This protocol will allow researchers to take advantage of available chemical and genetic tools to study the mechanisms of liver regeneration in zebrafish. Partial hepatectomy allows the precise removal of tissue to stimulate liver regeneration without the need for the body-wide effects of toxic liver injury. Only by understanding the molecular and cellular mechanisms of liver regeneration can we design new therapies to treat patients with advanced liver disease.
To begin, remove the fish from the Tricaine tank using forceps and embed them in the groove of a Tricaine soaked sponge with the ventral side up. Then place the sponge under a dissecting microscope with top-down illumination. Pinch the skin and scales just posterior to the heart with fine forceps and create a hole in the body cavity by making a cut under the forceps with the spring loaded scissor.
Rotate the sponge and make a three to four millimeter incision along the abdomen by cutting posterially toward the pelvic fins. Then rotate the sponge back. Squeeze the sides of the sponge with one hand to force the visceral organs out of the body cavity.
The ventral lobe of the liver is present on top of the intestine, and the liver is a pink or orange structure spread out over the golden brown intestine, and can be clearly visualized with CFP fluorescence. Squeeze the fine forceps so that the tines are touching each other and slide the tines between the liver and the intestine. Then slowly separate the tines to break the portal vein attachments between the liver and the intestine.
Repeat this process until all the portal connections between the liver and intestine have been severed, and pull the ventral lobe from the intestine using fine forceps. Cut the ventral lobe free from the rest of the liver. Examination of the animal with CFP fluorescence demonstrates that the ventral lobe has been removed.
When finished, carefully transfer the fish from the sponge into the tank containing system water and stream the system water over the gills for a few minutes until the fish recovers and swims on its own. After euthanizing the fish, remove it from ice water. Place it in the groove of a sponge with the ventral side up and make an incision of the ventral body wall at the anterior posterior position of the heart.
Then make two more incisions from the edges of the first incision that run along the anterior posterior axis to the pelvic fins, and peel back the skin and muscle to reveal the visceral organs. The visceral organs can now be visualized with both bright field and fluorescence microscopy. Liver recovery after partial hepatectomy was estimated by calculating the liver to body weight ratio, which was 1.8%for males and 3.3%for females.
The liver to body weight ratio of both sexes was determined at post-surgery day zero, and day seven in partial hepatectomy and sham controls. At post-surgery day zero, sham animals have a visible ventral lobe, but in partial hepatectomy animals, the ventral lobe was completely absent, which resulted in a 30%reduction in males and 20%reduction in females of liver to body weight ratios. At post-surgery day seven in partial hepatectomy animals, the ventral lobe was not regenerated, and the liver to body weight ratios in partial hepatectomy and sham controls were comparable, indicating regained liver mass after partial hepatectomy.
The effect of time on ventral lobe regeneration was determined by performing a full 1/3 partial hepatectomy and measuring the ventral lobe to the intestine length ratio. The ventral lobe occupied 50 to 100%of the intestine length in sham controls, but was severely reduced in 1/3 partial hepatectomy animals. Partial hepatectomy animals at post-surgery day 36 had an increased ventral lobe to intestine ratio as compared to post-surgery day one.
A statistically significant increased size of the ventral lobe was observed in animals recovering from partial hepatectomy at post-surgery day 36 as compared to day one, but not in sham controls. Some animals regenerated the ventral lobe, but some did not, indicating variation in surgery response. The most critical part of this protocol is to cleanly separating the liver and the intestine so that the ventral lobe can be entirely removed.
Partial hepatectomy in zebrafish can be performed in mutant or transgenic lines to assay that genetic requirements for liver regeneration.
