The scientific advice of Drs. Timothy McMahon and Siqing Shan is appreciated. The presenters thank Drs. David Kirsch and Patricia Steeg for the generous gift of the fluorescently labeled Mouse Sarcoma and metastatic MDAMB-231 cells, respectively. This work was funded in part by the U.S. Defense Advanced Research Projects Agency (DARPA) Prime Award Number N66001-10-C-2134, and in part by the Department of Radiation Oncology, Duke University Medical Center.

NOTA: Todos los procedimientos relacionados con los animales descritos en este protocolo han sido previamente aprobado por el animal de Atención Institucional y el empleo Comisión de la Universidad de Duke (DUIACUC). Cultivo Celular y Inyección 1. Cáncer <ol><li> Cultivar las células cancerígenas metastásicas marcados con fluorescencia (por ejemplo, células de cáncer de mama MDAMB231-GFP humanos, regalo del doctor Patricia Steeg, NCI, y las células del sarcoma de ratón marcado con YFP, regalo del doctor David Kirsch, Duke University Medical Center, Departamento de Oncología Radioterápica) en medio de cultivo adecuado (por ejemplo, medio Eagle modificado de Dulbecco (DMEM) con suero bovino fetal al 10% y 1% de penicilina / estreptavidina) a 37 ° C hasta aproximadamente 90% de confluencia. </li><li> Tripsinizar las células, lavar 2 veces con PBS, contar, y luego inyectarlos en las venas de la cola de 10 semanas de edad ratas hembra desnudos anestesiados con isoflurano a los 5 millones de células por animal, usando una jeringa con una aguja 27 G. Anesthes nivel quirúrgicosia es verificada por la falta de reacción ante pizca dedo del pie. </li></ol> 2. El seguimiento de las metástasis Usando MicroCT <ol><li> Examinar las ratas una vez a la semana utilizando un micro-CT / micro-irradiador, para detectar la presencia de metástasis por encima de aproximadamente 2 mm de diámetro de diámetro. El micro-CT es comisionado como se describió anteriormente 12. <ol><li> Ratas sujetas a 3% anestesia con isoflurano antes de la imagen. Confirmar profunda anestesia por pellizco dedo del pie. </li><li> Después de inicio de la anestesia, las ratas transferir rápidamente a la base de formación de imágenes en la cámara de formación de imágenes y conectarse a través de una nariz de cono a una mezcla de isoflurano al aire a 2,5-3%. Ajustar la posición de la rata en la cuna de una manera que el tórax se coloca en el haz de fotones del escáner MicroCT, usando los controles de posición externos y delimitador de láser en la base de formación de imágenes. Asegúrese de que la puerta de la cámara de formación de imágenes está cerrada, para proteger el investigador de los rayos gamma. </li><li> Controlar la posición del animal usando de nuevola cámara de vídeo en color. Realizar una baja resolución prueba de imagen de TC, y utilizar la imagen resultante para ajustar el campo de visión a las dimensiones xyz de la cavidad torácica. </li><li> Imagen del tórax rata utilizando un filtro Al 2 mm a 40 kVp, 2,5 mA, y 0.008 voxel a las 7 FPS y devolver el animal a su jaula. Obtención de imágenes de un animal debe tener no más de 15 o 20 min. No devuelva un animal que ha sido sometido a cirugía para la compañía de otros animales hasta que se recupere completamente. No deje a un animal sin vigilancia hasta que se haya recuperado el conocimiento suficiente para mantener decúbito esternal. </li><li> Confirme metástasis por la aparición de objetos relativamente de radio-opaco que no puede ser explicado por los vasos sanguíneos intratorácicos (Figura 1) </li></ol></li></ol> Cirugía 3. Cámara Ventana <ol><li> Anestesia, signos vitales y cateterización de la vena de la cola <ol><li> Seleccione