This work was supported by National Institutes of Health Grant 5-P20GM103629 to WLM and the Tulane Center for Aging. We would like to thank Matthew Nice for his help with editing the protocol text.

Todos los experimentos y procedimientos con animales fueron aprobados por el Comité de Cuidado y Uso de Animales Institucional de la Universidad de Tulane (IACUC). 1. Procedimiento de configuración quirúrgica <ol><li> instrumentos quirúrgicos, equipos de autoclave, y suministros de cultivo antes de la cirugía. suministros quirúrgicos para cada rata incluyen: 1 sábana, 1 sábana con orificio precortado (0,5 pulgadas x 1,5 pulgadas) en el centro, gasas, y 1 almohadilla inferior absorbente. Los instrumentos quirúrgicos incluyen: 1 escalpelo con una cuchilla de número 10, 2 pares de pinzas, y un par de tijeras finas. Los suministros de cultivo incluyen: 1 sábana, 1 par de pinzas, y se prepararon para placas de 6 pocillos con filtros de policarbonato. </li><li> Esterilizar una plataforma de plexiglás, una etapa quirúrgica y un espacio de mesa quirúrgica con 70% de etanol. Mantenga la etapa quirúrgica en un recipiente estéril hasta su uso. <ol><li> Crear una etapa quirúrgica mediante la perforación de una de aproximadamente 2 en por 1 en el agujero en el centro de una placa de cultivo de 100 mm. A continuación, utilice papel de lija para suavizarlos bordes afilados y añaden una capa de pegamento de silicona a los bordes del agujero para crear una superficie elevada para los tejidos. </li><li> Por otra parte, el diseño de la etapa quirúrgica utilizando software de CAD y hacer un 3-D de impresión (Figura 1). </li></ol></li><li> Coloque una almohadilla inferior absorbente estéril y extienda una plataforma de plexiglás en la parte superior de la misma. Coloque la sábana, sin un agujero pre-corte, a través de una almohadilla caliente al lado de la almohadilla inferior absorbente. </li><li> Pre-caliente solución salina estéril tamponada con fosfato (PBS), medios de comunicación y solución salina a 37 ° C. Coloque los medios de comunicación y PBS en placas de cultivo separadas encima de la resistencia de calentamiento y el lugar de solución salina en un tubo cónico de 50 ml al lado de la configuración quirúrgica. </li><li> Asegúrese de que todos los paquetes se abren antes del comienzo de la cirugía para asegurar una manipulación estéril de todos los materiales. Una lista completa de las herramientas comunes que se utilizan en este procedimiento se muestran en el Tabla de materiales quirúrgicos y herramientas específicas. </li></ol> 2. MesenterLa recolección de tejidos y <ol><li> Uso de adultos ratas Wistar machos (350 ± 25 g; 6 - 8 semanas de edad). Otras cepas y edades de las ratas pueden ser sustituidos. </li><li> Anestesiar la rata a través de una inyección intramuscular de ketamina (80 mg / kg de peso corporal) y xilazina (8 mg / kg de peso corporal). Confirmar la rata está bajo anestesia apretando entre los dedos para comprobar si hay una respuesta refleja; no debería haber ninguna. analgesia preventiva para este procedimiento terminal no es necesario. </li><li> Afeitarse la región abdominal y eliminar el vello restante usando crema de depilación. Limpiar la piel abdominal dos veces con alcohol isopropílico al 70%, seguido de povidona-yodo. Para las toallitas el cirujano debe comenzar en el centro de la zona quirúrgica y se mueven hacia el exterior de la zona preparada de una manera circular para no solapar