Investigación para Prevención de la Ceguera; Bartly J. Mondino MD, Director del Jules Stein Eye Institute de UCLA, y Ramiro Ramírez-Solís, Jacqui Blanca, y Jeanne Estabel en el Instituto Sanger, Wellcome Trust Genoma Campus. Esta investigación cumple con la Declaración de ARVO para el Uso de Animales en Investigación Oftalmológica y Visual.

1. Disección Blunt: Enucleación del ojo del ratón en las muestras no fijadas <ol><li> Separar los párpados para mejorar la exposición y el acceso al mundo posterior (globo ocular) superficie. </li><li> Coloque un vendaje fórceps curvo detrás (en) el mundo en la órbita (cuenca del ojo). El Mahajan Sharptip aderezo fórceps es un instrumento de medida con puntas de hacer este paso más fácil (ver materiales y reactivos de mesa). </li><li> Cierre la pinza y agarrar el tejido conectivo orbital y el nervio óptico detrás del globo mientras que teniendo cuidado de evitar apretar el globo. </li><li> Retire con cuidado la pinza hacia arriba y coger el globo ocular de la órbita. El blanco de hilo tejido es el nervio óptico. </li><li> Coloque el ojo en PBS. Usando una aguja de calibre 30-, hacer una punción única herida inmediatamente posterior al limbo mediante la inserción de la aguja 1-2 mm en el globo ocular. Esto permite fijadores, tales como glutaraldehído, que de otro modo son incapaces de penetrar en el tejido del ojo, a directamente enter el ojo. La creación de esta herida de punción puede no ser necesario si un fijador, tal como paraformaldehído o alcohol, es capaz de difundirse en los tejidos oculares de interés. </li><li> Coloque todo el ojo inmediatamente en fijador para la fijación de inmersión. </li></ol> 2. Disección de Sharp: Enucleación de la fijación del ojo del ratón perfusión siguiente <ol><li> Utilice un fórceps curvo colibri y mantiene los párpados lejos del mundo. </li><li> Orient curvas tijeras de Westcott disección paralelas al mundo, mientras apuntando hacia la parte posterior de la órbita. </li><li> Cortar el tejido conectivo que rodea fijo el mundo, desde las inferiores, a los lados medial, superior y lateral. </li><li> Coloque la pinza curvada detrás del globo, sujete el tejido conectivo sin presionar en el mundo, y tire hacia adelante la enucleación del ojo. Dado que el tejido conjuntivo orbital es rígido en fijación por perfusión, de corte adicional de los tejidos orbitales puede ser necesaria para liberar completamente la globe. </li></ol> 3. La fijación y embalaje Envío de los tejidos fijos deben seguir sus requerimientos de servicios institucionales y los servicios postales, incluyendo las etiquetas y envases apropiados. El protocolo siguiente es un ejemplo de envío de una muestra biológica no infecciosa a temperatura ambiente. Esto requiere 3 capas de embalaje, pero no de clase Sustancia / B biológico o etiquetas de nitrógeno líquido. <ol><li> Coloque el ojo en un vial de vidrio de 5-mL de centelleo con al menos 3-ml fijador, o tampón de post-fijador si la fijación es corto. Escribir el número de seguimiento en la tapa del vial y sellarlo con Parafilm. </li><li> Colocar los viales de centelleo en una muestra biológica aprobada contenedor de envío. En nuestro instituto, por ejemplo, contenedores aprobados requieren tres capas: un recipiente sellado en el que se coloca la muestra, una capa intermedia absorbente, y una capa protectora externa. </li><li> Colocar el recipiente en un paquete de burbuja y luego en una aplicaciónlaboratorio Roved contenedor de transporte junto con la documentación apropiada. </li></ol> 4. Los resultados representativos El examen histológico de los ojos puede ser realizada por una variedad de métodos que incluyen hematoxilina y eosina tinción, la detección de la expresión enzimática, microscopía electrónica de transmisión, y la inmunohistoquímica. Después de la fijación de inmersión en paraformaldehído al 4%, el tejido se embebió en parafina y se seccionaron con un microtomo. En alto rendimiento fenotipificación, se analiza óptica de alumnos por secciones nerviosas que muestra todos los tipos de tejidos en el ojo (Figura 1). Las secciones de tejido también se examinaron para la expresión de lacZ (Figura 2), microscopía electrónica de transmisión de los orgánulos celulares (Figura 3), y la expresión de moléculas específicas con anticuerpos (Figura 4). En nuestro método de cribaje fenotipo, que no era importante para mantener la orientación del ojo con respecto a la nasal (canto medial) y temporal (Lateral) canto lados. Hay por lo menos dos opciones si la orientación del ojo es necesario. Después de la enucleación, hemos aplicado cauterización luz en la córnea temporal en la posición de las tres con un dispositivo de cauterio de mano. La lesión es evidente en la histología y no se pierde durante el procesamiento como tintas histológicos. La desventaja es que los daños de cauterización de la córnea, que puede ser un tejido crítico para su examen. La otra opción requiere un anatomista experto que pueda identificar a las inserciones de los músculos extraoculares. Bajo un microscopio estereoscópico, la identificación de la inserción del músculo oblicuo inferior y superior marca el aspecto inferior y superior y del globo. 3 Con cualquier método de orientación, es importante realizar el seguimiento del espécimen, tal como un ojo derecho o izquierdo. En conjunto, esto permitirá orientación globo exacto antes de cortar. <img src="/files/ftp_upload/3184/3184fig1.jpg" alt="Figura 1" /> Figura 1. Imágenes histológicas ratón ojo afte<html> r adquisición remota. ojos fueron enucleados ratón por disección aguda posterior a la fijación de perfusión con paraformaldehído al 4%. A. alumnos por sección del nervio óptico se tiñeron con hematoxilina y eosina demuestra la preservación de todas las subestructuras de tejidos. ON, nervio óptico. (Barra de escala = 500 micras verde) B. Una visión un aumento mayor de la retina normal muestra su estructura laminar: las células ganglionares de la retina, RGC; capa IPL, plexiforme interna, capa INL, nuclear interna, capa OPL, plexiforme externa; ONL, exterior capa nuclear (células fotorreceptoras) (flecha); IS, segmentos fotorreceptores interiores (cabeza de flecha); OS, fotorreceptoras segmentos externos; RPE, epitelio pigmentario de la retina, coroides, c. (Barra de escala = verde 50 micras) C. En comparación con la retina normal, esta muestra es más delgada debido a la degeneración de los fotorreceptores. La ONL, IS y OS están completamente ausentes (flecha). (Barra verde escala = 50 micras) &quot;Alt =&quot; Figura 2 &quot;/&gt; Figura 2. Expresión de lacZ en ratones transgénicos. Los ojos fueron enucleados mediante disección roma y se sumergieron en 1 mg / ml de X-gal solución (5 mM K3Fe (CN) 6, 5 mM K4Fe (CN) 6, 2 mM MgCl2, 0,02% de NP40 , y 0,1% de desoxicolato de sodio) durante la noche a 37 ° C. Ojos se fijaron en glutaraldehído al 2% formaldehyde/0.2% durante 30 minutos y se seccionaron con un microtomo. X-gal de productos (azul) etiquetas: el epitelio ciliar A., B. epitelio corneal, y la célula C. ganglionares de la retina (RGC) de capa en la retina. INL, la capa nuclear interna, capa ONL, nuclear externa. <img src="/files/ftp_upload/3184/3184fig3.jpg" alt="Figura 3" /> Figura 3. Microscopía electrónica de transmisión de epitelio pigmentario de la retina (RPE). Después de la disección roma y la creación de una herida por punción, ojos eran de inmersión fijos en 2,5% de paraformaldehído / 2,5% de glutaraldehído en 0,1 M de tampón fosfato de sodio. Los ojos eran de segundadarily fijado en textroxide de osmio al 1%, se deshidrataron gradualmente, y se incluyeron en resina de Spurr. El tejido se seccionó a 90 nm y se colocaron en rejillas de ranura de cobre Formvar y cubiertos en imágenes utilizando un microscopio electrónico de transmisión. La micrografía muestra, los segmentos externos de los fotorreceptores de la retina neurosensorial, melanosomas (M) en el epitelio pigmentario de la retina, y la membrana de Bruch. <img src="/files/ftp_upload/3184/3184fig4.jpg" alt="Figura 4" /> Figura 4. Marcaje inmunohistoquímico de superóxido dismutasa-3 (verde) en el vítreo del ratón. Los ojos fueron enucleados mediante disección roma y se sometieron a fijación inmersión en paraformaldehído al 4%. Ellos fueron incluidos en parafina y se seccionaron utilizando un micrótomo. Las secciones de tejido se incubaron con antisuero policlonal de conejo diluido 1:50 SOD3. SOD3 expresión se detectó usando un anticuerpo de cabra anti-conejo IgG (H + L)-Alexa Fluor 488 de anticuerpo secundario conjugado. Etiquetado de SOD3 se muestra en green.

