Development of a new, simple rat model of early alcohol-induced liver injury based on sensitization of kupffer cells
✍ Scribed by Nobuyuki Enomoto; Shunhei Yamashina; Hiroshi Kono; Peter Schemmer; Chantal A. Rivera; Ayako Enomoto; Teruhiro Nishiura; Tetsuro Nishimura; David A. Brenner; Dr. Ronald G. Thurman
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 898 KB
- Volume
- 29
- Category
- Article
- ISSN
- 0270-9139
No coin nor oath required. For personal study only.
✦ Synopsis
The continuous intragastric in vivo enteral feeding model in the rat developed by Tsukamoto and French has been very useful; however, it requires surgical expertise. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hours later. Accordingly, these experiments were designed to determine if a new, simple animal model of ethanol hepatotoxicity could be developed based on Kupffer cell sensitization. Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hours intragastrically. Livers were stained with hematoxylin-eosin to assess steatosis, inflammation, and necrosis, and tissue triglycerides, serum transaminases, and plasma endotoxin were measured. Kupffer cells were isolated 0 to 24 hours after one intragastric dose of ethanol daily, and intracellular Ca 2؉ ([Ca 2؉ ] i ) was measured using fura-2, while tumor necrosis factor ␣ (TNF-␣) was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western and Northern analysis. Ethanol caused steatosis, necrosis, and inflammation in only a few weeks, and after 8 weeks, serum aspartate transaminase (AST) levels were doubled. Values were similar to levels achieved in the enteral feeding model.
Triglycerides were also increased significantly by ethanol as expected, and endotoxin levels were increased to 70 to 80 pg/mL. This latter increase was prevented (F20 pg/mL) by antibiotics implicating endotoxin. In isolated Kupffer cells from untreated control rats, [Ca 2؉ ] i increased to 82 ؎ 7 nmol/L after addition of lipopolysaccharide (LPS) (100 ng/mL), and levels were elevated about twofold by ethanol given 24 hours earlier ( 174؎ 15 nmol/L). In addition, TNF-␣ production by Kupffer cells was increased fourfold in cells isolated from rats treated with ethanol 24 hours earlier. Sterilization of the gut with antibiotics blocked all effects of ethanol on [Ca 2؉ ] i and TNF-␣ release completely. Moreover, 4 weeks after ethanol, CD14 in Kupffer cells was elevated about twofold. A new, simple chronic model of ethanol hepatotoxicity has been developed here based on sensitization of Kupffer cells to endotoxin. (HEPATOLOGY 1999;29:1680-1689.)
Although it is well known that alcoholic liver disease results from the dose-and time-dependent consumption of ethanol, mechanisms remain unclear. For a long time, a significant drawback for research in this area was the lack of appropriate animal models. For example, feeding ethanol chronically with the diet causes only fatty liver. 1 The establishment of a continuous intragastric in vivo enteral feeding model in the rat by Tsukamoto and French represented a major development in this area. 2,3 With this model, it has been shown that inactivation of Kupffer cells with gadolinium chloride (GdCl 3 ) prevented early alcohol-induced liver injury. 4 Moreover, intestinal sterilization with antibiotics (polymyxin B and neomycin) 5 or decreasing endotoxin with lactobacillus 6 prevented alcohol-induced liver injury. These experiments pointed to a role for activation of Kupffer cells by gut-derived endotoxin as a primary event in mechanisms of alcohol-induced liver injury. Although this chronic enteral ethanol model has been very useful, it is both timeconsuming and expensive, and requires surgical expertise.
Recently, we found that Kupffer cells isolated from rats treated once with ethanol 24 hours earlier exhibited enhanced sensitivity to lipopolysaccharide (LPS) as a result of up-regulation of the endotoxin receptor, CD14. 7 Furthermore, Su et al. 8 showed that message for the LPS/LPS binding protein receptor CD14 was up-regulated in Kupffer cells from rats exposed to chronic ethanol using the Tsukamoto-French enteral delivery model. Moreover, female rats had higher endotoxin levels after alcohol and exhibited greater susceptibility to early alcohol-induced liver injury than males. 9 Accordingly, the purpose of this study was to determine if a new, simple model of early ethanol-induced liver injury could be developed based on sensitization of Kupffer cells to gut-derived endotoxin by daily intragastric administration of ethanol to female rats.
MATERIALS AND METHODS
Animals and Treatments.
Female Wistar rats weighing between 200 and 250 g were fed ad libitum a liquid diet (Dyets, Bethlehem, PA; #710270) in which 35% of the calories were from corn oil and 47% Abbreviations: LPS, lipopolysaccharide; AST, aspartate transaminase; ALT, alanine transaminase; [Ca 2ϩ ] i , intracellular Ca 2ϩ ; TNF-␣, tumor necrosis factor ␣.