zonas que han sido lavadas previamente con la misma pieza de gasa estéril o bastoncillo de algodón estéril. A continuación, traslado de los animales a la configuración quirúrgica estéril y colocar encima de la plataforma de plexiglásformar. </li><li> El uso de una hoja de bisturí, hacer un 0,75 en - 1,25 en la incisión en el intestino a partir del 1 de debajo del esternón. Tenga cuidado de no perforar el intestino o mesenterio (1 capa de la piel, 1 capa de tejido conectivo, y 1 capa de músculo). </li><li> Colocar una sábana con un agujero pre-corte sobre la incisión y colocar una etapa quirúrgica estéril encima de la cortina. Asegúrese de que los alinea con la apertura de la incisión. Utilizar bastoncillos de algodón estériles para localizar y extraer el íleon a través de la boca del escenario quirúrgico. </li><li> Tire de 6 - 8 ventanas mesentéricas a través de la fase usando bastoncillos de algodón, y tenga cuidado de no tocar las ventanas (Figura 1). Los tejidos se recogen típicamente de la región del íleon del intestino delgado comenzando cerca del ciego. Mantenga tejidos expuestos húmedo con solución salina estéril calentado según sea necesario con una jeringa estéril a gotear la solución. </li><li> La eutanasia a las ratas mediante la inyección intracardíaca de pentobarbital sódico (0,2 ml por rata). Antes de retirar misenteric ventanas, aseguran que la rata se sacrificó palpando el corazón; no debería haber ningún pulso. </li><li> Retire los tejidos mesenterio deseados mediante el uso de pinzas para agarrar la almohadilla de grasa y unas tijeras finas para cortar la ventana. Deja un borde de grasa (2 mm) alrededor de la ventana. Lavar los tejidos una vez en PBS estéril calentado y una vez en los medios de comunicación. </li><li> Volver íleon exteriorizada a la cavidad abdominal y disponer de animales de acuerdo con las directrices institucionales. </li></ol> 3. Mesenterio cultivo tisular para estudios con lapso de tiempo <ol><li> Transferencia de suministros de cultivo tratadas en autoclave (ver sección 1.1) y los tejidos a una campana de flujo laminar estéril. </li><li> Use pinzas para transferir cada tejido encima de una membrana de filtro de policarbonato. tejidos para discapacitados cerca de la almohadilla de grasa para evitar daños en el sistema vascular. </li><li> propagar rápidamente el tejido usando la almohadilla de grasa, teniendo cuidado de no tocar la ventana. Invertir el inserto con el tejido en la parte inferior de una placa de 6 pocillos y cubrir con 3 ml de medio (Figura 1 </strong&gt;). medios típicos utilizados para este procedimiento incluye medio esencial mínimo (MEM) con 1% de penicilina estreptomicina (PenStrep) y 10% de suero bovino fetal (FBS). Los medios pueden complementarse con otros sueros y / o factores de crecimiento para estimular la angiogénesis. </li><li> Repita los pasos 3.2 a 3.3 para cada tejido y la cultura en condiciones estándar de la incubadora (5% CO2, 37 ° C) durante un máximo de 5 días. </li></ol> 4. Time-lapse de Mesenterio Tissue <ol><li> En el día de formación de imágenes, complementar los medios de comunicación en cada pocillo con conjugado BSI-lectina y se incuba bajo condiciones de cultivo estándar para 30 min. Lavar tejidos dos veces con medio libre de lectina. BSI-lectina mancha permanecerá visible en el tejido del mesenterio para un máximo de 3 días de cultivo. </li><li> La transferencia de la placa a una platina del microscopio. Identificar