Mouse model of chronic and binge ethanol feeding (the NIAAA model)

Abstract

Chronic alcohol consumption is a leading cause of chronic liver disease worldwide, leading to cirrhosis and hepatocellular carcinoma. Currently, the most widely used model for alcoholic liver injury is ad libitum feeding with the Lieber-DeCarli liquid diet containing ethanol for 4-6 weeks; however, this model, without the addition of a secondary insult, only induces mild steatosis, slight elevation of serum alanine transaminase (ALT) and little or no inflammation. Here we describe a simple mouse model of alcoholic liver injury by chronic ethanol feeding (10-d ad libitum oral feeding with the Lieber-DeCarli ethanol liquid diet) plus a single binge ethanol feeding. This protocol for chronic-plus-single-binge ethanol feeding synergistically induces liver injury, inflammation and fatty liver, which mimics acute-on-chronic alcoholic liver injury in patients. This feeding protocol can also be extended to chronic feeding for longer periods of time up to 8 weeks plus single or multiple binges. Chronic-binge ethanol feeding leads to high blood alcohol levels; thus, this simple model will be very useful for the study of alcoholic liver disease (ALD) and of other organs damaged by alcohol consumption.

Figure 1

Overview of the NIAAA model procedure. Mice are initially fed the control Lieber-DeCarli diet ad libitum for 5 d to acclimatize them to liquid diet and tube feeding. Afterward, ethanol (EtOH)-fed groups are allowed free access to the ethanol Lieber-DeCarli diet containing 5% (vol/vol) ethanol for 10 d, and control groups are pair-fed with the isocaloric control diet. At day 11, ethanol-fed and pair-fed mice are gavaged in the early morning with a single dose of ethanol (5 g kg⁻¹ body weight) or isocaloric maltose dextrin, respectively, and euthanized 9 h later. This model can be extended to longer periods of chronic feeding (up to 8 weeks) plus single or multiple binges. All of the animal experiments were approved by the NIAAA Animal Care and Use Committee.

Figure 2

Cage and feeding-tube setup. (a,b) Photos showing modified mouse cages. A hole is drilled at the bottom of the front side of the cage to insert the mouth of the feeding tube into the cage. The feeding tube is held using a rubber band tied to steel wire hooks. (c,d) Photos showing commercial feeding-tube holders designed to hang over the lip of the cage. Note that it is much easier to change the feeding tubes in the modified mouse cages (a,b) than those in tube holders (c,d). Perforations made in cages must be consistent in their location to ensure that the diet in the feeding tube remains accessible after placement.

Figure 3

Oral gavage. (a) Measurement of the length of the gavage needle against the animal’s body. (b,c) Administration of the solution upon verification of proper placement of the gavage needle. All of the animal experiments were approved by the NIAAA Animal Care and Use Committee.

Figure 4

Body weight changes during the chronic-plus-binge ethanol feeding model. (a) Evolution of body weight of C57BL/6J female mice (n = 17) during the course of the chronic-plus-single-binge ethanol feeding. (b) Body weight of pair-fed (n = 7) and ethanol-fed (n = 34) C57BL/6 female mice was measured on the first day of feeding (initial), after the 5-d acclimatization period and at day 11 before gavage (final). Data represent means ± s.e.m. All of the animal experiments were approved by the NIAAA Animal Care and Use Committee.

Figure 5

Hepatic expression of CYP2E1 and plasma ethanol levels post ethanol feeding. (a) Representative immunostaining of CYP2E1 in the liver tissues from 10-d pair-fed and ethanol-fed mice (chronic feeding without binge). Scale bars, 200 µm. (b) Mice were fed the ethanol diet for 10 d. At day 11, blood was collected in the early morning and plasma ethanol concentration was measured (chronic without binge, 0 h after gavage). The mice were then gavaged with 5 g kg⁻¹ ethanol, and plasma ethanol concentration was measured 1 and 2 h after gavage. Data represent mean ± s.e.m. All of the animal experiments were approved by the NIAAA Animal Care and Use Committee.

Figure 6

Comparison of liver injury induced by the chronic-plus-single-binge ethanol feeding model and other feeding models. C57BL/6 mice were subjected to 4 weeks of chronic feeding alone, 4–6 weeks of chronic feeding plus multiple binges (twice a week during the 4–6 week feeding) and 10 d of chronic feeding plus a single binge. Mice were euthanized 9 h after gavage. Liver injury was assessed by measuring serum ALT and AST levels. Numbers in parentheses in graphs denote the number of mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t test). Note that chronic feeding (4–6 weeks) plus multiple binges (twice a week during the 4–6 week feeding), which was associated with a mortality rate of 10–40% in male C57BL/6N mice, was not tested in female C57BL/6 mice because a higher mortality rate is expected. The values in this figure were obtained from multiple independent experiments. All of the animal experiments were approved by the NIAAA Animal Care and Use Committee.