Pesquisa para prevenir a cegueira; Bartly J. Mondino MD, Diretor do Jules Stein Eye Institute, UCLA, e Ramiro Ramirez-Solis, Jacqui Branco, e Jeanne Estabel no Instituto Sanger, Wellcome Trust Genome Campus. Esta pesquisa está em conformidade com a Declaração de ARVO para o Uso de Animais em Oftalmologia e Pesquisa Visual.

1. Dissecção Blunt: Enucleação do olho do rato em amostras não corrigidos <ol><li> Separar as pálpebras para melhorar a exposição e o acesso ao mundo posterior (globo ocular) de superfície. </li><li> Coloque uma pinça de vestir curva por trás (abaixo) o globo na órbita (órbita). O Mahajan Sharptip vestir fórceps é um instrumento personalizado com pontas para facilitar essa etapa (ver materiais e reagentes de mesa). </li><li> Feche a pinça e captar o tecido conjuntivo orbital e nervo óptico atrás do globo, tendo o cuidado para evitar apertar o globo. </li><li> Com cuidado, puxe a pinça para cima e arrancar o globo ocular da órbita. O tecido do tipo fio branco é o nervo óptico. </li><li> Colocar o olho em PBS. Utilizando uma agulha de calibre 30, fazer uma punção única ferida imediatamente posterior ao limbo, inserindo a agulha de 1-2 mm dentro do globo ocular. Isso permite que os fixadores, tais como glutaraldeído, que são de outro modo incapazes de penetrar no tecido do olho, para directamente enter o olho. Criação desse ferimento pode não ser necessário se um fixador, tal como paraformaldeído ou álcool, é capaz de difundir-se tecidos oculares de interesse. </li><li> Coloque todo o olho imediatamente em fixador para fixação de imersão. </li></ol> 2. Dissecção aguda: Enucleação do mouse fixação perfusão olho seguinte <ol><li> Use uma curva colibri forcep para segurar a pálpebra para longe do globo. </li><li> Tesoura Westcott Orient curvas dissecação paralelas ao mundo, com o objectivo para a parte traseira da órbita. </li><li> Corte o tecido fixo conjuntivo que envolve o mundo dos inferiores, mediais, laterais superiores e lateral. </li><li> Coloque a pinça curva atrás do globo, segure o tecido conjuntivo, sem pressionar no globo, e puxe para a frente a enuclear o olho. Uma vez que o tecido conjuntivo orbital é rígida de fixação da perfusão, o corte de tecidos orbitais adicional pode ser necessária para libertar completamente o globe. </li></ol> 3. Fixação e Embalagem Envio de tecidos fixas devem seguir os seus requisitos de serviços institucionais e correios, incluindo etiquetas e embalagens apropriadas. O protocolo seguinte é um exemplo de envio de uma amostra biológica não infecciosa, à temperatura ambiente. Isto requer três camadas de embalagem, mas não de classe A substância / B biológica ou rótulos de azoto líquido. <ol><li> Colocar o olho para um frasco de cintilação de 5 ml de vidro com, pelo menos, fixador de 3 ml, ou pós-fixador tampão, se a fixação for curto. Escreva o número de rastreamento na tampa do frasco e feche-o com Parafilm. </li><li> Frascos de cintilação lugar em uma amostra biológica aprovado contêiner de transporte. No nosso instituto, por exemplo, contentores aprovados requerem três camadas: um recipiente vedado no qual a amostra é colocada, uma camada intermédia absorvente, e uma camada protectora exterior. </li><li> Colocar o recipiente num pacote de plástico-bolha e, em seguida, para uma aplicaçãocontêiner percorreram laboratório junto com a documentação adequada. </li></ol> 4. Resultados representativos O exame histológico dos olhos pode ser efectuada por uma variedade de métodos incluindo hematoxilina-eosina, a detecção de expressão enzimática, a microscopia electrónica de transmissão, e imuno-histoquímica. Após fixação imersão em paraformaldeído a 4%, o tecido foi embebido em parafina e seccionados em micrótomo. No alto rendimento fenotipagem, analisamos seções aluno nervo óptico que todos os tipos de amostras de tecido do olho (Figura 1). Secções de tecido foram também examinados para expressão de lacZ (Figura 2), microscopia electrónica de transmissão de organelos celulares (Figura 3), e a expressão de moléculas específicas com anticorpos (Figura 