Forschung zur Erblindung verhindern; Bartly J. Mondino MD, Direktor des Jules Stein Eye Institute, UCLA, und Ramiro Ramirez-Solis, Jacqui White, and Jeanne Estabel am Sanger Institute, Wellcome Trust Genome Campus. Diese Forschung entspricht der ARVO Statement für die Verwendung von Tieren in Ophthalmic und Visual Research.

Ein. Blunt Dissection: Enukleation der Maus Auge fixierten Proben <ol><li> Auseinanderziehen die Augenlider, um die Exposition und den Zugang zu den hinteren Globus (Augapfel) Oberfläche zu verbessern. </li><li> Legen Sie eine gebogene Verband forcep hinter (unter) den Globus in der Umlaufbahn (Augenhöhle). Die Mahajan Sharptip Verband forcep ist ein Brauch Instrument mit Spitzen um diesen Schritt zu erleichtern (siehe Materialien und Reagenzien Tabelle). </li><li> Schließen Sie die Zange und fassen Sie den Orbital Bindegewebe und Sehnerv hinter dem Globus, während sie vorsichtig, um zu vermeiden, drückte den Globus. </li><li> Ziehen Sie die Zange nach oben und reißen den Augapfel aus der Augenhöhle. Die weißen Faden-ähnliches Gewebe ist der Sehnerv. </li><li> Legen Sie das Auge in PBS. Mit einer 30-Gauge-Nadel, machen eine einzige Stichwunde unmittelbar hinter dem Limbus durch Einführen der Nadel 1-2 mm in den Augapfel. Dies ermöglicht Fixiermittel, wie Glutaraldehyd, die sonst nicht in der Lage, um das Auge Gewebe zu durchdringen sind, um direkt enter das Auge. Schaffung dieses Einstichwunde eventuell nicht notwendig, wenn ein Fixiermittel wie Paraformaldehyd oder Alkohol, in der Lage ist Diffundieren in Augengeweben von Interesse. </li><li> Legen Sie das gesamte Auge sofort in Fixativ für Tauch-Fixierung. </li></ol> 2. Sharp Dissection: Enukleation der Maus eye folgenden Perfusionsfixierung <ol><li> Verwenden Sie eine gekrümmte colibri forcep um das Augenlid entfernt von der Welt zu halten. </li><li> Orient gekrümmten Westcott Dissektion Schere parallel zu den Globus beim Zielen in Richtung der Rückseite der Bahn. </li><li> Schneiden Sie die feste Bindegewebe, das die Welt umgibt aus den minderwertigen, medial, überlegen, und Seitenflächen. </li><li> Legen Sie die gebogenen Pinzette hinter den Globus, fassen das Bindegewebe, ohne Druck auf der ganzen Welt, und nach vorne ziehen, um das Auge entkernen. Da die Orbital Bindegewebe steif Perfusionsfixierung können zusätzliche Schneiden von Orbital Gewebe notwendig sein, komplett loslassen globE. </li></ol> 3. Fixation und Verpackung Versand von festen Geweben folgen müssen Ihren institutionellen und Postdienst Anforderungen einschließlich geeigneter Etiketten und Verpackungen. Das folgende Protokoll ist ein Beispiel des Versendens eines nicht-infektiösen biologischen Probe bei Umgebungstemperatur. Dies erfordert 3 Schichten der Verpackung, aber keine Klasse A / B biologische Substanz oder flüssigem Stickstoff Etiketten. <ol><li> Legen Sie das Auge in einer 5-mL Glasszintillationsröhrchen mit mindestens 3-mL Fixativ, oder post-Fixateur-Puffer, wenn die Fixierung ist kurz. Schreiben Sie die Tracking-Nummer auf dem Deckel des Fläschchens und verschließen Sie diese mit Parafilm. </li><li> Ort Szintillationsgefäße in einer biologischen Probe geeigneten Behälter. Im Institut, erfordern zum Beispiel zugelassenen Behältern drei Schichten: einen abgedichteten Behälter, in dem die Probe platziert ist, eine absorbierende mittlere Schicht und eine schützende Deckschicht. </li><li> Stellen Sie den Behälter in eine Luftpolsterfolie Paket und dann in einer Appschweiften Labor Transportbehälter zusammen mit den entsprechenden Unterlagen. </li></ol> 4. Repräsentative Ergebnisse Die histologische Untersuchung der Augen können durch eine Vielfalt von Verfahren einschließlich Hämatoxylin und Eosin-Färbung, enzymatische Nachweis Expression, Transmissionselektronenmikroskopie und Immunhistochemie erfolgen. Nach dem Eintauchen Fixierung in 4% Paraformaldehyd wurde Gewebe in Paraffin und geschnitten auf einem Mikrotom eingebettet. In Hochdurchsatz-Phänotypisierung analysieren wir Schüler-Sehnervs Abschnitte, die Probe alle Gewebearten im Auge (Abbildung 1). Gewebestücke wurden auch für lacZ-Expression (Abbildung 