Partial Hepatectomy in Adult Zebrafish

Summary

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

Abstract

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

Introduction

Among the solid organs in humans, the liver is the only organ capable of regeneration¹. This is critical, as the liver is an essential organ, responsible for key metabolic functions, energy storage, blood detoxification, secretion of plasma proteins, and bile production². Hepatocytes lost due to toxic or inflammatory damage are replaced primarily via division of the remaining hepatocytes¹. One classical experimental model for studying liver regeneration is partial hepatectomy, where individual lobes of the liver are removed, leaving the remaining lobes intact³. This procedure was initially developed in rats, in which approximately two-thirds of the liver mass is removed. After partial hepatectomy in mammals, compensatory regeneration occurs in the remaining lobes until the liver recovers its initial mass. Notably, the mammalian liver does not replace the missing lobes.

Zebrafish (Danio rerio) represent a tractable model for studying adult organ regeneration⁴. The zebrafish liver, while structurally different from the mammalian liver, is made up of the same cell types and serves the same function as in mammals². It is composed of three lobes, with two dorsal lobes and a single ventral lobe that are flattened along the intestine. Partial hepatectomy has previously been performed in zebrafish, with conflicting accounts as to the precise mode of regeneration. Typically, a one-third partial hepatectomy is performed by removal of the entire ventral lobe. Initial reports indicated that after removal of the ventral lobe, it was fully regenerated within a week^5,6,7, suggesting that in contrast to the mammalian liver, the zebrafish liver is capable of epimorphic regeneration. Subsequent studies demonstrated that removal of the ventral lobe resulted in compensatory regeneration in the dorsal lobes, rather than the regeneration of the missing ventral lobe, and ultimately the recovery of liver mass within a week^8,9. Transcriptomic profiling of the dorsal lobes following resection of the ventral lobe revealed significant changes associated with compensatory regeneration¹⁰. Given that the mode of liver regeneration can vary with the extent of the injury⁸, we speculated that the discrepancies in results may be due to technical variation in the partial hepatectomy protocol between research groups.

This protocol describes a procedure for performing a one-third partial hepatectomy on adult zebrafish by removing the ventral lobe. This technique will be valuable for assessing mechanisms of liver regeneration.

Protocol

Zebrafish were raised and bred according to standard procedures. Experiments were approved by the Brigham and Women's Hospital's Institutional Animal Care and Use Committee (2016N000405). Adult zebrafish were fasted for 24 h prior to the start of the protocol. System water refers to the water in zebrafish housing tanks in the aquatic facility.

1. Preparation and anesthetization

1. Prepare 0.016% Tricaine solution in system water.
   CAUTION: Tricaine is an irritant if it comes in contact with the eyes, skin, or respiratory tract.
2. Prepare a sponge to hold anesthetized zebrafish during the dissection protocol. Cut a full sponge into quarters. Using a razor blade, remove a thin wedge of sponge that runs parallel to the long axis of the sponge quarter.
   1. The slit should be long enough to accommodate an adult fish (this will vary between different sizes of fish). For example, for an adult fish 35 mm in length, the length of the slit should be 20 mm. The head and body are snugly held in the sponge, but the tail runs past the edge of the sponge (Figure 1B).
3. Soak the sponge in 0.016% Tricaine solution.
4. Place adult zebrafish (either male or female) in 625 mL of 0.016% Tricaine solution.
5. Incubate for 6 min or until the fish is unresponsive to touch.
6. Using forceps, carefully remove the fish from the Tricaine tank and place the fish ventral side up in the groove of the sponge (Figure 1A - B).
7. Place the sponge under a dissecting microscope with top-down illumination.

2. Surgery

1. Using fine forceps, pinch the skin and scales medially, just posterior to the heart (Figure 1B).
2. Using spring-loaded scissors, make a cut under the forceps to create a hole in the body cavity (Figure 1C - D). Take care not to injure the heart or a major blood vessel, as this will result in increased mortality.
3. Using spring-loaded scissors, make a 3-4 mm incision along the abdomen, processing posteriorly until the incision arrives at the pelvic fins (Figure 1D - E). By this point, the ventral lobe of the liver may be visible through the incision.
4. 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 will be visible on top of the intestine (Figures 1F,2A-B). The liver will appear as a pink or orange structure spread out over the golden-brown intestine. Animals designated as sham controls are recovered at this point.
5. Squeeze the fine forceps so that the two tines are touching. While keeping pressure on the sponge, slide the tines of the fine forceps in between the liver and the intestine (Figure 1G). Take care to not puncture the intestine, as this will result in increased mortality.
6. Slowly relax the pressure on the forceps so that the tines move away from one another (Figure 1H). This sliding action severs the numerous portal vein attachments between the ventral lobe and the intestine (Figure 2B), and is necessary to cleanly remove the ventral lobe. Repeat this process until all of the portal connections between the liver and intestine have been severed.
7. Peel back the ventral lobe from the intestine using fine forceps and cut the ventral lobe free from the rest of the liver (Figure 1I).
8. This procedure results in a one-third partial hepatectomy (Figure 1J).