animales con presencia de enfermedad metastásica. Inyectar animal con un d intraperitonealose de 50 mg / kg de pentobarbital. Confirme anestesia nivel quirúrgico por pizca dedo del pie antes de proceder. Nota: Los protocolos de anestesia deben ser adaptadas a la respectiva configuración experimental. Pentobarbital fue elegido aquí como un anestésico de acción prolongada, con el fin de inducir la anestesia profunda para largos procedimientos, al tiempo que ofrece la opción de re-facile de dosificación. Sin embargo, la pérdida de animales para la sobredosificación es un problema común con anestesia con pentobarbital. Otra opción que conserva reflejos autonómicos en un grado mayor que pentobarbital es la ketamina en combinación con sedantes tales como xilazina o medetomidina, que sin embargo permite sólo para un único ciclo de re-dosificación. </li><li> Afeitado animales en el lado del cuerpo que tiene la enfermedad metastásica, y en la zona del cuello, usando una maquinilla. Limpie todo el pelo suelto que queda de la piel. Después se elimina el pelo suelto, aplique ungüento veterinario para los ojos, para evitar que se sequen. NOTA: ratas desnudas Athymic pueden tener el pelo residual que REQUIeliminación res antes de proceder con los procedimientos quirúrgicos. Es muy importante eliminar todo el pelo a fondo, ya que puede interferir con los procedimientos quirúrgicos y de formación de imágenes. </li><li> Fijar el animal en posición supina sobre una placa de metal que se coloca en una almohadilla de calefacción 37 ° C el agua recirculada. Las extremidades delanteras y traseras son fijos en el plato con cinta. NOTA: Es útil para controlar y registrar los signos vitales, tales como la frecuencia cardíaca y la oxigenación de la sangre arterial mediante el uso de un oxímetro de pulso, a través de procedimientos quirúrgicos y experimentales. </li></ol></li><li> La intubación traqueal <ol><li> Para colocar un catéter para la ventilación del animal, primero hacer una incisión transversal de la piel cervical, seguido por la separación mediana de la musculatura longitudinal ventral a la tráquea. </li><li> Utilice acción repetida-apertura al cierre con un afilados fórceps para crear un paso para la sutura a través de la cara dorsal de la tráquea. </li><li> Hacer una pequeña incisión en la tráquea en tél lado ventral, no más de semi-circular, aproximadamente entre el segundo y el tercer anillo traqueal. Deja un parte suficientemente larga de la tráquea expuesta en la superficie dorsal, para permitir la fijación del catéter traqueal. </li><li> Inserte un 2,5 a 3,0 mm &quot;Y&quot; cánula traqueal en la tráquea, y apriete con una sutura monofilamento 4-0. Asegúrese de que la cánula está conectado a un ventilador ciclado de presión con una botella conectada al conducto de caducidad que se llena de 6 cm de agua, para mantener la presión pulmonar positivo. Corriente de llenado de gas debe ser de 100% de oxígeno, a menos experimentalmente deseado lo contrario. </li><li> Insertar un catéter con una aguja G 25-27, y llena con solución salina heparinizada en una de las venas de la cola de la rata, y fijar en su lugar con cinta adhesiva. NOTA: Asegúrese de que la permeabilidad del catéter vena de la cola durante todo el procedimiento mediante inyecciones repetidas de una pequeña cantidad de solución salina heparinizada en la vena de la cola. Además, la intubación oro-traqueal, es decir, la orientación de un traquealtubo a través de la boca del animal anestesiado y más allá de la laringe dentro de la tráquea, es una posible alternativa al procedimiento de traqueotomía que se describe aquí. Sin embargo, este método