2), Transmissionselektronenmikroskopie von Zellorganellen (Abbildung 3), und die Expression von spezifischen Molekülen mit Antikörpern (Abbildung 4) geprüft. In unserem Phänotyp Screeningverfahren, war es nicht wichtig, Orientierung des Auges in Bezug auf die Nasen (medialen Augenwinkel) und zeitliche aufrechtzuerhalten (lateral Augenwinkel) Seiten. Es gibt mindestens zwei Optionen, wenn Auges Orientierung erforderlich ist. Nach Enukleation, haben wir Licht Kauter auf die zeitliche Hornhaut am 03.00 Position mit einem Handheld-Kauter Gerät. Die Läsion ist offensichtlich auf Histologie und nicht während der Verarbeitung, wie histologische Tinten verloren gehen. Der Nachteil ist, dass Kauter schädigt die Hornhaut, die eine kritische Gewebe für eine Untersuchung sein kann. Die andere Option erfordert einen erfahrenen Anatomen, die die Augenmuskeln Insertionen identifizieren können. Unter einem Stereomikroskop, markiert Identifizierung des unteren und oberen schrägen Muskel Einsetzen der unteren und oberen und Aspekt des Globus. 3 Mit jeder Orientierung Verfahrens ist es wichtig, die Probe als rechte oder linke Auge verfolgen. Gemeinsam werden diese Exact Globe Orientierung vor lassen Schneidens. <img src="/files/ftp_upload/3184/3184fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Maus Auge histologische Bilder afte<html> r Remote-Akquisition. Maus Augen waren durch scharfe Dissektion nach Perfusionsfixierung mit 4% Paraformaldehyd enukleiert. A. Schüler-Sehnervs Abschnitt mit Hämatoxylin und Eosin gefärbt zeigt Erhaltung aller Gewebe Unterkonstruktionen. ON, Sehnerv. (Grün Maßstab = 500 Mikron) B. Eine stärkere Vergrößerung Ansicht des normalen Netzhaut zeigt seine Schichtstruktur: RGC, retinalen Ganglienzellen, IPL, inneren plexiformen Schicht; INL, innere Körnerschicht; OPL, plexiformen Schicht; ONL, äußere Körnerschicht (Sehzellen) (Pfeil); IS, Photorezeptor inneren Segmente (Pfeilspitze); OS, Photorezeptor äußeren Segmente, RPE, Pigmentepithel; C, Aderhaut. (Grün-Skala bar = 50 Mikron) C. der normalen Netzhaut ist im Vergleich dieser Probe dünner aufgrund Photorezeptordegeneration. Die ONL, IS und OS fehlen völlig (Pfeil). (Grün-Skala bar = 50 Mikron) &quot;Alt =&quot; Bild 2 &quot;/&gt; Abbildung 2. LacZ-Expression in transgenen Mäusen. Augen wurden stumpf enukleiert und eingetaucht in 1 mg / ml X-gal-Lösung (5 mM K3Fe (CN) 6, 5 mM K4Fe (CN) 6, 2 mM MgCl2, 0,02% NP40 und 0,1% Natriumdesoxycholat) über Nacht bei 37 ° C. Augen wurden in 2% formaldehyde/0.2% Glutaraldehyd für 30 Minuten und auf einem Mikrotom geschnitten fixiert. X-gal Produkt (blau) Etiketten: A. Ziliarepithel, B. Hornhautepithel, und C. die retinalen Ganglienzellen (RGC) in der Netzhaut. INL, innere Körnerschicht; ONL, äußeren Körnerschicht. <img src="/files/ftp_upload/3184/3184fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Transmissionselektronenmikroskopie des retinalen Pigmentepithels (RPE). Nach stumpf und die Schaffung einer Stichwunde, waren Augen Eintauchen in 2,5% Paraformaldehyd / 2,5% Glutaraldehyd in 0,1 M Natriumphosphatpuffer fixiert. Augen waren gebrauchtdarily in 1% Osmium textroxide fixiert, schrittweise entwässert, und in Spurr das Harz. Gewebe wurde bei 90 nm geschnitten und gelegt auf Kupfer Schlitz Gittern in Formvar abgedeckt und abgebildet mit einem Transmissions-Elektronen-Mikroskop. Die Aufnahme zeigt, Photorezeptor äußeren Segmente des Neuroretina, Melanosomen (M) innerhalb des retinalen Pigmentepithels und der Bruch-Membran. <img src="/files/ftp_upload/3184/3184fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Immunhistochemische Kennzeichnung von Superoxiddismutase-3 (grün) in der Maus Glaskörper. Augen wurden durch stumpfe Dissektion entkernt und unterzog Eintauchen Fixierung in 4% Paraformaldehyd. Sie wurden in Paraffin und unter Verwendung eines Mikrotoms geschnitten eingebettet. Gewebeschnitte wurden mit polyklonalem Kaninchen-Antiserum inkubiert SOD3 1:50 verdünnt. SOD3 Expression wurde unter Verwendung eines Ziegen-Anti-Kaninchen-IgG (H + L)-Alexa Fluor 488 konjugierten sekundären Antikörper. Kennzeichnung von SOD3 in gre angezeigten.