3. Recovery

1. Carefully remove the fish from the sponge and place it in a tank of system water.
2. Pipette system water over the gills for a few minutes until the fish is swimming on its own (Figure 1K).
3. Monitor fish for 2-4 hours before placing them back onto the system. Do not feed the fish for a full 24 h after the surgery.
4. Monitor fish daily for the duration of the experiment.
5. With time, the incision in the body wall will heal naturally without the need for sutures (Figure 1L,2C).

4. Ventral lobe to intestine length analysis

1. Euthanize all animals destined for analysis in ice water for 10 min until all opercular movements cease.
2. Remove the fish from ice water and place it ventral side up in the groove of a sponge.
3. Using spring-loaded scissors, make an incision in the ventral body wall at the anterior-posterior position of the heart. Then make two more incisions that run along the anterior-posterior axis from the first incision all the way to the pelvic fins. (Figure 2A).
4. Peel back the skin and muscle to reveal the visceral organs (Figure 2A).
5. Acquire bright-field and fluorescent images of the visceral organs using an epifluorescence microscope. This field of view will include the area where the ventral lobe was resected. Because animals are euthanized prior to analysis, this kind of analysis precludes long-term imaging of the same fish.

5. Liver to body weight ratio analysis

1. Euthanize all animals destined for analysis in ice water for 10 min until all opercular movements cease.
2. Place fish in a 50 mL conical tube.
3. Add 25 mL of 4% paraformaldehyde in 1x PBS and 0.3% Tween to the tube.
   CAUTION: Formaldehyde is toxic, and solutions containing formaldehyde should always be processed in a chemical hood.
4. Nutate for 48 h at 4 °C.
5. Perform four 10 min washes in 1x PBS and 0.3% Tween.
6. Retrieve fish with forceps, and blot dry on a paper towel.
7. Record the weight of the entire fish.
8. Using spring-loaded scissors, make an incision in the ventral body wall at the anterior-posterior position of the heart. Then, make two more incisions that run along the anterior-posterior axis from the first incision all the way to the pelvic fins. (Figure 2A).
9. Peel back the skin and muscle to reveal the visceral organs (Figure 2A).
10. Acquire bright-field images of the liver using an epifluorescence microscope.
11. Dissect out the liver, placing the pieces of liver on a weighing boat.
12. Record the weight of the liver.

Results

In order to examine the regenerative potential of the adult zebrafish liver, we performed partial hepatectomy (PHX) in adult zebrafish. In general, large adults (30-40 mm in length) were selected, ranging from 1.5-2.5 years old. Within individual experiments, animals were selected from the same tank, and were age- and size-matched. As an appropriate control, we utilized sham surgeries in which the animal was both anesthetized and received a large incision in the ventral body wall but was recovered without removing any ti...

Discussion

The anatomical differences between zebrafish and mammalian models for liver regeneration present unique challenges to liver resection. The liver in zebrafish is in close proximity to the heart and the intestine; inadvertently damaging either organ results in increased mortality. The zebrafish liver is not encapsulated, making it more difficult to separate from the intestine. The liver receives nutrient-rich blood from the intestine through portal veins. In mammals, veins leaving the intestine converge on a primary portal...

Materials

Name	Company	Catalog Number	Comments
16% Paraformaldehyde Aqueous Solution, EM Grade	Electron Microscopy Sciences	15700
50 mL Falcon Centrifuge Tubes, Polypropylene, Sterile	Corning	352098
AS 82/220.R2 PLUS Analytical Balance	Bay State Scale & Systems, INC.	WL-104-1051
Dumont #55 Forceps	Fine Science Tools	11295-51
EMS Kuehne Coverglass/Specimen Forceps	Electron Microscopy Sciences	72997-07
Epifluorescence microscope	Zeiss	Discovery.V8
Mastertop Cellulose Cleaning Scrub Sponge	Amazon	B07CBSM53Z
PBS10X Liquid Conc 4L	EMD Millipore	6505-4L
Super Fine Micro Scissors, 3 1/4" straight	Biomedical Research Instruments	11-1020
Tricaine methanesulfonate	Syndel	TRIC-M-GR-0010
Tween 20, Fisher BioReagents	Fischer Scientific	BP337-500