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

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. 

1. Preparation of Solutions

1. Prepare 20 L egg water (interchangeably used with ‘embryo medium’)  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.
2. 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.
3. 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 °C. Thaw before use.
   CAUTION: Tricaine can induce a loss of sensation. Prepare and use in accordance with appropriate handling guidelines.
4. 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.
5. 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.
6. 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.
7. 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.

2. Preparation of Larval Zebrafish

1. 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.
2. 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.
3. Keep no more than 100 embryos per Petri dish. Under a stereomicroscope, remove unfertilized eggs using a glass pipette. Grow embryos at 28 °C and add phenylthiourea (PTU) to a final concentration of 0.003% after 10 hours-post-fertilization (hpf) to inhibit pigment development.

3. Ethanol, Metronidazole Treatment and Liver Regeneration in Larval Zebrafish

1. Prepare fresh EtOH solution by adding ethanol into embryo medium to a final concentration of 1.5%. Do not add PTU.
2. 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.
3. 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.
4. 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 °C. Cover Petri dishes with plastic wrap to maintain EtOH concentration and with aluminum foil to protect from light.
5. 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.
6. 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.
7. 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 °C for further analyses.

4. Chemical Screens in EtOH/MTZ-treated Larval Zebrafish

1. 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 °C freezer. Cover with aluminum foil to prevent photocatalytic degradation of chemicals. Thaw the stock solutions at RT with gentle rocking.
2. Prepare chemical solutions by diluting  10 mM stock solutions with embryo medium to a final concentration of 50 µM (in 96 well plates: initial screen; in 1.5 ml tubes: retesting). Control larvae were treated with equal concentrations of DMSO in embryo medium. 
3. 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)  plates prefilled with embryo medium at a density of 5 larvae per well. Use duplicate wells for each chemical.
4. Remove embryo medium and add either 250 µl (96-well plates) or 500 µl (24-well plates)  chemical solutions to each well. Cover plates with aluminum foil and treat larvae for 50 hr at 28 °C. Inspect chemical-soaking larvae daily and remove any dead larvae from individual wells.
5. 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.
6. Preserve larvae by formaldehyde fixation for confocal imaging as described in section 5.
7. 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 ('retesting').

5. Larval Zebrafish Fixation, Immunostaining, and Confocal Imaging

1. 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 °C.
   CAUTION: Formaldehyde causes irritation and is known to be a human carcinogen. Handle in a chemical fume hood.
2. Discard fixing solution properly and then wash 3 times with PBS at RT. Fixed larvae can be kept in PBS at 4 °C for up to several days. Proceeding to the next step promptly gives better immunostaining results.
3. Carefully remove yolk and skin covering the liver area using a #55 forceps under a stereomicroscope.
4. Permeabilize and block larvae in Block buffer (4% bovine serum albumin and 0.3% Triton X-100 in PBS) O/N at 4 °C.
5. Incubate with rabbit anti-Collagen I antibody at a dilution of 1:100 in Block buffer O/N at 4 °C. Wash 5 times for 15 min each with Wash buffer (0.3% Triton X-100 in PBS).
6. Incubate with AlexaFluor 647 conjugated donkey anti-rabbit antibody at a dilution of 1:200 in Block buffer O/N at 4 °C. Wash 5 times for 15 min each with Wash buffer. Then wash once with PBS.
7. 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.
8. 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 µm intervals.

6. Preparation of Adult Zebrafish

1. 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.
2. Keep no more than 100 embryos per Petri dish. Under a stereomicroscope, remove unfertilized eggs using a glass pipette. Grow embryos at 28 °C and add PTU to a final concentration of 0.003% after 10 hpf to inhibit pigment development.
3. 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 °C until they achieve normal heart rate of 120-180 beats per minute¹⁸.
4. Raise sorted larvae to 6-12 months old based on the standard procedure¹⁹.

7. Ethanol, Metronidazole Treatment, and Liver Regeneration in Adult Zebrafish

1. 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.
2. 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 °C incubator.
3. Transfer adult fish to fresh EtOH solution daily, discard dead fish properly as a biohazard. Continuously treat animals for 72 hr before MTZ treatment.
4. Prepare fresh 10 mM MTZ solution in system water in 0.1% DMSO. Pre-warm the MTZ solution in a 28 °C incubator. Treat fish with 500 ml MTZ solution in 1 L crossing tank for 8 hr at 28 °C, protect from light using aluminum foil.
5. 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.
6. Allow animals to recover and maintain them in an incubator at 28 °C. Feed and transfer fish to fresh system water daily until the time point for analyses.

8. Adult Zebrafish Liver Fixation, Immunostaining, and Confocal Imaging 

1. 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.
2. 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.
3. 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 °C.
4. Discard fixing solution properly, and wash 3 times with PBS at RT. Fixed samples can be kept in PBS at 4 °C for several days. Proceeding to the next step immediately gives better immunostaining results.
5. 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.
6. Use a vibratome to cut samples into transverse sections at 50 µm intervals in ice cold PBS, starting from the anterior end. Transfer sections to 6-well plate with PBS using a #1 paint brush.
7. Permeabilize and block sections in Block buffer O/N at 4 °C.
8. Incubate with rabbit anti-Collagen I antibody at a dilution of 1:100 in Block buffer O/N at 4 °C. Wash 5 times, 15 min each with Wash buffer.
9. Incubate with AlexaFluor 647 conjugated donkey anti-rabbit antibody at a dilution of 1:200 in Block buffer O/N at 4 °C. Wash 5 times, 15 min each with Wash buffer and once with PBS.
10. 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.
11. 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 µm intervals.