**2. The CDE model**

when there is a massive cell loss or a continuous damage to mature hepatic cells, overwhelming the replicative capacity of the remaining hepatocytes, expansion of immature-like cells is observed at the interface between the portal area and the parenchyma in a process called ductular reaction (DR). Expression of biliary markers is a hallmark of DR cells, but nevertheless, DR constitutes a heterogeneous population of proliferating cells ranging from immature stem-like cells to more committed cells with an intermediate hepatobiliary phenotype [1–4]. Cells of the DR are also called liver progenitor cells (LPC) as they have been shown to differentiate into both hepatocytes and cholangiocytes lineages in culture (reviewed in Ref. [5]). In normal livers, no DR are usually observed and LPC are seen, in two-dimensional tissue sections, as single cells located mainly in the canal of Hering, which represents the connection between the smallest ramifications of the biliary tree and the hepatocyte canalicular system [6, 7]. DR/LPC and biliary cells cannot strictly be distinguished at the histological level but based on their location and morphological differences [8]. In a three-dimensional viewpoint, DR and the biliary tree constitute together a contiguous heterogeneous epithelial structure [9]. In humans as in rodents, the histological and morphological patterns of DR vary according to injurious settings and their lineage commitment toward hepatocytes or cholangiocytes has

Over the past decade, there has been a considerable interest in understanding DR/LPC biology. LPC are indeed seen as a potential reservoir for mature hepatocytes. Understanding the nature and differentiation process of LPC may generate cells for liver-cell therapy, which is increasingly under demand due to organ shortage for liver transplantation. Moreover, DR has also been postulated to trigger portal fibrosis [11]. Unraveling the potential mediators of DR could therefore be of great interest to modulate progression of profibrogenic reaction observed in

Several rodent models of liver injury associate with a DR and are instrumental to study the LPC response and its implication in liver regeneration and wound healing. These models, as in human liver diseases, exhibit a large variety of DR/LPC patterns with different morphological features, kinetics of response, and differentiation potential. The models of liver injury with DR generally combine the damage and loss of epithelial cells (hepatocytes and/or cholangiocytes) with the inhibition of the proliferative capacity (replicative senescence) of mature epithelial cells. Toxins [12, 13], carcinogens [14, 15], or modified diets [16, 17] have been used to induce cell injury, either alone or associated with surgical removal of part of the liver to amplify liver cell depletion. Ethionine, 2-acetylaminofluorene (AAF), and retrorsine are used to block the ability of mature epithelial cells to divide and prevent them from contributing to the liver regeneration process. In mice, dietary manipulations are regarded as convenient, efficient, and reproducible models to induce a robust DR, without need for animal handling, repeated injections, or surgical manipulation. The two most popular dietary DR models are a cholinedeficient diet supplemented with ethionine in the drinking water (CDE) or a diet enriched in

In the literature, the DDC and CDE models are often used equivalently to study the LPC response and their role in tissue repair. However, DR in those two models exhibits major

been related to the primary site of cell loss or dysfunction [10].

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

many chronic liver diseases.

3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC).

#### **2.1. The CDE model of hepatocellular injury: pathophysiological mechanisms and DR phenotype**

The CDE model consists of *ad libitum* administration of a choline-deficient diet together with procurement of ethionine in the drinking water. Choline is provided by food intake and contributes to the structural integrity and signaling function of cell membranes. A choline withdrawal leads to a decreased synthesis of phosphatidylcholine, a phospholipid crucial for cell membrane and a major building stone of the very low-density lipoprotein particles produced by hepatocytes to export triglycerides. Choline deficiency causes intracytoplasmic fat accumulation, hepatocyte dysfunction, and cell damage [18, 19]. Such (extensive) hepatocellular damage results in high hepatocyte replication ratio, causing their exhaustion and restraining the production of hepatic drug metabolism-related enzymes [20]. Ethionine, a synthetic amino acid, specifically targets the hepatocytes in which, when provided in large excess, it competes with its naturally occurring analog methionine. Competition of ethionine with methionine favors the synthesis of S-adenosyl ethionine (SAE) instead of S-adenosyl methionine (SAM). Consequently, an ethyl group is transferred instead of a methyl group in methylation reactions hereby generating abnormal proteins, lipids, RNA, and DNA molecules, which results in hepatocytic cell damage [21]. Prolonged feeding with ethionine produces liver tumors with extensive LPC proliferation [22]. However, administration of ethionine in supplement to a choline-deficient diet greatly shortens the time required for LPC proliferation [22]. Although, the exact mechanism of action of CDE-induced injury is not well known, it appears that the combined administration of ethionine with choline-deficient chow induces a liver injury in which the hepatocytes are specifically targeted and the replication of the surviving hepatocytes is inhibited [23]. Hepatocyte proliferation to replace damaged liver cells is prevented and activation of the LPC compartment ensues. Several publications characterized the kinetics of the LPC response and liver damage to CDE [24–27]. Briefly, short-term CDE feeding results in steatosis, inflammation, LPC expansion (DR), and fibrosis that progress in parallel. Cirrhosis and hepatocellular carcinoma may be observed in long-term studies. We intend to describe and analyze in depth the morphology and differentiation capacity of DR after 3 weeks of CDE (except when specified otherwise), at a time when pathological damages are installed and DR robustly established.

