Pathogenesis of Liver Fibrosis and Regeneration in the Japanese Medaka (Oryzias latipes)

Animal models of human diseases are essential to elucidate the molecular mechanisms of disease and to develop and evaluate diagnostic and therapeutic approaches. Fish are attractive model organisms due to the easy and economical maintenance, large number of embryos produced, external development of transparent embryos, low background incidence of neoplasia, well annotated genome, availability of inbred strains and established techniques to manipulate gene expression and create mutant lines. Consequently, it is not surprising that fish are increasingly used in biomedical research, high-throughput screening of chemicals, and environmental monitoring. It is known that notable mechanistic differences in disease pathogenesis often exist between humans and the relevant animal models used. These differences do not preclude using a species as a model of human disease but it is important to know and understand these differences and the limitations/special characteristics that apply. In fact, the degree of similarity of fish models have with appropriate mouse models and humans has become a critical issue. Although it is established that certain aspects of tissue and cellular organization, reaction to injury, and neoplasms are remarkably similar histologically between humans and fish, little is known regarding the specific molecular mechanisms leading to liver pathologies in piscine models. In this dissertation, the medaka fish was used as a model for investigating liver fibrosis and regeneration following injury.;In chapter 2, we developed a dimethylnitrosamine (DMN)-induced fish model of hepatic injury in Japanese Medaka (Oryzias latipes) and anchored expression of key genes involved in the pathogenesis of fibrosis with the development of hepatic fibrosis and neoplasia. We demonstrated that the main cellular and molecular events in the pathogenesis of hepatic fibrosis in mammals and medaka fish exposed to DMN are conserved. Hepatocellular injury is followed by activation of hepatic stellate cells, TGF-β pathway activation, change in the balance between matrix metalloproteinases and tissue inhibitors of metalloproteinases and increase in collagen production with the end result of excessive deposition of collagenous extracellular matrix. These data also confirm the medaka as a useful animal model of hepatic fibrosis. In chapter 3, the immunohistochemical and ultrastructural characteristics of the putative piscine oval cell/progenitor cell compartment after acute and chronic toxic hepatic injury were determined in order to characterize the cellulars lineages and differentiation processes in medaka. The study demonstrated that fish oval cells/hepatic progenitor cells (HPC) shared similar morphology and immunoreactivity with rodent oval cells and human HPCs, and had similar bipotential lineage pathways. This provided additional evidence of the striking morphological similarities in the hepatic regenerative process between fish and mammals. In chapter 4, a label retention cell assay was performed to identify the location of the HPCs in the Japanese Medaka liver. Labeling was performed either during liver development in embryos and physiological growth was used to dilute the label in hepatic cells, or following acute hepatic necrosis in adult medaka and the subsequent hepatic regeneration phase was used to wash out the label. Phenotypic characteristics, location and double immunolabelling were used to confirm the identity of the BrdU retaining cells. Label retention was observed in three possible HPC niches: hepatocytes, bile preductular epithelial cells, and cholangiocytes. These data demonstrated that the process of hepatic regeneration is a complex system relying on multiple HPC niches as observed in mice.;Collectively, the results of our investigations improve comparative understanding of the liver’s response to chronic injury across taxa and demonstrate the utility of the medaka model for hepatic injury studies.