A modified choline-deficient, ethionine-supplemented diet reduces morbidity and retains a liver progenitor cell response

Abstract

The choline-deficient, ethionine-supplemented (CDE) dietary model induces chronic liver damage, and stimulates liver progenitor cell (LPC)-mediated repair. Long-term CDE administration leads to hepatocellular carcinoma in rodents and lineage-tracing studies show that LPCs differentiate into functional hepatocytes in this model. The CDE diet was first modified for mice by our laboratory by separately administering choline-deficient chow and ethionine in the drinking water (CD+E diet). Although this CD+E diet is widely used, concerns with variability in weight loss, morbidity, mortality and LPC response have been raised by researchers who have adopted this model. We propose that these inconsistencies are due to differential consumption of chow and ethionine in the drinking water, and that incorporating ethionine in the choline-deficient chow, and altering the strength, will achieve better outcomes. Therefore, C57Bl/6 mice, 5 and 6 weeks of age, were fed an all-inclusive CDE diet of various strengths (67% to 100%) for 3 weeks. The LPC response was quantitated and cell lines were derived. We found that animal survival, LPC response and liver damage are correlated with CDE diet strength. The 67% and 75% CDE diet administered to mice older than 5 weeks and greater than 18 g provides a consistent and acceptable level of animal welfare and induces a substantial LPC response, permitting their isolation and establishment of cell lines. This study shows that an all-inclusive CDE diet for mice reproducibly induces an LPC response conducive to in vivo studies and isolation, whilst minimizing morbidity and mortality.

Relevance of the CDE and DDC Mouse Models to Study Ductular Reaction in Chronic Human Liver Diseases

“…In parallel, if they are too young or too little (<15 g) at the time of dietary exposure, toxicity and ensuing mortality might be excessively high. With the administration of the CDE diet to mice of 6 weeks of age and with a body weight between 18 and 20 g, we and others show a substantial and reasonably reproducible LPC response while maintaining the well-being of the animals [24,28,29,35]. Mice are experiencing the most severe effects of the diet during the first week of administration.…”

“…This makes it difficult to control effective ethionine intake. Addition of 5% sucrose, choline-free orange juice, or fruit syrup is sometimes used to increase the attractiveness of the drink and this is most of the time not reported in the experimental protocol [9,23,28,29,[32][33][34]. Ethionine smell increases with exposure to the ambient air and we found that we could maintain stable water intake by replacing ethionine-containing water by a fresh solution every day.…”

“…In the CDE model, DR expansion, seen on two-dimensional (2D) sections by staining with a biliary marker such as cytokeratin (CK) 19, is observed arising from the portal area and invading progressively the parenchyma ( Figure 1G). First observable after approximately 1 week of CDE feeding, the DR progressively amplifies to a maximum around 3-4 weeks [23,24,29]. DR cells are small cells with a high nuclear-to-cytoplasm ratio, usually uniform in size with a fusiform shape and oval nuclei.…”

“…Although this sounds trivial, control over ethionine solution consumption is crucial as both variation in ethionine intake and (severe) dehydration may influence LPC response and induce large interindividual variation in the model. To circumvent this, Passman et al also propose to include ethionine in the chow [29]. Pathophysiological mechanisms and DR phenotype in the CDE and DDC models.…”

“…Mice are experiencing the most severe effects of the diet during the first week of administration. Following the first few days, significant weight loss is observed, often associated with mortality [29,36]. Approximately 1 week after the onset of the CDE treatment, the mice adapt, regain weight, and show (normal) physical activity and behavior.…”