Research to Prevent Blindness; Bartly J. Mondino MD, directeur de l&#39;Institut Jules Stein Eye, UCLA, et Ramiro Ramirez-Solis, Jacqui blanc, et Jeanne Estabel à l&#39;Institut Sanger, Wellcome Trust Genome Campus. Cette recherche est conforme à la Déclaration ARVO pour l&#39;utilisation des animaux en ophtalmologie et de la recherche visuelle.

1. Dissection Blunt: énucléation de l&#39;œil de la souris dans les échantillons non fixés <ol><li> Séparez les paupières pour améliorer la visibilité et l&#39;accès au globe postérieure (œil) de surface. </li><li> Placez un pansement pince courbée derrière (sous) le monde dans l&#39;orbite (orbite de l&#39;œil). Le Mahajan Sharptip pansement pince est un instrument de mesure avec bouts pointus pour faciliter cette étape (voir matériels et réactifs de table). </li><li> Fermez la pince et saisir le tissu conjonctif orbitale et le nerf optique derrière le globe tout en faisant attention à ne pas écraser le monde. </li><li> Tirez doucement sur le pince vers le haut et arracher le globe oculaire hors de l&#39;orbite. Le fil blanc-comme le tissu est le nerf optique. </li><li> Placez l&#39;œil dans du PBS. En utilisant une aiguille de calibre 30, faire une ponction unique liquidée immédiatement en arrière du limbe en insérant l&#39;aiguille 1-2 mm dans le globe oculaire. Ceci permet fixateurs, tels que le glutaraldéhyde, qui sont dans l&#39;impossibilité de pénétrer dans le tissu oculaire, directement enter l&#39;œil. Création de cette piqûre peut ne pas être nécessaire si un fixateur comme le paraformaldéhyde ou l&#39;alcool, est capable de diffuser dans les tissus oculaires d&#39;intérêt. </li><li> Placez l&#39;œil entier immédiatement dans fixateur pour la fixation d&#39;immersion. </li></ol> 2. Dissection Sharp: énucléation de la fixation de la souris oeil perfusion suivante <ol><li> Utilisez une pince courbe colibri de tenir la paupière loin du monde. </li><li> Orienter les courbes ciseaux à dissection Westcott parallèles au monde, tout en visant vers l&#39;arrière de l&#39;orbite. </li><li> Coupez le tissu conjonctif qui entoure fixe le monde des inférieures, médianes, les côtés supérieurs et latéraux. </li><li> Placez la pince courbée derrière le globe, tenez le tissu conjonctif sans appuyer sur le globe, et tirer vers l&#39;avant pour énucléer l&#39;œil. Puisque le tissu conjonctif orbitale est raide de fixation par perfusion, coupe supplémentaire des tissus orbitaires peut être nécessaire pour libérer complètement le globe. </li></ol> 3. Fixation et d&#39;emballage Livraison de tissus fixés doivent respecter vos obligations de service postal et institutionnels, y compris des étiquettes et des emballages. Le protocole suivant est un exemple de l&#39;expédition d&#39;un échantillon non biologique infectieux à température ambiante. Cela nécessite 3 couches de l&#39;emballage mais pas de classe A substance / B biologiques ou des étiquettes d&#39;azote liquide. <ol><li> Placez l&#39;oeil dans un flacon de 5 ml à scintillation en verre avec au moins 3 ml de fixateur, ou post-fixateur de mémoire tampon si la fixation est courte. Écrivez le numéro de suivi sur le couvercle du flacon et le sceller avec du parafilm. </li><li> Placer les flacons à scintillation dans un échantillon biologique approuvé conteneur d&#39;expédition. Dans notre institut, par exemple, des contenants approuvés besoin de trois couches: un conteneur étanche dans lequel l&#39;échantillon est placé, une couche intermédiaire absorbante et une couche externe protectrice. </li><li> Placer le récipient dans un emballage plastique-bulles, puis dans une applicationparcouraient conteneur d&#39;expédition de laboratoire ainsi que la documentation appropriée. </li></ol> 4. Les résultats représentatifs L&#39;examen histologique des yeux peut être effectuée par une variété de méthodes, y compris l&#39;hématoxyline et coloration à l&#39;éosine, détection de l&#39;expression enzymatique, microscopie électronique à transmission, et l&#39;immunohistochimie. Après fixation par immersion dans du paraformaldéhyde 4%, le tissu a été inclus dans la paraffine et sectionnés au microtome. Dans le phénotypage à haut débit, nous analysons les sections nerveuses élève optique que l&#39;échantillon tous les types de tissus de l&#39;œil (figure 1). Des morceaux de tissu ont également été examinées pour l&#39;expression de lacZ (figure 2), la microscopie électronique à transmission des organites cellulaires (figure 3), et l&#39;expression