Name: histological analyses of acute alcoholic liver injury in zebrafish
Uploaded: 2017-05-25T13:00:00.000+00:00
Duration: 10 min 45 s
Description: Watch this Scientific Journal Video about Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish at JoVE.com

Os autores gostariam de agradecer a Dra. Katy Murray no Zebrafish International Resource Center; Dr. Stacey Huppert e Kari Huppert no CCHMC, pelos seus conselhos úteis sobre o protocolo; E serviço veterinário CCHMC, para o cuidado com peixes. Este trabalho foi apoiado pelo NIH subvenção R00AA020514 e uma bolsa de investigação do Centro de Genómica Pediátrica no CCHMC (para CY). Foi também apoio em parte pelo NIH concessão P30 DK078392 (Integrative Morphology Core) do Centro de Pesquisa de Doenças Digestivas Core em Cincinnati.

<ol>
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L., Passeri, M., Sadler, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drinks+like+a+fish:+using+zebrafish+to+understand+alcoholic+liver+disease.">Drinks like a fish: using zebrafish to understand alcoholic liver disease.</a> Alcohol Clin Exp Res. 35 (5), 826-829 (2011).</li><li>Howarth, D. L., Yin, C., Yeh, K., Sadler, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+hepatic+dysfunction+parameters+in+two+models+of+fatty+liver+disease+in+zebrafish+larvae.">Defining hepatic dysfunction parameters in two models of fatty liver disease in zebrafish larvae.</a> Zebrafish. 10 (2), 199-210 (2013).</li><li>Lin, J. N., 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=Development+of+an+Animal+Model+for+Alcoholic+Liver+Disease+in+Zebrafish.">Development of an Animal Model for Alcoholic Liver Disease in Zebrafish.</a> Zebrafish. , (2015).</li><li>Passeri, M. J., Cinaroglu, A., Gao, C., Sadler, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+steatosis+in+response+to+acute+alcohol+exposure+in+zebrafish+requires+sterol+regulatory+element+binding+protein+activation.">Hepatic steatosis in response to acute alcohol exposure in zebrafish requires sterol regulatory element binding protein activation.</a> Hepatology. 49 (2), 443-452 (2009).</li><li>Tsedensodnom, O., Vacaru, A. M., Howarth, D. L., Yin, C., Sadler, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+metabolism+and+oxidative+stress+are+required+for+unfolded+protein+response+activation+and+steatosis+in+zebrafish+with+alcoholic+liver+disease.">Ethanol metabolism and oxidative stress are required for unfolded protein response activation and steatosis in zebrafish with alcoholic liver disease.</a> Dis Model Mech. 6 (5), 1213-1226 (2013).</li><li>Yin, C., Evason, K. J., Maher, J. J., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bHLH+transcription+factor+Hand2+marks+hepatic+stellate+cells+in+zebrafish:+Analysis+of+stellate+cell+entry+into+the+developing+liver.">The bHLH transcription factor Hand2 marks hepatic stellate cells in zebrafish: Analysis of stellate cell entry into the developing liver.</a> Hepatology. , (2012).