4). No nosso método de rastreio fenótipo, não foi importante para manter a orientação do olho em relação à nasal (canto medial) e temporal (Lateral) canto lados. Existem pelo menos duas opções, se a orientação do olho é necessário. Após enucleação, temos aplicado cautério luz na córnea temporais na posição das três horas com um cautério handheld. A lesão é óbvio na histologia e não ficar perdido durante o processamento, como tintas histológicas. A desvantagem é que os danos cautério a córnea, o que pode ser um tecido crucial para exame. A outra opção requer um anatomista qualificados que possam identificar as inserções dos músculos extraoculares. Sob um microscópio estereoscópico, a identificação da inserção do músculo oblíquo superior e inferior marca o aspecto inferior e superior e do mundo. 3 Com qualquer método de orientação, é importante acompanhar a amostra como um olho direito ou esquerdo. Juntos, isso vai permitir que a orientação exata mundo antes do corte. <img src="/files/ftp_upload/3184/3184fig1.jpg" alt="Figura 1" /> Figura 1. Imagens do mouse olho histológicas afte<html> r aquisição remota. olhos de camundongos foram enucleados por dissecção aguda após a fixação de perfusão com paraformaldeído 4%. A. Aluno-secção do nervo óptico corados com Hematoxilina e Eosina mostra preservação de todas as subestruturas de tecido. ON, nervo óptico. (Barra de escala verde = 500 microns) B. Uma visão maior ampliação da retina normal mostra sua estrutura laminar: RGC, as células ganglionares da retina; IPL, camada plexiforme interna; INL, camada interna nuclear; OPL, camada plexiforme externa; ONL, exterior camada nuclear (células fotorreceptoras) (seta); IS, segmentos fotorreceptoras internas (cabeça de seta); OS, fotorreceptoras segmentos exteriores; RPE, epitélio pigmentar da retina, c, coróide. (Verde barra de escala = 50 microns) C. Em comparação com a retina normal, este espécime é mais fina devido à degeneração dos fotorreceptores. O ONL, É e OS estão completamente ausentes (seta). (Verde barra de escala = 50 microns) &quot;Alt =&quot; Figura 2 &quot;/&gt; Figura 2. Expressão de lacZ em murganhos transgénicos. Os olhos foram enucleados por dissecção romba e imersos em 1 mg / ml de solução de X-gal (5 mM K3Fe (CN) 6, 5 mM de K4Fe (CN) 6, 2 mM de MgCl2, 0,02% de NP40 , e desoxicolato de sódio a 0,1%) durante a noite a 37 ° C. Os olhos foram fixados em glutaraldeído a 2% formaldehyde/0.2% durante 30 minutos e seccionados em micrótomo. X-gal do produto (azul) etiquetas: A. epitélio ciliar, B. epitélio da córnea, e C. a camada de células ganglionares da retina (RGC) na retina. INL, camada nuclear interna; ONL, camada nuclear externa. <img src="/files/ftp_upload/3184/3184fig3.jpg" alt="Figura 3" /> Figura 3. Microscopia eletrônica de transmissão do epitélio pigmentar da retina (EPR). Após dissecção romba e criação de um ferimento, os olhos estavam fixos imersão em paraformaldeído 2,5% / 2,5% glutaraldeído em 0,1 M de tampão fosfato de sódio. Olhos eram secondarily fixado em textroxide de ósmio a 1%, gradualmente desidratados e embebidos em resina Spurr a. Tecido foi seccionado em 90 nm e colocados em grades de cobre cobertas de caça-níqueis e Formvar fotografada usando um microscópio eletrônico de transmissão. A micrografia mostra, os segmentos exteriores fotorreceptoras da retina neurosensorial, melanossomas (M) no interior do epitélio pigmentado da retina e membrana de Bruch. <img src="/files/ftp_upload/3184/3184fig4.jpg" alt="Figura 4" /> Figura 4. Imunohistoquímica de superóxido dismutase-3 (verde) no vítreo do rato. Olhos foram enucleados por dissecção romba e foi submetido a fixação de imersão em paraformaldeído a 4%. Eles foram incluídos em parafina e seccionados usando um micrótomo. As secções de tecido foram incubadas com anti-soro policlonal de coelho diluído 1:50 SOD3. SOD3 expressão foi detectada utilizando um anticorpo de cabra anti-coelho IgG (H + L)-Alexa Fluor 488 anticorpo secundário conjugado. Rotulagem de SOD3 é mostrado na 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>

