前駆細胞が媒介する肝細胞の再生を研究するためにゼブラフィッシュにおけるエタノール誘発性線維症肝臓モデルの開発

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.