This work was supported in part by grants from the GTEC (2731336 and 1411318), the NIH (K01DK081351), and the NSF (1354837) to C. H. S. We thank Alem Giorgis for critical reading of the manuscript.

Zebrafish were raised and bred using a standard protocol that meets the criteria of the National Institutes of Health and approved by the Georgia Institute of Technology Institutional Animal Care and Use Committee.&#160;
1. Preparation of Solutions
<ol>
	<li>Prepare 20 L egg water (interchangeably used with &#8216;embryo medium&#8217;)&#160;&#160;to maintain embryonic/larval zebrafish. Dissolve 1.5 g CaSO4 and 6 g instant ocean sea salt in 250 ml distilled water. Pour into a carboy filled with 20 L di...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+liver+fibrosis+in+rodents.">Modeling liver fibrosis in rodents.</a> Methods Mol Med. 117, 237-250 (2005).</li><li>Gabele, E., 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+new+model+of+interactive+effects+of+alcohol+and+high-fat+diet+on+hepatic+fibrosis.">A new model of interactive effects of alcohol and high-fat diet on hepatic fibrosis.</a> Alcohol Clin Exp Res. 35 (7), 1361-1367 (2011).</li><li>Lieber, C. 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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=Metabolic+profiling+of+an+alcoholic+fatty+liver+in+zebrafish+(Danio+rerio).">Metabolic profiling of an alcoholic fatty liver in zebrafish (Danio rerio).</a> Mol Biosyst. 8 (7), 2001-2009 (2012).</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>Yin, C., Evason, K. J., Maher, J. J., Stainier, D. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitroreductase-mediated+cell/tissue+ablation+in+zebrafish:+a+spatially+and+temporally+controlled+ablation+method+with+applications+in+developmental+and+regeneration+studies.">Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies.</a> Nat Protoc. 3 (6), 948-954 (2008).</li><li>Parsons, M. J., 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=Notch-responsive+cells+initiate+the+secondary+transition+in+larval+zebrafish+pancreas.">Notch-responsive cells initiate the secondary transition in larval zebrafish pancreas.</a> Mech Dev. 126 (10), 898-912 (2009).</li><li>Baker, K., Warren, K. S., Yellen, G., Fishman, M. 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R., Saxena, M. T., Mumm, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+zebrafish+chemical+screening+technologies.">Advances in zebrafish chemical screening technologies.</a> Future Med Chem. 4 (14), 1811-1822 (2012).</li><li>Chen, C. H., Durand, E., Wang, J., Zon, L. I., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=zebraflash+transgenic+lines+for+in+vivo+bioluminescence+imaging+of+stem+cells+and+regeneration+in+adult+zebrafish.">zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish.</a> Development. 140 (24), 4988-4997 (2013).</li><li>Westhoff, J. 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=Development+of+an+automated+imaging+pipeline+for+the+analysis+of+the+zebrafish+larval+kidney.">Development of an automated imaging pipeline for the analysis of the zebrafish larval kidney.</a> PLoS One. 8 (12), e82137 (2013).</li><li>Perlman, Z. E., 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=Multidimensional+drug+profiling+by+automated+microscopy.">Multidimensional drug profiling by automated microscopy.</a> Science. 306 (5699), 1194-1198 (2004).</li><li>Chu, J., 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=New+school+in+liver+development:+lessons+from+zebrafish.">New school in liver development: lessons from zebrafish.</a> Hepatology. 50 (5), 1656-1663 (2009).</li><li>Choi, T. Y., Ninov, N., Stainier, D. Y., Shin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+conversion+of+hepatic+biliary+epithelial+cells+to+hepatocytes+after+near+total+loss+of+hepatocytes+in+zebrafish.">Extensive conversion of hepatic biliary epithelial cells to hepatocytes after near total loss of hepatocytes in zebrafish.</a> Gastroenterology. 146 (3), 776-788 (2014).</li><li>He, J., Lu, H., Zou, Q., Luo, L. <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+liver+after+extreme+hepatocyte+loss+occurs+mainly+via+biliary+transdifferentiation+in+zebrafish.">Regeneration of liver after extreme hepatocyte loss occurs mainly via biliary transdifferentiation in zebrafish.</a> Gastroenterology. 146 (3), 789-800 (2014).</li><li>Yao, 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=Fine+structure,+enzyme+histochemistry,+and+immunohistochemistry+of+liver+in+zebrafish.">Fine structure, enzyme histochemistry, and immunohistochemistry of liver in zebrafish.</a> Anat Rec (Hoboken). 295 (4), 567-576 (2012).</li><li>Yovchev, M. 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The authors declare that they have no competing financial interests.

