The authors would like to acknowledge Dr. Katy Murray at the Zebrafish International Resource Center; Dr. Stacey Huppert and Kari Huppert at CCHMC, for their helpful advice on the protocol; and CCHMC veterinary service, for the fish care. This work was supported by NIH grant R00AA020514 and a research grant from the Center for Pediatric Genomics at CCHMC (to C.Y.). It was also support in part by NIH grant P30 DK078392 (Integrative Morphology Core) of the Digestive Disease Research Core Center in Cincinnati.

<ol><li>Yoon, Y. H., Chen, C. M., Yi, H. Y. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aYoon%2C+YH+%22Surveillance+report+%23100%3A+Liver+cirrhosis+mortality+in+the+United+States%3A+National%2C+State%2C+and+regional+trends%22">Surveillance report #100: Liver cirrhosis mortality in the United States: National, State, and regional trends</a>. , National Institute on Alcohol Abuse and Alcoholism. Bethesda, MD. 2000-2011 (2014).</li>
<li>Singal, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evolving+frequency+and+outcomes+of+liver+transplantation+based+on+etiology+of+liver+disease%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Evolving frequency and outcomes of liver transplantation based on etiology of liver disease.</a> Transplantation. 95 (5), 755-760 (2013).</li>
<li>Louvet, A., Mathurin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alcoholic+liver+disease%3A+mechanisms+of+injury+and+targeted+treatment%5BTitle%5D)+AND+%22Nat+Rev+Gastroenterol+Hepatol%22%5BJournal%5D)">Alcoholic liver disease: mechanisms of injury and targeted treatment.</a> Nat Rev Gastroenterol Hepatol. 12 (4), 231-242 (2015).</li>
<li>Ki, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin-22+treatment+ameliorates+alcoholic+liver+injury+in+a+murine+model+of+chronic-binge+ethanol+feeding%3A+role+of+signal+transducer+and+activator+of+transcription+3%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">Interleukin-22 treatment ameliorates alcoholic liver injury in a murine model of chronic-binge ethanol feeding: role of signal transducer and activator of transcription 3.</a> Hepatology. 52 (4), 1291-1300 (2010).</li>
<li>Tsuchiya, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interstrain+differences+in+liver+injury+and+one-carbon+metabolism+in+alcohol-fed+mice%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">Interstrain differences in liver injury and one-carbon metabolism in alcohol-fed mice.</a> Hepatology. 56 (1), 130-139 (2012).</li>
<li>Koteish, A., Yang, S., Lin, H., Huang, X., Diehl, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+ethanol+exposure+potentiates+lipopolysaccharide+liver+injury+despite+inhibiting+Jun+N-terminal+kinase+and+caspase+3+activation%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Chronic ethanol exposure potentiates lipopolysaccharide liver injury despite inhibiting Jun N-terminal kinase and caspase 3 activation.</a> J Biol Chem. 277 (15), 13037-13044 (2002).</li>
<li>Leo, M. A., Lieber, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hepatic+fibrosis+after+long-term+administration+of+ethanol+and+moderate+vitamin+A+supplementation+in+the+rat%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">Hepatic fibrosis after long-term administration of ethanol and moderate vitamin A supplementation in the rat.</a> Hepatology. 3 (1), 1-11 (1983).</li>
<li>Tsukamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Severe+and+progressive+steatosis+and+focal+necrosis+in+rat+liver+induced+by+continuous+intragastric+infusion+of+ethanol+and+low+fat+diet%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">Severe and progressive steatosis and focal necrosis in rat liver induced by continuous intragastric infusion of ethanol and low fat diet.</a> Hepatology. 5 (2), 224-232 (1985).</li>
<li>Tsukamoto, H., Mkrtchyan, H., Dynnyk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intragastric+ethanol+infusion+model+in+rodents%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Intragastric ethanol infusion model in rodents.</a> Methods Mol Biol. 447, 33-48 (2008).</li>
