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

- Dissolve metronidazole in egg water containing DMSO, a solvent, and add phenylthiourea, which prevents pigment formation in embryos. Transfer a few larvae of the appropriate age to treatment and control plates. Next, add the desired concentration of the metronidazole solution to the test plate and egg water containing DMSO to the control plate.

Let the larvae in both the plates grow at 28 degrees Celsius. The transgenic larvae produce nitroreductase only in hepatocytes, which converts the metronidazole into a cytotoxic drug, resulting in the death of only the targeted hepatocytes. After treatment, remove the metronidazole solution from the treatment plate and wash the embryos with egg water.

Add tricaine to anesthetize the larvae and examine them under an epifluorescence microscope for severe hepatocyte ablation-- the destruction of liver cells. You will observe a much smaller liver in treated individuals than in control group individuals. In the example protocol, we will use metronidazole for hepatocyte ablation in transgenic zebrafish larvae to study liver regeneration.

- Transfer up to 100 embryos into a 100 millimeter Petri dish containing 25 milliliters of egg water and place the dish at 28 degrees Celsius. To inhibit pigmentation, add PTU to the culture up to 10 hours post-fertilization. At 80 hours post fertilization, anesthetize the larvae with tricaine and use an epifluorescence microscope to collect the CFP positive larvae, keeping only the animals with similarly sized livers

Then replace the tricaine egg water with fresh egg water supplemented with PTU and return the CFP positive zebrafish larvae to the 28 degrees Celsius incubator. To treat the zebrafish larvae with MTZ, first, divide the appropriate number of larvae into two different culture vessels. Keep the experimental larvae group in a solution of freshly prepared MTZ and the control group in egg water supplemented with DMSO.

Cover the experimental group with aluminum foil to prevent photo inactivation of the MTZ and incubate both groups of zebrafish larvae at 28 degrees Celsius. At the end of the ablation period, remove the MTZ solution from the plates and wash the MTZ-treated lavae two to three times with 25 milliliters of egg water and swirling, discarding the egg water after each wash. After the last wash, immobilize the larvae with half the concentration of tricaine previously used to avoid heart edema and death in the MTZ-treated larvae. Then use the CFP filter on an epifluorescence microscope to assess the hepatocyte ablation levels based on the relative liver size of the MTZ-treated lavae. Consider the zebrafish with large livers representing 0% to 5% of the larvae to be nonablated, those with medium sized livers representing 10% to 20% of the larvae to be partially ablated, and those with very small livers representing 80% to 90% of the larvae to be fully ablated.