Los autores desean agradecer a Keith Michel del Anderson Pequeño Fondo imágenes Animal MD por su asistencia técnica a micro-TC, Edward TH Yeh, MD, para la asistencia quirúrgica y Rebecca Bartow, PhD, por su asistencia editorial. Este trabajo fue apoyado en parte por la Asociación Americana del Corazón.

1. Cebado de la bomba osmótica <ol><li> Utilizar técnicas estériles (por ejemplo, guantes, campana de flujo laminar) en la preparación de las bombas. Retire la bomba y el moderador de flujo del embalaje. Pesar la bomba de vacío y registre el peso (mg). </li><li> Llene la bomba lentamente, para evitar la creación de burbujas de aire, mediante el uso de una jeringa pequeña de 1 cc y de punta roma tubo de llenado 27G. Cuando la solución alcanza la parte superior de la bomba, pare de llenado. </li><li> Limpie el exceso de solución y pesar la bomba de llenado. Para la mayoría de soluciones acuosas, el peso en mg es el mismo que el volumen en l. La diferencia en el peso en vacío frente llena debe ser más de 90% del volumen de llenado media especificada. Si no, evacuar la bomba con la jeringa y volver a llenar. Si tiene algún problema técnico, por favor consulte la sección de solución de problemas de las instrucciones de la bomba en http://www.alzet.com/products/guide_to_use/filling.html </li><li> Use un par de pinzas para romper el ala blanca de la moderat flujoo, tenga cuidado de no doblar o aplastar el tubo. Adjuntar un catéter P50 a cualquiera de los extremos del moderador, que abarca alrededor de 3-4 mm. Usar una jeringa para llenar el catéter y adjunta moderador y salir de la jeringa unida a la parte distal del catéter. </li><li> Insertar el moderador de flujo en la bomba hasta que el catéter esté a ras con la superficie de la bomba. A continuación, retirar la jeringa desde el extremo distal del catéter. Coloque la bomba en un 0,9% de solución salina estéril o solución de PBS en una incubadora de 37 ° C durante un mínimo de 6 horas (preferiblemente O / N) para cebar la bomba antes de implantarlo en el ratón. El caudal de la bomba varía con el número de modelo (por ejemplo, Modelo 1002 = 0,25 l / h durante 14 días). </li></ol> 2. Creación de isquemia del miembro posterior unilateral en el ratón <ol><li> Se anestesia a los ratones con isoflurano conjunto entre 2-4% en 100% de O 2 a una velocidad de flujo de 0,4 L / min y eliminar el vello (preferentemente el día antes de la cirugía) en la zona de extremidad posterior y la parte posterior con un depilatocrema ry. </li><li> Esterilice todos los instrumentos quirúrgicos antes de la cirugía. Coloque el ratón en la cámara de la inducción anestésica. Usar la misma configuración de la anestesia en la Sección 2.1. </li><li> Espere hasta que el ratón no responde a los estímulos externos y luego se coloca en decúbito prono sobre la mesa de operaciones con un cono de nariz amueblada que ofrece un flujo continuo de isoflurano (1,5 a 2,0%). </li><li> Inyectar la dosis recomendada de anestésico (bupivacaína; 0,25% (&lt;1 ml / kg)) en el sitio de la incisión; tratar la piel con una solución de povidona-yodo seguido por etanol al 70%. Hacer una incisión de 4-6 mm en la parte posterior (en el lado de la columna vertebral, en la región de la escápula). Separe la membrana subcutánea con unas pinzas finas, luego gire el ratón a la posición supina tratar la piel con una solución de yodo-povidona seguido de etanol al 70% y hacer 0.8-1 cm incisión con tijeras micro-quirúrgica sobre la rodilla hacia el muslo, siguiendo el camino de la arteria femoral. </li><li> Túnel de lalado del torso con un tubo de vaina pequeña (6-francés) de la parte media del muslo a la incisión en la parte posterior. </li><li> Coloque el ratón en la posición prona. Retire la bomba / catéter osmótica cebado de la solución de PBS y guiar el catéter, a través de la tubería de revestimiento, para el muslo. </li><li> Coloque la bomba en el interior de la parte trasera y cerrar la incisión de vuelta con 6-0 sutura de prolene (tratamiento de la piel con una solución de yodo-povidona seguido de etanol al 70%). Coloque la parte posterior del ratón en la posición de decúbito supino; quitar el tubo de vaina, dejando el catéter expuesto a través de la incisión en el muslo. Asegure la pierna con cinta quirúrgica. </li><li> Separe el tejido subcutáneo de grasa para revelar la vasculatura. Para visualizar mejor la vasculatura, utilizar un retractor para abrir la incisión. </li><li> Pierce la vaina femoral membranosa suavemente con