que permite la visualización completa de la red vascular coronaria - arterias, capilares y venas por igual.

Watch this Scientific Journal Video about Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging at JoVE.com

Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

La visualización de los vasos coronarios es fundamental para avanzar en nuestra comprensión de las enfermedades cardiovasculares. Aquí se describe un método para la perfusión vascular coronaria murino con una goma de silicona radiopaco (Microfil), en preparación para la micro-tomografía axial computerizada (μCT) de imágenes.

Perfusión retrógrada y llenado de la vasculatura coronaria del ratón, como preparación para la Micro tomografía computarizada

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for ...

retrograde-perfusion-filling-mouse-coronary-vasculature-as

Research

JoVE Journal

Medicine

32.3K vistas.  University of Washington. La visualización de los vasos coronarios es fundamental para avanzar en nuestra comprensión de las enfermedades cardiovasculares. Aquí se describe un método para la perfusión vascular coronaria murino con una goma de silicona radiopaco (Microfil), en preparación para la micro-tomografía axial computerizada (μCT) de imágenes.

Video: Retrograde Perfusion and Filling of Mouse Coronary Vasculature as Preparation for Micro Computed Tomography Imaging

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

La segmentación y la medición de los volúmenes de grasa en modelos murinos de obesidad Usando rayos X de tomografía computarizada

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Investigación

Medicina

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

Osmótica Drug Delivery de miembros posteriores isquémicos y la perfusión de la vasculatura con Microfil para Micro-tomografía computarizada

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Evaluación de la viabilidad de la inyección de grasa humana en ratones desnudos con micro-tomografía computarizada

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

El uso de la tomografía computarizada-Micro para la evaluación del desarrollo del tumor y Seguimiento de la respuesta al tratamiento en un modelo de ratón de cáncer de pulmón

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Imagen Fluorescente combinado del infrarrojo cercano y la tomografía computarizada-Micro para visualizar de manera directa tromboembolia cerebral

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Identifying Coronary Artery Calcification on Non-gated Computed Tomography Scans

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed tomography (CT) scans with a high correlation with gated cardiac CT scans. This standardized protocol takes a step-wise approach to not only optimizing an image for the identification of calcification but also to distinguishing CAC from other common causes of calcification in the cardiac silhouette. Recognition of CAC on non-gated CT scans helps to identify a very powerful prognostic factor that can influence therapeutic interventions or downstream diagnostic testing without requiring a gated cardiac scan. These non-gated CT scans are often acquired as part of the routine care of the patient, and this data is readily available without another dose of&#160;ionizing radiation. This protocol allows for the precise and accurate extraction of this data for the purposes of retrospective data analysis in clinical research studies, but also in the clinical evaluation and management of patients.

Here, we present a protocol to reliably and systematically identify coronary artery calcification (CAC) on non-gated computed tomography (CT) scans of the chest or abdomen. CAC provides an objective measure of coronary artery disease for both research and clinical purposes.

Identificación de la calcificación coronaria en cerrada no computado exploraciones de la tomografía

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed ...

Continuous Blood Sampling in Small Animal Positron Emission Tomography/Computed Tomography Enables the Measurement of the Arterial Input Function

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal blood time-activity concentration also known as arterial input function (AIF) is a key point, especially for the characterization of animal disease models and the introduction of newly developed radiotracers. The knowledge of radiotracer availability in the blood helps to interpret PET/CT-derived data of tissue activity. For this purpose, online blood sampling during the PET/CT imaging is advisable to measure the AIF. In contrast to manual blood sampling and image-derived approaches, continuous online blood sampling has several advantages. Besides the minimized blood loss, there is an improved resolution and a superior accuracy for the blood activity measurement. However, the major drawback of online blood sampling is the costly and time-consuming preparation to catheterize the femoral vessels of the animal. Here, we describe an easy and complete workflow for catheterization and continuous blood sampling during small animal PET/CT imaging and compared it to manual blood sampling and an image-derived approach. Using this highly-standardized workflow, the determination of the fluorodeoxyglucose ([18F]FDG) AIF is demonstrated. Further, this procedure can be applied to any radiotracer in combination with different animal models to create fundamental knowledge of tracer kinetic and model characteristics. This allows a more precise evaluation of the behavior of pharmaceuticals, both for diagnostic and therapeutic approaches in the preclinical research of oncological, neurodegenerative and myocardial diseases.

Here a protocol for continuous blood sampling during PET/CT imaging of rats to measure the arterial input function (AIF) is described. The catheterization, the calibration and setup of the system and the data analysis of the blood radioactivity are demonstrated. The generated data provide input parameters for subsequent bio-kinetic modeling.

El muestreo sanguíneo continuo en tomografía por emisión de positrones de animales pequeños permite la medición de la función de entrada arterial

For quantitative analysis and bio-kinetic modeling of positron emission tomography/computed tomography (PET/CT) data, the determination of the temporal ...

Tissue Preparation Techniques for Contrast-Enhanced Micro Computed Tomography Imaging of Large Mammalian Cardiac Models with Chronic Disease

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 &#181;m) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

Here, we present a protocol to obtain high resolution micro computed tomography images of healthy and pathological large mammalian whole hearts with collagen-selective contrast enhancement.

Técnicas de preparación de tejidos para imágenes de microtomografía computarizada mejoradas con contraste de modelos cardíacos de mamíferos grandes con enfermedades crónicas

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

DUCT: Fundición de doble resina seguida de tomografía microcomputarizada para análisis hepático 3D

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

Sample Preparation for Computed Tomography-based Three-dimensional Visualization of Murine Hind-limb Vessels

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ function. Clarifying the mechanism of blood vessel formation is therefore extremely useful for elucidating developmental processes and pathological mechanisms. Murine hind-limb vessels are often used as a model for physiological and pathological angiogenesis. Evaluation is mainly performed via a two-dimensional method using tissue sections. However, methods for evaluating three-dimensional (3D) vascular morphology are particularly limited. This paper introduces a method for visualizing murine hind-limbs using computed tomography (CT). Radiation-opaque resin is injected through the descending aorta, and whole vessels are filled with dye. By adjusting the time of dye injection, arterial-specific filling is also possible, and samples can be obtained with any micro-X-ray CT device. This contrast method provides a basic technique for the 3D evaluation of murine blood vessels in the lower extremities. Furthermore, this method can be used to visualize all blood vessels below the diaphragm and evaluate blood vessels in the abdominal organs.

Here, we describe a visualization and quantification method for murine hind-limb vessels using micro-X-ray computed tomography.

Preparación de muestras para la visualización tridimensional basada en tomografía computarizada de vasos murinos de las extremidades posteriores

Blood vessels are complex networks with tree-like structures, and vascular networks are essential for maintaining both circulation and maintaining organ ...