los vasos sanguíneos y linfáticos en función de su estructura de la morfología y de la red. </li><li> Localizar una región de red deseada en cada tejido y tomar imágenes. Tomar nota de la proyección de imagenubicación para garantizar la misma región será capturado por las imágenes subsiguientes. Si se utiliza una plataforma motorizada, documentar las coordenadas. </li><li> Devolver los tejidos a la incubadora y continuar con la cultura hasta el punto final deseado. Repita los pasos 4.1 a 4.3, según sea necesario en función de los puntos de tiempo experimentales deseados. </li></ol> 5. Tejido Immunolabeling <ol><li> Etiquetado BSI-lectina <ol><li> Incubar los tejidos durante 30 min a 37 ° C con 1:40 lectina conjugada con FITC en los medios (2,5 ml solución de anticuerpo por pocillo en placa de 6 pocillos), seguido de dos lavados con medios de comunicación. Para enjuagues, añadir medios y luego vuelva a colocar inmediatamente. </li></ol></li><li> Etiquetado Live / Dead <ol><li> Incubar los tejidos durante 10 min a 37 ° C con 1: 500 de etidio 2 mM homodímero-1 y 1: 500 1 mM de calceína AM en los medios (2,5 ml solución de anticuerpo por pocillo en placa de 6 pocillos), seguido de dos lavados con medios de comunicación. </li></ol></li><li> BSI-lectina / Etiquetado NG2 <ol><li> tejidos separadas en un microscopio deslizaronE (1 - 2 tejidos / diapositiva) y dejar secar. Eliminar el exceso de grasa con un bisturí presionando firmemente para extirpar la grasa. </li><li> Para reparar tejidos en metanol frío durante 30 minutos a -20 ° C. Lavar los tejidos con PBS (3 x 10 min). </li><li> Para el etiquetado de anticuerpo primario incubar tejidos para 1 h a temperatura ambiente con 1: anticuerpo policlonal de conejo y de NG2 100 5% de suero de cabra normal (NGS). Lavar los tejidos con PBS (3 x 10 min). </li><li> Para el etiquetado de anticuerpo secundario se incuba tejidos para 1 h a temperatura ambiente con 1: 100 de cabra anti-conejo conjugado con CY2 anticuerpo (GAR-CY2) y 5% NGS. Lavar los tejidos con PBS (3 x 10 min). </li><li> Incubar los tejidos durante 30 min a temperatura ambiente con 01:40 lectina conjugada con FITC en PBS seguido de dos lavados con PBS. Para enjuagues, añadir PBS y luego reemplace inmediatamente. </li><li> Para montar las diapositivas, con cobertura de tejidos 50:50 PBS y solución de glicerol y el cubreobjetos en la parte superior. Sellar los bordes de diapositivas utilizando el esmalte de uñas. </li></ol></li><li> LYVE-1 / Etiquetado PECAM<ol><li> Difundir los tejidos en un portaobjetos de microscopio (1 - 2 tejidos / diapositiva) y dejar secar. Eliminar el exceso de grasa con un bisturí presionando firmemente para extirpar la grasa. </li><li> Para reparar tejidos en metanol frío durante 30 minutos a -20 ° C. Lavar los tejidos con PBS + 0,1% de saponina (3 x 10 min). </li><li> Para el etiquetado de anticuerpo primario incubar tejidos para 1 h a temperatura ambiente con 1: monoclonal biotinilado anticuerpo CD31 200 del ratón y 1: 100 de conejo policlonal LYVE-1 de anticuerpos en + 0,1% de saponina + 2% de albúmina de suero bovino PBS (BSA) + 5% NGS . Lavar los tejidos con PBS + 0,1% de saponina (3 x 10 min). </li><li> Para el etiquetado de anticuerpo secundario, se incuban los tejidos durante 1 h a temperatura ambiente con 1: anticuerpo de estreptavidina 500 conjugado con Cy3 y 1: 100 GAR-CY2 en PBS + 0,1% de saponina + 2% BSA + 5% NGS. Lavar los tejidos con PBS + 0,1% de saponina (3 