We observed HPC-mediated hepatocyte regeneration in the EtOH/MTZ-treated recovering livers, suggesting that even in the presence of substantial amount of ECM proteins including fibrillar type I collagen, the HPCs retain their competency to regenerate as hepatocytes. The MTZ only-treatment did not increase deposition of ECM proteins significantly, whereas the EtOH only-treatment did not induce HPC activation15. By utilizing the combined EtOH/MTZ treatment, we were able to investigate HPC-driven regeneration in ...

Despite the remarkable regeneration capacity of hepatocytes1, which are the major parenchymal cell type of the liver, chronic liver failure impairs this ability, leading to hepatic progenitor cell (HPC)-dependent regeneration2.
Chronic liver damage is mainly derived from alcohol abuse, chronic hepatitis C virus (HCV) infection3 and non-alcoholic fatty liver disease (NAFLD)4. It leads to sustained liver fibrosis, which is associated with the accumulation of extracellular matrix (ECM) proteins. Persisting ECM accumulation distorts intact hepatic architecture by forming a fibrous scar tissue5, subsequently resulting in cirrhosis with high morbidity and mortality. Many attempts have been made to mitigate the fibrotic response mainly by focusing on inhibiting profibrogenic cytokines and activated myofibroblasts6. The latter is primarily derived from hepatic stellate cells (HSCs), the principle hepatic non-parenchymal cells responsible for liver scar formation4. Nevertheless, regenerative therapies that stimulate endogenous cellular sources including HPCs to regenerate hepatocytes in the presence of sustained fibrogenic insults await further investigation.
Many experimental models of hepatic fibrosis have been described in mammals. Repetitive injection of carbon tetrachloride (CCl4) has been widely used to induce liver fibrosis in murine and rat models7. When combined with a high-fat (HF) diet, alcohol led to a substantial upregulation of profibrogenic gene expression and hepatic fibrosis8. While steatosis (lipid accumulation) results from acute alcohol exposure, it makes the liver susceptible to more severe hepatic injury9.
The zebrafish, Danio rerio, has emerged as an invaluable vertebrate model system for studying regeneration. Though other lower vertebrates such as newts and axolotls have a remarkable capacity for regeneration, the zebrafish has advantages over other model systems in regards to the gene manipulation and visualization strategies needed to manipulate potential regenerative factors10. The zebrafish also represents an attractive vertebrate model for studying alcoholic liver disease (ALD) by simply adding ethanol (EtOH) to their water. Acute EtOH exposure to larval and adult zebrafish caused hepatic steatosis11-13. When adult zebrafish received extended EtOH exposure, collagen deposition was observed with upregulation of fibrosis-related genes14. However, a need exists for developing models to study liver regeneration in response to EtOH as a fibrogenic stimulus.