</li><li>Lassen, N., 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=Molecular+cloning,+baculovirus+expression,+and+tissue+distribution+of+the+zebrafish+aldehyde+dehydrogenase+2.">Molecular cloning, baculovirus expression, and tissue distribution of the zebrafish aldehyde dehydrogenase 2.</a> Drug Metab Dispos. 33 (5), 649-656 (2005).</li><li>Reimers, M. J., Hahn, M. E., Tanguay, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+zebrafish+alcohol+dehydrogenases+share+common+ancestry+with+mammalian+class+I,+II,+IV,+and+V+alcohol+dehydrogenase+genes+but+have+distinct+functional+characteristics.">Two zebrafish alcohol dehydrogenases share common ancestry with mammalian class I, II, IV, and V alcohol dehydrogenase genes but have distinct functional characteristics.</a> J Biol Chem. 279 (37), 38303-38312 (2004).</li><li>Zhang, C., Ellis, J. L., Yin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+vascular+endothelial+growth+factor+signaling+facilitates+liver+repair+from+acute+ethanol-induced+injury+in+zebrafish.">Inhibition of vascular endothelial growth factor signaling facilitates liver repair from acute ethanol-induced injury in zebrafish.</a> Dis Model Mech. , (2016).</li><li>Vonghia, L., 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=Acute+alcohol+intoxication.">Acute alcohol intoxication.</a> Eur J Intern Med. 19 (8), 561-567 (2008).</li><li>Wittekind, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+staining+for+routine+diagnostic+pathology+including+the+role+of+tannic+acid.+1.+Value+and+limitations+of+the+hematoxylin-eosin+stain.">Traditional staining for routine diagnostic pathology including the role of tannic acid. 1. Value and limitations of the hematoxylin-eosin stain.</a> Biotech Histochem. 78 (5), 261-270 (2003).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio Rerio). , (2007).</li><li>Theise, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+alcoholic+liver+disease.">Histopathology of alcoholic liver disease.</a> Clinical Liver Disease. 2 (2), (2013).</li><li>Lorent, 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=Inhibition+of+Jagged-mediated+Notch+signaling+disrupts+zebrafish+biliary+development+and+generates+multi-organ+defects+compatible+with+an+Alagille+syndrome+phenocopy.">Inhibition of Jagged-mediated Notch signaling disrupts zebrafish biliary development and generates multi-organ defects compatible with an Alagille syndrome phenocopy.</a> Development. 131 (22), 5753-5766 (2004).</li><li>Huang, M., Xu, J., Shin, C. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+PCR-ready+genomic+DNA+from+zebrafish+tissues.">Method for isolation of PCR-ready genomic DNA from zebrafish tissues.</a> Biotechniques. 43 (5), 610-614 (2007).</li><li>van der Velden, Y. U., 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=The+serine-threonine+kinase+LKB1+is+essential+for+survival+under+energetic+stress+in+zebrafish.">The serine-threonine kinase LKB1 is essential for survival under energetic stress in zebrafish.</a> Proc Natl Acad Sci U S A. 108 (11), 4358-4363 (2011).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

