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>

<ol>
	<li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+cancer+and+other+diseases.">Angiogenesis in cancer and other diseases.</a> Nature. 407 (6801), 249-257 (2000).</li><li>Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+life,+disease+and+medicine.">Angiogenesis in life, disease and medicine.</a> Nature. 438 (7070), 932-936 (2005).</li><li>Kaunas, R., Kang, H., Bayless, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+Regulation+of+Angiogenic+Sprouting+by+Biochemical+Factors+and+Wall+Shear+Stress.">Synergistic Regulation of Angiogenic Sprouting by Biochemical Factors and Wall Shear Stress.</a> Cell Mol Bioeng. 4 (4), 547-559 (2011).</li><li>Pitulescu, M. E., Schmidt, I., Benedito, R., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+gene+targeting+in+the+neonatal+vasculature+and+analysis+of+retinal+angiogenesis+in+mice.">Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice.</a> Nat Protoc. 5 (9), 1518-1534 (2010).</li><li>Song, J. W., Munn, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid+forces+control+endothelial+sprouting.">Fluid forces control endothelial sprouting.</a> Proc Natl Acad Sci U S A. 108 (37), 15342-15347 (2011).</li><li>Chan, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+in+vitro+3D+capillary+beds+by+self-directed+angiogenic+sprouting.">Engineering of in vitro 3D capillary beds by self-directed angiogenic sprouting.</a> PLoS One. 7 (12), e50582 (2012).</li><li>Peirce, S. M., Mac Gabhann, F., Bautch, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+experimental+and+computational+approaches+to+sprouting+angiogenesis.">Integration of experimental and computational approaches to sprouting angiogenesis.</a> Curr Opin Hematol. 19 (3), 184-191 (2012).</li><li>Stapor, P. C., Azimi, M. S., Ahsan, T., Murfee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+angiogenesis+model+for+investigating+multicellular+interactions+across+intact+microvascular+networks.">An angiogenesis model for investigating multicellular interactions across intact microvascular networks.</a> Am J Physiol Heart Circ Physiol. 304 (2), H235-H245 (2013).</li><li>Azimi, M. S., 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=An+ex+vivo+model+for+anti-angiogenic+drug+testing+on+intact+microvascular+networks.">An ex vivo model for anti-angiogenic drug testing on intact microvascular networks.</a> PLoS One. 10 (3), e0119227 (2015).</li><li>Nicosia, R. F., Ottinetti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+microvessels+in+serum-free+matrix+culture+of+rat+aorta.+A+quantitative+assay+of+angiogenesis+in+vitro.">Growth of microvessels in serum-free matrix culture of rat aorta. A quantitative assay of angiogenesis in vitro.</a> Lab Invest. 63 (1), 115-122 (1990).</li><li>Hutter-Schmid, B., Kniewallner, K. M., Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures+as+a+model+to+study+angiogenesis+of+brain+vessels.">Organotypic brain slice cultures as a model to study angiogenesis of brain vessels.</a> Front Cell Dev Biol. 3, 52 (2015).</li><li>Sawamiphak, S., Ritter, M., Acker-Palmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+retinal+explant+cultures+to+study+ex+vivo+tip+endothelial+cell+responses.">Preparation of retinal explant cultures to study ex vivo tip endothelial cell responses.</a> Nat Protoc. 5 (10), 1659-1665 (2010).</li><li>Unoki, N., Murakami, T., Ogino, K., Nukada, M., Yoshimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+imaging+of+retinal+angiogenesis+reveals+decreased+development+and+progression+of+neovascular+sprouting+by+anecortave+desacetate.">Time-lapse imaging of retinal angiogenesis reveals decreased development and progression of neovascular sprouting by anecortave desacetate.</a> Invest Ophthalmol Vis Sci. 51 (5), 2347-2355 (2010).</li><li>Norrby, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+models+of+angiogenesis.">In vivo models of angiogenesis.</a> J Cell Mol Med. 10 (3), 588-612 (2006).</li><li>Kelly-Goss, M. R., Sweat, R. S., Azimi, M. S., Murfee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+islands+during+microvascular+regression+and+regrowth+in+adult+networks.">Vascular islands during microvascular regression and regrowth in adult networks.</a> Front Physiol. 4, 108 (2013).</li><li>Kelly-Goss, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+proliferation+along+vascular+islands+during+microvascular+network+growth.">Cell proliferation along vascular islands during microvascular network growth.</a> BMC Physiol. 12, 7 (2012).</li><li>Skalak, T. C., Price, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mechanical+stresses+in+microvascular+remodeling.">The role of mechanical stresses in microvascular remodeling.</a> Microcirculation. 3 (2), 143-165 (1996).</li><li>Kadohama, T., Nishimura, K., Hoshino, Y., Sasajima, T., Sumpio, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+different+types+of+fluid+shear+stress+on+endothelial+cell+proliferation+and+survival.">Effects of different types of fluid shear stress on endothelial cell proliferation and survival.</a> J Cell Physiol. 212 (1), 244-251 (2007).</li><li>Milkiewicz, M., Brown, M. D., Egginton, S., Hudlicka, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+shear+stress,+angiogenesis,+and+VEGF+in+skeletal+muscles+in+vivo.">Association between shear stress, angiogenesis, and VEGF in skeletal muscles in vivo.</a> Microcirculation. 8 (4), 229-241 (2001).</li><li>Corliss, B. A., Azimi, M. S., Munson, J. M., Peirce, S. M., Murfee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages:+An+Inflammatory+Link+Between+Angiogenesis+and+Lymphangiogenesis.">Macrophages: An Inflammatory Link Between Angiogenesis and Lymphangiogenesis.</a> Microcirculation. 23 (2), 95-121 (2016).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="679">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:165px;">Drape</td>
			<td style="width:183px;">Cardinal Health</td>