Recently, we developed an EtOH-induced fibrotic liver model in zebrafish15. We combined a hepatocyte-specific genetic ablation system with EtOH treatment in larval and adult zebrafish. We generated two transgenic lines, Tg(fabp10a:CFP-NTR)gt1 and Tg(fabp10a:mCherry-NTR)gt2, in which E.coli nitroreductase (NTR) are fused to the cyan and mCherry fluorescent protein, respectively, under the control of the hepatocyte-specific fatty acid binding protein 10a, liver basic (fabp10a) promoter. In this system, NTR converts a nontoxic prodrug metronidazole (MTZ) into a DNA inter-strand cross-linking agent16, inducing explicit death of hepatocytes. Using this model, we demonstrated that a population of hepatic cells, which are responsive to Notch signaling, converted into hepatocytes in the near absence of hepatocytes and in the excess of ECM. We designated these cells as HPCs. Furthermore, through chemical screens, we identified small molecule activators of Wnt signaling and inhibitors of Notch signaling that augment hepatocyte regeneration in the fibrotic liver. Therefore, our fibrotic liver model in zebrafish represents a superb chemical screening system compared to cell culture- or mammalian-based screening system. It is an in vivo system with significant cost- and time-saving benefits. Here we describe the detailed procedures for establishing an EtOH-induced fibrotic liver model and for performing chemical screens using this model in zebrafish. Furthermore, time-course analyses were performed to investigate how hepatocyte regeneration occurs in the fibrotic liver. This protocol will provide an invaluable tool to study the mechanisms and strategies of enhancing hepatocyte regeneration in the fibrotic liver.

<table>
	<tbody>
		<tr>
			<td>Calcium sulfate hemihydrate (CaSO4)</td>
			<td>Acros</td>
			<td>AC385355000</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>EMD</td>
			<td>MX0075</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-Piperazinediethanesulfonic acid (PIPES)</td>
			<td>Sigma-Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td>200 proof</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher Scientific</td>
			<td>F79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Metronidazole (MTZ)</td>
			<td>Sigma-Aldrich</td>
			<td>M3761</td>
			<td></td>
		</tr>
		<tr>
			<td>1-phenyl-2-thiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>3-amino benzoic acid ethyl ester (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) tablet</td>
			<td>Amresco</td>
			<td>E404</td>
			<td>Dissolve one tablet with 100 ml distilled water</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Fisher Scientific</td>
			<td>BP1600</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-melting agarose&#160;</td>
			<td>Amresco</td>
			<td>BP165</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem Cell Signaling Compound Library</td>
			<td>Selleck Chemicals</td>
			<td>L2100</td>
			<td>10mM stock in DMSO</td>
		</tr>
		<tr>
			<td>ActiProbe-1K Library</td>
			<td>Timtec</td>
			<td>ActiProbe-1K</td>
			<td>10mM stock in DMSO</td>
		</tr>
		<tr>
			<td>SB 415286</td>
			<td>Selleck Chemicals</td>
			<td>S2729</td>
			<td>Dissolve with DMSO to 10mM</td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>Selleck Chemicals</td>
			<td>S2924</td>
			<td>Dissolve with DMSO to 10mM</td>
		</tr>
		<tr>
			<td>Anti-Collagen I antibody</td>
			<td>Abcam</td>
			<td>ab23730</td>
			<td>Use at 1:100 for immunostaining, reacts with fish</td>
		</tr>
		<tr>
			<td>AlexaFluor 647 Donkey anti-rabbit IgG (H+L)</td>
			<td>Molecular Probes</td>
			<td>A31573</td>
			<td>Use at 1:200 for immunostaining</td>
		</tr>
		<tr>
			<td>Mounting media (Vectorshield)</td>
			<td>Vector Laboratories</td>
			<td>H-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>VWR</td>
			<td>10062-896</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td>Dumont #55</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>75x25 mm</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>48366-045</td>
			<td>18 mm</td>
		</tr>
		<tr>
			<td>Plastic wrap</td>
			<td>Fisher Scientific</td>
			<td>22305654</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher Scientific</td>
			<td>1213100</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrotome</td>
			<td>Leica</td>
			<td>VT1000 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica</td>
			<td>M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluoresent microscope</td>
			<td>Leica</td>
			<td>M205 FA</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocol microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 1 shows the development of an EtOH-induced fibrotic liver model in larval zebrafish. To optimize a protocol for exposing zebrafish larvae to EtOH, we first assessed EtOH toxicity. 2.5 days-post-fertilization (dpf) larvae were exposed to EtOH concentration 1%, 1.5%, or 2% for 24 hr followed by a concurrent 24 hr EtOH/MTZ treatment. Exposure to 2% EtOH caused high mortality, while nearly all larvae exposed to 1% EtOH or less showed minimal fibrogenic changes with rar...