unas pinzas de punta fina para acceder al paquete vascular. Diseccionar y separar la arteria femoral de la vena femoral y el nervio cerca de la ingle, con cuidado de no causar exsangrado excesivo. </li><li> Coloque dos 8-0 suturas de prolene adyacentes para interrumpir la arteria femoral 2-3 mm proximal a la bifurcación femoral (el origen de la femoral profunda y arterias epigástrica). </li><li> Cortar el catéter a la longitud adecuada y coloque la punta a la región anterolateral del muslo, fijándolo con una sutura de prolene 6-0 a la fascia superficial para asegurar la entrega específica de la sustancia. Cierre la incisión de la pierna con una sutura continua 6-0. Administrar una última inyección subcutánea de analgésico (buprenorfina, 0,1 mg / kg), el tratamiento de la piel con una solución de povidona yodada seguido de etanol al 70% y devolver el ratón a la jaula. Utilice una lámpara de calentamiento durante la recuperación. </li><li> Después de un período de recuperación de 30 minutos, prepare el ratón por láser Doppler para confirmar la creación de la isquemia de extremidades posteriores. </li><li> Controle el animal dos veces al día durante 5 días después de la cirugía. La movilidad del animal se observó para analizar el dolor; residuos en la jaula servirá como un Indicdor de comer. Además, durante el período de monitor de determinar si el animal está experimentando dolor o malestar y dar una inyección subcutánea de buprenorfina (0.1mg/kg) dos veces al día. </li></ol> 3. Imágenes Preparación de Muestras <ol><li> Anestesiar el ratón como se indica en el apartado 2.1. Transferir el ratón sobre la mesa de operaciones en posición supina. Inyectar 1.000 UI de heparina por vía subcutánea para prevenir la coagulación en la vasculatura. Mientras que el ratón está unido a la ojiva, hacer una incisión lo suficientemente grande como para exponer la cavidad torácica y la parte inferior del abdomen. Mover los órganos abdominales hacia el lado izquierdo del animal para exponer la aorta abdominal infrarrenal (IAA). Separe cuidadosamente la aorta de la vena cava inferior. </li><li> Ligar el IAA proximal para reducir la hemorragia durante la inserción del catéter. A continuación, hacer una pequeña incisión en el IAA. Inserte un 27 ½ T contundente aguja (unido a un catéter P10) en el IAA y fijarlo con una sutura de 6-0. </li><li> Llenar dosJeringas de 20 cc con solución salina heparinizada y 10% neutros formalina tamponada (MNB). Llene una jeringa de 5 cc con un compuesto de caucho de silicona radiopaca contraste. Mezclar los tres ingredientes de MV-compuesto: MV-disolvente: MV agente de curado de los ratios de trabajo de 1:1.25:0.5. Dado que la mezcla (Microfil) sólo tiene un tiempo de trabajo de 20 minutos antes de la polimeriza el agente, se recomienda no agregar el agente MV-curado hasta justo antes de la inyección en el sistema vascular. </li><li> Haga un corte en la aurícula derecha y administrar una sobredosis de isoflurano para inducir la eutanasia y para permitir el drenaje del exceso de líquido. Conecte la jeringa llena de solución salina a la punta de la aguja y comenzar a limpiar los vasos durante 5 min. Asegúrese de que toda la sangre se inunda de los vasos; apagar el isoflurano y desconecte la ojiva, una vez que comience el lavado. A continuación, conecte la jeringa llena de formol al catéter detallado anteriormente (3.2) y perfuse presión durante otros 5 min. Por último, la perfusión de la última jeringuilla, con el contrasteagente, poco a poco en el ratón durante 5 min. </li><li> Opcional: Fotografía del Microfil en la vasculatura con una cámara para asegurarse de que sólo la estructura arterial es perfundido. </li><li> Coloque el ratón en un recipiente lleno con NBF al 10% y se almacena a 4 ° CO / N para la polimerización de agente de contraste. Después de 24 horas, quitar la piel en la mitad inferior del torso (distal a la ligadura de la aorta abdominal inferior) mediante el uso de herramientas de disección, actualizar la NBF 10%, y colocar el ratón en el contenedor durante 4 días más a TA. </li><li> Diseccionar las extremidades posteriores y contralateral isquémica, y colocar las muestras en una solución de ácido fórmico (Cal-Ex II) durante 48 horas para facilitar la descalcificación del hueso. A continuación, lavar las muestras con agua corriente durante 1 hora y volver al 10% NBF y proceder con Faxitron (sistema de rayos x utiliza para asegurarse que el hueso está descalcificada) y micro-TC. </li></ol>