			<td style="width:132px;">4012</td>
			<td style="width:199px;">12&#8221;x12&#8221; Bio-Shield Regular Sterilization Wraps</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Scalpel Handle</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-9843</td>
			<td style="width:199px;">Scalpel Handle, #3; Solid; 4&#34; Length</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Surgical Blade</td>
			<td>Cincinnati Surgical</td>
			<td>0110</td>
			<td>Stainless Steel; Size 10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Culture Dish (60mm)</td>
			<td>Thermo Scientific</td>
			<td>130181</td>
			<td>10/Sleeve</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:165px;">Graefe Forcep (curved tweezers)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5135</td>
			<td style="width:199px;">Micro Dissecting Forceps; Serrated; Slight Curve; 0.8mm Tip Width; 4&#34; Length</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Graefe Forcep (straight tweezers)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5130</td>
			<td style="width:199px;">Micro Dissecting Forceps; Serrated, Straight; 0.8mm Tip Width; 4&#34; Length</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:165px;">Noyes Micro Scissor</td>
			<td style="width:183px;">Roboz Surgical Instrument</td>
			<td style="width:132px;">RS-5677</td>
			<td style="width:199px;">Noyes Micro Dissecting Spring Scissors; 
			Straight, Sharp-Blunt Points; 13mm Cutting Edge; 0.25mm Tip Width, 4 1/2&#34; Overall Length</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Gauze Pads</td>
			<td>FisherBrand</td>
			<td>13-761-52</td>
			<td style="width:199px;">Non-Sterile Cotton Gauze Sponges; 4&#34;x4&#34; 12-Ply</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Cotton-Tippled Applicators</td>
			<td>FisherBrand</td>
			<td>23-400-124</td>
			<td style="width:199px;">6&#34; Length; Wooden Shaft; Single Use Only</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">6-Well Plate</td>
			<td>Fisher Scientific</td>
			<td>08-772-49</td>
			<td style="width:199px;">Flat Bottom with Low Evaporation Lid; Polystyrene; Non-Pyrogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Syring 5ml</td>
			<td>Fisher Scientific</td>
			<td>14-829-45</td>
			<td>Luer-Lok Tip</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Sterile Bowl</td>
			<td>Medical Action Industries Inc.</td>
			<td>01232</td>
			<td style="width:199px;">32 oz. Peel Pouch; Blue; Sterile Single Use</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:165px;">6-Well Plate Inserts (CellCrown Inserts)</td>
			<td>SIGMA</td>
			<td>Z681792-3EA</td>
			<td>6-Well Plate Inserts; Non-Sterile</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Polycarbonate Filter Membrane</td>
			<td>SIGMA</td>
			<td>TMTP04700</td>
			<td style="width:199px;">Isopore Membrane Filter; Polycarbonate; Hydrophilic; 5.0&#160;&#181;m, 47&#160;mm, White Plain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Beuthanasia</td>
			<td style="width:183px;">Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 011168</td>
			<td style="width:199px;">Active Ingredient: Per 100mL, 390 mg pentobarbital sodium, 50mg phenytoin sodium&#160;</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;">Ketamine</td>
			<td style="width:183px;">Fort Dodge Animal Health (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 000680</td>
			<td>Kateset 100 mg/ml</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Xylazine</td>
			<td style="width:183px;">LLOYD. Inc. (Ordered from MWI Veterinary Supply)</td>
			<td>MWI #: 000680</td>
			<td>Anased 100 mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline</td>
			<td style="width:183px;">Baxter</td>
			<td>2F7122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Invitrogen</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM</td>
			<td>Invitrogen</td>
			<td>11095080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PenStrep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Jackson ImmunoResearch</td>
			<td>001-000-162</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Saponin&#160;</td>
			<td>SIGMA</td>
			<td>S7900-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Isopropyl Alcohol</td>
			<td>Fisher Scientific</td>
			<td>S25372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Povidone-Iodine</td>
			<td>Operand</td>
			<td>82-226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Hydrochloric Acid</td>
			<td>SIGMA</td>
			<td>320331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Methanol</td>
			<td>Fisher Scientific</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td>56-81-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:165px;">FITC-conjugated Lectin</td>
			<td>SIGMA</td>
			<td>L9381-2MG</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody</td>
			<td>SIGMA</td>
			<td>AB5320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PECAM (CD31) Antibody</td>
			<td>BD Biosciences</td>
			<td>555026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LYVE-1 Antibody</td>
			<td>AngioBio Co.</td>
			<td>11-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rabbit Cy2-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-585-144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Mouse Cy3-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-227-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Cy3-conjugated Antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>016-160-084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Bromo-2&#39;-Deoxyuridine</td>
			<td>SIGMA</td>
			<td>B5002</td>
			<td></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:165px;">Monoclonal Mouse Anti-Bromodeoxyuridine&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Clone Bu20a</td>
			<td>Dako</td>
			<td style="width:132px;">M074401-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Anti-Rat CD11b&#160;</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

