FIPE / HCPA - Fundo de Incentivo à Pesquisa e Eventos CAPES - <a href="http://www.capes.gov.br/">Coordenação de Aperfeiçoamento de Pessoal de Nível Superior</a>

1. Texto Protocolo <ol><li> Antes de recolha de sangue do peixe-zebra, é necessário para preparar a água anestesiar. Verter ~ 200 ml de água de aquário em um recipiente com uma capacidade de 500 ml. Adicionar ~ 200 g de pedaços de gelo. A temperatura deve ser de cerca de 4 ° C. À medida que os pedaços de gelo derreter, será necessário adicionar mais fichas de gelo para manter uma temperatura constante próximo de 4 ° C. </li><li> Quando a água anestesiar está pronto, preparar os materiais necessários para a coleta de sangue. Coloque uma ponta de retenção baixa em uma pipeta P20 e deixar o pipetador onde ele pode ser facilmente acessado. Não permita que a ponta da pipeta entrar em contato com quaisquer fontes de contaminação. </li><li> Cobrir uma placa de Petri com um pedaço de gaze seca. Uma lâmina de aço e outro pedaço de gaze deve ser colocado em um local facilmente acessível. </li><li> Uma centrífuga adaptado para tubos de plástico será necessário. </li><li> Quando os referidos materiais são preparados, capturar o peixe-zebra primeiro a ser sagacidade anestesiadosha rede de pesca e solte-o na água que foi preparado para a anestesia. Zebrafish requerem 3-6 s em água gelada para ser anestesiados, dependendo do peixe. Mantenha o peixe na água fria até que já não responde a estímulos externos. </li><li> Usando a rede de pesca, coloque o peixe anestesiados sobre um pedaço de gaze preparada, deixando a cauda para fora da gaze. Dobre a gaze sobre a cabeça do peixe e do corpo deixando de fora apenas a cauda. Coloque o peixe coberto com a gaze na placa de Petri. </li><li> Use a lâmina de aço para fazer uma incisão diagonal apenas entre a barbatana anal e da barbatana caudal. O sangue vai começar a sair. Neste ponto, é necessário trabalhar rapidamente. </li><li> Gentilmente aspirar o sangue que sai com o pipetador P20 (pré-carregado com uma ponta de retenção baixa). A quantidade de sangue que pode ser recolhida depende do tamanho do peixe e em que medida que era anestesiados correctamente. É geralmente varia de 5 a 20 uL. Quando o sangue deixa de coming para fora, suavemente transferir o sangue para um tubo de aspiração. </li><li> Para evitar hemólise, é crítico que o tubo com sangue em que ser manuseados com muito cuidado, sem quaisquer movimentos drásticas até que seja colocada na centrífuga. </li><li> Para evitar hemólise, é também importante que a amostra de sangue ser fixada na centrífuga no prazo de 10 minutos de recolha de sangue. </li><li> Se necessário, é possível combinar o sangue a partir de mais de um animal, tornando uma piscina. Amostras combinadas irá funcionar bem, desde que o atraso entre a colheita de sangue a partir do primeiro peixe e centrifugação não exceda 10 minutos. </li><li> Quando a recolha de sangue é feito, centrifugar o sangue durante 10 minutos a 0,5 g (Eppendorf Centrífuga 5415D). </li><li> Após centrifugação, o soro é na camada de topo do tubo. Com uma pipeta, aspirar o soro, certificando-se para obter apenas o soro, mantendo ambas as camadas bem dividido e estável. </li><li> Transferir o soro para um microtubo novo e seestá pronto para ser utilizado em análises bioquímicas. O soro pode ser armazenado em gelo enquanto se esperar para iniciar as análises