requiere entrenamiento y experiencia especial, para evitar daños a la tráquea, y también para evitar la canulación accidental del esófago. </li></ol></li><li> Aplicación de ventana pulmonar <ol><li> Quitar la piel desde el lado del pecho donde se encuentra la enfermedad metastásica, mediante la creación de una incisión y, a continuación, separando la piel mediante disección roma. Nota: La piel puede ser extirpado y se retira posterior al desprendimiento </li><li> Proceder mediante la disección de las dos capas de la superposición de la musculatura (pectoral, serrato, y dorsal ancho), pero dejando los músculos intercostales intacto. Crear una perforación en la cavidad del pecho de aproximadamente 1,5 cm de diámetro, mediante la eliminación de porciones de típicamente dos costillas adyacentes. Lo ideal es ubicar la perforación de la región de la sexta y séptima ribs. </li><li> Osteotomía: <ol><li> Para minimizar el sangrado y el daño a la superficie del pulmón, sujeta fuertemente la costilla que se corta con un fórceps quirúrgico dentadas durante el corte. Usando tijeras quirúrgicas, corte el lado medial de la primera costilla, en un ángulo de aproximadamente 45 °, dejando el lado puntiagudo del hueso de la costilla restante apuntando hacia el exterior. </li><li> Posteriormente, cortar el lado lateral de la costilla de una manera similar, dejando de nuevo el lado puntiagudo del hueso de la costilla apunta hacia afuera, para evitar daños en la superficie del pulmón. </li><li> Repita el procedimiento para la costilla adyacente, a continuación, cortar los músculos intercostales y retire la pieza extirpada. Durante este procedimiento, mantener la presión pulmonar de una manera que la interacción mecánica entre la superficie del pulmón y la caja torácica se reduce al mínimo. Para ello, la regulación apropiada de presión inspiración en el ventilador. </li></ol></li><li> La inserción de la ventana: <ol><li> Inserte una ventana de pulmón por encargo, que consta de un cubreobjetos que esunido a una toma de Plexiglas (Figura 1B). Coloque la ventana a la toma por encolado, o mediante la aplicación de una cantidad muy pequeña de grasa de vacío. Inserte la ventana de manera que las metástasis de superficie se encuentran cerca del centro de la ventana. Si es necesario, ajustar el agujero insertado para llevar la micrometástasis en el centro de la ventana mediante la ampliación del agujero ligeramente hacia el lado respectivo. NOTA: La enfermedad metastásica en la superficie pleural se puede identificar como puntos blancos claramente reconocibles o áreas dentro de las partes sanas de color rosa de lo contrario-a-salmón de la superficie pulmonar que aparecen predominantemente a lo largo de las fisuras. Mientras micrometástasis pueden ocurrir en otras áreas del exterior pleural, las líneas celulares investigadas casi siempre muestran la superficie micrometástasis en el área perforada, una vez que la enfermedad metastásica puede ser detectada radiológicamente. </li><li> Después de insertar el enchufe en la perforación, y la creación de un contacto directo con la pleura visceral de la lung, la sutura de los bordes del marco de la ventana en el músculo intercostal circundante, utilizando 4,0 sutura de monofilamento (Figura 1C). Utilice un ligero aumento de la presión de inspiración en el ventilador para ayudar a aire residual escapar y para crear un sello. Nota: La tasa de respiración de ratas puede variar mucho, dependiendo del estado de la anestesia, el estado de excitación o la ansiedad, la concentración de oxígeno en el aire inspirado (FiO 2), etc. Es recomendable ajustar la tasa de respiración de 70 a 90 latidos por minuto. La presión de inspiración se debe ajustar con precaución y no se puede ajustar a más de aprox. 