Name: repeated blood collection for blood tests in adult zebrafish
Uploaded: 2015-08-30T14:00:00.000+00:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Repeated Blood Collection for Blood Tests in Adult Zebrafish at JoVE.com

This work was supported by JSPS KAKENHI Grant Number 25860294 and 25590073. We would like to thank Ms. Yui Namie for the hand-drawn illustration, and Mr. Koshi Kataoka and Ms. Sayuri Ichikawa for assistance with the zebrafish maintenance.

Todos os procedimentos com animais foram aprovados pelo Comitê de Ética da Universidade de Mie o, e foram realizados de acordo com a regulamentação bem-estar animal japonês &#39;Ato de Bem-Estar e Gestão de Animais&#39; (Ministério do Meio Ambiente do Japão) e tenha cumprido as normas internacionais. 1. Preparação da agulha NOTA: Todos os experimentos foram realizados sob anestesia, e foram feitos todos os esforços para minimizar o sofrimento. Para a eutanásia, os peixes foram imersos num banho de gelo-água (5 partes de gelo / 1 parte de água em ≤4 ° C) durante ≥20 min. <ol><li> Prepare as agulhas microcapilares vidro puxando um capilar de vidro de 1.0 mm de diâmetro exterior, com um puxador de agulha (Figura 1A). </li><li> Corte as pontas das agulhas obliquamente utilizando uma tesoura fina (Figura 1B). O diâmetro da ponta ideal deve ser de aproximadamente 100 - 200 um (Figura 1C). Se o diamete pontar é demasiado estreito, o sangue não vai entrar na agulha. </li><li> Dissolve-se heparina em solução salina a uma concentração de 5 mg / ml. </li><li> Coloque uma agulha pré-cortados no final nosepiece de um conjunto do tubo de aspirador e mantenha o bocal na boca, ou se conectar a agulha para um dispensador de lâmpada. Mergulhe a ponta da agulha para a solução de heparina, e heparinizar a agulha por sucção e soprando através da solução (Figura 1D e 1E). Nota: O conjunto do tubo de aspirador foi usado para a criopreservação de esperma de peixe-zebra 16. Tubo de borracha longa pode parar qualquer sangue voando na boca. A definição de um filtro no meio do caminho do tubo pode evitar os perigos. </li><li> Armazenar as agulhas heparinizados em um prato de Petri de 10 cm e seco ao ar durante pelo menos 1 h. Um grande número de agulhas pode ser preparado antecipadamente. </li></ol> 2. Anestesia <ol><li> Preparar a solução de anestésico numa pequena caixa de plástico através da mistura de 200 ml de água de peixe com 100 ul de 2-fenoxietanol (2-PE). A concentração final dos anestésicos é de 500 ppm. CUIDADO! 2-PE tem um efeito rápido. </li><li> Remover número desejado de peixe (estirpe AB) a partir do sistema de circulação. </li><li> Use uma rede para transferir o peixe para os anestésicos para 1-2 min (Figura 1F). Observar os peixes nadar gradualmente, espalhar as barbatanas peitorais horizontalmente, suspiro, e tem movimentos rápidos opérculo dentro de 1 min. </li><li> À medida que o tempo passa, observar o peixe estava deitado no fundo do caso e finalmente parar de nadar. O plano cirúrgico de anestesia chegará quando o peixe pára para respirar e os movimentos são lentos opérculo. Neste ponto, o peixe está pronto para a coleta de sangue. </li><li> Usando uma escumadeira levantar o peixe anestesiados a partir do 2-PE e suavemente coloque-o sobre uma toalha de papel embebido com os anestésicos (Figura 1G). Cubra a cabeça do peixe com papel de tecido macio também embebido com solução de 2-PE para prevenir o ressecamento dos olhos e usar outro papel de tecido macio e seco para gentilmentesecar a superfície do corpo. </li></ol> 