Name: Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration (Video) | JoVE Journal
Uploaded: 2016-05-13T14:00:00.000+00:00
Duration: 10 min 42 s
Description: Learn to establish an ethanol-induced fibrotic liver model in zebrafish for studying hepatocyte regeneration mechanisms. This cost-effective method enables chemical screening and single-cell visualization.

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5 minutes

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 distilled water and agitate.</li>
	<li>Prepare 1 L 1-phenyl-2-thiourea (PTU) stock solution (20x). Dissolve 0.6 g PTU powder in 1 L distilled water. Add 1 ml of stock solution per 20 ml embryo medium to a final concentration of 0.003% as a working solution. 
	CAUTION: PTU may cause systemic toxicity following inhalation or dermal exposure. Prepare and use in accordance with appropriate handling guidelines.</li>
	<li>Prepare 1 L ethyl 3-aminobenzoate methanesulfonate (Tricaine) stock solution (20x). Dissolve 4 g Tricaine powder in distilled water. Adjust pH to 7 by adding 1 M Tris buffer (pH 9), and bring the final volume to 1 L with distilled water. Aliquot into 50 ml and store at -20 &#176;C. Thaw before use. 
	CAUTION: Tricaine can induce a loss of sensation. Prepare and use in accordance with appropriate handling guidelines.</li>
	<li>Prepare 100 ml larval metronidazole (MTZ) working solution. Make fresh 15 mM MTZ solution by dissolving 0.25 g MTZ powder in 0.1% dimethyl sulfoxide (DMSO) in 100 ml embryo medium. Dissolve MTZ powder completely by vigorous shaking. Make fresh MTZ solution and use aluminum foil to prevent photocatalytic degradation of MTZ. 
	CAUTION: MTZ may cause irritation. Prepare and use in accordance with appropriate handling guidelines.</li>
	<li>Prepare 100 ml larval ethanol/metronidazole (EtOH/MTZ) working solution. Make fresh MTZ solution and use aluminum foil to prevent photocatalytic degradation of MTZ. Add 1.5 ml 100% ethanol into 100 ml MTZ solution to a final concentration of 1.5% just before use.</li>
	<li>Prepare 500 ml adult MTZ working solution. Make fresh 10 mM MTZ solution by dissolving 0.83 g MTZ powder in 0.1% DMSO in 500 ml system water. Dissolve MTZ powder completely by vigorous shaking. Make fresh MTZ solution and use aluminum foil to prevent photocatalytic degradation of MTZ.</li>
	<li>Prepare 1 L PEM buffer. Dissolve 30.2 g PIPES, 0.74 g EGTA, and 0.12 g MgSO4 in distilled water. Adjust pH to 7 with NaOH. Bring final volume to 1 L with distilled water.</li>
</ol>
2. Preparation of Larval Zebrafish
<ol>
	<li>Set up mating of [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11]15,17 adult zebrafish with Tg(hand2:EGFP)pd24 13 adult zebrafish in mating tanks. Use dividers to conduct timed mating.</li>
	<li>Remove the divider the following morning and allow fish to mate without disruption. Harvest embryos 2 hr later by straining system water and transferring the embryos into 100 mm Petri dishes with embryo medium.</li>
	<li>Keep no more than 100 embryos per Petri dish. Under a stereomicroscope, remove unfertilized eggs using a glass pipette. Grow embryos at 28 &#176;C and add phenylthiourea (PTU) to a final concentration of 0.003% after 10 hours-post-fertilization (hpf) to inhibit pigment development.</li>
</ol>
3. Ethanol, Metronidazole Treatment and Liver Regeneration in Larval Zebrafish
<ol>
	<li>Prepare fresh EtOH solution by adding ethanol into embryo medium to a final concentration of 1.5%. Do not add PTU.</li>
	<li>Treat embryos with 20 ml ethanol solution per Petri dish from 56 to 80 hpf. Cover Petri dishes with plastic wrap to prevent ethanol evaporation.</li>
	<li>Sort out [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11; Tg(hand2:EGFP)pd24] larvae with similar liver size, as determined by CFP expression, at 80 hpf. Do not use Tricaine when sorting the EtOH-treated larvae because this will cause high mortality rate with subsequent EtOH/MTZ treatment. Keep sorted larvae at a density of 30 embryos per Petri dish.</li>
	<li>Prepare fresh 15 mM MTZ in embryo medium in 0.1% DMSO. Add proper amount of EtOH into MTZ solution to make a final concentration of 1.5%. Incubate larvae in 20 ml EtOH/MTZ solution per Petri dish for 24 hr at 28 &#176;C. Cover Petri dishes with plastic wrap to maintain EtOH concentration and with aluminum foil to protect from light.</li>