8 cm H 2 O (0,6 mmHg), para evitar daños en la superficie del pulmón. </li></ol></li><li> Coloque el animal en un limitador de diseño personalizado que está diseñado para eliminar la dirección Z movimiento (Figura 1D) en una placa de acero que se coloca en un termostática (eléctrica) manta de calefacción, bajo un microscopio de fluorescencia. Controlar el cuerpo del animalla temperatura usando un termistor rectal. Ajuste los tornillos de la inmovilización de los animales, y la presión de inspiración en el ventilador para lograr un control óptimo de movimiento lateral. Nota: la respiración natural en mamíferos involucra a los tres sentidos de circulación pulmonar y la extensión del pecho:, dorso-ventral, y cráneo-caudal bilateral. Con el fin de preservar el movimiento de la respiración natural tanto como sea posible, es importante para reducir al mínimo la compresión de la dirección Z en la medida necesaria. Debido a la restricción-Z dimensiones tiene el potencial de introducir artefactos que puedan afectar el flujo de sangre y otros parámetros, es aconsejable mantener condiciones constantes durante la serie de mediciones repetidas en el mismo animal. </li></ol></li></ol> 4. Imaging y medición de flujo sanguíneo en la microcirculación <ol><li> Recoger las células rojas de la sangre a través de punción cardíaca y etiquetarlos con DiI (1,1 = -dioctadecyl-3,3,3 perclorato =, 3 = tetrametil-indocarbocianina), como se describió anteriormente &lt;sup&gt; 10. </li><li> Inyectar 300 l de glóbulos rojos marcados en la vena de la cola de la rata antes de realizar la cirugía de la cámara de ventana, para evitar efectos de adhesión pase primero a la ventana de cristal. Eliminar cualquier burbuja de aire en la jeringa o un catéter, como la introducción de aire en la vena inhibirá cualquier inyecciones adicionales. </li><li> Image flujo de sangre con una cámara CCD microscopio a -40 ° C chip de temperatura de enfriamiento, y aproximadamente 100X resolución global (es decir, con un objetivo de microscopio 10X, 10X y pre-cámara ocular). Utilice conjuntos de filtros Rodamina / TRITC estándar (450-490 nm de excitación, emisión&gt; 515 nm). Registre la resolución real de las secuencias de imágenes resultantes velocidad de fotogramas y pixel. Consigue al menos 200 (idealmente alrededor de 300) imágenes por pila, para garantizar el éxito del análisis de la velocidad del flujo. </li><li> Reponer la pérdida de líquidos en el animal mediante la inyección de aproximadamente 1 ml de solución salina IP cada hr. NOTA: Los valores experimentales que implican una intervención, por ejemplo,un fármaco que modifica la velocidad del flujo sanguíneo en una metástasis del cáncer pulmonar, requiere mediciones repetidas de la velocidad del flujo de sangre en el cáncer metastásico pulmonar. Para estos experimentos prolongados, es importante para reponer el animal con suficiente líquido. </li><li> La eutanasia a los animales por infusión de 3 ml de 3N KCl en la vena de la cola </li><li> Evaluar las pilas de imágenes utilizando un publicada, disponible públicamente algoritmo informático basado en Matlab que va a crear el flujo de escala de grises de velocidad y mapas de color codificado para todo el flujo de sangre rastros 10,11. Posteriormente evaluar las imágenes en escala de grises resultantes por el uso de software de análisis de imagen comercial o de acceso público, tales como la imagen J, después de thresholding de valores que indican ningún movimiento de las células de la sangre, es decir, sin vasculatura activo. </li></ol>

