**3. The DDC model**

#### **3.1. The DDC model of biliary injury: pathophysiological mechanisms and DR phenotype**

The DDC model consists of *ad libitum* administration of a diet enriched with the porphyrinogenic agent 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) with normal water. Exposure to a DDC diet provokes the inhibition of the mitochondrial enzyme ferrochelatase, catalyst of the insertion of ferrous iron into protoporphyrin IX to form heme, leading to progressive accumulation of protoporphyrin. This brown pigment first accumulates in the cytoplasm of parenchymal cells and in Kupffer cells. Because of its hydrophobic nature, the excess of protoporphyrin can only exit the liver through biliary excretion, leading to precipitation of this poorly soluble molecule in bile canaliculi and bile ducts, forming crystals increasing in size and number [37]. After 3 weeks of DDC diet, accumulated pigments plug and obstruct the lumen of the smaller branches of the biliary tree and confer a dark coloration to the liver (**Figure 1C**). Bile ducts, usually recognizable as monolayer rings of small cuboidal cholangiocytes delineating a central lumen, show profound morphological alterations while hepatocytes have a normal appearance except for pigment coloration (**Figure 1H**). This indicates that the DDC dietary regimen mostly damage the biliary system, which is additionally supported by increased serum bilirubin (**Figure 1E**). We observed moderately elevated transaminases levels (two to threefold time, **Figure 1D**) although another group reports higher transaminase levels after DDC feeding [16]. In later stages, livers in DDC-fed mice develop pericholangitis and periductal onion skin-like fibrosis. Our discussion here analyzes DR morphology and microenvironment after 3 weeks of DDC feeding.

In the DDC livers, bile duct damage is associated with a biliary response in which dysmorphic cholangiocytes proliferate in the portal area. In all portal tracts, DR expands as multiple small pseudo-ducts arising next to the larger preexisting bile ducts (**Figure 1H**). These newly formed ductular structures are composed of small cuboidal or more cylindrical cells, irregular in size and shape, assembled in tube-like structures outlining a lumen in most cases, sometime plugged by porphyrin crystals. In contrast to infiltrating DR in CDE livers, DR expansion observed in DDC livers remains enclosed within the portal mesenchyme. No parenchymal invasion crossing the boundaries of portal mesenchyme was observed nor did those reactive cells, always observed as a cluster, adopt a phenotype supporting migration. However, the portal mesenchyme extends and may bridge distant portal spaces (**Figure 1H**). 3D biliary analysis of DDC livers identifies branches randomly directed around the portal vein, connected to the biliary tree but forming apparent distinct structures from the large-diameter bile ducts [9]. Moreover, 3D plastination of the DDC-fed mouse confirms slight focal dilatation of intrahepatic bile ducts and porphyrin plugs while biliary network seems to be less dense [16] (**Figure 1K**).

Finally, concerning LPC capacity of differentiation, upon DDC-induced injury, there is no evidence that cells of the neo ducts undergo hepatocytic cell differentiation [23, 28, 30]. When animals are reversed to a standard chow after DDC diet, the degree of DR expansion decreases, but still with no evidence that DR cells differentiate into hepatocytes. Because a specific LPC marker, that is, exclusively expressed in LPC and not in cholangiocytes, is lacking, we are currently unable to experimentally address the contribution of LPC to biliary regeneration *in vivo* during disease evolution. We can, however, hypothesize that, if not entirely supported by proliferation of mature cholangiocytes, LPC located at the most proximal part of the biliary tree contribute to neo duct formation in the DDC model [9].

### **3.2. The DDC model: practical aspects**

*2.2.2. Mouse variables*

fied animals.

**phenotype**

**3. The DDC model**

LPC response and morbidity vary according to weight and age of the mice at the time of introduction of the CDE diet. Mice above 25–30 g will be quite resistant to the diet and if they are too old, perhaps because of loss of cell plasticity, LPC response will be discrete. 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. 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. Thus, by respecting the simple rules proposed above, body weight loss may be limited to 10% of the starting body weigh during the first week with weight stabilization thereafter. Importantly, sensitivity to the dietary regimen and magnitude of liver damage and LPC reaction largely vary according to the genetic background of the mice [17]. This imposes the use of an appropriate control group (best being littermates) when comparing the effect of gene deletion or addition in genetically modi-

148 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

**3.1. The DDC model of biliary injury: pathophysiological mechanisms and DR** 

The DDC model consists of *ad libitum* administration of a diet enriched with the porphyrinogenic agent 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) with normal water. Exposure to a DDC diet provokes the inhibition of the mitochondrial enzyme ferrochelatase, catalyst of the insertion of ferrous iron into protoporphyrin IX to form heme, leading to progressive accumulation of protoporphyrin. This brown pigment first accumulates in the cytoplasm of parenchymal cells and in Kupffer cells. Because of its hydrophobic nature, the excess of protoporphyrin can only exit the liver through biliary excretion, leading to precipitation of this poorly soluble molecule in bile canaliculi and bile ducts, forming crystals increasing in size and number [37]. After 3 weeks of DDC diet, accumulated pigments plug and obstruct the lumen of the smaller branches of the biliary tree and confer a dark coloration to the liver (**Figure 1C**). Bile ducts, usually recognizable as monolayer rings of small cuboidal cholangiocytes delineating a central lumen, show profound morphological alterations while hepatocytes have a normal appearance except for pigment coloration (**Figure 1H**). This indicates that the DDC dietary regimen mostly damage the biliary system, which is additionally supported by increased serum bilirubin (**Figure 1E**). We observed moderately elevated transaminases levels (two to threefold time, **Figure 1D**) although another group reports higher transaminase levels after DDC feeding [16]. In later stages, livers

Contrasting with the CDE model, the DDC model is robust and reproducible and has little impact on animal welfare. In all studies, diet (standard rodent chow) is supplemented with 0.1% (wt/wt) of DDC. Similarly, the different mouse strains tested so far develop comparable hepatic phenotype to DDC feeding [16] although differences in susceptibility and kinetics of the response might be expected according to strains. Of note, DDC diet applied to rats does not induce any LPC response [38].