<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>

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Die meisten transgenen Mäusen gibt es in Laboratorien, die nicht prüfen müssen die Augen. Unsere Video-Technik stellt eine einfache, standardisierte Methode zur Remote-chirurgischen Fähigkeiten Übertragung von Gewebe Akquisition von Laboratorien mit wenig Erfahrung mit den Augen zu optimieren. Dieses Video-Technik hilft bei der Bewältigung einer großen Sünden in High-Throughput-Phänotypisierung, die Verwendung einer begrenzten Anzahl von Experten Standorten aufgrund der nicht-standardisierten Gewebe Sammlung un...

<table border="1"><tbody><tr><th> Name </th><th> Firma </th><th> Catalog Number </th><th> Kommentare </th></tr><tr><td> Curved Dressing Forcep </td><td> Storz Ophthalmics </td><td> E1408 </td><td></td></tr><tr><td> Mahajan Sharptip Verband forcep </td><td> Storz Ophthalmics </td><td> E1406 (REF SP7-64520) </td><td></td></tr><tr><td> Curved Westcott Schere </td><td> Storz Ophthalmics </td><td> E3321 WH </td><td></td></tr><tr><td> 15 ° BD Beaver Mikrochirurgische Klinge </td><td> Becton-Dickinson </td><td> 374881 </td><td></td></tr><tr><td> 0,22 Fine-Castroviejo Naht-Zange </td><td> Storz Ophthalmics </td><td> E1805 </td><td></td></tr><tr><td> 0,12 Colibri Pinzette </td><td> Storz Ophthalmics </td><td> 2/132 </td><td></td></tr><tr><td> 30-Gauge-Nadel </td><td> Becton-Dickinson </td><td> 305128 </td><td></td></tr><tr><td> Biohazard Mailer </td><td> Fischer </td><td> 03-523-4 </td><td></td></tr><tr><td> Parafilm </td><td> Fischer </td><td> 13-374-10 </td><td></td></tr><tr><td> Glasszintillationsröhrchen </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% Paraformaldehyd </td><td> Electron Microscopy Sciences </td><td> 15700 </td><td></td></tr><tr><td> 2,5% Paraformaldehyd / 2,5% Glutaraldehyd in 0,1 M Natriumphosphatpuffer </td><td> Electron Microscopy Sciences </td><td> 15700 &amp; 16300 </td><td> Gemischt im Labor </td></tr><tr><td> 50% Glutaraldehyd </td><td> Electron Microscopy Sciences </td><td> 16300 </td><td></td></tr><tr><td> 0,25% Formvar </td><td> Electron Microscopy Sciences </td><td> 15810 </td><td></td></tr><tr><td> Polizistpro Slot Grid </td><td> Electron Microscopy Sciences </td><td> M2010-CR </td><td></td></tr><tr><td> 4% Osmiumtetroxid </td><td> Electron Microscopy Sciences </td><td> 19140 </td><td></td></tr><tr><td> Anti-SOD3 Antikörper </td><td> Abcam </td><td> Ab21974 </td><td></td></tr><tr><td> Ziege anti-Kaninchen Alexa Fluor 488 </td><td> Invitrogen </td><td> A11070 </td><td></td></tr><tr><td> Spurr die Einbettung von Harz </td><td> Electron Microscopy Sciences </td><td> 14300 </td><td></td></tr></tbody></table>