<ol>
	<li>Billingsley, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+metastases+from+soft+tissue+sarcoma:+analysis+of+patterns+of+diseases+and+postmetastasis+survival.">Pulmonary metastases from soft tissue sarcoma: analysis of patterns of diseases and postmetastasis survival.</a> Ann Surg. 229, 602-610 (1999).</li><li>Rashid, O. M., Takabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+the+role+of+surgery+in+the+management+of+breast+cancer+lung+metastasis.">The evolution of the role of surgery in the management of breast cancer lung metastasis.</a> J Thorac Dis. 4, 420-424 (2012).</li><li>Mayer, A., Vaupel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia,+lactate+accumulation,+and+acidosis:+siblings+or+accomplices+driving+tumor+progression+and+resistance+to+therapy.">Hypoxia, lactate accumulation, and acidosis: siblings or accomplices driving tumor progression and resistance to therapy.</a> Advances in experimental medicine and biology. 789, 203-209 (2013).</li><li>Okunieff, P., O'Dell, W., Zhang, M., Zhang, L., Maguire, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+oxygen+measurements+and+personalized+medicine.">Tumor oxygen measurements and personalized medicine.</a> Advances in experimental medicine and biology. 765, 195-201 (2013).</li><li>Palmer, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+optical+molecular+imaging+and+analysis+in+mice+using+dorsal+window+chamber+models+applied+to+hypoxia,+vasculature+and+fluorescent+reporters.">In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent reporters.</a> Nature protocols. 6, 1355-1366 (2011).</li><li>Palmer, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+tumor+hypoxia+dynamics.">Optical imaging of tumor hypoxia dynamics.</a> Journal of biomedical optics. 15, (2010).</li><li>Funakoshi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+lung+metastasis+for+intravital+studies.">A new model of lung metastasis for intravital studies.</a> Microvasc Res. 59, 361-367 (2000).</li><li>Kuebler, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+assessment+of+pulmonary+vascular+responses.">Real-time imaging assessment of pulmonary vascular responses.</a> Proc Am Thorac Soc. 8, 458-465 (2011).</li><li>Tabuchi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precapillary+oxygenation+contributes+relevantly+to+gas+exchange+in+the+intact+lung.">Precapillary oxygenation contributes relevantly to gas exchange in the intact lung.</a> Am J Respir Crit Care Med. 188, 474-481 (2013).</li><li>Hanna, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+measurement+of+blood+flow+velocity+and+direction+and+hemoglobin+oxygen+saturation+in+the+rat+lung+using+intravital+microscopy.+American+journal+of+physiology.">Automated measurement of blood flow velocity and direction and hemoglobin oxygen saturation in the rat lung using intravital microscopy. American journal of physiology.</a> Lung cellular and molecular physiology. 304, 86-91 (2013).</li><li>Fontanella, A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mapping+of+hemodynamics+in+the+lung,+brain,+and+dorsal+window+chamber-grown+tumors+using+a+novel,+automated+algorithm.">Quantitative mapping of hemodynamics in the lung, brain, and dorsal window chamber-grown tumors using a novel, automated algorithm.</a> Microcirculation. , (2013).</li><li>Newton, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commissioning+a+small-field+biological+irradiator+using+point,+2D,+and+3D+dosimetry+techniques.">Commissioning a small-field biological irradiator using point, 2D, and 3D dosimetry techniques.</a> Medical physics. 38, 6754-6762 (2011).</li><li>Vaupel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironmental+physiology+and+its+implications+for+radiation+oncology.">Tumor microenvironmental physiology and its implications for radiation oncology.</a> Seminars in radiation oncology. 14, 198-206 (2004).</li><li>Tabuchi, A., Mertens, M., Kuppe, H., Pries, A. R., Kuebler, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+murine+pulmonary+microcirculation.">Intravital microscopy of the murine pulmonary microcirculation.</a> J Appl Physiol. 104, 338-346 (2008).</li><li>Fingar, V. H., Taber, S. W., Wieman, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+for+the+study+of+pulmonary+microcirculation:+determination+of+pulmonary+edema+in+rats.">A new model for the study of pulmonary microcirculation: determination of pulmonary edema in rats.</a> J Surg Res. 57, 385-393 (1994).</li><li>Manzoor, A. A., Schroeder, T., Dewhirst, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-stop-shop+tumor+imaging:+buy+hypoxia,+get+lactate+free.">One-stop-shop tumor imaging: buy hypoxia, get lactate free.</a> The Journal of clinical investigation. 118, 1616-1619 (2008).</li><li>Evans, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+analytical+methods+as+applied+to+the+evaluation+of+vasculature+and+hypoxia+in+human+brain+tumors.">Imaging and analytical methods as applied to the evaluation of vasculature and hypoxia in human brain tumors.</a> Radiation research. 170, 677-690 (2008).</li><li>Radiloff, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combination+of+theophylline+and+endothelin+receptor+antagonism+improves+exercise+performance+of+rats+under+simulated+high+altitude.">The combination of theophylline and endothelin receptor antagonism improves exercise performance of rats under simulated high altitude.</a> Journal of applied physiology. 113, 1243-1252 (2012).</li></ol>