	<li>At the end of the treatment, remove EtOH/MTZ solution by transferring larvae to new Petri dishes with fresh embryo medium. Wash larvae 3 times with embryo medium and keep them in 20 ml embryo medium without PTU.</li>
	<li>To sort out the larvae with near-complete ablation of hepatocytes, observe fluorescence under an epifluorescence microscope with the CFP filter at a magnification of 80X. Successful ablation results in minimal CFP fluorescence in the liver and significantly reduced liver volume.</li>
	<li>To avoid death of the EtOH/MTZ-treated larvae, do not use Tricaine for sorting. Fix the larvae for immunostaining (refer to section 5) or keep sorted larvae in Petri dishes at 28 &#176;C for further analyses.</li>
</ol>
4. Chemical Screens in EtOH/MTZ-treated Larval Zebrafish
<ol>
	<li>Bring the 96-well plates containing 10 mM chemical stocks in DMSO (refer to the Materials List for details regarding the commercial chemical libraries) from the -80 &#176;C freezer. Cover with aluminum foil to prevent photocatalytic degradation of chemicals. Thaw the stock solutions at RT with gentle rocking.</li>
	<li>Prepare chemical solutions by diluting &#160;10 mM stock solutions with&#160;embryo medium to a final concentration of 50 &#181;M&#160;(in 96 well plates: initial screen; in 1.5 ml tubes: retesting). Control larvae were treated with equal concentrations of DMSO in embryo medium.&#160;</li>
	<li>Transfer larvae with near-complete hepatocyte ablation, which were treated with 1.5% EtOH/15 mM MTZ and sorted as aforementioned, to either 96-well (initial screen) or 24-well (retesting)&#160;&#160;plates prefilled with embryo medium at a density of 5 larvae per well. Use duplicate wells for each chemical.</li>
	<li>Remove embryo medium and add either 250 &#181;l (96-well plates) or 500 &#181;l (24-well plates)&#160;&#160;chemical solutions to each well. Cover plates with aluminum foil and treat larvae for 50 hr at 28 &#176;C. Inspect chemical-soaking larvae daily and remove any dead larvae from individual wells.</li>
	<li>At the end of the chemical treatment, observe the number and/or intensity of CFP-expressing hepatocytes under an epifluorescence microscope with the CFP filter at a magnification of 80X. Photograph live larvae showing enhanced or reduced number and/or intensity of CFP-expressing hepatocytes with a digital camera for records.</li>
	<li>Preserve larvae by formaldehyde fixation for confocal imaging as described in section 5.</li>
	<li>To retest the chemicals that facilitate hepatocyte regeneration in the initial screen, examine their potency at higher and lower concentrations to find the most efficacious concentration with the procedures described in 4.1-4.5 (&#39;retesting&#39;).</li>
</ol>
5. Larval Zebrafish Fixation, Immunostaining, and Confocal Imaging
<ol>
	<li>Anesthetize larvae by adding Tricaine to a final concentration of 0.02%. Transfer 10-15 larvae to a 1.5 ml tube and wash once with phosphate-buffered saline (PBS). Remove PBS and add 1 ml of freshly prepared 2% formaldehyde in PEM buffer to fix O/N at 4 &#176;C. 
	CAUTION: Formaldehyde causes irritation and is known to be a human carcinogen. Handle in a chemical fume hood.</li>
	<li>Discard fixing solution properly and then wash 3 times with PBS at RT. Fixed larvae can be kept in PBS at 4 &#176;C for up to several days. Proceeding to the next step promptly gives better immunostaining results.</li>
	<li>Carefully remove yolk and skin covering the liver area using a #55 forceps under a stereomicroscope.</li>
	<li>Permeabilize and block larvae in Block buffer (4% bovine serum albumin and 0.3% Triton X-100 in PBS) O/N at 4 &#176;C.</li>
	<li>Incubate with rabbit anti-Collagen I antibody at a dilution of 1:100 in Block buffer O/N at 4 &#176;C. Wash 5 times for 15 min each with Wash buffer (0.3% Triton X-100 in PBS).</li>
	<li>Incubate with AlexaFluor 647 conjugated donkey anti-rabbit antibody at a dilution of 1:200 in Block buffer O/N at 4 &#176;C. Wash 5 times for 15 min each with Wash buffer. Then wash once with PBS.</li>
	<li>Gently transfer larvae to glass slides using a glass pipette, and orient fixed larvae with the left lateral side facing up. Remove excess PBS using Kimwipes. Add a drop of mounting media, and seal cover glass with nail polish. Conducting confocal imaging immediately is greatly recommended.</li>
	<li>Use a confocal system equipped with a 40X/1.3 oil lens to capture images. Use laser strength at 20-50%. Capture Z stack images at 1 &#181;m intervals.</li>