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

La captura de Reparación de Tejidos en pez cebra larvas con Time-lapse Brightfield Estereomicroscopía

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Investigación

Biología del Desarrollo

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

analizando<em&gt; En Vivo</em&gt; Migración de células usando trasplantes de células y time-lapse en embriones de pez cebra

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

Análisis para el Desarrollo de riñón de pez cebra con time-lapse de uso de un microscopio de disección equipado para seccionamiento óptico

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Un filtro de papel sumergido Sandwich para el largo plazo<em&gt; Ex Ovo</em&gt; Time-lapse de pollo temprano embriones

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

La proyección de imagen de lapso tiempo multi-Photon para visualizar el desarrollo en tiempo real: visualización de la migración de las células de cresta Neural en embriones de pez cebra

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Cultivo de células epiteliales del pigmento retiniano en un modelo Ex Vivo de la membrana de Bruch humano envejecido

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Un método In Vivo para el estudio de integridad de barrera Hematotesticular ratón

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

Análisis de la dinámica de Actomyosin en escalas celulares y de tejidos locales utilizando películas de lapso de tiempo de cámaras de huevo de Drosophila cultivada

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Ex Vivo Perfusion of the Rodent Placenta

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An assay is described here to perfuse an isolated rat placenta and evaluate the maternal-to-fetal translocation of xenobiotics ex vivo. In addition, the evaluation of physiological processes such as fluid flow to the fetus and placental metabolism may be conducted with this methodology. This technique is suitable for evaluating maternal-to-fetal kinetics of pharmaceutical candidates or environmental contaminants. In contrast to current alternative approaches, this methodology allows the evaluation of the isolated maternal-fetal vasculature, with the systemic neural or immune involvement removed, allowing any observed changes in physiological function to be attributable to local factors within the isolated tissue.

Here is presented a protocol of ex vivo maternal-fetal vascular perfusion to enable the administration of a test article into maternal vasculature and to evaluate placental transfer of xenobiotic particles or pharmacological agents in addition to alterations in placental physiology.

Ex vivo perfusion de la placenta de roedores

The placenta is a key organ during pregnancy that serves as a barrier to fetal xenobiotic exposure and mediates the exchange of nutrients for waste. An ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Un método de montaje en capas para la microscopía confocal de lapso de tiempo extendido de embriones enteros de pez cebra

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...