La ricerca per prevenire la cecità; Bartly J. Mondino MD, direttore del Jules Stein Eye Institute, UCLA, e Ramiro Ramirez-Solis, Jacqui bianco, e Jeanne Estabel presso l&#39;Istituto Sanger, Wellcome Trust Genome Campus. Questa ricerca è conforme con la dichiarazione ARVO per l&#39;uso di animali in oftalmica e di ricerca visiva.

1. Scollamento: enucleazione dell&#39;occhio mouse in campioni non fissati <ol><li> Separare le palpebre per migliorare l&#39;esposizione e l&#39;accesso al mondo posteriore (bulbo oculare) di superficie. </li><li> Posizionare una pinza curva medicazione dietro (sotto) il globo in orbita (cavità oculare). Il Mahajan Sharptip medicazione pinza è uno strumento personalizzato con punte appuntite per rendere questo passaggio più facile (vedi materiali e reagenti tabella). </li><li> Chiudere la pinza e afferrare il tessuto connettivo orbitale e del nervo ottico dietro il globo facendo attenzione a evitare di comprimere il mondo. </li><li> Estrarre delicatamente la pinza verso l&#39;alto e cogliere il bulbo oculare dall&#39;orbita. Il bianco filiforme tessuto è il nervo ottico. </li><li> Posizionare l&#39;occhio in PBS. Utilizzando un ago da 30 gauge, effettuare una singola puntura ferita immediatamente posteriormente al limbus, inserendo l&#39;ago di 1-2 mm nel bulbo oculare. Questo permette fissativi, come glutaraldeide, che sono comunque in grado di penetrare il tessuto oculare, direttamente a enter l&#39;occhio. Creazione di questo puntura può non essere necessario se un fissativo, come paraformaldeide o alcool, è in grado di diffondere nei tessuti oculari di interesse. </li><li> Posizionare l&#39;intero occhio immediatamente in fissativo per la fissazione immersione. </li></ol> 2. Sharp Dissection: enucleazione della fissazione del mouse perfusione degli occhi seguenti <ol><li> Utilizzare una curva colibri pinza per tenere la palpebra lontano dal mondo. </li><li> Orient curve Westcott forbici dissezione parallele al mondo, mentre volti verso la parte posteriore dell&#39;orbita. </li><li> Tagliare il tessuto fissato connettivo che circonda il globo dagli inferiori, mediale, i lati superiore e laterale. </li><li> Posizionare la pinza curva dietro il globo, afferrare il tessuto connettivo senza premere sul globo, e tirare avanti per enucleare l&#39;occhio. Poiché il tessuto connettivo orbitale è rigida da fissazione perfusione, taglio aggiuntivo di tessuti orbitali può essere necessario rilasciare completamente la globe. </li></ol> 3. Fissazione e imballaggio Spedizione dei tessuti fissi deve seguire le vostre esigenze di servizio istituzionali e postali, tra cui etichette e imballaggi appropriati. Il protocollo seguito è un esempio di spedizione di un non infettiva campione biologico a temperatura ambiente. Ciò richiede 3 strati di imballaggi, ma non di classe Una sostanza / B biologica o etichette di azoto liquido. <ol><li> Posizionare l&#39;occhio in un 5 ml flaconcino di vetro scintillazione con almeno 3 ml di fissativo, o post-fissativo buffer se la fissazione è breve. Scrivere il numero di inseguimento sul coperchio del flaconcino e sigillarla con Parafilm. </li><li> Inserire fiale di scintillazione in un campione biologico approvato container. Nel nostro istituto, per esempio, contenitori approvati richiedono tre strati: un contenitore chiuso in cui è posto il campione, uno strato intermedio assorbente e uno strato protettivo esterno. </li><li> Mettere il recipiente in una bolla-wrap pacchetto e poi in un appvagava container laboratorio con documentazione appropriata. </li></ol> 4. Risultati rappresentativi Esame istologico degli occhi può essere eseguita da una varietà di metodi compresi ematossilina ed eosina colorazione, rilevamento espressione enzimatica, microscopia elettronica a trasmissione, e immunoistochimica. Seguendo fissazione immersione in paraformaldeide al 4%, il tessuto è stato incorporato in paraffina e sezionati con un microtomo. In high-throughput fenotipizzazione, analizziamo pupilla ottica sezioni nervose che tutti i tipi di campioni dei tessuti oculari (Figura 1). Sezioni di tessuto sono stati esaminati per l&#39;espressione lacZ (Figura 2), microscopia elettronica a trasmissione di organelli cellulari (Figura 3), e l&#39;espressione di molecole