Los autores no tienen nada que revelar.

Se presenta un modelo que es factible cambios de imagen en el flujo sanguíneo en la microcirculación y otros procesos dinámicos en las metástasis pulmonares de ratas, usando microscopía intravital y análisis de flujo de sangre computacional. Si bien existen otros métodos para llevar a cabo la microscopía en pulmones expuestos en ribcages abiertos de los roedores, este modelo también es la primera imagen de metástasis pulmonares a través de una perforación de la pared del pecho en un entorno de pecho cerrado....

The lung is one of the most important target organs of metastatic disease, and because this condition is difficult to treat successfully with chemo- and radiation therapy, a cure is still rare1,2. Specific pathophysiological and microcirculatory features of solid primary and metastatic tumors, such as microregional hypoxia, diffusion limitation and inefficient tumor vasculature, greatly contribute to their resistance to anticancer treatment3,4. Due to the microscopic scale and dynamic nature of parameters such as microvascular blood flow, intravital microscopy of the tumor in the living animal has become a very important research tool in the field5. While intravital microscopy models have been applied to tumors in different organ sites, including the metastatic lung within an open rib cage, no protocol has been developed yet for the research of pulmonary metastases in a physiologically preserving, closed-chest environment6,7. Such an endeavor is particularly hampered by the necessity to surgically access the rib cage without affecting the overall function of the lung7-9. Recently, a method was introduced to image pulmonary microcirculatory blood flow in a close-chest setting in live rats, using fluorescence intravital microscopy10. This protocol enables the systematic quantification of blood flow velocity from injected, fluorescently labeled red blood cells, using computerized analysis, while keeping the animal physiologically stable and preserving the integrity of the lung11. In this present study, it is shown how this technology can be modified to image and quantify microcirculatory blood flow in tail vein-inoculated pulmonary metastases on the pleural surface in the immunocompromised rat. This model is also the first one to study metastatic lung tumors in a closed-chest intravital microscopy setting.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Athymic nude rats</td>
			<td style="width:71px;">Charles River</td>
			<td style="width:119px;">Strain code 316</td>
			<td style="width:167px;">Female 10 week-old athymic nude rats</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micro-CT/micro-Irradiator&#160;</td>
			<td style="width:71px;">Precision X-ray Inc.</td>
			<td style="width:119px;">Xrad 225Cx</td>
			<td>Use MicroCT to detect metastases</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">DiI (1,1=-dioctadecyl-3,3,3=,3=-tetramethyl-indocarbocyanine perchlorate)</td>
			<td style="width:71px;">Sigma Aldrich</td>
			<td style="width:119px;">468495-100MG</td>
			<td>Mix 100 ul packed red blood cells with 100 ul of 0.5 mg/ml DiI in 200 proof ethanol, 2 ml of 5% dextrose solution in water, and fill up to a 10-ml final volume with saline</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Rodent ventilator</td>
			<td style="width:71px;">Kent Scientific</td>
			<td style="width:119px;">TOPO Small Animal Ventilator</td>
			<td>Device is important to maintain positive lung pressure after application of pneumothorax</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axioskop fluorescence microscope upright</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Microscope for intravital imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Andor CCD camera</td>
			<td style="width:71px;">Andor</td>
			<td style="width:119px;">iXonEM 885</td>
			<td>CCD camera for live imaging of blood flow</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pulse oximeter</td>
			<td style="width:71px;">StarrLife</td>
			<td style="width:119px;">MouseOx</td>
			<td>Pulse oximeter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluorescence microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">Axioskop</td>
			<td>Fluorescence microscope</td>
		</tr>
	</tbody>
</table>

automated measurement of microcirculatory blood flow velocity in pulmonary metastases of rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

La vasculatura en tumores sólidos se sabe que difieren significativamente de suministro normal de sangre, mostrando un mayor grado de tortuosidad y mayores distancias intervasculares 13. En consecuencia, las pistas del flujo sanguíneo en metástasis de cáncer de mama y el sarcoma pulmonar experimental tienen formas irregulares y grandes lagunas intervasculares (Figura 2A, dos paneles inferiores) cuando se compara con la microcirculación pulmonar normal (Figura 2A, panel s...

Watch this Scientific Journal Video about Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats at JoVE.com

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Medición Automatizada de la microcirculación de la sangre Velocidad del caudal en metástasis pulmonar de ratas

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

automated-measurement-microcirculatory-blood-flow-velocity-pulmonary

Research

JoVE Journal

Medicine

8.4K vistas.  Duke University Medical Center. A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

Video: Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

Medición in vivo de la capa de ratón pulmonar superficie endotelial

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Investigación

Medicina

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

Medición automática de enfisema pulmonar y la pequeña vía aérea Remodelación de cigarrillos sin humo Ratones expuestos

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Increasing Pulmonary Artery Pulsatile Flow Improves Hypoxic Pulmonary Hypertension in Piglets

