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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Partial Hepatectomy in Adult Zebrafish

Published: April 4th, 2021

DOI:

10.3791/62349

1Harvard Medical School, 2Brigham and Women’s Hospital, 3Massachusetts General Hospital

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

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.

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 procedu....

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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 c.......

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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.......

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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.......

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I.M.O. is supported by the NIAAA (F32AA027135). W.G. is supported by R01DK090311, R01DK105198, R24OD017870, and the Claudia Adams Barr Program for Excellence in Cancer Research. W.G. is a Pew Scholar in Biomedical Sciences.

....

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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

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  9. Zhu, Z., Chen, J., Xiong, J. W., Peng, J. Haploinsufficiency of Def activates p53-dependent TGFβ signalling and causes scar formation after partial hepatectomy. PLoS One. 9, (2014).
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