</ol>
6. Preparation of Adult Zebrafish
<ol>
	<li>Set up mating of [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11]15,17 adult zebrafish in mating tanks. Harvest embryos the next morning by straining system water and transferring the embryos into 100 mm Petri dishes with embryo medium.</li>
	<li>Keep no more than 100 embryos per Petri dish. Under a stereomicroscope, remove unfertilized eggs using a glass pipette. Grow embryos at 28 &#176;C and add PTU to a final concentration of 0.003% after 10 hpf to inhibit pigment development.</li>
	<li>At 4 dpf, use Tricaine to anesthetize larvae and sort out [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11] larvae. Wash out Tricaine by changing to fresh embryo medium several times. Allow larvae to recover at 28 &#176;C until they achieve normal heart rate of 120-180 beats per minute18.</li>
	<li>Raise sorted larvae to 6-12 months old based on the standard procedure19.</li>
</ol>
7. Ethanol, Metronidazole Treatment, and Liver Regeneration in Adult Zebrafish
<ol>
	<li>Transfer 6-12 months old [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11] adult zebrafish to mating tanks using a net. Keep fish at a density of no more than 10 fish per tank.</li>
	<li>Prepare fresh EtOH solution by adding EtOH in system water to a final concentration of 1%. Treat adult fish with 500 ml EtOH solution in each tank and maintain them in a 28 &#176;C incubator.</li>
	<li>Transfer adult fish to fresh EtOH solution daily, discard dead fish properly as a biohazard. Continuously treat animals for 72 hr before MTZ treatment.</li>
	<li>Prepare fresh 10 mM MTZ solution in system water in 0.1% DMSO. Pre-warm the MTZ solution in a 28 &#176;C incubator. Treat fish with 500 ml MTZ solution in 1 L crossing tank for 8 hr at 28 &#176;C, protect from light using aluminum foil.</li>
	<li>Observe hourly during the treatment and remove dead fish immediately. Discard dead fish properly as a biohazard. At the end of the MTZ treatment, wash out MTZ by transferring animals to fresh system water twice.</li>
	<li>Allow animals to recover and maintain them in an incubator at 28 &#176;C. Feed and transfer fish to fresh system water daily until the time point for analyses.</li>
</ol>
8. Adult Zebrafish Liver Fixation, Immunostaining, and Confocal Imaging&#160;
<ol>
	<li>Anesthetize adult fish with 0.02% Tricaine and euthanize in ice water for 15 min. Pat the fish dry on a paper towel and place it on a dissecting mat.</li>
	<li>Expose gastrointestinal organs by removing skin and muscles as previously described20. Use scissors to cut the skin and underlying muscle along the belly from the anal fin to the operculum, and then posteriorly along the side of the fish back to the anal fin.</li>
	<li>Put the fish in a 15 ml conical tube and wash once with PBS. Remove PBS and add 4 ml of freshly prepared 2% formaldehyde in PEM buffer to fix O/N at 4 &#176;C.</li>
	<li>Discard fixing solution properly, and wash 3 times with PBS at RT. Fixed samples can be kept in PBS at 4 &#176;C for several days. Proceeding to the next step immediately gives better immunostaining results.</li>
	<li>Dissect the whole gut with liver from the body cavity of the fish by cutting the esophagus. Embed the gastrointestinal organs with 4% low-melting agarose in a sectioning mold.</li>
	<li>Use a vibratome to cut samples into transverse sections at 50 &#181;m intervals in ice cold PBS, starting from the anterior end. Transfer sections to 6-well plate with PBS using a #1 paint brush.</li>
	<li>Permeabilize and block sections in Block buffer O/N at 4 &#176;C.</li>
	<li>Incubate with rabbit anti-Collagen I antibody at a dilution of 1:100 in Block buffer O/N at 4 &#176;C. Wash 5 times, 15 min each with Wash buffer.</li>
	<li>Incubate with AlexaFluor 647 conjugated donkey anti-rabbit antibody at a dilution of 1:200 in Block buffer O/N at 4 &#176;C. Wash 5 times, 15 min each with Wash buffer and once with PBS.</li>
	<li>Gently transfer sections to glass slides using a #1 paint brush. Remove excess PBS using Kimwipes, add a drop of mounting media, and seal cover glass with nail polish. Conducting confocal imaging immediately is highly suggested.</li>
	<li>Use a confocal system equipped with a 40X/1.3 oil lens to capture images. Use laser strength at 20-50%. Capture Z stack images at 1 &#181;m intervals.</li>
</ol>