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular failure. Current treatments are limited. Physiologically, pulsatile blood flow is detrimental to the vasculature. In response to sustained pulsatile stress, vessels release nitric oxide (NO) to induce vasodilation for self-protection. Based on this observation, this study developed a protocol to assess whether an artificial pulmonary pulsatile blood flow could induce an NO-dependent decrease in pulmonary artery pressure. One group of piglets was exposed to chronic hypoxia for 3 weeks and compared to a control group of piglets. Once a week, the piglets underwent echocardiography to assess PAH severity. At the end of hypoxia exposure, the piglets were subjected to a pulsatile protocol using a pulsatile catheter. After being anesthetized and prepared for surgery, the jugular vein of the piglet was isolated and the catheter was introduced through the right atrium, the right ventricle and the pulmonary artery, under radioscopic control. Pulmonary artery pressure (PAP) was measured before (T0), immediately after (T1) and 30 min after (T2) the pulsatile protocol. It was demonstrated that this pulsatile protocol is a safe and efficient method of inducing a significant reduction in mean PAP via an NO-dependent mechanism. These data open up new avenues for the clinical management of PAH.

Pulmonary hypertension is associated with a significant reduction in pulmonary artery pulsatility, contractility and elasticity, contributing to an increase in pulmonary artery pressure and pulmonary resistance. Using a hypoxic piglet model, this study demonstrated that improving pulmonary artery plasticity using a newly developed pulsatile catheter improves hypoxic pulmonary hypertension.

El aumento de la arteria pulmonar de flujo pulsátil Mejora hipóxico hipertensión pulmonar en lechones

Pulmonary arterial hypertension (PAH) is a disease affecting distal pulmonary arteries (PA). These arteries are deformed, leading to right ventricular ...

Transcutaneous Microcirculatory Imaging in Preterm Neonates

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest capillaries, arterioles and venules. The magnifying effect visualizes the flow pattern of erythrocytes through these vessels.
It's non-invasive character makes it suitable to apply in (preterm) neonates, even in cardiorespiratory unstable patients. In adults and children, MI is mainly performed sublingually, but this is not possible in preterm infants as these cannot cooperate and the size of the probe is problematic. In preterm infants, MI is therefore performed transcutaneously. Their thin skin makes it possible to obtain high quality images of peripheral microcirculation. 
In this manuscript we will demonstrate the method of transcutaneous MI in preterm infants. We will focus on the different techniques and provide tips to optimize image quality. The highlights of software settings, safety and offline analysis are also addressed.

Microcirculatory imaging (MI) is used to monitor peripheral perfusion in critically ill or preterm neonates. This manuscript and video demonstrates the optimal approach for obtaining high-quality images.

Transcutánea microcirculatoria Imaging en neonatos prematuros

Microcirculatory imaging (MI) is a relatively new research tool mainly used in the intensive care setting. MI provides a clear view of the smallest ...

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 ...

Simultaneous Electrocardiography Recording and Invasive Blood Pressure Measurement in Rats

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. Research focusing on cardiovascular disease models, potential cardiovascular therapeutic targets or pharmaceutical agents requires assessment of systemic arterial pressure and heart rhythm changes. In situations where radio telemetry systems are not available or affordable, the technique of femoral artery cannulation is an alternative way to obtain intra-arterial pressure waveform recordings and systemic BP measurements. This technique is economical and can be performed with standard equipment in animal facilities. However, invasive arterial pressure recording requires cannulation of small arteries, which can be a challenging surgical skill. Here, we present step-by-step protocols for femoral artery cannulation procedures. Key procedures include the calibration of the data acquisition system, tissue dissection and femoral artery cannulation, and setup of the arterial cannulation system for pressure recording. Surface electrocardiography recording procedures are also included. We also present examples of BP recordings from normotensive and hypertensive rats. This protocol allows reliable direct recordings of systemic BP with simultaneous electrocardiography.

Here, we describe a setup for simultaneous recording of electrocardiography and intra-arterial blood pressure (BP) in experimental rats, which can be done with standard equipment in animal facilities and can be applied to physiological or pharmacological studies to investigate pathogenic or therapeutic mechanisms in cardiovascular medicine.

Grabación simultánea de electrocardiografía y sangre invasiva presión medida en ratas

For studies related to cardiovascular physiology or pathophysiology, blood pressure (BP) and electrocardiography are basic observational parameters. ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

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

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

Evaluación de la Reserva de Flujo Coronario Después de la Reperfusión de Isquemia Miocárdica en Ratas

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

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

Pruebas de hemocompatibilidad de implantes que contactan con la sangre en un modelo de bucle de flujo que imita el flujo sanguíneo humano

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.