con anticorpi specifici (Figura 4). Nel nostro metodo di screening fenotipo, non era importante mantenere l&#39;orientamento dell&#39;occhio rispetto al nasale (canto mediale) e temporale (Lateral canto) lati. Ci sono almeno due opzioni se orientamento occhio è necessaria. Dopo enucleazione, abbiamo applicato cauterizzazione luce alla cornea temporale nella posizione 03:00 con un dispositivo portatile cauterio. La lesione è evidente all&#39;esame istologico e non perdersi durante la lavorazione come gli inchiostri istologici. Il rovescio della medaglia è che i danni cauterio la cornea, che può essere un tessuto fondamentale per l&#39;esame. L&#39;altra opzione richiede un anatomista esperto in grado di identificare le inserzioni muscolari extraoculari. Allo stereomicroscopio, identificazione di inserimento inferiore e superiore muscolo obliquo segna l&#39;aspetto inferiore e superiore e del globo. 3 Con entrambi i metodi di orientamento, è importante monitorare il campione come un occhio destro o sinistro. Insieme, questo permetterà l&#39;orientamento esatto mondo prima del sezionamento. <img src="/files/ftp_upload/3184/3184fig1.jpg" alt="Figura 1" /> Figura 1. Immagini dell&#39;occhio mouse istologici afte<html> r acquisizione remota. occhi del mouse sono stati enucleati dalla dissezione netta seguendo fissazione perfusione con paraformaldeide al 4%. A. alunni-sezione del nervo ottico colorato con ematossilina e eosina mostra la conservazione di tutte le sottostrutture dei tessuti. ON, nervo ottico. (Barra della scala verde = 500 micron) B. Un punto di vista più alto ingrandimento della retina normale mostra la sua struttura laminare: RGC, cellule gangliari della retina, IPL, strato interno plessiforme, INL, strato nucleare interno, OPL, strato esterno plessiforme; ONL, esterno strato nucleare (cellule fotorecettrici) (freccia), IS, segmenti interni dei fotorecettori (punta di freccia), sistema operativo, segmenti esterni dei fotorecettori, RPE, epitelio pigmentato retinico, C, coroide. (Barra della scala verde = 50 micron) C. Rispetto alla retina normale, questo modello è più sottile a causa della degenerazione dei fotorecettori. Il ONL, IS e OS sono completamente assenti (freccia). (Scala bar verde = 50 micron) &quot;Alt =&quot; Figura 2 &quot;/&gt; Figura 2. Espressione lacZ in topi transgenici. Occhi sono stati enucleati per via smussa e immerso in 1 mg / ml di X-gal soluzione (5 mM K3Fe (CN) 6, 5 mM K4Fe (CN) 6, 2 mM MgCl2, 0,02% NP40 , e sodio desossicolato 0,1%) per una notte a 37 ° C. Occhi sono stati fissati in glutaraldeide al 2% formaldehyde/0.2% per 30 minuti e sezionato con un microtomo. X-gal prodotto (blu) etichette: A. epitelio ciliare, epitelio corneale B., C. e la cellule gangliari della retina (RGC) strato nella retina. INL, strato nucleare interno; ONL, strato nucleare esterno. <img src="/files/ftp_upload/3184/3184fig3.jpg" alt="Figura 3" /> Figura 3. Microscopia elettronica a trasmissione di epitelio pigmentato retinico (RPE). Dopo scollamento e la creazione di una puntura, gli occhi fissi ad immersione nel 2,5% paraformaldeide / 2,5% glutaraldeide in tampone fosfato di sodio 0.1M. Gli occhi erano di secondadarily fissato textroxide osmio 1%, a poco a poco disidratati, ed inclusi in resina di Spurr. Tessuto era sezionati a 90 nm e posto su griglie di rame rivestite in scanalatura formvar e ripreso con un microscopio elettronico a trasmissione. La micrografia mostra, segmenti esterni dei fotorecettori della retina neurosensoriale, melanosomi (M) all&#39;interno dell&#39;epitelio pigmentato retinico e della membrana di Bruch. <img src="/files/ftp_upload/3184/3184fig4.jpg" alt="Figura 4" /> Figura 4. Etichettatura immunoistochimica di superossido dismutasi-3 (verde) nel vitreo mouse. Gli occhi sono stati enucleati per via smussa ed ha subito la fissazione immersione in paraformaldeide al 4%. Sono stati inclusi in paraffina e sezionati con un microtomo. Le sezioni di tessuto sono state incubate con un anticorpo policlonale di coniglio SOD3 antisiero diluito 1:50. SOD3 espressione è stata rilevata usando un anti-rabbit IgG di capra (H + L)-Alexa Fluor 488 anticorpo secondario coniugato. Etichettatura di SOD3 è mostrato in 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>