<ol>
	<li>Song, B. J., Tsang, S. H., Lin, C. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+models+of+retinal+degeneration+and+targets+for+gene+therapy.">Genetic models of retinal degeneration and targets for gene therapy.</a> Gene Ther. Mol. Biol. 11, 229-262 (2007).</li><li>Mahajan, V. B., Mondino, B. J., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+Mouse+Eye+Phenomics+System.">A high-throughput Mouse Eye Phenomics System.</a> Cold Spring Harbor Laboratories, Mouse Genetics Meeting. , (2010).</li><li>Smith, R. S., John, S. W. M., Nishina, P. M., Sundberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In Research Methods For Mutant Mice. , (2002).</li><li>Chang, B., Hawes, N. L., Hurd, R. E., Davisson, M. T., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+mutants+in+the+mouse.">Retinal degeneration mutants in the mouse.</a> Vision Res. 42, 517-525 (2002).</li><li>Anderson, M. G., Smith, R. S., Hawes, N. L., Zabaleta, A., Chang, B., Wiggs, J. L., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+genes+encoding+melanosomal+proteins+cause+pigmentary+glaucoma+in+DBA/2J+mice.">Mutations in genes encoding melanosomal proteins cause pigmentary glaucoma in DBA/2J mice.</a> Nat. Genet. 30, 81-85 (2002).</li><li>Hawes, N. L., Smith, R. S., Chang, B., Davisson, M., Heckenlively, J. R., John, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+fundus+photography+and+angiography:+a+catalogue+of+normal+and+mutant+phenotypes.">Mouse fundus photography and angiography: a catalogue of normal and mutant phenotypes.</a> Mol. Vis. 5, 22-22 (1999).</li><li>Won, J., Shi, L. Y., Hicks, W., Wang, J., Hurd, R., Naggert, J. K., Chang, B., Nishina, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+resources+for+vision+research.">Mouse model resources for vision research.</a> J. Ophthalmol. 2011, 391384-391384 (2011).</li><li>Pinto, L. H., Vitaterna, M. H., Siepka, S. M., Shimomura, K., Lumayag, S., Baker, M., Fenner, D., Mullins, R. F., Sheffield, V. C., Stone, E. M., Heffron, E., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+from+screening+over+9000+mutation-bearing+mice+for+defects+in+the+electroretinogram+and+appearance+of+the+fundus.">Results from screening over 9000 mutation-bearing mice for defects in the electroretinogram and appearance of the fundus.</a> Vision. Res. 44, 3335-3345 (2004).</li><li>Heckenlively, J. R., Winston, J. V., Roderick, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+mouse+retinal+degenerations.+I.+Correlation+of+indirect+ophthalmoscopy,+electroretinograms,+and+histology.">Screening for mouse retinal degenerations. I. Correlation of indirect ophthalmoscopy, electroretinograms, and histology.</a> Doc. Ophthalmol. 71, 229-239 (1989).</li><li>Tsang, S. H., Gouras, P., Yamashita, C. K., Kjeldbye, H., Fisher, J., Farber, D. B., Goff, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+in+mice+lacking+the+gamma+subunit+of+the+rod+cGMP+phosphodiesterase.">Retinal degeneration in mice lacking the gamma subunit of the rod cGMP phosphodiesterase.</a> Science. 272, 1026-1029 (1996).</li><li>Tsang, S. H., Woodruff, M. L., Jun, L., Mahajan, V., Yamashita, C. K., Pedersen, R., Lin, C. S., Goff, S. P., Rosenberg, T., Larsen, M., Farber, D. B., Nusinowitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+carrying+the+H258N+mutation+in+the+gene+encoding+the+beta-subunit+of+phosphodiesterase-6+(PDE6B)+provide+a+model+for+human+congenital+stationary+night+blindness.">Transgenic mice carrying the H258N mutation in the gene encoding the beta-subunit of phosphodiesterase-6 (PDE6B) provide a model for human congenital stationary night blindness.</a> Hum. Mutat. 28, 243-254 (2007).</li></ol>

No hay conflictos de interés declarado.