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	<li>Epstein, S. E., Fuchs, S., Zhou, Y. F., Baffour, R., Kornowski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+interventions+for+enhancing+collateral+development+by+administration+of+growth+factors:+basic+principles,+early+results+and+potential+hazards.">Therapeutic interventions for enhancing collateral development by administration of growth factors: basic principles, early results and potential hazards.</a> Cardiovasc. Res. 49, 532-542 (2001).</li><li>Esaki, J., Marui, A., Tabata, Y., Komeda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+release+systems+of+angiogenic+growth+factors+for+cardiovascular+diseases.">Controlled release systems of angiogenic growth factors for cardiovascular diseases.</a> Expert Opin. Drug Deliv. 4, 635-649 (2007).</li><li>Bentley, M. D., Ortiz, M. C., Ritman, E. L., Romero, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+microcomputed+tomography+to+study+microvasculature+in+small+rodents.">The use of microcomputed tomography to study microvasculature in small rodents.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 282, 1267-1279 (2002).</li><li>Golomb, B. A., Dang, T. T., Criqui, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+arterial+disease:+morbidity+and+mortality+implications.">Peripheral arterial disease: morbidity and mortality implications.</a> Circulation. 114, 688-699 (2006).</li><li>Hiatt, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+therapy+for+peripheral+arterial+disease+and+claudication.">Pharmacologic therapy for peripheral arterial disease and claudication.</a> J. Vasc. Surg. 36, 1283-1291 (2002).</li><li>Hirsch, A. T., 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=Peripheral+arterial+disease+detection,+awareness,+and+treatment+in+primary+care.">Peripheral arterial disease detection, awareness, and treatment in primary care.</a> JAMA. 286, 1317-1324 (2001).</li><li>McDonald, D. M., Choyke, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+angiogenesis:+from+microscope+to+clinic.">Imaging of angiogenesis: from microscope to clinic.</a> Nat. Med. 9, 713-725 (2003).</li><li>Hirsch, A. T., 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=ACC/AHA+Guidelines+for+the+Management+of+Patients+with+Peripheral+Arterial+Disease+(lower+extremity,+renal,+mesenteric,+and+abdominal+aortic):+a+collaborative+report+from+the+American+Associations+for+Vascular+Surgery/Society+for+Vascular+Surgery,+Society+for+Cardiovascular+Angiography+and+Interventions,+Society+for+Vascular+Medicine+and+Biology,+Society+of+Interventional+Radiology,+and+the+ACC/AHA+Task+Force+on+Practice+Guidelines+(writing+committee+to+develop+guidelines+for+the+management+of+patients+with+peripheral+arterial+disease)--summary+of+recommendations.">ACC/AHA Guidelines for the Management of Patients with Peripheral Arterial Disease (lower extremity, renal, mesenteric, and abdominal aortic): a collaborative report from the American Associations for Vascular Surgery/Society for Vascular Surgery, Society for Cardiovascular Angiography and Interventions, Society for Vascular Medicine and Biology, Society of Interventional Radiology, and the ACC/AHA Task Force on Practice Guidelines (writing committee to develop guidelines for the management of patients with peripheral arterial disease)--summary of recommendations.</a> J. Vasc. Interv. Radiol. 17, 1383-1397 (2006).</li><li>Zagorchev, L., Mulligan-Kehoe, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+vessels+in+mouse+models+of+disease.">Molecular imaging of vessels in mouse models of disease.</a> Eur. J. Radiol. 70, 305-311 (2009).</li><li>Losordo, D. W., Dimmeler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+angiogenesis+and+vasculogenesis+for+ischemic+disease.+Part+I:+angiogenic+cytokines.">Therapeutic angiogenesis and vasculogenesis for ischemic disease. Part I: angiogenic cytokines.</a> Circulation. 109, 2487-2491 (2004).</li><li>Waters, R. E., Terjung, R. L., Peters, K. G., Annex, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+models+of+human+peripheral+arterial+occlusive+disease:+implications+for+investigation+of+therapeutic+agents.">Preclinical models of human peripheral arterial occlusive disease: implications for investigation of therapeutic agents.</a> J. Appl. Physiol. 97, 773-780 (2004).</li></ol>