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

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 ...

The overall goal of this protocol is to establish an Ethanol-induced Fibrotic Liver Model in Zebrafish and to perform chemical screens using this model. This method can help answer key questions in the liver regeneration field. Such as the molecular and cellular mechanisms of how hepatocytes regenerate in the fibrotic liver.The main advantage of this technique, is that it is a cost effective and time saving in vevo chemical screening. Furthermore, a detailed time course analysis can be performed through visualization of a single cell. Demonstrating the procedure will be Mianbo Huang, a postdoc, and Jin Xu, a graduate student from the laboratory.After preparing reagents according to the text protocol, set up mating tanks with dividers using adult transgenic Zebrafish containing fatty acid binding protein 10a:CFP-NTR with Tp1:mCherry and Tg(hand2:EGFP)Zebrafish. The following morning, two hours after removing the dividers, harvest the embryos by straining the system water and transferring the embryos into 100 millimeter petri dishes containing embryo medium. At 56 hours post fertilization, or HPF, add 1.5 percent ethanol to the embryos.Use plastic wrap to cover the plates to prevent an ethanol evaporation. Then incubate at 28 degrees Celsius for 24 hours. Prepare fresh 15 millimolar MTZ and embryo medium with 0.1 percent DMSO and add ethanol to a final concentration of 1.5 percent.Then, at 80 HPF, treat the sorted embryos with 20 milliliters of ethanol MTZ solution per petri dish. Use plastic wrap to cover the plates to prevent ethanol evaporation and cover with foil to protect from light. Then incubate at 28 degrees Celsius for 24 hours.The following day, observe the embryos under an epifluorescent microscope using a CFP filter and a magnification of 80x to sort out the larvae with near complete ablation of hepatocytes. To carry out chemical screens, remove the 96 well plates containing ten millimolar chemical stocks in DMSO from the negative 80 degree freezer. Use aluminum foil to cover the plates to prevent photocatalytic degradation of the chemicals and thaw with gentle rocking at room temperature.In 1.5 milliliter tubes, prepare chemical solutions by diluting five microliters of ten millimolar stock solutions with one milliliter of embryo medium to a final concentration of 50 micromolar. Next, transfer five larvae per well with near complete hapatocyte ablation to 24 well plates filled with embryo medium. Then, remove the embryo medium and add 500 microliters of the chemical solutions to each well.With aluminum foil, cover the plates and incubate the larvae at 28 degrees Celsius for 50 hours. After the incubation, observe the number and/or intensity of CFP-expressing hepatocytes under an epifluoesent microscope at 80x magnification. Image live larvae with enhanced or reduced numbers and/or intensity of CFP-expressing hapatocytes.Set up matings of transgenic Zebrafish containing fatty acid binding protein 10a:CFP-NTR with TP1:mCherry and raise the sorted larvae to six to 12 months old. Use a net to transfer the adult transgenic Zebrafish to mating tanks. Prepare fresh one percent ethanol in system water then use 500 milliliters of the solution to replace the system water in the mating tanks.Incubate the tanks at 28 degrees Celsius for 72 hours, transferring fish into fresh ethanol solution and removing any dead fish daily during the incubation. Prepare fresh ten millimolar MTZ solution in system water with 0.1 percent DMSO, then add 500 milliliters of pre-warmed MTZ solution to fish in one liter mating tanks. Use aluminum foil to cover the tanks.Incubate at 28 degrees Celsius for eight hours and analyze according to the text protocol. After anesthetizing according to the text protocol, expose the gastrointestinal organs by using scissors to cut the skin and underlining muscle along the belly from the anal fin to the operculum. Then cut posteriorly along the side of the fish back to the anal fin.Place the fish into a fifteen milliliter conical tube and use PBS to wash once. Then remove