O protocolo atual descreve um procedimento detalhado para tratamento de etanol agudo em larvas de peixe-zebra e as análises histopatológicas subsequentes com coloração de H &amp; E. O tratamento agudo com etanol deve ser realizado não antes de 96 h após a fecundação, pois esta é a fase em que o fígado zebrafish começa a expressar enzimas metabolizadoras de álcool 13 . 2% de etanol é a dose máxima que as larvas podem tolerar 13 , 1...

A doença hepática alcoólica (ALD), que é causada pelo consumo excessivo de álcool, é uma das principais causas de morbidade e mortalidade relacionadas ao álcool. Nos Estados Unidos, quase metade das mortes por doença hepática envolve álcool 1 , e a ALD é responsável por quase 1 em cada 3 transplantes de fígado 2 . ALD tem um amplo espectro. A esteatose, que se caracteriza pelo excesso de acumulação de lípidos nos hepatócitos, ocorre no estágio inicial de consumo excessivo de álcool e é reversível após a cessação do uso de álcool. Sob a influência de fatores genéticos e ambientais ea ingestão contínua de álcool, a esteatose hepática pode evoluir para hepatite alcoólica e, eventualmente, cirrose 3 . Estudos usando os modelos de ALD de roedores forneceram insights substanciais sobre a doença, mas eles têm limitações (revisado na referência 3 ). A alimentação oral de uma dieta com álcool só causa esteatose em roedores 4 , &lt;/ Sup&gt; 5 . O desenvolvimento de inflamação e fibrose requer um segundo insulto 6 , 7 ou infusão intragástrica crônica, que é invasiva e tecnicamente desafiadora 8,9 . O tele-peixe-zebra também desenvolve lesão hepática em resposta ao tratamento crônico e álcool agudo 10 , 11 , 12 , 13 , 14 , 15 . Em particular, o peixe-zebra larval representa um organismo modelo complementar atractivo no qual se estuda a lesão hepática alcoólica aguda 10 , 11 , 13 , 15 . O fígado zebrafish é funcional e produz enzimas-chave para o metabolismo de etanol por 4 dias(Dpf) 13,16,17. O etanol pode ser adicionado diretamente à água ea exposição a etanol a 2% por 24 h é suficiente para induzir esteatose hepática e respostas fibrogênicas em larvas de peixe-zebra 13,15. Tem sido relatado que o tratamento com etanol a 2% durante 24 horas resultou numa concentração de etanol tecidual de 80 mM em larvas de peixe-zebra 13 . Outros demonstraram que as larvas toleram esta concentração e que os fenotipos do fígado observados nos animais tratados são específicos da exposição ao etanol 11 , 13 , 15 , 18 . No entanto, como 80 mM é quase letal em seres humanos 19 , é importante avaliar a histologia do fígado do zebrafish tratado com etanol e deteImportância fisiológica para os seres humanos. O rápido desenvolvimento externo e translucência das larvas de peixe-zebra permitem caracterizar a ação do álcool no fígado em tempo real e em amostras fixas. A disponibilidade de linhas transgênicas fluorescentes específicas de células e os recentes avanços na microscopia confocal facilitam o estudo de como diferentes tipos de células hepáticas modificam sua morfologia e comportamento em resposta ao tratamento agudo com etanol 11,15. No entanto, a imagem confocal do peixe-zebra transgénico fluorescente não pode substituir completamente a coloração com Hematoxilina e Eosina (H &amp; E) quando se estuda a histologia do fígado. A marcação de todos os tipos de células hepáticas ao mesmo tempo utilizando peixes-zebra transgênicos requer a geração de linhas transgênicas individuais, cada uma rotulando um tipo de célula hepática com um fluoróforo único. A introdução de diferentes fundos transgênicos no mesmo peixe requerG gerações múltiplas, o que é demorado e oneroso. É necessária uma imunofluorescência adicional para detectar os componentes da matriz extracelular. A coloração de H &amp; E, por outro lado, rotula simultaneamente todos os tipos de células hepáticas e componentes de matriz extracelular, proporcionando assim uma visão geral do fígado 20 . Além disso, revela prontamente várias características histopatológicas de doenças hepáticas, tais como morte de hepatócitos, esteatose e fibrose. Embora H &amp; E é uma mancha de rotina na histologia do fígado de mamíferos, não é comumente usado na pesquisa de fígado zebrafish, eo protocolo é menos bem estabelecida. Este trabalho descreve um protocolo para tratamento de etanol agudo em larvas de peixe-zebra e para as análises histológicas de seguimento com coloração de H &amp; E. O protocolo de coloração de H &amp; E pode ser usado em todos os estudos de desenvolvimento e função do fígado. Além disso, as seções de parafina podem ser usadas para imuno-histoquímica, bem como para outrasIns na patologia hepática, incluindo a mancha tricrômica, mancha reticulina, etc.

