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

**Relevance of the CDE and DDC Mouse Models to Study** 

DOI: 10.5772/intechopen.69533

Laure-Alix Clerbaux, Noémi Van Hul, Annette S.H. Gouw, Rita Manco, Regina Español-Suñer and Isabelle A. Leclercq Annette S.H. Gouw, Rita Manco, Regina Español-Suñer and Isabelle A. Leclercq

Laure-Alix Clerbaux, Noémi Van Hul,

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69533

#### **Abstract**

The liver has the remarkable capacity to regenerate through cellular division of hepatocytes. However, following severe injuries that abrogates the replicative capacity of hepatocytes some immature-like cells proliferate around the portal area and invade the parenchyma in a process known as ductular reaction (DR). In humans, DR is observed in virtually all chronic liver disorders although the morphological patterns may vary. DR biology has gained considerable interest because of potential contribution to hepatic cell restoration, fibrosis or carcinogenesis. In humans, observational studies are available but experimental manipulations and lineage tracing are impossible. Animal models represent thus valuable tools to explore such questions. Feeding rodents a choline-deficient, ethionine-supplemented diet (CDE) or a diet enriched in 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC) are the most popular models to study DR. They are often used equivalently in the literature although the aspects and outcome of the DR are different and model-specific. Here, we describe experimental procedures and the pathophysiological mechanisms at play; we describe the hepatic lesions and highlight the unique character of DR phenotype, proliferation, lineage commitment and microenvironment in each model. We then compare the models with DR phenotype in human pathologies.

**Keywords:** liver progenitor cells, ductular reaction, CDE, DDC

### **1. Introduction**

In a healthy liver, hepatocytes are quiescent long-lived cells. Upon mild to moderate hepatocellular injury or depletion, hepatocytes self-duplicate to restore the liver mass. However,

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© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

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

etiological and phenotypical differences. Such differences recapitulate the specificity of the pathophysiological responses to distinct injurious processes. DR activation, expansion, and capacity for differentiation are dictated by the nature of the cellular injury and by the differ-

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

http://dx.doi.org/10.5772/intechopen.69533

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In this chapter, we will first describe the pathophysiological mechanisms at play in each model and the experimental procedures to induce DR with the CDE or DDC diet. A description of the hepatic lesions in terms of the cellular compartment injured after CDE and DDC feeding and highlight of the unique character of each model with regard to the DR phenotype, proliferation, lineage commitment, and microenvironment will be explored. Finally, the relevance of these models to study and understand the diversity of DR seen in human chronic

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

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

ential microenvironment changes.

liver diseases will be addressed.

**2. The CDE model**

**phenotype**

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 many chronic liver diseases.

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

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 etiological and phenotypical differences. Such differences recapitulate the specificity of the pathophysiological responses to distinct injurious processes. DR activation, expansion, and capacity for differentiation are dictated by the nature of the cellular injury and by the differential microenvironment changes.

In this chapter, we will first describe the pathophysiological mechanisms at play in each model and the experimental procedures to induce DR with the CDE or DDC diet. A description of the hepatic lesions in terms of the cellular compartment injured after CDE and DDC feeding and highlight of the unique character of each model with regard to the DR phenotype, proliferation, lineage commitment, and microenvironment will be explored. Finally, the relevance of these models to study and understand the diversity of DR seen in human chronic liver diseases will be addressed.
