Targeted Liver Ablation: Inducing Hepatocyte Death with Metronidazole in Transgenic Zebrafish Larvae

Overview

The article describes targeted liver ablation, a method for inducing hepatocyte death with metronidazole in transgenic zebrafish larvae. The sample protocol describes the process for generating, manipulating and assessing targeted liver ablation in zebrafish.

Protocol

1. Preparation of Embryos/larvae

1. Transfer 100 embryos into a 100 mm petri dish with 25 ml of egg water. Keep no more than 100 embryos per petri dish and raise them at 28 °C.
2. To inhibit pigmentation, add phenylthiourea (PTU) into the petri dishes prior to 10 hr post-fertilization (hpf), and raise embryos/larvae until 80 hpf. The final concentration of PTU is 0.2 mM.
   CAUTION: PTU is toxic. Use in accordance with appropriate handling guidelines.
3. Anesthetize the larvae by adding 3-amino benzoic acid ethyl ester (Tricaine), at a final concentration of 0.016%, into the petri dishes. Then, using an epifluorescence microscope, sort CFP-positive larvae. Use larvae with a similar liver size and discard those with a smaller or larger liver.
   NOTE: any CFP-positive larvae, regardless of intensity, can be used because there is no difference in the extent of hepatocyte ablation between the hemizygous and homozygous larvae.
   CAUTION: tricaine is toxic. Use in accordance with appropriate handling guidelines.
4. Remove the egg water containing Tricaine and add egg water supplemented with 0.2 mM PTU. Keep the embryos/larvae at 28 °C.

2. Mtz Treatment

1. Prepare fresh 10 mM Mtz solution by dissolving 0.171 g of Mtz powder in 100 ml egg water supplemented with 0.2% DMSO and 0.2 mM PTU. To completely dissolve the Mtz, mix the solution at RT with rapid stirring for 20-30 minutes. To help solubilize Mtz and to enhance Mtz permeation into the larvae, add DMSO when preparing Mtz.
   CAUTION: prolonged exposure or increased concentration of Mtz is toxic. Use in accordance with appropriate handling guidelines.
2. Separate the larvae into control and experimental groups by transferring desired number of larvae into two different 100 mm petri dish or multi-well plate. For the experimental group, keep the larvae in 10 mM Mtz solution; for the control group, keep them in egg water containing 0.2% DMSO.
3. Cover the plates or petri dishes containing the Mtz solution with aluminum foil to prevent the photo-inactivation of Mtz, and incubate at 28 °C. The duration of Mtz treatment depends on the larval stage and the transgenic line.

3. Analysis of Biliary-driven Liver Regeneration

1. At the end of the ablation period, remove the Mtz solution from the plates. Wash the Mtz-treated larvae by adding 25 ml of egg water. Swirl the plate 2-3 times to wash the larvae before discarding the egg water. Repeat three times and then immerse the larvae once more in egg water. To avoid heart edema and larval death in the Mtz-treated larvae, add a lower concentration of Tricaine, at a final concentration of 0.008%, to immobilize the larvae.
2. For liver analysis, examine the liver size of the Mtz-treated larvae under an epifluorescence microscope with the CFP filter. Assess hepatocyte ablation levels based on the relative liver size: large (non-ablated; 0-10% larvae), medium (partially ablated; 10-20%), and very small (fully ablated; 80-90%).

Materials

Name	Company	Catalog Number	Comments
3-amino benzoic acid ethyl ester (Tricaine) powder	Sigma-Aldrich	A5040	Make 0.4% (15 mM) tricaine stock : Bring 0.4 g tricaine to 100 mL with egg water. Adjust to pH 7.
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D8418
Metronidazole	Sigma-Aldrich	M1547
N-Phenylthiourea (PTU)	Sigma-Aldrich	P7629
100 x 15 mm petri dish	Denville Scientific Inc.	M5300
egg water			0.3 g/L of sea salts ‘Instant Ocean’ and 0.5 mM CaSO4 in distilled water.
epifluorescence microscope	Leica Microsystems	M205FA