3. Recolha de Sangue <ol><li> Coloque uma agulha heparinizado no final nosepiece do conjunto do tubo de aspiração (ou o bulbo dispensador) e segure a extremidade do bocal do conjunto do tubo de aspiração na boca. </li><li> Segure a extremidade nosepiece ea agulha juntos, e retire cuidadosamente as escalas que interferem com a ponta da agulha. Inserir a agulha em um 30 - ângulo de 45 ° para o local de coleta de sangue. Evitar a perfuração do tracto gastrointestinal (Figura 1H). Em caso de utilização de bulbo dispenser, pressione a lâmpada com o polegar eo dedo médio, e bloquear o buraco na ponta do bulbo com primeiro dedo, em seguida, inserir a agulha conforme descrito acima. Nota: O local para a recolha de sangue é ao longo do eixo do corpo e do posterior para o ânus na região da aorta dorsal. A aorta dorsal (DA) e a veia cardinal posterior (PCV) são apenas ventral para a coluna vertebral (Figura 2). </li><li> Começar a sugar obocal extremidade do conjunto do tubo de aspiração, quando a agulha é sentida tocar na coluna vertebral. Se o sangue não subir, mova a ponta da agulha sutilmente com a mão para estimular o fluxo sanguíneo. Note que quando o sangue está a aumentar na agulha, pare imediatamente a tremer e sugar delicadamente (Figura 1I). Em caso de utilização de bulbo dispensador, libertar a pressão do bolbo para aspirar o sangue. </li><li> Observar o sangue irá subir lentamente para dentro da agulha de uma maneira pulsátil, sem aspiração, o qual é provável, porque a pressão do sangue arterial. Assim, não é necessário para sugar se a agulha penetra correctamente a artéria. </li><li> Pare sucção depois o volume adequado de sangue é recolhido. Retire a agulha do peixe e pressione o local da punção usando lenço de papel macio para evitar qualquer sangramento. Observe a parada do sangramento após cerca de 10-20 segundos de pressão do dedo (Figura 1J). </li><li> Após o sangramento é interrompido, transferir imediatamente o peixe de volta a um cleum em água quente (~ 28 ° C) do tanque. Ajude o peixe para recuperar suavemente por água de roda para as brânquias até que ele começa a nadar. Nota: Mantenha-se 5 postsampling peixe em um tanque de 2 L e conecte o tanque para o sistema de circulação para oxigenar o peixe. A adição de antibióticos para o peixe de água não é necessária. Manter o peixe com caixa normal e alimentação. </li><li> Expulsar o sangue a partir da agulha para uma área limpa de uma folha de parafilme (Figura 1K). </li><li> Medir glicose no sangue usando qualquer glicosímetro portátil comercial (Figura 1D). Nota: O glucometer usa um eléctrodo de flavina-adenina-dinucleótido-desidrogenase de glucose e exige um volume de amostra de 0,6 ul. <ol><li> Insira uma tira de teste completamente no medidor e directamente tocar a gota de sangue. Observe a coleta de sangue na tira de teste automaticamente e obter a glicose no sangue resultado 5 segundos na área de exibição. Anote o resultado e descartar a tira de teste. Use umnova tira de teste para cada medição. </li></ol></li><li> (passo facultativo) Leve quantidades precisas de sangue por uma pipetagem e transferir o sangue para um tubo de microcentrífuga para análises posteriores (hemoglobina, triacilglicerol (TG), colesterol total, etc.). Se necessário, diluir o sangue total de um peixe com solução salina. Centrifugar as amostras de sangue durante 3 min a 680 xg à temperatura ambiente e recolher o plasma. Transfira o plasma para um novo tubo. Neste ponto, que está pronta para ser utilizada em análises bioquímicas. </li></ol>