After 3 weeks, CDE livers are pale with signs of steatosis throughout the parenchyma. Liver weight is comparable or slightly lower than the deep-red control livers (**Figure 1A** and **B**). Signs of hepatocellular injury are observed with necrotic and apoptotic hepatocytes while bile ducts appear normal within the portal triad (**Figure 1G**) [23, 28]. Also, serum alanine aminotransaminases are increased while bilirubin levels are in the near normal range, indicative of hepatocytic damage (**Figure 1D** and **E**).

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. On 2D liver sections, they are found as individual cells, grouped in multifocal clusters or organized in a single or double row of cells forming arborizing structures (**Figure 1**) [24]. Architectural three-dimensional (3D) analysis of the biliary tree remodeling in response to CDE reveal that DR are connected to the preexisting bile ducts and that biliary branches intricately split around the portal vein with a random directionality [9]. Moreover, plastination of the bile duct system reveal a denser biliary network after CDE feeding (**Figure 1J**).

Finally, with regard to LPC capacity of differentiation *in vivo*, DR cell-tracking experiments using different transgenic mouse models [23, 28, 30] indicate that, upon CDE diet, a small number of DR cells do differentiate into hepatocytes: in the process LPC lose biliary markers, grow in size, and acquire mature hepatocyte morphological features and functional proteins. Although differentiation is consistently reported, only few DR-derived hepatocytes are reported in this model (<2.5% of hepatocytes).

#### **2.2. The CDE model: practical aspects**

Although being widely used, the CDE model is difficult to handle and researchers are confronted with difficulties and ethical issues due to variability in the LPC response, well-being of the animals, morbidity, and mortality. Here, we will review several factors influencing the LPC response to the model. These parameters must be taken into account and controlled to strengthen the model and provide reproducibility.

Mice are not fond of ethionine (due to bad smell), and usually decrease water consumption. 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, 32–34]. 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. 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].

biliary network after CDE feeding (J), and dilatation of intrahepatic bile ducts in DDC-fed mice (K).

**Figure 1.** Pathophysiological mechanisms and DR phenotype in the CDE and DDC models. Livers retrieved from control mice (A), mice receiving the CDE (B), or DDC diet (C) for 3 weeks. Serum biochemical measurements for total aminotransferase (ALT) (D) and bilirubin (E) showed increased ALT and near normal bilirubin in the CDE model and increased serum bilirubin with slightly elevated ALT levels in DDC, indicative of hepatocellular damage upon CDE treatment and of primarily biliary injury after DDC diet. Liver sections stained with anticytokeratin 19 (CK19) in control (F), CDE (G), and DDC (H) livers after 3 weeks of diet. In control, CK19 staining reveals bile ducts and LPC as isolated cells close to the periportal tract. In CDE livers, besides bile ducts, DR CK19+ cells are strongly increased in number, forming cells organized in filaments expanding inside the lobule. After DDC feeding, in addition to the larger preexisting bile ducts, CK19+ newly formed DR structures are composed of small cuboidal cells, irregular in size and shape accumulated around the portal area. Plastination of the bile duct system reveals delicately structured biliary tree in control mice (I), a denser

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#### *2.2.1. Dietary variables*

Rodent food manufacturers can easily provide food in which the choline content is strictly controlled. Although low choline dietary content could be used [31], we will describe here a model using dietary choline deficiency. The second parameter to adjust for is ethionine supplementation. In the literature, the amount of ethionine in the water varies from 0.05 to 0.165% (wt/vol). Relevance of the CDE and DDC Mouse Models to Study Ductular Reaction in Chronic Human... http://dx.doi.org/10.5772/intechopen.69533 147

in parallel. Cirrhosis and hepatocellular carcinoma may be observed in long-term studies. We intend to describe and analyze in depth the morphology and differentiation capacity of DR after 3 weeks of CDE (except when specified otherwise), at a time when pathological damages