Mayoría de los ratones transgénicos que existen en laboratorios que no examinan los ojos. Nuestra técnica de vídeo ilustra un método simple estandarizado para la transferencia remota habilidad quirúrgica para optimizar la adquisición de tejido de laboratorios con poca experiencia con los ojos. Esta técnica ayuda a vídeo superar un escollo importante en alto rendimiento fenotipificación, que es el uso de un número limitado de sitios de expertos por razones de métodos no normalizados de recogida de tejido y la...

<table border="1"><tbody><tr><th> Nombre </th><th> Empresa </th><th> Número de Catálogo </th><th> Comentarios </th></tr><tr><td> Fórceps curvo Vestir </td><td> Storz Ophthalmics </td><td> E1408 </td><td></td></tr><tr><td> Mahajan Sharptip aderezo fórceps </td><td> Storz Ophthalmics </td><td> E1406 (REF SP7-64520) </td><td></td></tr><tr><td> Curvas Tijeras Westcott </td><td> Storz Ophthalmics </td><td> E3321 WH </td><td></td></tr><tr><td> 15 ° BD Beaver Microsurgical hoja </td><td> Becton-Dickinson </td><td> 374881 </td><td></td></tr><tr><td> 0,22 Fine-Castroviejo Pinza para Sutura </td><td> Storz Ophthalmics </td><td> E1805 </td><td></td></tr><tr><td> 0,12 Colibri fórceps </td><td> Storz Ophthalmics </td><td> 2/132 </td><td></td></tr><tr><td> 30-aguja de calibre </td><td> Becton-Dickinson </td><td> 305128 </td><td></td></tr><tr><td> Biohazard Mailer </td><td> Pescador </td><td> 03-523-4 </td><td></td></tr><tr><td> Parafilm </td><td> Pescador </td><td> 13-374-10 </td><td></td></tr><tr><td> Viales de centelleo de vidrio </td><td> Wheaton </td><td> 4500413033 </td><td></td></tr><tr><td> PBS, pH 7,4 </td><td> Invitrogen </td><td> 70011-044 </td><td></td></tr><tr><td> 16% de paraformaldehído </td><td> Microscopía Electrónica de Ciencias </td><td> 15700 </td><td></td></tr><tr><td> 2,5% de paraformaldehído / 2,5% de glutaraldehído en tampón de fosfato sódico 0,1 M </td><td> Microscopía Electrónica de Ciencias </td><td> 15700 y 16300 </td><td> Mezclado en laboratorio </td></tr><tr><td> 50% de glutaraldehído </td><td> Microscopía Electrónica de Ciencias </td><td> 16300 </td><td></td></tr><tr><td> 0,25% Formvar </td><td> Microscopía Electrónica de Ciencias </td><td> 15810 </td><td></td></tr><tr><td> Policíapor cuadrícula Slot </td><td> Microscopía Electrónica de Ciencias </td><td> M2010-CR </td><td></td></tr><tr><td> 4% de tetróxido de osmio </td><td> Microscopía Electrónica de Ciencias </td><td> 19140 </td><td></td></tr><tr><td> Anticuerpo anti-SOD3 </td><td> Abcam </td><td> Ab21974 </td><td></td></tr><tr><td> De cabra anti-conejo Alexa Fluor 488 </td><td> Invitrogen </td><td> A11070 </td><td></td></tr><tr><td> Incrustación de resina de Spurr </td><td> Microscopía Electrónica de Ciencias </td><td> 14300 </td><td></td></tr></tbody></table>

mouse eye enucleation for remote high-throughput phenotyping

El ojo del ratón es un modelo genético importante para el estudio de traslación de la enfermedad oftálmica humano. Enfermedades que causan ceguera en los seres humanos, como la degeneración macular, degeneración de los fotorreceptores, las cataratas, el glaucoma, el retinoblastoma y la retinopatía diabética se han recapitulado en ratones transgénicos. 1-5 mayoría de los ratones transgénicos y knock-out han sido generados por los laboratorios para estudiar las enfermedades no oftalmológicas, pero conservación genética entre los sistemas de órganos sugiere que muchos de los mismos genes también puede desempeñar un papel en el desarrollo y la enfermedad ocular. Por lo tanto, estos ratones representan un recurso importante para el descubrimiento de nuevas correlaciones genotipo-fenotipo en el ojo. Debido a que estos ratones se encuentran dispersos en todo el mundo, es difícil de adquirir, mantener y fenotipo ellos de una manera eficiente y rentable manera. Así, la mayoría de alto rendimiento pantallas fenotipificación oftálmicas se limita a unos pocos lugares que requieren en el lugar, la experiencia oftálmica para examinar los ojos en ratones vivos. 6-9 Un enfoque alternativo desarrollado por nuestro laboratorio es un método para la adquisición remota de tejido que se puede utilizar en grandes encuestas o en pequeña escala de los ojos de ratones transgénicos. Los procedimientos estandarizados de vídeo basado en la transferencia de habilidades quirúrgicas, la fijación del tejido, y el envío permitir que cualquier laboratorio para recolectar los ojos enteros de animales mutantes y enviarlos por fenotipificación molecular y morfológica. En este artículo, video, presentamos las técnicas de la enucleación y transferir los ojos fijos tanto no fijadas y la perfusión de ratón para el análisis fenotípicos remotos.