<li>Howarth, D. L., Passeri, M., Sadler, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drinks+like+a+fish%3A+using+zebrafish+to+understand+alcoholic+liver+disease%5BTitle%5D)+AND+%22Alcohol+Clin+Exp+Res%22%5BJournal%5D)">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/pubmed?term=((Defining+hepatic+dysfunction+parameters+in+two+models+of+fatty+liver+disease+in+zebrafish+larvae%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">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/pubmed?term=((Development+of+an+Animal+Model+for+Alcoholic+Liver+Disease+in+Zebrafish%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">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/pubmed?term=((Hepatic+steatosis+in+response+to+acute+alcohol+exposure+in+zebrafish+requires+sterol+regulatory+element+binding+protein+activation%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">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/pubmed?term=((Ethanol+metabolism+and+oxidative+stress+are+required+for+unfolded+protein+response+activation+and+steatosis+in+zebrafish+with+alcoholic+liver+disease%5BTitle%5D)+AND+%22Dis+Model+Mech%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3407311/">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/pubmed?term=((Molecular+cloning%2C+baculovirus+expression%2C+and+tissue+distribution+of+the+zebrafish+aldehyde+dehydrogenase+2%5BTitle%5D)+AND+%22Drug+Metab+Dispos%22%5BJournal%5D)">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/pubmed?term=((Two+zebrafish+alcohol+dehydrogenases+share+common+ancestry+with+mammalian+class+I%2C+II%2C+IV%2C+and+V+alcohol+dehydrogenase+genes+but+have+distinct+functional+characteristics%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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/pubmed?term=((Inhibition+of+vascular+endothelial+growth+factor+signaling+facilitates+liver+repair+from+acute+ethanol-induced+injury+in+zebrafish%5BTitle%5D)+AND+%22Dis+Model+Mech%22%5BJournal%5D)">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/pubmed?term=((Acute+alcohol+intoxication%5BTitle%5D)+AND+%22Eur+J+Intern+Med%22%5BJournal%5D)">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/pubmed?term=((Traditional+staining+for+routine+diagnostic+pathology+including+the+role+of+tannic+acid.+1.+Value+and+limitations+of+the+hematoxylin-eosin+stain%5BTitle%5D)+AND+%22Biotech+Histochem%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWesterfield%2C+M+%22The+Zebrafish+Book%3A+A+Guide+for+the+Laboratory+Use+of+Zebrafish+%28Danio+Rerio%29%22">The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio Rerio)</a>. , University of Oregon Press. (2007).</li>
<li>Theise, N. D. <a target="_blank" href="http://onlinelibrary.wiley.com/doi/10.1002/cld.172/full">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/pubmed?term=((Inhibition+of+Jagged-mediated+Notch+signaling+disrupts+zebrafish+biliary+development+and+generates+multi-organ+defects+compatible+with+an+Alagille+syndrome+phenocopy%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+an+Ethanol-induced+Fibrotic+Liver+Model+in+Zebrafish+to+Study+Progenitor+Cell-mediated+Hepatocyte+Regeneration%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration.</a> J Vis Exp. (111), (2016).</li>
<li>Paredes, J. F., Lopez-Olmeda, J. F., Martinez, F. J., Sanchez-Vazquez, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Daily+rhythms+of+lipid+metabolic+gene+expression+in+zebra+fish+liver%3A+Response+to+light%2Fdark+and+feeding+cycles%5BTitle%5D)+AND+%22Chronobiol+Int%22%5BJournal%5D)">Daily rhythms of lipid metabolic gene expression in zebra fish liver: Response to light/dark and feeding cycles.</a> Chronobiol Int. 32 (10), 1438-1448 (2015).</li>
<li>Meeker, N. D., Hutchinson, S. A., Ho, L., Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Method+for+isolation+of+PCR-ready+genomic+DNA+from+zebrafish+tissues%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">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/pubmed?term=((The+serine-threonine+kinase+LKB1+is+essential+for+survival+under+energetic+stress+in+zebrafish%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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>