bioquímicas. </li><li> Se o soro não irá ser imediatamente utilizado, ele pode ser congelada a -18 ° C até cerca de 3 meses. </li></ol> 2. Os resultados representativos Foi possível recolher 5-20 ul de sangue total a partir de cada peixe que representa o sangue até 4 vezes mais do que as técnicas anteriormente descrito (Tabela 2). A análise bioquímica do colesterol total, HDL-colesterol, LDL-colesterol e triglicérides foram realizadas após a coleta de sangue, utilizando esta técnica. Dois grupos de ambos os sexos peixes foram mantidos em jejum durante 24 horas antes da recolha de sangue para evitar a interferência ingestão de alimentos. As análises foram feitas com pequena escala testes colorimétricos (Labtest Diagnóstica SA, Brasil) para análises de colesterol total e triglicérides, 3 uL de soro foram utilizados. Para o LDL-colesterol e HDL-colesterolanálise, 4 uL e 10 uL de soro foram utilizados, respectivamente. Estas análises foram realizadas em amostras combinadas de 10 peixes-zebra por amostra. Os níveis séricos lipídicos foram comparados entre os peixes que acessou seus próprios ovos e aquelas que, em um aquário de fundo coberto, não têm acesso a seus próprios ovos por um período experimental de 2 semanas. Análise de soro mostraram que os níveis séricos de colesterol total (com ovos de 362 ± 42 mg / dL e sem ovos de 357 ± 13 mg / dL), HDL-colesterol (com ovos de 91,22 ± 1,79 mg / dL e sem ovos de 72,14 ± 2,89 mg / dL) e LDL-colesterol (com ovos de 55,68 ± 10,88 mg / dL e sem ovos de 44,18 ± 9,84 mg / dL) não diferiram significativamente entre os grupos. No entanto, os níveis de triglicéridos foram significativamente menores no grupo experimental (sem ovos 292 ± 64 mg / dL) do que no grupo de controlo (com ovos 457 ± 25 mg / dL, P = 0,03). <table border="1" ><tbody><tr><td> &amp; Nbsp; </td><td> Com acesso aos ovos </td><td> Sem acesso aos ovos </td></tr><tr><td> Colesterol total (mg / dL) </td><td> 362,82 ± 73,11 </td><td> 357,69 ± 23,08 </td></tr><tr><td> LDL - colesterol (mg / dL) </td><td> 55,69 ± 18,84 </td><td> 44,19 ± 17,05 </td></tr><tr><td> HDL - colesterol (mg / dL) </td><td> 91,23 ± 3,11 </td><td> 72,14 ± 5,01 </td></tr><tr><td> Triglicérides (mg / dL) </td><td> 457,64 ± 43,78 * </td><td> 292,36 ± 111,28 </td></tr></tbody></table> Tabela 1. Colesterol e triglicérides níveis séricos para ambos os grupos estudados (com acesso aos ovos e sem acesso aos ovos), expresso em média ± desvio padrão. * Estatisticamente significativo (P = 0,03). Teste t de Student. <table border="1"><tbody><tr><td> Aut.<html> hors </td><td> Local de incisão </td><td> Método de colheita </td><td> Anestesia </td><td> Quantidade de sangue coletado </td></tr><tr><td> Jagadeeswaran et. ai., 1999 Murtha et al., 2003 </td><td> Posterior dissecção Micro para nadadeira dorsal </td><td> Micropipeta </td><td> Não é mencionado MS222 3% em água fria </td><td> 1 uL um 5 5 um uL 10 </td></tr><tr><td> Eames et al., 2010 </td><td> Decapitação </td><td> Micro capilares tubo </td><td> MS222 0,02% 28 ° C de água </td><td> 5 um uL 10 </td></tr><tr><td> Presente Estudo </td><td> Incisão entre barbatana anal e nadadeira caudal </td><td> Micropipeta e dicas de retenção baixa </td><td> Fichas de água e gelo </td><td> 5 um uL 20 </td></tr></tbody></table>e_content &quot;&gt; Análise Comparativa Tabela 2. entre as técnicas de sangue previamente descritos de coleta com a descrita no presente estudo.