<ol>
	<li>Lieschke, G., Currie, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat Rev Genet. 8 (5), 353-367 (2007).</li><li>Penberthy, W. T., Shafizadeh, E., Lin, S. <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+as+a+model+for+human+disease.">The zebrafish as a model for human disease.</a> Front Biosci. 7, d1439-d1453 (2002).</li><li>Stachura, D. L., Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+dissection+of+zebrafish+hematopoiesis.">Cellular dissection of zebrafish hematopoiesis.</a> Methods Cell Biol. 101, 75-110 (2011).</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 (3-4), 239-249 (1999).</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 (4), 731-738 (1999).</li><li>Lieschke, G. J., Oates, A. C., Crowhurst, M. O., Ward, A. C., Layton, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+and+functional+characterization+of+granulocytes+and+macrophages+in+embryonic+and+adult.">Morphologic and functional characterization of granulocytes and macrophages in embryonic and adult.</a> Blood. 98 (10), 3087-3096 (2001).</li><li>Iwanami, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+for+understanding+the+evolution+of+the+vertebrate+immune+system+and+human+primary+immunodeficiency.">Zebrafish as a model for understanding the evolution of the vertebrate immune system and human primary immunodeficiency.</a> Exp Hematol. 42 (8), 697-706 (2014).</li><li>Babin, P. J., Goizet, C., Raldua, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+of+human+motor+neuron+diseases:+advantages+and+limitations.">Zebrafish models of human motor neuron diseases: advantages and limitations.</a> Prog Neurobiol. 118, 36-58 (2014).</li><li>Seth, A., Stemple, D. L., Barroso, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+use+of+zebrafish+to+model+metabolic+disease.">The emerging use of zebrafish to model metabolic disease.</a> Dis Mod Mech. 6 (5), 1080-1088 (2013).</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 (2), 205-213 (2010).</li><li>Moss, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+the+pancreas+in+adult+zebrafish.">Regeneration of the pancreas in adult zebrafish.</a> Diabetes. 58 (8), 1844-1851 (2009).</li><li>Oka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diet-induced+obesity+in+zebrafish+shares+common+pathophysiological+pathways+with+mammalian+obesity.">Diet-induced obesity in zebrafish shares common pathophysiological pathways with mammalian obesity.</a> BMC Physiol. 10 (21), (2010).</li><li>Chu, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+Akt1+enhances+adipogenesis+and+leads+to+lipoma+formation+in+zebrafish.">Overexpression of Akt1 enhances adipogenesis and leads to lipoma formation in zebrafish.</a> PLoS One. 7 (5), e36474 (2012).</li><li>Velasco-Santamar&#237;a, Y. M., Korsgaard, B., Madsen, S. S., Bjerregaard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bezafibrate,+a+lipid-lowering+pharmaceutical,+as+a+potential+endocrine+disruptor+in+male+zebrafish+(Danio+rerio).">Bezafibrate, a lipid-lowering pharmaceutical, as a potential endocrine disruptor in male zebrafish (Danio rerio).</a> Aquat Toxicol. 105 (1-2), 107-118 (2011).</li><li>Zang, L., Shimada, Y., Nishimura, Y., Tanaka, T., Nishimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+reliable+method+for+repeated+blood+collection+from+aquarium+fish.">A novel, reliable method for repeated blood collection from aquarium fish.</a> Zebrafish. 10 (3), 425-432 (2013).</li><li>Carmichael, C., Westerfield, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+and+in+vitro+fertilization+at+the+zebrafish+international+resource+center.">Cryopreservation and in vitro fertilization at the zebrafish international resource center.