Los autores declaran no tener ningún conflicto de intereses. Todos los procedimientos con animales se realizaron de conformidad con las Directrices para el Cuidado y Uso de Animales de Laboratorio y fueron aprobados por el Cuidado de Animales institucional y el empleo Comisión (IACUC) de la Universidad de Texas Health Science Center en Houston.

Aquí presentamos un método para la administración de fármacos / sustancias osmótica en un modelo murino de isquemia de extremidad posterior. Además, se describe una técnica de colada en el que hemos utilizado Microfil para producir una reconstrucción 3D para el análisis de la red vascular. El nivel o la gravedad de la isquemia varía según el lugar donde se realiza la ligadura / resección arterial. Hemos creado una doble ligadura en la arteria femoral común proximal al origen de ...

La enfermedad arterial periférica (PAD) es una enfermedad aterosclerótica que causa suministro insuficiente de sangre en las piernas 4. Afecta 8-12000000 estadounidenses, y los tratamientos médicos actuales sólo ofrecen un alivio limitado 5,6. Nuevos agentes terapéuticos que mejoran la circulación sanguínea en las piernas no sólo se frene la progresión de la enfermedad, sino también mejorar la calidad de vida. La incidencia de la PAD es mayor en personas mayores de 50 años, la terapia farmacológica tan local es una modalidad de tratamiento más deseable debido a que la reducción de la función hepática y renal a menudo en pacientes de mayor edad pueden disminuir el metabolismo de drogas y aumentar los efectos secundarios con la administración sistémica. Por lo tanto, hemos creado un modelo de ratón de la EAP para examinar si los agentes administrados localmente aliviar la isquemia miembro posterior al promover la angiogénesis y el remodelado microvascular. Específicamente, se utilizó una bomba osmótica sondaje para suministrar continuamente el agente terapéutico a laisquémica del músculo del muslo de ratones. Utilizando el sistema de entrega, hemos sido capaces de mantener concentraciones óptimas de la droga en el entorno local; este enfoque permite una bioactividad medicamento apropiado, evita los efectos secundarios sistémicos, y supera la desventaja de acceso limitado local de drogas asociados con la administración sistémica. Además, para evaluar si los agentes administrados localmente promueven la revascularización, se utilizó fundición avanzada y las técnicas de imagen de alta definición que permiten la cuantificación de los cambios en la microvasculatura. En conjunto, la combinación de las metodologías utilizadas en este artículo de vídeo es útil en estudios preclínicos para ayudar en la comprensión de revascularización inducida farmacológicamente en los pacientes con PAD 7-9.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent/Material</td>
		 </tr>
		 <tr>
			<td>Surgical tools</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Puritan sterile cotton swabs</td>
			<td>Fisher Scientific</td>
			<td>22-029-499</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Betadine (povidone-iodine)</td>
			<td>Fisher Scientific</td>
			<td>19-065534</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>70% Alcohol pads</td>
			<td>Fisher Scientific</td>
			<td>NC9926371</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Phosphate buffered saline</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>6-0 prolene suture</td>
			<td>Cardinal Health</td>
			<td>8709</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>8-0 prolene suture</td>
			<td>Cardinal Health</td>
			<td>2775</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Depilatory cream</td>
			<td>Nair</td>
			<td></td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Osmotic pump</td>
			<td>ALZET</td>
			<td>1002</td>
			<td>Type: Tool, 14 day release</td>
		 </tr>
		 <tr>
			<td>Vinyl catheter</td>
			<td>ALZET</td>
			<td>7760</td>
			<td>Type: Tool</td>
		 </tr>
		 <tr>
			<td>Heparinized saline (0.9%)</td>
			<td>Baxter</td>
			<td>2B0944</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Neutral buffered formalin</td>
			<td>Richard-Allan Scientific</td>
			<td>5705</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Microfil (silicone rubber contrast agent)</td>
			<td>Flowtech</td>
			<td>MV-112</td>
			<td>Type Reagent, Microfil White</td>
		 </tr>
		 <tr>
			<td>Cal-Ex II (formic acid solution)</td>
			<td>Fisher Scientific</td>
			<td>CS511-1D</td>
			<td>Type: Reagent</td>
		 </tr>
		 <tr>
			<td>Buprenex</td>
			<td>CIII</td>
			<td>7571</td>
			<td>Type: Analgesic</td>
		 </tr>
		 <tr>
			<td>Bupivicaine</td>
			<td>Hospira, Inc.</td>
			<td>381</td>
			<td>Type: Analgesic</td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>Dissecting microscope</td>
			<td>Carl Zeiss Microimaging</td>
			<td>Zeiss Stemi 2000-C</td>
			<td>Type:Equipment</td>
		 </tr>
		 <tr>
			<td>Laser Doppler perfusion imager</td>
			<td>Perimed Inc.</td>
			<td>Periscan PIM3 </td>
			<td>Type:Equipment</td>
		 </tr>
		 <tr>
			<td>Micro-CT imaging system</td>
			<td>GE Healthcare</td>
			<td>Explore Locus SP </td>
			<td>Type:Equipment</td>
		 </tr>
		</tbody>
 </table>