<ol>
	<li>Schneider, A. C. R., dos Santo, J. L., Porawski, M., Schaefer, P. G., Maurer, R. L., Matte, U., da Silveira, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementa&#231;&#227;o+de+um+novo+modelo+de+experimenta&#231;&#227;o+animal+Zebrafish.">Implementa&#231;&#227;o de um novo modelo de experimenta&#231;&#227;o animal Zebrafish.</a> Rev. HCPA. 29, 100-103 (2009).</li><li>Briggs, 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=The+zebrafish:+a+new+model+organism+for+integrative+physiology.">The zebrafish: a new model organism for integrative physiology.</a> Am. J. Physiol. Regulatory Integrative Comp. Physiol. 282, 3-9 (2002).</li><li>Menke, A. L., Sptsbergen, J. M., Wolterbeek, A. P. M., Woutersen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+anatomy+and+histology+of+adult+Zebrafish.">Normal anatomy and histology of adult Zebrafish.</a> Toxicologic Pathology. 000, 1-16 (2011).</li><li>Murtha, J. M., Qi, W., Keller, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematologic+and+serum+biochemical+values+for+Zebrafish.">Hematologic and serum biochemical values for Zebrafish.</a> Comp. Med. 53, 37-41 (2003).</li><li>Jagadeeswaran, P., Sheehan, J. P., Craig, F. E., Troyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+zebrafish+thrombocytes.">Identification and characterization of zebrafish thrombocytes.</a> Br. J. Haematol. 107, 731-738 (1999).</li><li>Jagadeeswaran, P., Sheehan, 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=Analysis+of+blood+coagulation+in+the+zebrafish.">Analysis of blood coagulation in the zebrafish.</a> Blood Cells Mol. Dis. 25, 239-249 (1999).</li><li>Eames, S. C., Philipson, L. H., Prince, V. E., Kinkel, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sugar+measurement+in+zebrafish+reveals+dynamics+of+glucose+homeostasis.">Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis.</a> Zebrafish. 7, 205-213 (2010).</li><li>Kilpatrick, E. S., Rumley, A. G., Rumley, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+haemolysis+on+blood+glucose+meter+measurement.">The effect of haemolysis on blood glucose meter measurement.</a> Diabet. Med. 12, 341-343 (1995).</li><li>Gupta, T., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Organs+from+the+Adult+Zebrafish.">Dissection of Organs from the Adult Zebrafish.</a> J. Vis. Exp. (37), e1717 (2010).</li><li>Kinkel, M. D., Eames, S. C., Philipson, L. H., Prince, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraperitoneal+Injection+into+Adult+Zebrafish.">Intraperitoneal Injection into Adult Zebrafish.</a> J. Vis. Exp. (42), e2126 (2010).</li><li>Pugach, E. K., Li, P., White, R., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+Injection+in+Adult+Zebrafish.">Retro-orbital Injection in Adult Zebrafish.</a> J. Vis. Exp. (34), e1645 (2009).</li></ol>

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Este artigo apresenta uma técnica simples que permite que mais sangue e análise de soro em experimentos de zebrafish. Esta técnica tem o potencial de contribuir para estudos futuros zebrafish hematológicas que exigem dados de parâmetros sanguíneos. Deve também permitir para aplicações maiores do peixe-zebra como um modelo experimental. Esta técnica não exige habilidades especiais ou de aplicação de uma técnica precisa. Além disso, permite-se o dobro da quantidade de sangue a s...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Pontas de baixa retenção </td><td> Applied Biosystems </td><td> 022493020 </td><td></td></tr><tr><td> Eppendorf Centrífuga 5415D </td><td> Eppendorf </td><td> Descontinuado </td><td></td></tr></tbody></table>