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<li>Yao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fine+structure%2C+enzyme+histochemistry%2C+and+immunohistochemistry+of+liver+in+zebrafish%5BTitle%5D)+AND+%22Anat+Rec+%28Hoboken%29%22%5BJournal%5D)">Fine structure, enzyme histochemistry, and immunohistochemistry of liver in zebrafish.</a> Anat Rec (Hoboken). 295 (4), 567-576 (2012).</li>
<li>Yovchev, M. I., Xue, Y., Shafritz, D. A., Locker, J., Oertel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repopulation+of+the+fibrotic%2Fcirrhotic+rat+liver+by+transplanted+hepatic+stem%2Fprogenitor+cells+and+mature+hepatocytes%5BTitle%5D)+AND+%22Hepatology%22%5BJournal%5D)">Repopulation of the fibrotic/cirrhotic rat liver by transplanted hepatic stem/progenitor cells and mature hepatocytes.</a> Hepatology. 59 (1), 284-295 (2014).</li>
</ol>

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 (CaSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Acros</td><td>AC385355000</td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</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)-&lt;br /&gt;&lt;em&gt;N,N,N&amp;prime;,N&amp;prime;&lt;/em&gt;-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&amp;nbsp;</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>10&amp;nbsp;mM stock in DMSO</td></tr><tr><td>ActiProbe-1K Library</td><td>Timtec</td><td>ActiProbe-1K</td><td>10&amp;nbsp;mM stock in DMSO</td></tr><tr><td>SB 415286</td><td>Selleck Chemicals</td><td>S2729</td><td>Dissolve with DMSO to 10&amp;nbsp;mM</td></tr><tr><td>CHIR-99021</td><td>Selleck Chemicals</td><td>S2924</td><td>Dissolve with DMSO to 10&amp;nbsp;mM</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>Epifluoresence 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

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...

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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Research

JoVE Journal

Developmental Biology

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.

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

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.

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

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.

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

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.

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

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.

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

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

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

Partial Hepatectomy in Adult Zebrafish

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.

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

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Learn to establish an ethanol-induced fibrotic liver model in zebrafish for studying hepatocyte regeneration mechanisms. This cost-effective method enables chemical screening and single-cell visualization.

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

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