the PBS and add four milliliters of freshly prepared three percent formaldehyde in PEM buffer. Incubate at four degrees Celsius overnight to fix the fish.The following day, after washing the samples according to the text protocol, cut the esophagus to dissect the whole gut with the liver from the body cavity. Then place the gastrointestinal organs in a sectioning mold and use four percent low melting agarose to embed the tissue. Next, trim the block into a trapezoid shape and glue it to the base.Then use a vibratome and starting from the interior end of the tissue, cut samples into transverse sections at fifty micrometer intervals to ice-cold PBS. Use a number one paintbrush to transfer the sections to a six well plate with PBS. After immunostaining the sections according to the text protocol, use a number one paintbrush to gently transfer the sections to glass slides.Use kimwipes to remove excess PBS before adding a drop of mounting medium and a cover slip. Then use nail polish to seal the cover glass. Finally, using a confocal system equipped with a 40x 1.3 NA oil lens, capture Z-Stack images at 20 to 50 percent strength and one microammeter intervals.This figure shows that ethanol MTZ treated larvae develop upward curvature of the trunk and tail, paricardialidema, and failure to inflate the swim bladder. As seen here, DMSO treated control livers showed no fibular type one collagen deposition. Whereas ethanol MTZ treated livers displayed elevated type one collagen accumulation at 25 and 50 hours post ablation.In addition, compared to DMSO treated controlled livers, HSC's increased in number and adopted a myofibrillblast-like shape lacking cytoplasmic processes in the ethanol MTZ treated regenerating livers. This figure shows the development of an ethanol induced fibrotic liver model in adult Zebrafish treated with one percent ethanol followed by MTZ. From these time course analyses, significantly elevated type one collagen deposition in the ethanol MTZ treated regenerating livers at two, three, and four days post ablation was observed, as compared to DSMO treated livers.In this chemical screening experiment, hepatocytes were ablated in the presence of 1.5 percent ethanol and 15 millimolar MTZ. And the larvae were then exposed to chemicals for 50 hours. A number of wint agonists, such as inhibitors of glycogen synthase kinase-3 and a wint activator, promoted hepatocyte regeneration compared to DMSO.These data suggest that this sustained fibrotic model provides an invaluable tool to discover small molecules that enhance hepatocyte regeneration. Once mastered, this technique can be done in one week if it is performed properly. While attempting this procedure, it's important to remember to achieve near complete ablation of hepatocytes and to maintain the ethanol concentration throughout the procedure.Following this procedure, the efficiency of the identified chemical compounds can be tested in mammalian liver injury models. After its development, this technique can open up new avenues in the field of cellular regeneration therapy to explore the potential discovery of novel therapeutic strategies for chronic liver failure patients. After watching this video, you should have a good understanding of how to develop an ethanol induced fibrotic liver model in Zebrafish and how to perform chemical screens.Don't forget that working with formaldehyde can be extremely hazardous and precautions, such as wearing gloves and handling the reagent in a chemical fume hood, should always been taken while performing this procedure.

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9.1K Görüntüleme.  Georgia Institute of Technology. The overall goal of this protocol is to establish an Ethanol-induced Fibrotic Liver Model in Zebrafish and to perform chemical screens using this model. This method can help answer key questions in the liver regeneration field. Such as the molecular and cellular mechanisms of how hepatocytes regenerate in the fibrotic liver.The main advantage of this technique, is that it is a cost effective and time saving in vevo chemical screening. Furthermore, a detailed time course analysis can be...