<table><tbody><tr><td>1.5 mL centrifuge tubes</td><td>E &amp;amp; K Scientific</td><td>280150</td><td></td></tr><tr><td>15 mL conical tubes</td><td>VWR International</td><td>89039-664</td><td></td></tr><tr><td>50 mL conical tubes</td><td>VWR International</td><td>89039-658</td><td></td></tr><tr><td>95% ethanol (EtOH)</td><td>Decon Labs, Inc.</td><td>2801</td><td>Flammable</td></tr><tr><td>Acetic acid</td><td>Newcomer Supply</td><td>10010A</td><td>Irritant</td></tr><tr><td>Agarose</td><td>Research Products International</td><td>9012-36-6</td><td></td></tr><tr><td>Aluminum jar rack holder</td><td>Newcomer Supply</td><td>5300JRK</td><td></td></tr><tr><td>Bacteriological petri dishes with lid</td><td>Corning</td><td>351029</td><td></td></tr><tr><td>Biopsy pads</td><td>Simport</td><td>M476.1</td><td></td></tr><tr><td>Charged slides</td><td>Fisher Scientific</td><td>12-550-16</td><td></td></tr><tr><td>Clear mounting media</td><td>Fisher Scientific</td><td>8310-16</td><td>Can be substituted with other clear mounting media</td></tr><tr><td>Commercial sea salts</td><td>Instant Ocean</td><td>SS15-10</td><td></td></tr><tr><td>Disposable microtome blades</td><td>Fisher Scientific</td><td>4280L</td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica Biosystems</td><td>Leica Mz 95</td><td></td></tr><tr><td>Enclosed tissue processor</td><td>Leica Biosystems</td><td>ASP300 S</td><td></td></tr><tr><td>Eosin-Phloxine stain set</td><td>Newcomer Supply</td><td>1082A</td><td></td></tr><tr><td>Ethyl alcohol</td><td>Sigma-Aldrich</td><td>E7023</td><td>Flammable</td></tr><tr><td>Formaldehyde solution, ACS reagent, 37 WT. % in H&lt;sub&gt;2&lt;/sub&gt;0, contains 10 - 15% methanol as stabilizer (to prevent polymerization)</td><td>Sigma-Aldrich</td><td>252549</td><td>A suspected carcinogen; irritant</td></tr><tr><td>Formalin, Buffered, 10%</td><td>Fisher Scientific</td><td>SF100-4</td><td>A suspected carcinogen; irritant</td></tr><tr><td>Graduated media bottle</td><td>VWR International</td><td>16159-520</td><td></td></tr><tr><td>Harris hematoxylin</td><td>Poly Scientific R&amp;amp;D Corp.</td><td>s212</td><td>Irritant</td></tr><tr><td>Histology molds</td><td>Sakura Finetek USA Inc</td><td>4557</td><td></td></tr><tr><td>Hot plate/Stirrer</td><td>VWR International</td><td>47751-148</td><td></td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Fisher Scientific</td><td>A144</td><td>Irritant</td></tr><tr><td>Incubator</td><td>VWR International</td><td>97058-220</td><td></td></tr><tr><td>Insulin syringes</td><td>BD Medical</td><td>BD-309301</td><td></td></tr><tr><td>Inverted compound microscope</td><td>Carl Zeiss Microscopy</td><td>491912-9850-000</td><td></td></tr><tr><td>Isopropanol</td><td>Newcomer Supply</td><td>12094E</td><td>Flammable</td></tr><tr><td>Methylene blue</td><td>Sigma-Aldrich</td><td>M9140</td><td>Irritant</td></tr><tr><td>Microtome</td><td>Leica Biosystems</td><td>Leica Jung BioCut 2035&amp;nbsp;</td><td></td></tr><tr><td>Nutating mixer</td><td>VWR International</td><td>82007-202</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>P6148-1KG</td><td>A suspected carcinogen; irritant</td></tr><tr><td>Pasteur pipet</td><td>VWR International</td><td>53283-916</td><td></td></tr><tr><td>Pipette pump (10 mL)</td><td>VWR International</td><td>53502-233</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>Potassium phosphate, monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P9791</td><td></td></tr><tr><td>Razor blades</td><td>Grainger</td><td>4A807</td><td></td></tr><tr><td>Slide Staining Kit</td><td>Newcomer Supply</td><td>5300KIT</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Fisher BioReagents</td><td>S318-500</td><td>Very hazardous</td></tr><tr><td>Sodium phosphate, dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S3264</td><td></td></tr><tr><td>Stainless steel strainer (5 inch diameter)</td><td>Adaptive Science Tools</td><td>L0906045in</td><td></td></tr><tr><td>Tissue cassettes</td><td>Simport</td><td>M505.12</td><td></td></tr><tr><td>Tissue embedding center</td><td>Sakura Finetek USA Inc</td><td>#5100</td><td></td></tr><tr><td>Tissue wipers, 1-Ply</td><td>Fisher Scientific</td><td>06666A</td><td></td></tr><tr><td>Transfer pipets</td><td>Fisher Scientific</td><td>137117M</td><td></td></tr><tr><td>Tricaine powder/Ethyl 3-aminobenzoate methanesulfonate salt</td><td>Sigma-Aldrich</td><td>A5040</td><td>Irritant</td></tr><tr><td>Tris base, primary standard and buffer</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Wash bottle, low-density polyethylene, wide mouth</td><td>Nalge Nunc International</td><td>2402-0750</td><td></td></tr><tr><td>Xylenes</td><td>Fisher Scientific</td><td>X3S-4</td><td>Irritant</td></tr></tbody></table>

