Development of a new, simple rat model of early alcohol-induced liver injury based on sensitization of Kupffer cells

The continuous intragastric in vivo enteral feeding model in the rat develo ped by Tsukamoto and French has been very useful; however, it requires surg ical expertise. Recently, we found that Kupffer cells isolated from rats tr eated only once with ethanol were sensitized to endotoxin 24 hours later. A ccordingly, these experiments were designed to determine if a new, simple a nimal model of ethanol hepatotoxicity could be developed based on Kupffer c ell sensitization. Female Wistar rats were given ethanol (5 g/kg body weigh t) once every 24 hours intragastrically. Livers were stained with hematoxyl in-eosin to assess steatosis, inflammation, and necrosis, and tissue trigly cerides, serum transaminases, and plasma endotoxin were measured. Kupffer c ells were isolated 0 to 24 hours after one intragastric dose of ethanol dai ly, and intracellular Ca2+ ([Ca2+](i)) was measured using fura-2, while tum or necrosis factor alpha (TNF-alpha) was measured by enzyme-linked immunoso rbent assay. CD14 was evaluated by Western and Northern analysis. Ethanol c aused steatosis, necrosis, and inflammation in only a few weeks, and after 8 weeks, serum aspartate transaminase (AST) levels were doubled. Values wer e similar to levels achieved in the enteral feeding model. Triglycerides we re also increased significantly by ethanol as expected, and endotoxin level s were increased to 70 to 80 pg/mL. This latter increase was prevented (<20 pg/mL) by antibiotics implicating endotoxin. In isolated Kupffer cells fro m untreated control rats, [Ca2+](i) increased to 82 +/- 7 nmol/L after addi tion of lipopolysaccharide (LPS) (100 ng/mL), and levels were elevated abou t twofold by ethanol given 24 hours earlier (174 +/- 15 nmol/L). In additio n, TNF-alpha production by Kupffer cells was increased fourfold in cells is olated from rats treated with ethanol 24 hours earlier. Sterilization of th e gut with antibiotics blocked all effects of ethanol on [Ca2+](i) and TNF- alpha release completely. Moreover, 4 weeks after ethanol, CD14 in Kupffer cells was elevated about twofold. A new, simple chronic model of ethanol he patotoxicity has been developed here based on sensitization of Kupffer cell s to endotoxin.