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Camundongos transgênicos mais existem em laboratórios que não examinam os olhos. Nossa técnica vídeo mostra um método simples e padronizada para a transferência remota de habilidade cirúrgica para otimizar a aquisição de tecidos a partir de laboratórios com pouca experiência com os olhos. Esta técnica de vídeo ajuda a superar um grande armadilha em alto rendimento fenotipagem, que é a utilização de um número limitado de locais especializados devido a métodos não normalizados de recolha de tecidos e q...

<table border="1"><tbody><tr><th> Nome </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> Pinça curva Vestir </td><td> Storz Ophthalmics </td><td> E1408 </td><td></td></tr><tr><td> Mahajan Sharptip vestir fórceps </td><td> Storz Ophthalmics </td><td> E1406 (REF SP7-64520) </td><td></td></tr><tr><td> Curvas Tesoura Westcott </td><td> Storz Ophthalmics </td><td> E3321 WH </td><td></td></tr><tr><td> 15 ° BD Beaver Microsurgical Lâmina </td><td> Becton-Dickinson </td><td> 374881 </td><td></td></tr><tr><td> 0,22 Fine-Castroviejo Fórceps suturando </td><td> Storz Ophthalmics </td><td> E1805 </td><td></td></tr><tr><td> 0,12 Colibri fórceps </td><td> Storz Ophthalmics </td><td> 2/132 </td><td></td></tr><tr><td> Calibre 30 agulha </td><td> Becton-Dickinson </td><td> 305128 </td><td></td></tr><tr><td> Biohazard Mailer </td><td> Pescador </td><td> 03-523-4 </td><td></td></tr><tr><td> Parafilm </td><td> Pescador </td><td> 13-374-10 </td><td></td></tr><tr><td> Vidro de cintilação frascos </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> Paraformaldeído 16% </td><td> Electron Microscopy Sciences </td><td> 15700 </td><td></td></tr><tr><td> 2,5% paraformaldeído Glutaraldeído / 2,5% em 0,1 M de tampão fosfato de sódio </td><td> Electron Microscopy Sciences </td><td> 15700 e 16300 </td><td> Misturados em laboratório </td></tr><tr><td> O glutaraldeído a 50% </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> PolicialGrade por slot </td><td> Electron Microscopy Sciences </td><td> M2010-CR </td><td></td></tr><tr><td> Tetróxido de ósmio a 4% </td><td> Electron Microscopy Sciences </td><td> 19140 </td><td></td></tr><tr><td> Anti-anticorpo SOD3 </td><td> Abcam </td><td> Ab21974 </td><td></td></tr><tr><td> Cabra anti-coelho Alexa Fluor 488 </td><td> Invitrogen </td><td> A11070 </td><td></td></tr><tr><td> Resina Spurr incorporação de </td><td> Electron Microscopy Sciences </td><td> 14300 </td><td></td></tr></tbody></table>

mouse eye enucleation for remote high-throughput phenotyping

O olho do rato é um modelo genético importante para o estudo da doença de translação oftálmica humana. Doenças que causam cegueira em humanos, tais como a degeneração macular, a degeneração de fotorreceptores, cataratas, glaucoma, retinoblastoma, e retinopatia diabética foram recapitulado em ratinhos transgénicos. 1-5 ratinhos transgénicos e knockout maioria foram gerados por laboratórios para estudar doenças não oftálmicas, mas conservação genética entre os sistemas de órgãos sugere que muitos dos mesmos genes podem também desempenhar um papel no desenvolvimento ocular e doenças. Assim, estes ratos representam um recurso importante para a descoberta de novas correlações genótipo-fenótipo no olho. Porque esses ratos estão espalhados por todo o mundo, é difícil de adquirir, manter e fenótipo-los de forma eficiente, boa relação custo-benefício. Assim, a maioria de alto rendimento telas fenotipagem oftálmicas são restritos a alguns locais que necessitam no local, a perícia oftalmológica para examinar os olhos em camundongos vivos. 6-9 Uma abordagem alternativa desenvolvida pelo nosso laboratório é um método para o controle remoto de aquisição tecido que pode ser utilizado em estudos de grandes dimensões ou de pequena escala de olhos de ratinhos transgénicos. Procedimentos padronizados para vídeo baseado em transferência de habilidade cirúrgica, fixação do tecido, e transporte permitem que qualquer laboratório para coletar olhos inteiros de animais mutantes e enviá-las para fenotipagem molecular e morfológica. Neste artigo de vídeo, nós apresentamos técnicas para enuclear e transferir ambos não corrigidos e perfusão olhos de rato fixos para análises fenotípicas remotos.

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

Mouse Eye Enucleation for Remote High-throughput Phenotyping

A técnica de dissecção ilustra enucleação do olho do rato para fixação do tecido para realizar fenotipagem em alto rendimento telas.

Enucleação Rato Olho para fenotipagem de alta capacidade remota

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 visualizações.  University of Iowa. A técnica de dissecção ilustra enucleação do olho do rato para fixação do tecido para realizar fenotipagem em alto rendimento telas.

Vídeo: 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.

Uma plataforma de alta capacidade automatizada para o Desenvolvimento de linhagens celulares para a fabricação de proteínas Therapeutics

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

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.

Um modelo do rato do Ensaio de bolso Córnea para o Estudo da angiogênese

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.

Hiperinsulinêmico euglicêmico-Clamps em Consciente, Ratos Unrestrained

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.

Navegação Magnetic remoto para Exato, o posicionamento do cateter em tempo real e Ablação em Eletrofisiologia Cardíaca Procedimentos

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 ELISA sequencial para a validação dos biomarcadores da doença enxerto-versus-hospedeiro aguda

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 caracterização de células-tronco adultas modificadas para liberação de fatores terapêuticos como estratégia de neuroproteção

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.

Etiquetagem de corantes fluorescentes de eritrócitos e leucócitos para estudar a dinâmica do fluxo na circulação da retina do 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.

Tratamento cirúrgico para a hiperplasia prostática benigna: Holmium Laser Enucleação da próstata (HoLEP).

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

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

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

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

Induzida por punção neovascularização de Iris como um modelo do rato da 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.