After 3 weeks, CDE livers are pale with signs of steatosis throughout the parenchyma. Liver weight is comparable or slightly lower than the deep-red control livers (**Figure 1A** and **B**). Signs of hepatocellular injury are observed with necrotic and apoptotic hepatocytes while bile ducts appear normal within the portal triad (**Figure 1G**) [23, 28]. Also, serum alanine aminotransaminases are increased while bilirubin levels are in the near normal range, indicative of

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. On 2D liver sections, they are found as individual cells, grouped in multifocal clusters or organized in a single or double row of cells forming arborizing structures (**Figure 1**) [24]. Architectural three-dimensional (3D) analysis of the biliary tree remodeling in response to CDE reveal that DR are connected to the preexisting bile ducts and that biliary branches intricately split around the portal vein with a random directionality [9]. Moreover, plastination of the bile duct system reveal a denser

Finally, with regard to LPC capacity of differentiation *in vivo*, DR cell-tracking experiments using different transgenic mouse models [23, 28, 30] indicate that, upon CDE diet, a small number of DR cells do differentiate into hepatocytes: in the process LPC lose biliary markers, grow in size, and acquire mature hepatocyte morphological features and functional proteins. Although differentiation is consistently reported, only few DR-derived hepatocytes are

Although being widely used, the CDE model is difficult to handle and researchers are confronted with difficulties and ethical issues due to variability in the LPC response, well-being of the animals, morbidity, and mortality. Here, we will review several factors influencing the LPC response to the model. These parameters must be taken into account and controlled to

Rodent food manufacturers can easily provide food in which the choline content is strictly controlled. Although low choline dietary content could be used [31], we will describe here a model using dietary choline deficiency. The second parameter to adjust for is ethionine supplementation. In the literature, the amount of ethionine in the water varies from 0.05 to 0.165% (wt/vol).

are installed and DR robustly established.

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

hepatocytic damage (**Figure 1D** and **E**).

biliary network after CDE feeding (**Figure 1J**).

reported in this model (<2.5% of hepatocytes).

strengthen the model and provide reproducibility.

**2.2. The CDE model: practical aspects**

*2.2.1. Dietary variables*

**Figure 1.** Pathophysiological mechanisms and DR phenotype in the CDE and DDC models. Livers retrieved from control mice (A), mice receiving the CDE (B), or DDC diet (C) for 3 weeks. Serum biochemical measurements for total aminotransferase (ALT) (D) and bilirubin (E) showed increased ALT and near normal bilirubin in the CDE model and increased serum bilirubin with slightly elevated ALT levels in DDC, indicative of hepatocellular damage upon CDE treatment and of primarily biliary injury after DDC diet. Liver sections stained with anticytokeratin 19 (CK19) in control (F), CDE (G), and DDC (H) livers after 3 weeks of diet. In control, CK19 staining reveals bile ducts and LPC as isolated cells close to the periportal tract. In CDE livers, besides bile ducts, DR CK19+ cells are strongly increased in number, forming cells organized in filaments expanding inside the lobule. After DDC feeding, in addition to the larger preexisting bile ducts, CK19+ newly formed DR structures are composed of small cuboidal cells, irregular in size and shape accumulated around the portal area. Plastination of the bile duct system reveals delicately structured biliary tree in control mice (I), a denser biliary network after CDE feeding (J), and dilatation of intrahepatic bile ducts in DDC-fed mice (K).

Mice are not fond of ethionine (due to bad smell), and usually decrease water consumption. 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, 32–34]. 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. 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].

#### *2.2.2. Mouse variables*

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 modified animals.

in DDC-fed mice develop pericholangitis and periductal onion skin-like fibrosis. Our discussion

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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

here analyzes DR morphology and microenvironment after 3 weeks of DDC feeding.

and porphyrin plugs while biliary network seems to be less dense [16] (**Figure 1K**).

tree contribute to neo duct formation in the DDC model [9].

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

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

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].

**4. Microenvironment-regulating DR expansion and differentiation**

microenvironment accompanying DR in the CDE versus DDC model.

The literature brings every day new evidence that the orchestrated interplay between proliferating hepatocytes or cholangiocytes, extracellular matrix-producing myofibroblasts, inflammatory cells (such as macrophages, neutrophils, or lymphocytes), and endothelial cells is pivotal in the regulation of DR expansion and differentiation. We will thus compare the