de molécules spécifiques avec des anticorps (figure 4). Dans notre méthode de dépistage phénotype, ce n&#39;était pas important pour maintenir l&#39;orientation de l&#39;oeil par rapport au nasal (canthus interne) et temporelle (lateral) canthus côtés. Il ya au moins deux options si l&#39;orientation des yeux est nécessaire. Après énucléation, nous avons appliqué la cautérisation lumière sur la cornée temporelle à la position trois heures avec un dispositif de cautérisation de poche. La lésion est évident sur l&#39;histologie et de ne pas se perdre pendant le traitement, comme les encres histologiques. L&#39;inconvénient est que les dommages cautérisation de la cornée, ce qui peut être un tissu essentiel pour examen. L&#39;autre option nécessite un anatomiste habile qui permet d&#39;identifier les insertions musculaires extra-oculaires. Sous un stéréomicroscope, l&#39;identification de l&#39;insertion du muscle oblique inférieur et supérieur constitue l&#39;aspect inférieur et supérieur et de la planète 3. Que soit la méthode d&#39;orientation, il est important de suivre le spécimen comme l&#39;œil droit ou gauche. Ensemble, ce qui permettra l&#39;orientation exacte monde avant la coupe. <img src="/files/ftp_upload/3184/3184fig1.jpg" alt="Figure 1" /> Figure 1. Images des yeux de souris histologiques afte<html> r distance acquisition. yeux de souris ont été énucléés par dissection après la fixation de perfusion avec du paraformaldéhyde 4%. A. Nombre d&#39;élèves par section du nerf optique colorées à l&#39;hématoxyline et à l&#39;éosine montre préservation de toutes les structures tissulaires. ON, le nerf optique. (Barre d&#39;échelle verte = 500 microns) B. Une vue plus fort grossissement de la rétine normale montre sa structure laminaire: RGC, les cellules ganglionnaires de la rétine; IPL, couche interne plexiforme; INL, couche nucléaire interne; OPL, couche externe plexiforme; ONL, extérieur la couche nucléaire (cellules photoréceptrices) (flèche); IS, les segments des photorécepteurs internes (tête de flèche); OS, photorécepteurs segments extérieurs; RPE, l&#39;épithélium pigmentaire rétinien; C, choroïde. (Barre d&#39;échelle verte = 50 microns) C. Par rapport à la rétine normale, ce spécimen est plus mince en raison de la dégénérescence des photorécepteurs. L&#39;ONL, IS et OS sont totalement absents (flèche). (Échelle bar vert = 50 microns) &quot;Alt =&quot; Figure 2 &quot;/&gt; Figure 2. L&#39;expression de lacZ dans des souris transgéniques. Yeux ont été énucléés par dissection et immergé dans 1 mg / ml solution X-gal (5 mM K3Fe (CN) 6, 5 mM K4Fe (CN) 6, 2 mM de MgCl2, 0,02% de NP40 , et le désoxycholate de sodium 0,1%) pendant une nuit à 37 ° C. Les yeux étaient fixés dans du glutaraldéhyde formaldehyde/0.2% 2% pendant 30 minutes et en coupe au microtome. X-gal produits (bleu) étiquettes: A. épithélium ciliaire, B. épithélium de la cornée, et C. de la cellule rétinienne ganglion (RGC) couche de la rétine. INL, couche nucléaire interne; ONL, couche nucléaire externe. <img src="/files/ftp_upload/3184/3184fig3.jpg" alt="Figure 3" /> Figure 3. Microscopie électronique à transmission de l&#39;épithélium pigmentaire rétinien (EPR). Après dissection et la création d&#39;une plaie perforante, les yeux étaient fixés par immersion dans du paraformaldéhyde à 2,5% / 2,5% de glutaraldéhyde dans un tampon 0,1 M phosphate de sodium. Les yeux étaient de secondedarily fixé dans textroxide osmium à 1%, peu à peu déshydraté, et noyé dans une résine de Spurr. Tissu a été coupé à 90 nm et placés sur des grilles de cuivre sous couvert de Formvar et imagée à l&#39;aide d&#39;un microscope électronique à transmission. La micrographie montre, segments externes des photorécepteurs de la rétine neurosensorielle, mélanosomes (M) dans l&#39;épithélium pigmentaire de la rétine et la membrane de Bruch. <img src="/files/ftp_upload/3184/3184fig4.jpg" alt="Figure 4" /> Figure 4. Marquage immunohistochimique de la superoxyde dismutase-3 (verte) dans le corps vitré de la souris. Yeux ont été énucléés par dissection et a subi une fixation immersion dans du paraformaldéhyde à 4%. Ils ont été inclus dans la paraffine et sectionnés à l&#39;aide d&#39;un microtome. Les coupes de tissu ont été incubées avec un antisérum polyclonal de lapin SOD3 dilué à 1:50. SOD3 expression a été détectée en utilisant un anticorps de chèvre anti-IgG de lapin (H + L)-Alexa Fluor 488 anticorps secondaire conjugué. L&#39;étiquetage des SOD3 est indiqué dans grefr.