x 10 min). </li><li> Para montar diapositivas, tejidos cubierta con 50:50 solución de PBS y glicerol y colocar un cubreobjetos en la parte superior. Sellar los bordes de diapositivas utilizando el esmalte de uñas. </li></ol></li><li> BrdU / BSI-lectina de etiquetado <ol><li> Añadir 1 mg / ml de BrdU a los medios de comunicación y la sustitución de los medios de tejido con una solución de BrdU. Incubar durante 2 horas a 37 ° C. </li><li> Difundir los tejidos en un portaobjetos de microscopio (1 - 2 tejidos / diapositiva) y dejar secar. Eliminar el exceso de grasa con un bisturí presionando firmemente para extirpar la grasa. </li><li> Para reparar tejidos en metanol frío durante 30 minutos a -20 ° C. Lavar los tejidos con PBS (3 x 10 min). </li><li> Desnaturalizar el ADN de tejidos en HCl 2 M durante 1 hora a 37 ° C. Lavar los tejidos en PBS + 0,1% de saponina (3 x 10 min). </li><li> Para el marcaje del anticuerpo primario, se incuban los tejidos durante 1 h a temperatura ambiente con 1: 100 de ratón monoclonal anti-BrdU en PBS + 0,1% de saponina + 2% BSA + 5% NGS. Lavar los tejidos con PBS + 0,1% de saponina (3 x 10 min). </li><li> Para el etiquetado de anticuerpo secundario, se incuban los tejidos durante 1 h a temperatura ambiente con 1: anticuerpo 100 de cabra anti-ratón conjugado con Cy3 (GAM-Cy3) en PBS + 0,1% de saponina + 2% BSA + 5% NGS. Lavar los tejidos con PBS + 0,1% de saponina (3 x 10 min). </li><li&gt; Incubar tejidos para 30 min a temperatura ambiente con una y cuarenta lectina conjugada con FITC en PBS seguido de dos lavados con PBS. </li><li> Para montar las diapositivas, los tejidos de la cubierta con PBS 50:50 y solución de glicerol y el cubreobjetos en la parte superior. Sellar los bordes de diapositivas utilizando el esmalte de uñas. </li></ol></li><li> BSI-lectina etiquetado / CD11b <ol><li> Difundir los tejidos en un portaobjetos de microscopio (1 - 2 tejidos / diapositiva) y dejar secar. Eliminar el exceso de grasa con un bisturí presionando firmemente para extirpar la grasa. </li><li> Para reparar tejidos en metanol frío durante 30 minutos a -20 ° C. Lavar los tejidos con PBS + 0,1% de saponina (3 x 10 min). </li><li> Para el etiquetado de anticuerpo primario incubar tejidos para 1 h a temperatura ambiente con 1: 100 de ratón anti-rata CD11b en PBS + 0,1% de saponina + 2% BSA + 5% NGS. Lavar los tejidos con PBS + 0,1% de saponina (3 x 10 min). </li><li> Para el etiquetado de anticuerpo secundario se incuba tejidos para 1 h a temperatura ambiente con 1: 100 GAM-Cy3 en PBS + 0,1% de saponina + 2% BSA + 5% NGS. Lavar los tejidos con PBS+ 0,1% de saponina (3 x 10 min). </li><li> Incubar los tejidos durante 30 min a temperatura ambiente con 01:40 lectina conjugada con FITC en PBS seguido de dos lavados con PBS. </li><li> Para montar las diapositivas, los tejidos de la cubierta con PBS 50:50 y solución de glicerol y el cubreobjetos en la parte superior. Sellar los bordes de diapositivas utilizando el esmalte de uñas. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Este protocolo se documenta un método para utilizar el modelo de cultivo de mesenterio de rata como una herramienta ex vivo para time-lapse de crecimiento de la red microvascular. El trabajo previo en nuestro laboratorio ha establecido el uso de nuestro modelo para 1) la angiogénesis 8, 2) linfagiogénesis 8, 3) la interacción de células endoteliales de pericitos-8, y 4) las pruebas de drogas anti-angiogénico 9....