</a> Methods Mol Biol. 546, 45-65 (2009).</li><li>Thorson, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+partitioning+of+body+water+in+Osteichthyes:+phylogenetic+and+ecological+implications+in+aquatic+vertebrates.">The partitioning of body water in Osteichthyes: phylogenetic and ecological implications in aquatic vertebrates.</a> Biol Bull-US. 120, 238-254 (1961).</li><li>Conte, F. P., Wagner, H. H., Harris, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+blood+volume+in+the+fish+(Salmo+gairdneri+gairdneri).">Measurement of blood volume in the fish (Salmo gairdneri gairdneri).</a> Am J Physiol. 205, 533-540 (1963).</li><li>Diehl, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+good+practice+guide+to+the+administration+of+substances+and+removal+of+blood,+including+routes+and+volumes.">A good practice guide to the administration of substances and removal of blood, including routes and volumes.</a> J Appl Toxicol. 21 (1), 15-23 (2001).</li><li>Nahas, K., Provost, J. -. P., Baneux, P. H., Rabemampianina, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+blood+removal+via+the+sublingual+vein+on+haematological+and+clinical+parameters+in+Sprague-Dawley+rats.">Effects of acute blood removal via the sublingual vein on haematological and clinical parameters in Sprague-Dawley rats.</a> Lab Anim. 34 (4), 362-371 (2000).</li><li>Curado, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+targeted+cell+ablation+in+zebrafish:+a+new+tool+for+regeneration+studies.">Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies.</a> DevDyn. 236 (4), 1025-1035 (2007).</li><li>Andersson, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine+signaling+promotes+regeneration+of+pancreatic+beta+cells+in+vivo.">Adenosine signaling promotes regeneration of pancreatic beta cells in vivo.</a> Cell Metab. 15 (6), 885-894 (2012).</li><li>Hiramitsu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eriocitrin+ameliorates+diet-induced+hepatic+steatosis+with+activation+of+mitochondrial+biogenesis.">Eriocitrin ameliorates diet-induced hepatic steatosis with activation of mitochondrial biogenesis.</a> Sci Rep-UK. 4, 3708 (2014).</li><li>Zang, L., Shimada, Y., Kawajiri, J., Tanaka, T., Nishimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Yuzu+(Citrus+junos+Siebold+ex+Tanaka)+peel+on+the+diet-induced+obesity+in+a+zebrafish+model.">Effects of Yuzu (Citrus junos Siebold ex Tanaka) peel on the diet-induced obesity in a zebrafish model.</a> J Funct Foods. 10, 499-510 (2014).</li><li>Schlegel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+non-alcoholic+fatty+liver+disease+with+zebrafish:+a+confluence+of+optics,+genetics,+and+physiology.">Studying non-alcoholic fatty liver disease with zebrafish: a confluence of optics, genetics, and physiology.</a> Cell Mol Life Sci. 69 (23), 3953-3961 (2012).</li><li>Stoletov, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+lipid+accumulation,+lipoprotein+oxidation,+and+macrophage+lipid+uptake+in+hypercholesterolemic+zebrafish.">Vascular lipid accumulation, lipoprotein oxidation, and macrophage lipid uptake in hypercholesterolemic zebrafish.</a> Circ Res. 104 (8), 952-960 (2009).</li><li>Thomas, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrient+balance+and+energy+expenditure+during+ad+libitum+feeding+of+high-fat+and+high-carbohydrate+diets+in+humans.">Nutrient balance and energy expenditure during ad libitum feeding of high-fat and high-carbohydrate diets in humans.</a> Am J Clin Nutr. 55 (5), 934-942 (1992).</li></ol>