Nessun conflitto di interessi dichiarati.

La maggior parte dei topi transgenici esistono in laboratori che non esaminano gli occhi. La nostra tecnica video illustra un metodo semplice e standardizzato per il trasferimento remoto abilità chirurgica per ottimizzare l&#39;acquisizione tessuti da laboratori con poca esperienza con gli occhi. Questa tecnica video aiuta superare un trabocchetto importante in high throughput fenotipizzazione, che è l&#39;uso di un numero limitato di siti esperti dovuti a non standardizzato di raccolta dei tessuti e fissazione metodi...

<table border="1"><tbody><tr><th> Nome </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Curvo Pinza Dressing </td><td> Storz Ophthalmics </td><td> E1408 </td><td></td></tr><tr><td> Mahajan Sharptip medicazione pinza </td><td> Storz Ophthalmics </td><td> E1406 (REF SP7-64520) </td><td></td></tr><tr><td> Curve Forbici Westcott </td><td> Storz Ophthalmics </td><td> E3321 WH </td><td></td></tr><tr><td> 15 ° BD Beaver microchirurgica Lama </td><td> Becton-Dickinson </td><td> 374881 </td><td></td></tr><tr><td> 0,22 Fine-Castroviejo sutura Pinze </td><td> Storz Ophthalmics </td><td> E1805 </td><td></td></tr><tr><td> 0,12 Colibri pinza </td><td> Storz Ophthalmics </td><td> 2/132 </td><td></td></tr><tr><td> Ago da 30 gauge </td><td> Becton-Dickinson </td><td> 305128 </td><td></td></tr><tr><td> Biohazard Mailer </td><td> Pescatore </td><td> 03-523-4 </td><td></td></tr><tr><td> Parafilm </td><td> Pescatore </td><td> 13-374-10 </td><td></td></tr><tr><td> Vetro fiale di scintillazione </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% paraformaldeide </td><td> Scienze della microscopia elettronica </td><td> 15700 </td><td></td></tr><tr><td> 2,5% paraformaldeide / glutaraldeide 2,5% in tampone fosfato di sodio 0,1 M </td><td> Scienze della microscopia elettronica </td><td> 15700 &amp; 16300 </td><td> Misto in laboratorio </td></tr><tr><td> 50% glutaraldeide </td><td> Scienze della microscopia elettronica </td><td> 16300 </td><td></td></tr><tr><td> 0,25% formvar </td><td> Scienze della microscopia elettronica </td><td> 15810 </td><td></td></tr><tr><td> PoliziottoSlot per Griglia </td><td> Scienze della microscopia elettronica </td><td> M2010-CR </td><td></td></tr><tr><td> Osmio diazoto 4% </td><td> Scienze della microscopia elettronica </td><td> 19140 </td><td></td></tr><tr><td> Anti-SOD3 anticorpo </td><td> Abcam </td><td> Ab21974 </td><td></td></tr><tr><td> Di capra anti-coniglio Alexa Fluor 488 </td><td> Invitrogen </td><td> A11070 </td><td></td></tr><tr><td> Resina incorporamento Spurr </td><td> Scienze della microscopia elettronica </td><td> 14300 </td><td></td></tr></tbody></table>

