Chronic liver disease is a major global health problem, accounting for ∼1 million deaths per year worldwide (Tapper and Parikh, 2023; Younossi et al., 2023). It is associated with reduced quality of life and an increased risk of mortality from infection, bleeding and cancer (Tapper and Parikh, 2023; Younossi et al., 2023). Most chronic liver disease is due to metabolic dysfunction [metabolic dysfunction-associated steatotic liver disease (MASLD)] and/or alcohol use [alcohol-related liver disease (ALD)], both of which are characterized by abnormal accumulation of lipids – steatosis – within hepatocytes (Rinella et al., 2024). Animal models are critical for determining how and why this abnormal lipid deposition occurs, and for defining the interplay between lipids, inflammation and fibrosis that characterizes the progressive form of steatotic liver disease known as steatohepatitis. Zebrafish are emerging as a highly useful tool for identifying mechanisms of hepatic steatosis and steatohepatitis, given their small size, amenability to pharmacologic and genetic manipulation, and optical translucency, which allows for the use of fluorescent reporters and dyes to visualize internal structures such as lipid droplets without the need for tissue removal. Many protocols have been developed to analyze hepatic accumulation in zebrafish, but it can be challenging for researchers to determine which method is best suited to their specific experimental needs.
Sadler, Cox and colleagues created a comparative resource to enable researchers to make informed decisions regarding the methods used to analyze lipid droplets in zebrafish larval livers. The authors performed a side-by-side characterization of hepatic steatosis using Oil Red O (ORO), Nile Red (NR), LipidTox and LipidSpot staining, as well as a genetic fusion model based on fluorescent perilipin 2 (PLIN2), a protein closely associated with lipid droplets. They provide detailed protocols, thorough analysis of lipid droplet number and size with different staining methods, and succinct tables listing the stains’ excitation and emission spectra, advantages and limitations. The authors show that fluorescent staining methods (NR, LipidTox and LipidSpot) are more sensitive in staining lipid droplets than traditional ORO staining in fixed tissue. In addition, these fluorescent dyes are conducive to the acquisition of cellular resolution imaging, which can reveal further details about the distribution and function of lipid droplets within the hepatocyte. The genetic EGFP-PLIN2 model facilitates live imaging of lipid dynamics in the same animal over time.
Zebrafish have facilitated the new discovery of genes that regulate adiposity and hepatic steatosis, the identification of mechanisms involved in ALD, and the dissection of lipid metabolic pathways involved in liver tumorigenesis (Salmi et al., 2019). By providing a clear roadmap for imaging lipid droplets in zebrafish larvae, this work from Sadler, Cox and colleagues promises to streamline future research using this experimental system and facilitate new discoveries in the field of steatotic liver disease.
DMM Research or Resource articles of particular interest or excellence may be accompanied by a short Editor's choice highlight, selected by a DMM editor and written by either members of the DMM in-house editorial team or an expert in the field. The Editor's choice aims to outline the challenges that the work addresses and how the work advances our insight into disease mechanism, therapy or diagnosis.