Crecimiento de la red microvascular y la remodelación son denominadores comunes para la función del tejido, cicatrización de heridas, y múltiples patologías y un proceso clave es la angiogénesis, que se define como el crecimiento de nuevos vasos sanguíneos a partir de los ya existentes 1, 2. Para la ingeniería de tejidos nuevos vasos o el diseño de terapias basadas angiogénicos, la comprensión de la importancia de la dinámica celulares implicados en la angiogénesis es crítica. Sin embargo, este proceso es complejo. Puede variar en lugares específicos dentro de una red microvascular y consiste en múltiples tipos de células (es decir, células endoteliales, células de músculo liso, pericitos, macrófagos, células madre) y sistemas múltiples (redes linfáticas y redes neuronales). Aunque los modelos in vitro han contribuido enormemente a examinar la relación entre las diferentes células implicadas en la angiogénesis 3, su relevancia fisiológica puede ser socavado debido a thei<html> r complejidad limitada y el hecho de que no reflejan estrechamente un escenario en vivo. Para superar estas limitaciones, los sistemas de cultivo tridimensional 3, ex vivo modelos tisulares 4, los sistemas de microfluidos 5, 6, 7 y modelos computacionales se han desarrollado e introducido en los últimos años. Sin embargo, todavía hay una necesidad de un modelo con capacidad de lapso de tiempo para investigar la angiogénesis en las redes microvasculares intactas ex vivo. El establecimiento de nuevos modelos de lapso de tiempo para los estudios de la angiogénesis con ese nivel de complejidad proporcionará una herramienta muy valiosa para comprender los mecanismos subyacentes que regulan la angiogénesis y para mejorar las terapias. Un posible modelo que permite la investigación ex vivo de la angiogénesis a través de una red microvascular intacto es el modelo de cultivo de mesenterio de rata&gt; 8. En un trabajo reciente, hemos demostrado que las redes microvasculares linfáticos sangre y permanecen viables después del cultivo. Más importante aún, el modelo de cultivo de mesenterio de rata se puede utilizar para investigar las interacciones funcionales de pericitos células endoteliales, la sangre y las conexiones de las células endoteliales linfáticas, y time-lapse. El objetivo de este trabajo es proporcionar a nuestro protocolo para el método de imagen de lapso de tiempo. Nuestros resultados representativos documentan los múltiples tipos de células que se mantienen viables después de la estimulación de la angiogénesis con suero y ofrecen ejemplos del uso de este método para la cuantificación de las respuestas angiogénicas específicos de tejido, así como estudios de seguimiento de células endoteliales.