mouse eye enucleation for remote high-throughput phenotyping

L&#39;occhio del mouse è un importante modello genetico per lo studio traslazionale di malattia oftalmica umana. Blinding malattie negli esseri umani, come la degenerazione maculare, degenerazione dei fotorecettori, cataratta, glaucoma, retinoblastoma, e la retinopatia diabetica sono stati ricapitolati in topi transgenici. 1-5 topi transgenici e knockout La maggior parte sono stati generati da laboratori per studiare non oftalmiche malattie, ma conservazione genetica tra sistemi di organi suggerisce che molti degli stessi geni possono giocare un ruolo nello sviluppo oculare e malattia. Quindi, questi topi rappresentano una risorsa importante per la scoperta di nuove correlazioni genotipo-fenotipo negli occhi. Poiché questi topi sono sparsi in tutto il mondo, è difficile da acquisire, mantenere e fenotipo in un efficiente e conveniente modo. Così, la maggior parte delle high-throughput schermi fenotipizzazione oftalmiche sono limitate a poche sedi che richiedono in loco, esperienza oftalmico per esaminare gli occhi in topi vivi. 6-9 Un approccio alternativo sviluppato dal nostro laboratorio è un metodo per l&#39;acquisizione remota tessuto che può essere utilizzato nelle indagini grandi o piccole dimensioni di occhi di topi transgenici. Procedure standardizzate per il video a base di trasferimento di competenze chirurgiche, fissaggio del tessuto, e la spedizione di consentire il laboratorio di raccogliere gli occhi interi di animali mutanti e li inviano per fenotipizzazione molecolare e morfologico. In questo articolo video, vi presentiamo le tecniche per enucleare e trasferire sia non fissata e la perfusione del mouse occhi fissi per le analisi fenotipizzazione remoti.

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 tecnica di dissezione illustra enucleazione dell&#39;occhio mouse per il fissaggio del tessuto per eseguire fenotipizzazione in alto throughput schermi.

Mouse enucleazione degli occhi per Remote ad alta velocità Fenotipizzazione

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 Views.  University of Iowa. La tecnica di dissezione illustra enucleazione dell'occhio mouse per il fissaggio del tessuto per eseguire fenotipizzazione in alto throughput schermi.

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.

A High-throughput piattaforma automatica per lo sviluppo di linee cellulari di produzione per Therapeutics Proteine

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

Ricerca

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 modello murino del saggio Cornea Pocket Study Angiogenesi

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.

Iperinsulinemico-euglicemico Morsetti in Cosciente, Mouse sfrenato

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.

Navigazione magnetica a distanza per Preciso, posizionamento del catetere in tempo reale e ablazione in Elettrofisiologia Cardiaca Procedure

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 sequenziale ELISA per la convalida dei biomarcatori di acuta 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.

High Throughput Caratterizzazione di cellule staminali adulte Engineered per consegna di fattori terapeutici per le strategie neuroprotettive

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.

Etichettatura fluorescente colorante di eritrociti e leucociti per studiare la dinamica del flusso nella circolazione retinica del mouse

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.

Trattamento chirurgico per l'iperplasia prostatica benigna: Holmium Laser Enucleation della 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.

Puntura-indotta di Neovascularization dell'iride come un modello murino di 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.