mouse eye enucleation for remote high-throughput phenotyping

Die Maus Auge ist ein wichtiges genetisches Modell für die translationale Erforschung der menschlichen Augenerkrankung. Blendende Erkrankungen beim Menschen, wie Makuladegeneration, Photorezeptordegeneration, Katarakt, Glaukom, Retinoblastom, und diabetische Retinopathie haben in transgenen Mäusen wurde rekapituliert. 1-5 meisten transgenen und Knockout-Mäuse von Laboratorien erzeugt wurden auf nicht-ophthalmologische Krankheiten zu studieren, aber Generhaltung zwischen Organsysteme lässt vermuten, dass viele der gleichen Gene können auch eine Rolle bei der Entwicklung und okulare Krankheit. Daher stellen diese Mäuse eine wichtige Ressource für die Entdeckung neuer Genotyp-Phänotyp-Korrelationen in die Augen. Da diese Mäuse auf der ganzen Welt verstreut sind, ist es schwierig, zu erwerben, zu halten und Phänotyp sie in eine effiziente, kostengünstige Weise. So sind die meisten High-Throughput-ophthalmologische Phänotypisierung Bildschirmen auf wenige Standorte, die vor Ort, ophthalmologische Know-how erfordern beschränkt Augen in lebenden Mäusen zu untersuchen. 6-9 Ein alternativer Ansatz in unserem Labor entwickelt wurde, ist eine Methode für Remote Gewebe-Übernahme, die in großen oder kleinen Erhebungen der transgenen Maus Augen verwendet werden können. Standardisierte Verfahren für Video-basierte chirurgische Fachwissen, Gewebefixierung und Versand ermöglichen jedem Labor zu ganzen Augen von mutierten Tiere zu sammeln und senden Sie sie für molekulare und morphologische Phänotypisierung. In diesem Video Artikel präsentieren wir Techniken, um entkernen und übertragen sowohl fixierten und Perfusion festen Mäuseaugen für Remote Phänotypisierung Analysen.

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

Die Dissektionstechnik zeigt Enukleation der Maus Auge für Gewebefixierung um Phänotypisierung in Hochdurchsatz-Screening durchzuführen.

Maus Eye Enukleation für Remote High-throughput Phänotypisierung

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 Aufrufe.  University of Iowa. Die Dissektionstechnik zeigt Enukleation der Maus Auge für Gewebefixierung um Phänotypisierung in Hochdurchsatz-Screening durchzuführen.

Serie: 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.

Ein automatisiertes Hochdurchsatz-Plattform für die Entwicklung von Manufacturing Zelllinien für Protein Therapeutics

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

Forschung

Medizin

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.

Ein Maus-Modell der Cornea Pocket-Assay für die Angiogenese-Studie

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.

Hyperinsulinämische-euglykämische Schellen in dem Bewusstsein, Unrestrained Mäuse

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.

Remote-Magnetic Navigation für präzise, ​​Echtzeit-Positionierung und Katheter Ablation in Cardiac Elektrophysiologie Procedures

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.

High Throughput Sequential ELISA zur Validierung von Biomarkern des akuten Graft-Versus-Host Disease

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.

Hochdurchsatz-Charakterisierung von adulten Stammzellen für die Zuführung von therapeutischen Faktoren für neuroprotektive Strategien Engineered

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.

Fluoreszenzfarbstoff-Markierung von Erythrozyten und Leukozyten zum Studieren der Flußdynamik bei der Maus-Retinal-Zirkulation

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.

Chirurgische Behandlung der benignen Prostatahyperplasie: Holmium Laser Enukleation der Prostata (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.

Punktion-induzierte Iris Neovaskularisation als einem Mausmodell der 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.