The authors declare that they have no competing financial interests.

The current protocol describes a detailed procedure for acute ethanol treatment in zebrafish larvae and the subsequent histopathological analyses with H&#38;E staining. Acute ethanol treatment should be conducted at no earlier than 96 h post-fertilization, as this is the stage at which the zebrafish liver starts to express alcohol-metabolizing enzymes13. 2% ethanol is the maximal dose that larvae can tolerate13,14. The ethanol-treated larv...

Alcoholic Liver Disease (ALD), which is caused by alcohol overconsumption, is a major cause of alcohol-related morbidity and mortality. In the United States, nearly half of liver disease deaths involve alcohol1, and ALD is responsible for almost 1 in 3 liver transplants2. ALD has a broad spectrum. Steatosis, which is characterized by excess lipid accumulation in hepatocytes, occurs in the early stage of heavy drinking and is reversible upon cessation of alcohol use. Under the influence of genetic and environmental factors and continuing alcohol intake, hepatic steatosis can progress to alcoholic hepatitis and, eventually, cirrhosis 3. Studies using the rodent ALD models have provided substantial insights into the disease, but they have limitations (reviewed in reference3). Oral feeding of an alcohol diet only causes steatosis in rodents4,5. Development of inflammation and fibrosis requires either a second insult6,7 or chronic intragastric infusion, which is invasive and technically challenging8,9. The teleost zebrafish also develops liver injury in response to both chronic and acute alcohol treatment10,11,12,13,14,15. In particular, the larval zebrafish represents an attractive complementary model organism in which to study acute alcoholic liver injury10,11,13,15. The zebrafish liver is functional and produces key enzymes for ethanol metabolism by 4 days post-fertilization (dpf)13,16,17.Ethanol can be directly added to the water, and exposure to 2% ethanol for 24 h is sufficient to induce hepatic steatosis and fibrogenic responses in zebrafish larvae13,15.
It has been reported that treatment with 2% ethanol for 24 h resulted in a tissue ethanol concentration of 80 mM in zebrafish larvae13. Others have shown that larvae tolerate this concentration and the liver phenotypes seen in the treated animals are specific to ethanol exposure11,13,15,18. However, because 80 mM is nearly lethal in humans19, it is important to evaluate the liver histology of the ethanol-treated zebrafish and determine the physiological relevance to humans.
The rapid external development and translucence of zebrafish larvae make it possible to characterize the action of alcohol within the liver in real-time and in fixed samples. The availability of cell type-specific fluorescent transgenic lines and the recent advances in confocal microscopy facilitate the study of how different liver cell types change their morphology and behavior in response to acute ethanol treatment11,15. However, confocal imaging of the fluorescent transgenic zebrafish cannot completely substitute for Hematoxylin and Eosin (H&#38;E) staining when studying liver histology. Marking all liver cell types at the same time using transgenic zebrafish requires the generation of individual transgenic lines, each labeling one liver cell type with a unique fluorophore. Introducing different transgenic backgrounds into the same fish requires breeding multiple generations, which is time-consuming and costly. Additional immunofluorescence staining is needed to detect extracellular matrix components. H&#38;E staining, on the other hand, simultaneously labels all liver cell types and extracellular matrix components, thus providing an overview of the liver20. Moreover, it readily reveals several histopathological features of liver diseases, such as hepatocyte death, steatosis, and fibrosis. Although H&#38;E is a routine stain in mammalian liver histology, it is not commonly used in zebrafish liver research, and the protocol is less well established.
This work describes a protocol for acute ethanol treatment in zebrafish larvae and for the follow-up histological analyses with H&#38;E staining. The H&#38;E staining protocol can be used in all studies of liver development and function. Moreover, the paraffin sections can be used for immunohistochemistry, as well as for other special stains in liver pathology, including the trichrome stain, reticulin stain, 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

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 morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

10% buffered formalin and 4% paraformaldehyde (PFA) are two of the most common fixatives used for histology practices. However, they do not give optimal fixation results for zebrafish liver tissue (Figure 1 and Table 1). Fixation with 10% formalin or 4% PFA often results in shrinkages, creating large gaps between the liver and surrounding tissues (Figure 1A,&#160;B; Figure 1B...

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

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

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 Views.  Cincinnati Children's Hospital Medical Center.  Dit protocol beschrijft histologische analyses van de levers van zebravislarven die 24 uur met 2% ethanol zijn behandeld. Een dergelijke acute ethanolbehandeling resulteert in hepatische steatosis en zwelling van de hepatische vasculatuur. 

Video: 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.

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

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.

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.

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.

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.

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

Medicine

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.

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

Cancer Research

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.

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.

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, ...

Behavior

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.

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 ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Samenvatting

Meer video's verkennen