<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>

Aucun conflit d&#39;intérêt déclaré.

La plupart des souris transgéniques existent dans les laboratoires qui n&#39;examinent pas les yeux. Notre technique vidéo illustre une méthode simple et standardisé pour le transfert de compétences à distance chirurgicale afin d&#39;optimiser l&#39;acquisition des tissus provenant de laboratoires ayant peu d&#39;expérience avec les yeux. Cette technique aide à surmonter vidéo un écueil majeur dans le phénotypage à haut débit, qui est l&#39;utilisation d&#39;un nombre limité de sites spécialisés en raiso...

<table border="1"><tbody><tr><th> Nom </th><th> Entreprise </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> Pince courbe Dressing </td><td> Storz Ophthalmics </td><td> E1408 </td><td></td></tr><tr><td> Mahajan Sharptip pansement pince </td><td> Storz Ophthalmics </td><td> E1406 (REF SP7-64520) </td><td></td></tr><tr><td> Ciseaux courbes Westcott </td><td> Storz Ophthalmics </td><td> E3321 WH </td><td></td></tr><tr><td> 15 ° BD Beaver microchirurgicale de la lame </td><td> Becton-Dickinson </td><td> 374881 </td><td></td></tr><tr><td> 0,22 Fine-Castroviejo Forceps suture </td><td> Storz Ophthalmics </td><td> E1805 </td><td></td></tr><tr><td> 0,12 pince Colibri </td><td> Storz Ophthalmics </td><td> 2/132 </td><td></td></tr><tr><td> 30-aiguille de calibre </td><td> Becton-Dickinson </td><td> 305128 </td><td></td></tr><tr><td> Biohazard Mailer </td><td> Pêcheur </td><td> 03-523-4 </td><td></td></tr><tr><td> Parafilm </td><td> Pêcheur </td><td> 13-374-10 </td><td></td></tr><tr><td> Verre flacons à scintillation </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> Paraformaldéhyde 16% </td><td> Sciences microscopie électronique </td><td> 15700 </td><td></td></tr><tr><td> 2,5% paraformaldéhyde / glutaraldéhyde 2,5% dans 0,1 M de tampon phosphate de sodium </td><td> Sciences microscopie électronique </td><td> 15700 &amp; 16300 </td><td> Mixé en laboratoire </td></tr><tr><td> Glutaraldéhyde à 50% </td><td> Sciences microscopie électronique </td><td> 16300 </td><td></td></tr><tr><td> 0,25% Formvar </td><td> Sciences microscopie électronique </td><td> 15810 </td><td></td></tr><tr><td> Flicpar grille fente </td><td> Sciences microscopie électronique </td><td> M2010-CR </td><td></td></tr><tr><td> Tétroxyde d&#39;osmium 4% </td><td> Sciences microscopie électronique </td><td> 19140 </td><td></td></tr><tr><td> Anti-SOD3 anticorps </td><td> Abcam </td><td> Ab21974 </td><td></td></tr><tr><td> Chèvre anti-lapin Alexa Fluor 488 </td><td> Invitrogen </td><td> A11070 </td><td></td></tr><tr><td> Résine de Spurr intégration </td><td> Sciences microscopie électronique </td><td> 14300 </td><td></td></tr></tbody></table>

