Establishment of a zebrafish model of alcohol-induced acute hepatic steatosis

Objective To establish a zebrafish model of hepatic steatosis induced by acute alcohol exposure as a model system that will be useful for investigations of disease pathogenesis and high-throughput screenings.Methods At 5 days post fertilization (dpf) , 288 juvenile zebrafish (wild-type strain AB) with normal liver development were selected and randomly divided into four groups (n=72 each) for culturing in water and Hank′s balanced salt solution alone (controls) or supplemented with ethanol at 2.0%, 2.5% and 3.0% concentrations.At 4, 24 and 32 h of culturing, the physical activity (swimming, circling) was observed, the survival rate was recorded, and the morphology was assessed by whole-mount and liver section microscopy.Oil Red O staining was used to determine the percent of steatosis and pathological features were assessed by hematoxylin-eosin staining.Differences between two groups were assessed by independent samples t-test.Results Starting at 24 h of ethanol exposure, the survival rates showed a decreasing trend that corresponded to increased alcohol concentration, so that by 32 h of exposure there were remarkable differences among the three alcohol-treated groups: 2.0% ethanol: 100% survival;2.5% ethanol: (91.21±1.61) % survival;and 3.0% ethanol: 0% survival.Abnormalities in liver morphology and physical activity were also observed at the 24 h time point and showed concentration-dependence: 2.0% ethanol: normal morphology, increased activity, abnormal circling;2.5% ethanol: some deformities;and 3.0% ethanol: bent spinal cord, pericardial and yolk sac edema, stationary behavior.Significant steatosis was observed in the 2.0% ethanol group at 32 h of exposure, but not at 24 h, as evidenced by the percent of steatosis[ (71.25±0.15) % vs.controls: (31.25±0.05) %, P=0.002] and pathological features (swollen size, lipid droplet-induced nucleus displacement) .Conclusion A model of acute alcohol-induced hepatic steatosis was successfully established in 5 dpf zebrafish by treating with 2.0% alcohol for 32 h.This model may represent a useful tool for investigating the disease pathogenesis and performing high-throughput screenings of potentially therapeutic drugs.