Watch this Scientific Journal Video about Mouse Eye Enucleation for Remote High-throughput Phenotyping at JoVE.com

Mouse Eye Enucleation for Remote High-throughput Phenotyping

La técnica de disección ilustra la enucleación del ojo del ratón para la fijación del tejido para realizar el fenotipado de alto rendimiento pantallas.

Ratón enucleación del ojo por remoto de alto rendimiento fenotipificación

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

mouse-eye-enucleation-for-remote-high-throughput-phenotyping

Research

JoVE Journal

Medicine

20.2K vistas.  University of Iowa. La técnica de disección ilustra la enucleación del ojo del ratón para la fijación del tejido para realizar el fenotipado de alto rendimiento pantallas.

Video: Mouse Eye Enucleation for Remote High-throughput Phenotyping

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

Una plataforma automatizada de alto rendimiento para el desarrollo de líneas celulares de fabricación de proteínas terapéuticas

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

Investigación

Medicina

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

Un modelo de ratón de la Prueba de bolsillo para el Estudio de la angiogénesis de córnea

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Hiperinsulinémico euglucémico abrazaderas en ratones conscientes, sin freno

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac substrates such as left atrial flutter. This protocol describes all the clinical and invasive interventional steps performed during a human electrophysiological study and ablation to assess the accuracy, safety and real-time navigation of the Catheter Guidance, Control and Imaging (CGCI) system. Patients who underwent ablation of a right or left atrium flutter substrate were included. Specifically, data from three left atrial flutter and two counterclockwise right atrial flutter procedures are shown in this report. One representative left atrial flutter procedure is shown in the movie. This system is based on eight coil-core electromagnets, which generate a dynamic magnetic field focused on the heart. Remote navigation by rapid changes (msec) in the magnetic field magnitude and a very flexible magnetized catheter allow real-time closed-loop integration and accurate, stable positioning and ablation of the arrhythmogenic substrate.

This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures.

Navegación magnética remota de información precisa, la posición del catéter en tiempo real y la ablación en Electrofisiología Cardiaca Procedimientos

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac ...

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Alto rendimiento secuencial ELISA para la Validación de Biomarcadores de aguda injerto contra huésped

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Alto rendimiento caracterización de células madre adultas de ingeniería para la entrega de los factores terapéuticos de estrategias neuroprotectoras

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

Etiquetado de colorantes fluorescentes de eritrocitos y leucocitos para estudiar la dinámica de flujo en la circulación de la retina del ratón

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the quality of life. BPH includes 2 major treatments: medication and surgery. Surgical treatment is often the most effective and final intervention. Holmium laser enucleation of the prostate (HoLEP), one of the most efficient surgical procedures for BPH, is conducted transurethrally. During surgery, the most important and most difficult part is to locate the surgical capsule of the prostate, which is consistent to the idea of anatomic surgery. Different skills may be needed in treating different parts of the prostate. HoLEP tends to be efficient and safe with good hemostatic properties, and able to treat bladder calculus, which may be the complications of BPH. The technique is particularly suitable for prostate of varying volumes and sizes. Indeed, HoLEP also presents certain disadvantages, such as a long learning curve and costly equipment. Regardless, this method may be the "new standard" and the excellent method for the surgical treatment of BPH.

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate HoLEP.

Here we present a safe and efficient protocol, holmium laser enucleation of the prostate, to treat benign prostatic hyperplasia.

Tratamiento quirúrgico de la hiperplasia prostática benigna: Holmium láser enucleación de la próstata (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

Neovascularization del diafragma como un modelo de ratón de iridis del rubeosis inducida por la punción

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.