Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Summary

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Abstract

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Introduction

Despite the remarkable regeneration capacity of hepatocytes¹, which are the major parenchymal cell type of the liver, chronic liver failure impairs this ability, leading to hepatic progenitor cell (HPC)-dependent regeneration².

Chronic liver damage is mainly derived from alcohol abuse, chronic hepatitis C virus (HCV) infection³ and non-alcoholic fatty liver disease (NAFLD)⁴. It leads to sustained liver fibrosis, which is associated with the accumulation of extracellular matrix (ECM) proteins. Persisting ECM accumulation distorts intact hepatic architecture by forming a fibrous scar tissue⁵

Protocol

Zebrafish were raised and bred using a standard protocol that meets the criteria of the National Institutes of Health and approved by the Georgia Institute of Technology Institutional Animal Care and Use Committee. 

1. Preparation of Solutions

1. Prepare 20 L egg water (interchangeably used with ‘embryo medium’)  to maintain embryonic/larval zebrafish. Dissolve 1.5 g CaSO₄ and 6 g instant ocean sea salt in 250 ml distilled water. Pour into a carboy filled with 20 L distilled water and agitate.
2. Prepare 1 L 1-phenyl-2-thiourea (PTU) stock solution (20x). Dissolve 0.6 g PTU powder in 1 L distilled water. Add....

Results

Figure 1 shows the development of an EtOH-induced fibrotic liver model in larval zebrafish. To optimize a protocol for exposing zebrafish larvae to EtOH, we first assessed EtOH toxicity. 2.5 days-post-fertilization (dpf) larvae were exposed to EtOH concentration 1%, 1.5%, or 2% for 24 hr followed by a concurrent 24 hr EtOH/MTZ treatment. Exposure to 2% EtOH caused high mortality, while nearly all larvae exposed to 1% EtOH or less showed minimal fibrogenic changes with rar.......

Discussion

We observed HPC-mediated hepatocyte regeneration in the EtOH/MTZ-treated recovering livers, suggesting that even in the presence of substantial amount of ECM proteins including fibrillar type I collagen, the HPCs retain their competency to regenerate as hepatocytes. The MTZ only-treatment did not increase deposition of ECM proteins significantly, whereas the EtOH only-treatment did not induce HPC activation¹⁵. By utilizing the combined EtOH/MTZ treatment, we were able to investigate HPC-driven regeneration in .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Calcium sulfate hemihydrate (CaSO4)	Acros	AC385355000
Magnesium sulfate (MgSO4)	EMD	MX0075
1,4-Piperazinediethanesulfonic acid (PIPES)	Sigma-Aldrich	P6757
Ethylene glycol-bis(2-aminoethylether)- N,N,N′,N′-tetraacetic acid (EGTA)	Sigma-Aldrich	E3889
Ethanol	Sigma-Aldrich	E7023	200 proof
Formaldehyde	Fisher Scientific	F79-500
Metronidazole (MTZ)	Sigma-Aldrich	M3761
1-phenyl-2-thiourea (PTU)	Sigma-Aldrich	P7629
3-amino benzoic acid ethyl ester (Tricaine)	Sigma-Aldrich	A5040
Phosphate-buffered saline (PBS) tablet	Amresco	E404	Dissolve one tablet with 100 ml distilled water
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D2438
Bovine serum albumin	Fisher Scientific	BP1600
Triton X-100	Fisher Scientific	BP151
Low-melting agarose	Amresco	BP165
Stem Cell Signaling Compound Library	Selleck Chemicals	L2100	10 mM stock in DMSO
ActiProbe-1K Library	Timtec	ActiProbe-1K	10 mM stock in DMSO
SB 415286	Selleck Chemicals	S2729	Dissolve with DMSO to 10 mM
CHIR-99021	Selleck Chemicals	S2924	Dissolve with DMSO to 10 mM
Anti-Collagen I antibody	Abcam	ab23730	Use at 1:100 for immunostaining, reacts with fish
AlexaFluor 647 Donkey anti-rabbit IgG (H+L)	Molecular Probes	A31573	Use at 1:200 for immunostaining
Mounting media (Vectorshield)	Vector Laboratories	H-1400
100 mm Petri dish	VWR	25384-088
24-well plate	VWR	10062-896
Forceps	Fine Science Tools	11255-20	Dumont #55
Glass slide	VWR	48312-003	75x25 mm
Cover glass	VWR	48366-045	18 mm
Plastic wrap	Fisher Scientific	22305654
Aluminum foil	Fisher Scientific	1213100
Kimwipes	Kimberly-Clark	34155
Vibrotome	Leica	VT1000 S
Stereo microscope	Leica	M80
Epifluoresence microscope	Leica	M205 FA
Confocol microscope	Zeiss	LSM700