osmotic drug delivery to ischemic hindlimbs and perfusion of vasculature with microfil for micro-computed tomography imaging

La investigación preclínica en modelos animales de enfermedad arterial periférica juega un papel vital en la prueba de la eficacia de los agentes terapéuticos diseñados para estimular la microcirculación. La elección del método de entrega para estos agentes es importante porque la vía de administración afecta profundamente a la bioactividad y la eficacia de estos agentes de 1,2. En este artículo se demuestra cómo administrar localmente una sustancia en miembros posteriores isquémicas mediante una bomba osmótica sondaje. Esta bomba puede suministrar un volumen fijo de solución acuosa de forma continua durante un período de tiempo asignado. También presentamos nuestro modelo de ratón de isquemia de extremidad posterior unilateral inducida por la ligadura de la arteria femoral común proximal al origen de la femoral profunda y arterias epigástrica en la pata trasera izquierda. Por último, se describe en la canulación in vivo y la ligadura de la aorta abdominal infrarrenal y la perfusión de la vasculatura del miembro posterior con Microfil, un agente radiopaco de fundición de silicona. Microfil puede perfundir y llenar todo el lecho vascular (arterial y venoso), y porque hemos ligó el conducto vascular mayor para la salida, el agente puede ser retenido en la vasculatura para el futuro de formación de imágenes ex vivo con el uso de la pequeña muestra de micro-CT 3 .

La animación demuestra la inserción de la bomba osmótica y la cirugía isquemia miembro posterior se detalla en el protocolo. Figura 1 muestra imágenes de láser Doppler de la perfusión de la extremidad posterior, lo que confirma la isquemia. Después de la colada vascular con Microfil, una imagen micro-CT 3D de la red vascular muestra que Microfil puede llenar los vasos efectivamente (Figura 2A), pero puede ocurrir que las discontinuidades (Figura 2B) debido a div...

Watch this Scientific Journal Video about Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging at JoVE.com

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

Mostramos aquí la<em&gt; In vivo</em&gt; Inserción de una bomba osmótica para la administración local de fármacos constante y la creación de isquemia de extremidad posterior en un modelo de ratón. Por otra parte, la vasculatura miembro posterior se perfunde con Microfil, un agente radiopaco de silicona, para prepararse para la tomografía micro-computarizada (micro-TC).

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 ...

osmotic-drug-delivery-to-ischemic-hindlimbs-perfusion-vasculature

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Medicine

12.2K vistas.  The Texas Heart Institute at St. Luke's Episcopal Hospital. Mostramos aquí la In vivo Inserción de una bomba osmótica para la administración local de fármacos constante y la creación de isquemia de extremidad posterior en un modelo de ratón. Por otra parte, la vasculatura miembro posterior se perfunde con Microfil, un agente radiopaco de silicona, para prepararse para la tomografía micro-computarizada (micro-TC).