blood collection for biochemical analysis in adult zebrafish

O peixe-zebra tem sido utilizado como um modelo animal para estudos de várias doenças humanas. Ela pode servir como uma plataforma poderosa pré-clínico para estudos de acontecimentos moleculares e estratégias terapêuticas, bem como para avaliar os mecanismos fisiológicos de cerca de 1 patologias. Há relativamente poucas publicações relacionadas à fisiologia do peixe-zebra adulto de órgãos e sistemas 2, que podem levar os pesquisadores a concluir que as técnicas básicas necessárias para permitir a exploração de sistemas de zebrafish faltam 3. Hematológicas valores bioquímicos do peixe-zebra foram relatados primeiramente em 2003 por Murtha e 4 colegas que empregou uma técnica de coleta de sangue primeiramente descrita por Jagadeeswaran e colegas em 1999. Resumidamente, o sangue foi recolhido através de uma ponta micropipeta através de uma incisão lateral, a aproximadamente 0,3 cm de comprimento, na região da aorta dorsal 5. Devido às dimensões hora envolvidos, estaé uma técnica de alta precisão requerendo um praticante altamente qualificado. A mesma técnica foi utilizada pelo mesmo grupo em outra publicação no mesmo ano 6. Em 2010, Eames e colegas avaliaram os níveis de glucose no sangue em zebrafish 7. Ganharam acesso ao sangue através da realização de decapitações com a tesoura e depois inserir um tubo de colheita heparinizado microcapilar na articulação peitoral. Eles mencionam dificuldades com hemólise que foram resolvidos com uma temperatura de armazenamento adequado com base no trabalho Kilpatrick et al. 8. Ao tentar usar a técnica Jagadeeswaran no nosso laboratório, verificou-se que era difícil de fazer a incisão no lugar precisamente a direita, como para não permitir que uma quantidade significativa de sangue a ser perdido antes da recolha pode ser iniciado. Recentemente, Gupta et al. 9 descreveu como dissecar órgãos zebrafish adulto, nozinho et al 10. Descrito como executar intraperitoninjeções EAL e Pugach et al. 11 descrito como realizar retro-orbital injeções. No entanto, mais estudos são necessários para explorar mais técnicas básicas para a investigação em zebrafish. O pequeno tamanho do zebrafish apresenta desafios para os pesquisadores que o utilizam como modelo experimental. Além disso, dado este pequenez de escala, é importante que as técnicas simples são desenvolvidos para permitir aos investigadores para explorar as vantagens do modelo de peixe-zebra.

Watch this Scientific Journal Video about Blood Collection for Biochemical Analysis in Adult Zebrafish at JoVE.com

Blood Collection for Biochemical Analysis in Adult Zebrafish

Este artigo apresenta uma técnica de coleta de sangue da aorta dorsal do peixe zebra. Ele também fornece instruções para a obtenção de soro para uso em análises bioquímicas, tais como testes para determinação dos níveis de colesterol e triglicérides.

Coleta de sangue para análise bioquímica em Zebrafish Adulto

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

blood-collection-for-biochemical-analysis-in-adult-zebrafish

Research

JoVE Journal

Biology

36.2K visualizações.  Centro de Pesquisa Experimental Laboratório de Hepatologia e Gastroenterologia Experimental. Este artigo apresenta uma técnica de coleta de sangue da aorta dorsal do peixe zebra. Ele também fornece instruções para a obtenção de soro para uso em análises bioquímicas, tais como testes para determinação dos níveis de colesterol e triglicérides.

Vídeo: Blood Collection for Biochemical Analysis in Adult Zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Transplante de Medula Renal inteiro em Zebrafish Adulto

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Biologia

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebrafish Injection Ventrículo Cerebral

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

Retro-orbital de injeção no Zebrafish Adulto

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Em Eletroporação vivo de Morpholinos na Retina Zebrafish Adulto

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Na eletroporação in vivo de Morpholinos no Adulto Regeneração Zebrafish Cauda Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging Zebrafish Adulto

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Um ensaio para a lateral de Regeneração de Linha em Zebrafish Adulto

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

Análise de Composição Nephron e função no rim adulto Zebrafish

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Colheita de sangue para testes repetidos de sangue em Zebrafish Adulto

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).