mouse eye enucleation for remote high-throughput phenotyping

L&#39;œil de la souris est un modèle important pour l&#39;étude génétique de translation de la maladie ophtalmique humaine. Prévenir la cécité chez l&#39;homme, telles que la dégénérescence maculaire, la dégénérescence des photorécepteurs, la cataracte, le glaucome, le rétinoblastome, et la rétinopathie diabétique ont été récapitulés dans les souris transgéniques. 5.1 La plupart des souris transgéniques et knock-out ont été générées par les laboratoires pour étudier les maladies non ophtalmiques, mais conservation des ressources génétiques entre les systèmes d&#39;organes suggère que bon nombre des mêmes gènes peuvent également jouer un rôle dans le développement oculaire et les maladies. Par conséquent, ces souris constituent une ressource importante pour la découverte de nouvelles corrélations génotype-phénotype dans les yeux. Parce que ces souris sont dispersés à travers le monde, il est difficile d&#39;acquérir, de conserver et de les phénotype d&#39;une manière efficace, rentable. Ainsi, la plupart des écrans à haut débit phénotypage ophtalmiques sont limités à quelques endroits qui ont besoin sur place, l&#39;expertise ophtalmologique pour examiner les yeux chez des souris vivantes. 9.6 Une approche alternative développée par notre laboratoire est une méthode de tissus acquisition à distance qui peut être utilisé dans les enquêtes à petite ou grande échelle des yeux de souris transgéniques. Les procédures standardisées pour la vidéo basée sur le transfert de compétences chirurgicales, la fixation des tissus, et l&#39;expédition permettre à tout laboratoire pour recueillir les yeux entiers à partir d&#39;animaux mutants et les envoyer pour le phénotypage moléculaire et morphologique. Dans cet article, vidéo, nous vous présentons des techniques pour énucléer et transférer à la fois non fixées et la perfusion yeux fixes souris pour les analyses de phénotypage à distance.

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 technique de dissection illustre énucléation de l&#39;oeil de la souris pour la fixation des tissus pour réaliser le phénotypage à haut débit écrans.

L&#39;énucléation des yeux de souris à distance pour le phénotypage à haut débit

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 vues.  University of Iowa. La technique de dissection illustre énucléation de l'oeil de la souris pour la fixation des tissus pour réaliser le phénotypage à haut débit écrans.

Vidéo: 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.

Une plate-forme à haut débit automatisé pour le développement de lignées cellulaires de fabrication des protéines thérapeutiques

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

Recherche

Médecine

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 modèle murin de l&#39;Assay Pocket cornée pour l&#39;étude de l&#39;angiogenèse

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.

Hyperinsulinémique euglycémique-pinces dans Conscient, Souris illimités

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.

Navigation magnétique à distance pour exacts, positionnement du cathéter en temps réel et en électrophysiologie cardiaque Ablation Procédures

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.

Haut débit séquentiel ELISA pour la validation des biomarqueurs de réaction aiguë du greffon contre l&#39;hôte

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.

High Throughput Caractérisation des cellules souches adultes d&#39;ingénierie pour la livraison des facteurs thérapeutiques pour les stratégies neuroprotectrices

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.

Étiquetage des colorants fluorescents des érythrocytes et des leucocytes pour étudier la dynamique du flux dans la circulation de la rétine de la souris

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.

Traitement chirurgical de l’hyperplasie bénigne de la prostate : énucléation Laser Holmium de la Prostate (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.

Induite par la ponction de néovascularisation Iris comme un modèle de souris de 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 ...

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.