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

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

Visualization of the vasculature is becoming increasingly important for understanding many different disease states. While several techniques exist for imaging vasculature, few are able to visualize the vascular network as a whole while extending to a resolution that includes the smaller vessels1,2. Additionally, many vascular casting techniques destroy the surrounding tissue, preventing further analysis of the sample3-5. One method which circumvents these issues is micro-Computed Tomography (&mu;CT). &mu;CT imaging can scan at resolutions &lt;10 microns, is capable of producing 3D reconstructions of the vascular network, and leaves the tissue intact for subsequent analysis (e.g., histology and morphometry)6-11. However, imaging vessels by ex vivo &mu;CT methods requires that the vessels be filled with a radiopaque compound. As such, the accurate representation of vasculature produced by &mu;CT imaging is contingent upon reliable and complete filling of the vessels. In this protocol, we describe a technique for filling mouse coronary vessels in preparation for &mu;CT imaging.
Two predominate techniques exist for filling the coronary vasculature: in vivo via cannulation and retrograde perfusion of the aorta (or a branch off the aortic arch) 12-14, or ex vivo via a Langendorff perfusion system 15-17. Here we describe an in vivo aortic cannulation method which has been specifically designed to ensure filling of all vessels. We use a low viscosity radiopaque compound called Microfil which can perfuse through the smallest vessels to fill all the capillaries, as well as both the arterial and venous sides of the vascular network. Vessels are perfused with buffer using a pressurized perfusion system, and then filled with Microfil. To ensure that Microfil fills the small higher resistance vessels, we ligate the large branches emanating from the aorta, which diverts the Microfil into the coronaries. Once filling is complete, to prevent the elastic nature of cardiac tissue from squeezing Microfil out of some vessels, we ligate accessible major vascular exit points immediately after filling. Therefore, our technique is optimized for complete filling and maximum retention of the filling agent, enabling visualization of the complete coronary vascular network &#x2013; arteries, capillaries, and veins alike.

Visualization of the coronary vessels is critical to advancing our understanding of cardiovascular diseases. Here we describe a method for perfusing murine coronary vasculature with a radiopaque silicone rubber (Microfil), in preparation for micro-Computed Tomography (&mu;CT) imaging.

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 ...

Investigación

Medicina

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 ...

Intraluminal Drug Delivery to the Mouse Arteriovenous Fistula Endothelium

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The simple murine AVF model was combined with intraluminal administration of drug solution to the venous endothelium at the same time as fistula creation. Technical aspects of this model are discussed. Under general anesthesia, an abdominal incision is made and the aorta and inferior vena cava (IVC) are exposed. The infra-renal aorta and IVC are dissected for clamping. After proximal and distal clamping, the puncture site is exposed and a 25 G needle is used to puncture both walls of the aorta and into the IVC. Immediately after the puncture, a reporter gene-expressing viral vector was infused in the IVC via the same needle, followed by 15 min of incubation. The intraluminal administration method enabled more robust viral gene delivery to the venous endothelium compared to administration by the external route. This novel method of delivery will facilitate studies that explore the role of the endothelium in AVF maturation and enable intraluminal drug delivery at the time of surgical operation.

After puncturing the aorta through the inferior vena cava (IVC) to create an aorto-caval fistula in the mouse, solution containing a drug is infused into the IVC via the same needle, followed by incubation. This method enables more robust drug delivery to the venous endothelium compared to the external route.

Intraluminal entrega de la droga a la fístula arteriovenosa ratón endotelio

Delivery of therapeutic agents to enhance arteriovenous fistula (AVF) maturation can be administered either via intraluminal or external routes. The ...

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 ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

Método de la entrega directa segmentaria intra-hepática utilizando un modelo de hígado de rata hiliar abrazadera

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Método optimizado de LC-MS/MS para el análisis de alto rendimiento de las muestras clínicas de Ivacaftor, sus principales metabolitos y Lumacaftor en fluidos biológicos de pacientes con Fibrosis quística

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Mejor método para el establecimiento de un In Vitro Barrera Blood - Brain modelo basado en las células endoteliales de cerebro porcino

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

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.