The authors have nothing to disclose.

Nós apresentamos aqui um protocolo detalhado para a obtenção de sangue em série a partir de peixe-zebra adulto. Este método é simples de realizar e usá-lo no laboratório, numa base diária. Este método de coleta de sangue é baseada na inserção de uma agulha capilar de vidro em aorta dorsal do peixe-zebra. Durante este procedimento, é crítico para ter o cuidado de não fazer a ablação da coluna porque é o critério para a busca de aorta dorsal. Reduzir a lesão da coluna irá melhorar a taxa de sobreviv?...

Zebrafish estão ganhando cada vez mais popularidade como um valioso modelo de doenças humanas porque os seus órgãos e genética são semelhantes aos dos seres humanos 1,2. No campo da biologia do desenvolvimento, muitos estudos têm demonstrado que o peixe-zebra e mostrar humana marcada semelhança na hematopoiese 3, hemostasia 4,5, e 6 myelopoiesis. Peixe-zebra adulto também são usados ​​para o estudo imunológico 7, 8 e neurodegenerativa doenças relacionadas à obesidade 9 porque este ações organismo modelo vias comuns com aqueles interrompido em doenças humanas. Para obesidade e doenças relacionadas à obesidade (diabetes, esteatose hepática não alcoólica e e esteatohepatite aterosclerose), glicemia peixe-zebra e níveis de lipídeos, foram exaustivamente investigados em vários modelos de obesidade transgênicos e induzida por dieta 10-13. Coleta de sangue repetidas de animais individuais irá reduzir o uso de animais e dezembroRease diferenças interindividuais. No entanto, a recolha repetida de amostra é tecnicamente difícil em pequenos animais, tais como peixe-zebra por causa do seu volume de sangue relativamente pequena e a falta de vasos facilmente acessíveis. Vários métodos para a recolha de sangue de uma só vez a partir de peixes-zebra foram desenvolvidos, embora estes métodos têm os seus próprios inconvenientes, incluindo a letalidade, danos nos tecidos associados e volume de sangue limitado. Por exemplo, 1-5 mL de sangue pode ser colhido a partir de uma incisão lateral de aproximadamente 0,3 cm de comprimento na região da aorta dorsal 5. Decapitação com uma tesoura de cortar o cinto peitoral pode coletar 5-10 ul sangue 10. Outro método de amostragem de sangue conveniente é a ablação da cauda 14. Punção cardíaca é um potencial método alternativo para a coleta de sangue repetidas do mesmo peixe, mas a quantidade muito pequena obtida (aproximadamente 50 nl) com este procedimento limita o número de análises que podem ser performed 11. Por conseguinte, um novo protocolo é necessário para permitir a recolha de amostras de sangue não letal repetida, o que seria um avanço crítico necessário para este organismo ser um organismo modelo padrão para doenças humanas. Esta técnica permitiria testar resposta farmacológica, a descoberta de biomarcadores moleculares para o diagnóstico, determinação do prognóstico e do acompanhamento de várias doenças, tais como doenças metabólicas, doenças degenerativas e vários tipos de doenças malignas. Assim, desenvolvemos um método minimamente invasivo para a obtenção de sangue de peixe-zebra em série 15. Aqui demonstramos o procedimento visualmente e fornecer um protocolo detalhado para esta técnica. Usando este método, foram avaliados o valor normal com base em vários parâmetros, incluindo a hemoglobina, glicemia de jejum, e lipídios no sangue de peixe-zebra adultos saudáveis. Além disso, também avaliamos se esse método é apropriado para estudos que exigem amostras de série por mONITORIZAÇÃO as mudanças temporais nos níveis de glicose no sangue durante as experiências superalimentação.

<table><tbody><tr><td>Glass capillaries with filament</td><td>Narishige</td><td>GD-1</td><td>1.0 mm outer diameter.</td></tr><tr><td>Needle puller</td><td>Narishige</td><td>PC-10</td><td>To produce the needles</td></tr><tr><td>Heparin</td><td>Wako Pure Chemical Industries</td><td>081-00136</td><td>For heparinization</td></tr><tr><td>Aspirator tube assembly</td><td>Drummond</td><td>2-040-000</td><td>For blood collection</td></tr><tr><td>Bulb dispenser</td><td>Drummond</td><td>1-000-9000</td><td>For blood collection</td></tr><tr><td>2-phenoxyethanol</td><td>Wako Pure Chemical Industries</td><td>163-12075</td><td>For anesthetizing the fish</td></tr><tr><td>DRI-CHEM3500V</td><td>Fujifilm</td><td>-</td><td>For hemoglobin measurement</td></tr><tr><td>DRI-CHEM Slides</td><td>Fujifilm</td><td>Hb-WII</td><td>For hemoglobin measurement</td></tr><tr><td>Glutest Neo Super</td><td>Sanwa Kagaku Kenkyusho</td><td>-</td><td>For bood glucose measurement</td></tr><tr><td>Wako L-type TG kit</td><td>Wako Pure Chemical Industries</td><td>464-44201</td><td>For TG measurement</td></tr><tr><td>Wako L-type CHO kit</td><td>Wako Pure Chemical Industries</td><td>460-44301</td><td>For total cholesterol measurement</td></tr><tr><td>Parafilm M</td><td>Alcan Packaging</td><td>PM996</td><td>To expel the blood on</td></tr></tbody></table>

repeated blood collection for blood tests in adult zebrafish

Recolha de sangue é repetida uma das técnicas mais comuns realizados em animais de laboratório. No entanto, um protocolo não-letal para a recolha de sangue do peixe-zebra não foi estabelecida. Os métodos anteriores para a recolha de sangue a partir de peixe-zebra são letais, tais como incisão lateral, decapitação e ablação cauda. Assim, temos desenvolvido um novo método de recolha de sangue &quot;repetida&quot;, e aqui presente um protocolo detalhado descrevendo esse procedimento. Este método é minimamente invasivo e resulta em uma taxa de mortalidade muito baixo (2,3%) para peixe-zebra, permitindo, assim, a amostragem de sangue repetidas do mesmo indivíduo. O volume máximo de recolha de amostras de sangue é dependente do peso do corpo do peixe. O volume de sangue para a amostragem repetida em intervalos deve ser ≤0.4% de peso corporal por semana ou ≤1% a cada 2 semanas, que foram avaliadas por medições de hemoglobina no sangue. Além disso, a hemoglobina, glicemia de jejum, triglicerídeos plasma (TG) e total cníveis holesterol em peixe-zebra adulto masculino e feminino foram medidos. Nós também aplicaram esse método para investigar a desregulação do metabolismo da glicose na obesidade induzida por dieta. Este método de coleta de sangue permitirá que muitas aplicações, incluindo glicose e metabolismo lipídico e estudos hematológicos, o que aumentará o uso de peixe-zebra como um modelo de doença organismo humano.