histological analyses of acute alcoholic liver injury in zebrafish

Doença hepática alcoólica (ALD) refere-se a danos ao fígado devido ao abuso de álcool agudo ou crônico. É uma das principais causas de morbidade e mortalidade relacionadas ao álcool e afeta mais de 2 milhões de pessoas nos Estados Unidos. Uma melhor compreensão dos mecanismos celulares e moleculares subjacentes à lesão hepática induzida pelo álcool é crucial para o desenvolvimento de tratamento eficaz para a ALD. As larvas de peixe-zebra exibem esteatose hepática e fibrogênese após apenas 24 horas de exposição a etanol a 2%, tornando-as úteis para o estudo da lesão hepática alcoólica aguda. Este trabalho descreve o procedimento para tratamento de etanol agudo em larvas de peixe-zebra e mostra que causa esteatose e inchaço dos vasos sanguíneos hepáticos. Um protocolo detalhado para hematoxilina e Eosina (H &amp; E) coloração que é otimizado para a análise histológica do zebrafish larval do fígado, também é descrito. A coloração de H &amp; E tem várias vantagens únicas em relação à imunofluorescência,Er células e componentes extracelulares simultaneamente e pode facilmente detectar lesão hepática, como esteatose e fibrose. Dado o uso crescente de peixe-zebra na modelagem de toxinas e lesões hepáticas induzidas por vírus, bem como doenças hepáticas hereditárias, este protocolo serve de referência para as análises histológicas realizadas em todos estes estudos.

10% de formalina tamponada e 4% de paraformaldeído (PFA) são dois dos fixadores mais comuns utilizados para as práticas histológicas. Contudo, não proporcionam resultados de fixação óptimos para o tecido hepático do peixe-zebra ( Figura 1 e Tabela 1 ). A fixação com formalina a 10% ou PFA a 4% resulta frequentemente em retracções, criando grandes lacunas entre o fígado e os tecidos circundantes ( Figura 1A<...

Watch this Scientific Journal Video about Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish at JoVE.com

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Este protocolo descreve análises histológicas dos fígados de larvas de peixe-zebra que foram tratadas com etanol a 2% durante 24 h. Tal tratamento com etanol agudo resulta em esteatose hepática e inchaço da vasculatura hepática.

Análises histológicas de lesão hepática alcoólica aguda em peixe-zebra

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

histological-analyses-of-acute-alcoholic-liver-injury-in-zebrafish

Research

JoVE Journal

Developmental Biology

14.2K visualizações.  Cincinnati Children's Hospital Medical Center.  Este protocolo descreve análises histológicas dos fígados de larvas de peixe-zebra que foram tratadas com etanol a 2% durante 24 h. Tal tratamento com etanol agudo resulta em esteatose hepática e inchaço da vasculatura hepática. 

Vídeo: Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

A regeneração renal em Zebrafish Adulto por lesão induzida pela gentamicina

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Biologia do Desenvolvimento

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Lesão Vessel Mecânica em embriões Zebrafish

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

Ablação específicas do hepatócito em Zebrafish para estudar a regeneração do fígado-driven biliar

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Desenvolvimento de um modelo de fígado fibrótica induzida por etanol no peixe-zebra para estudar Progenitor mediada por células de regeneração de hepatócitos

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Avaliar as alterações teratogênicos em um modelo de Zebrafish de exposição fetal ao álcool

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Medicina

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

Análise do Microambiente Hepático Durante a Progressão Precoce do Carcinoma Hepatocelular Associado à Doença Hepática Gordurosa Não Alcoólica em Peixe-Zebra.

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

Pesquisa do câncer

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

Quantificando o tamanho do fígado em zebrafish larval usando microscopia de campo brilhante

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

Quantificação dos níveis de etanol em embriões de zebrafish usando cromatografia de gás espacial da cabeça

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, and cognitive abnormalities are associated with FASD. Social difficulties are common behavioral abnormalities associated with FASD and often result in serious health issues. Animal models are critical to understanding the mechanisms responsible for ethanol-induced social defects. Zebrafish are social vertebrates that produce externally fertilized transparent eggs; these characteristics provide researchers with a precise yet simple procedure for creating the FASD phenotype and an innate behavior that can be leveraged to model the social deficits associated with FASD. Thus, zebrafish are ideal for characterizing the social deficits of FASD. The goal of the current protocol is to provide the user with a simple behavioral assay that can be used to characterize the consequences of a negative environment early during development and the effects it can have on social behavior in adulthood. The protocol can be used to characterize the effect mutations or teratogens have on adult social behavior. The protocol outlined here demonstrates how to characterize the social behavior of individual fish during a 20-min social assay. Furthermore, the data obtained using the current protocol provides evidence that the protocol can be used to characterize the effects of embryonic ethanol-induced social defects in adult zebrafish.

The goal of the current protocol is to outline the steps necessary to establish and use a social preference assay for adult zebrafish and demonstrate that it can be used to characterize ethanol-induced social defects.

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, ...

Comportamento

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

Hepatectomia Parcial em Zebrafish Adulto

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...