<table><tbody><tr><td>Drape</td><td>Cardinal Health</td><td>4012</td><td>12&amp;rdquo; x 12&amp;rdquo; Bio-Shield Regular Sterilization Wraps</td></tr><tr><td>Scalpel Handle</td><td>Roboz Surgical Instrument</td><td>RS-9843</td><td>Scalpel Handle, #3; Solid; 4&quot; Length</td></tr><tr><td>Sterile Surgical Blade</td><td>Cincinnati Surgical</td><td>0110</td><td>Stainless Steel; Size 10</td></tr><tr><td>Culture Dish (60 mm)</td><td>Thermo Scientific</td><td>130181</td><td>10/Sleeve</td></tr><tr><td>Graefe Forcep (curved tweezers)</td><td>Roboz Surgical Instrument</td><td>RS-5135</td><td>Micro Dissecting Forceps; Serrated; Slight Curve; 0.8 mm Tip Width; 4&quot; Length</td></tr><tr><td>Graefe Forcep (straight tweezers)</td><td>Roboz Surgical Instrument</td><td>RS-5130</td><td>Micro Dissecting Forceps; Serrated, Straight; 0.8 mm Tip Width; 4&quot; Length</td></tr><tr><td>Noyes Micro Scissor</td><td>Roboz Surgical Instrument</td><td>RS-5677</td><td>Noyes Micro Dissecting Spring Scissors;&lt;br/&gt; Straight, Sharp-Blunt Points; 13 mm Cutting Edge; 0.25 mm Tip Width, 4 1/2&quot; Overall Length</td></tr><tr><td>Gauze Pads</td><td>FisherBrand</td><td>13-761-52</td><td>Non-Sterile Cotton Gauze Sponges; 4&quot; x 4&quot; 12-Ply</td></tr><tr><td>Cotton-Tippled Applicators</td><td>FisherBrand</td><td>23-400-124</td><td>6&quot; Length; Wooden Shaft; Single Use Only</td></tr><tr><td>6-Well Plate</td><td>Fisher Scientific</td><td>08-772-49</td><td>Flat Bottom with Low Evaporation Lid; Polystyrene; Non-Pyrogenic</td></tr><tr><td>Sterile Syring 5 mL</td><td>Fisher Scientific</td><td>14-829-45</td><td>Luer-Lok Tip</td></tr><tr><td>Sterile Bowl</td><td>Medical Action Industries Inc.</td><td>01232</td><td>32 oz. Peel Pouch; Blue; Sterile Single Use</td></tr><tr><td>6-Well Plate Inserts (CellCrown Inserts)</td><td>SIGMA</td><td>Z681792-3EA</td><td>6-Well Plate Inserts; Non-Sterile</td></tr><tr><td>Polycarbonate Filter Membrane</td><td>SIGMA</td><td>TMTP04700</td><td>Isopore Membrane Filter; Polycarbonate; Hydrophilic; 5.0&amp;nbsp;&amp;micro;m, 47&amp;nbsp;mm, White Plain</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>Beuthanasia</td><td>Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply)</td><td>MWI #: 011168</td><td>Active Ingredient: Per 100 mL, 390 mg pentobarbital sodium, 50 mg phenytoin sodium&amp;nbsp;</td></tr><tr><td>Ketamine</td><td>Fort Dodge Animal Health (Ordered from MWI Veterinary Supply)</td><td>MWI #: 000680</td><td>Kateset 100 mg/mL</td></tr><tr><td>Xylazine</td><td>LLOYD. Inc. (Ordered from MWI Veterinary Supply)</td><td>MWI #: 000680</td><td>Anased 100 mg/mL</td></tr><tr><td>Saline</td><td>Baxter</td><td>2F7122</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14040-133</td><td></td></tr><tr><td>MEM</td><td>Invitrogen</td><td>11095080</td><td></td></tr><tr><td>PenStrep</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>16000-044</td><td></td></tr><tr><td>BSA</td><td>Jackson ImmunoResearch</td><td>001-000-162</td><td></td></tr><tr><td>Saponin&amp;nbsp;</td><td>SIGMA</td><td>S7900-100G</td><td></td></tr><tr><td>Isopropyl Alcohol</td><td>Fisher Scientific</td><td>S25372</td><td></td></tr><tr><td>Povidone-Iodine</td><td>Operand</td><td>82-226</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>SIGMA</td><td>320331</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>67-56-1</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>56-81-5</td><td></td></tr><tr><td>FITC-conjugated Lectin</td><td>SIGMA</td><td>L9381-2MG</td><td></td></tr><tr><td>Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody</td><td>SIGMA</td><td>AB5320</td><td></td></tr><tr><td>PECAM (CD31) Antibody</td><td>BD Biosciences</td><td>555026</td><td></td></tr><tr><td>LYVE-1 Antibody</td><td>AngioBio Co.</td><td>11-034</td><td></td></tr><tr><td>Goat Anti-Rabbit Cy2-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>111-585-144</td><td></td></tr><tr><td>Goat Anti-Mouse Cy3-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>115-227-003</td><td></td></tr><tr><td>Streptavidin Cy3-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>016-160-084</td><td></td></tr><tr><td>Live/Dead Viability/Cytotoxicity Kit</td><td>Invitrogen</td><td>L3224</td><td></td></tr><tr><td>Normal Goat Serum&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>005-000-121</td><td></td></tr><tr><td>5-Bromo-2&#039;-Deoxyuridine</td><td>SIGMA</td><td>B5002</td><td></td></tr><tr><td>Monoclonal Mouse Anti-Bromodeoxyuridine&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Clone Bu20a</td><td>Dako</td><td>M074401-8</td><td></td></tr><tr><td>Mouse Anti-Rat CD11b&amp;nbsp;</td><td>AbD Serotec</td><td>MCA275R</td><td></td></tr></tbody></table>