Este método de coleta de sangue causa lesão mínima de peixe-zebra (a &lt;1mm punção; Figura 1J) e produz uma taxa de mortalidade muito baixa de 2,3%. Foi examinado o volume máximo de sangue que pode ser recolhido a partir de um único peixe e avaliada a relação com o seu peso corporal (Figura 3). Descobrimos que o volume de sangue coletado máximo foi linearmente correlacionado com o peso corporal (R = 0,813). O maior volume de sangue coletado de um peixe indivíduo (peso corpor...

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

Repeated Blood Collection for Blood Tests in Adult Zebrafish

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 ...

repeated-blood-collection-for-blood-tests-in-adult-zebrafish

Research

JoVE Journal

Biology

34.9K visualizações.  Mie University. 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.

Vídeo: Repeated Blood Collection for Blood Tests in Adult Zebrafish

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

Sincronismo de replicação de DNA usando Zebrafish como um sistema modelo In Vivo de criação de perfil

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Genética

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 ...

Biologia

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

Usando citometria de fluxo para detectar e quantificar a formação sanguínea alterada no zebrafish em desenvolvimento

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

Biologia do Desenvolvimento

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

Examinando a regeneração muscular em modelos de zebrafish de doença muscular

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Optimized Intravenous Injection in Adult Zebrafish

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in mammalian research models. However, its application in adult zebrafish for drug delivery, stem cell transplantation, and regenerative and cancer studies has been limited due to the challenges posed by their small body size and intricate blood vessels. To overcome these limitations, alternative injection techniques such as intracardiac and retro-orbital (RO) injection have been explored in the past for stem cell transplantation in adult zebrafish. However, these techniques have their drawbacks, including the need for meticulous injection techniques or increased risk of mortality.
In this study, we have developed a refined and optimized IV injection procedure specifically tailored to adult zebrafish, addressing the challenges associated with their unique anatomy. To demonstrate the effectiveness of this technique, we performed successful IV injections of whole kidney marrow cells from Tg(mpo: EGFP) fish and FITC-dextran dye into adult Casper fish. The subsequent visualization of injected cells and dyes using a fluorescence microscope confirmed their successful delivery and engraftment within the zebrafish. Furthermore, we demonstrated that compared with the intracardiac and RO injections, the IV injection resulted in improved survival rates and engraftment efficiency in treated zebrafish. This approach enables precise delivery and localization of substances and holds great potential for large-scale drug and chemical screening using adult zebrafish. Additionally, the ability to visually track the injected cells and dyes provides invaluable insights into their engraftment, migration, and interactions with host tissues, enabling a more comprehensive evaluation of therapeutic effects and biological processes in zebrafish models.

Here, we present an optimized and effective protocol for the intravenous injection of cells or pharmacological agents into adult zebrafish, resulting in enhanced cell engraftment and increased survival rates of the treated zebrafish.

Injeção intravenosa otimizada em peixe-zebra adulto

Intravenous (IV) injection is widely recognized as the most effective and commonly utilized method for achieving systemic delivery of substances in ...

Pesquisa do câncer

Blood Collection for Biochemical Analysis in Adult Zebrafish

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 molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

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 ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Estudando a Diabetes através dos olhos de um peixe: Microdissection, visualização e análise do tg(fli:EGFP) adulto vasculatura da retina de Zebrafish

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Medicina

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 ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recuperação de Zebrafish Adulto Corações para aplicações de alto rendimento

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

Eletrocardiografia de superfície vivo para zebrafish adulto

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...