an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks

La angiogénesis, definida como el crecimiento de nuevos vasos sanguíneos a partir de vasos pre-existentes, implica células endoteliales, pericitos, células de músculo liso, células inmunes, y la coordinación con los vasos linfáticos y nervios. El multi-celda, las interacciones de múltiples sistemas requieren la investigación de la angiogénesis en un entorno fisiológicamente relevante. Por lo tanto, mientras que el uso de los modelos de cultivo celular in vitro han proporcionado ideas mecanicistas, una crítica frecuente es que no se recapitulan la complejidad asociada con una red microvascular. El objetivo de este protocolo es demostrar la capacidad de hacer comparaciones con lapso de tiempo de redes microvasculares intactas antes y después de la estimulación de la angiogénesis en los tejidos mesenterio de rata. tejidos cultivados contienen redes microvasculares que mantienen su jerarquía. marcaje inmunohistoquímico confirma la presencia de células endoteliales, células de músculo liso, pericitos, vasos sanguíneos y los vasos linfáticos. En unddition, el etiquetado de los tejidos con BSI-lectina permite la comparación de lapso de tiempo de las regiones locales de la red antes y después de la estimulación con suero o factor de crecimiento caracterizado por el aumento de la brotación y la densidad de los vasos capilares. En comparación con los modelos de cultivo celular comunes, este método proporciona una herramienta para estudios de linaje de células endoteliales y la evaluación de medicamentos angiogénico específico de tejido en las redes microvasculares fisiológicamente relevantes.

Después de 3 días de cultivo, los tejidos se marcaron con un kit de viabilidad / citotoxicidad en vivo / muerto para demostrar la viabilidad de la microvasculatura en el mesenterio de rata modelo de cultivo (Figura 2A). La mayoría de las células presentes en el mesenterio se mantuvo viable en la cultura en la que las células endoteliales fueron identificados en base a su ubicación en segmentos microvasculares. Proliferación de células endoteliales también se con...

Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

Un<em&gt; Ex Vivo</em&gt; Método para time-lapse de rata cultivadas mesentéricas microvasculares Redes

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

an-ex-vivo-method-for-time-lapse-imaging-cultured-rat-mesenteric

Research

JoVE Journal

Developmental Biology

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

6.8K vistas.  Tulane University. Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis. 

Video: An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In vitro e in vivo modelo para estudiar la adhesión bacteriana a la pared del recipiente en condiciones de flujo

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Investigación

Inmunología e Infección

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

Imaging neutrófilos y monocitos en mesentérica venas por Microscopia intravital en ratones anestesiados en Tiempo Real

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

Rata mesenterio ensayo angiogénesis

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Biología

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Rata Exteriorización Mesenterio: un modelo para investigar la dinámica celulares implicados en la angiogénesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

Evaluación de mecanismos de Control Vascular utilizando microscopia Video de arterias de resistencia aislada de ratas

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Medicina

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Sistema de Cultura ratón fetal Whole Intestino para<em&gt; Ex Vivo</em&gt; Manipulación de vías de señalización y tridimensional de imágenes en vivo del desarrollo de vellosidades

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

Medición dinámica e imagen de capilares, arterias y pericitos en el corazón del ratón

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

De dos fotones de imágenes de Ca intracelular<sup&gt; 2 +</sup&gt; Manejo y producción de óxido nítrico en el endotelio y las células del músculo liso de la aorta de rata aislada

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusión Técnica de investigar Reglamento de Microvessel Permeabilidad en Rata Mesenterio

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Desarrollo y caracterización de<em&gt; In Vitro</em&gt; Red de microvasos y mediciones cuantitativas de endotelial [Ca<sup&gt; 2+</sup&gt;]<sub&gt; i</sub&gt; Y la producción de óxido nítrico

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

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