Model of toxic fibrosis in Wistar rats: morphological and molecular-genetic parameters of the transition point to cirrhosis

Abstract

BACKGROUND: To date, when studying the cellular and molecular genetic mechanisms of liver fibrogenesis in experimental rat models, no attention is paid to the transition point of fibrosis to cirrhosis as a separate stage. As this pathology develops, changes in the cell phenotype and gene expression are dynamic in nature, so it is necessary to evaluate them in a long-term temporal dynamics.

AIM: The aim of this work is to study the morphological and molecular genetic changes in the liver of Wistar rats during nodular parenchymal rearrangement.

METHODS: Fibrosis and cirrhosis of the liver in male Wistar rats was induced with a freshly prepared solution of thioacetamide, which was administered intragastrically through a tube at a dose of 200 mg/kg of body weight 2 times a week for 13 weeks. The level of mRNA of the tweak (tnfsf12), fn14 (tnfrsf12a), ang, vegfa, cxcl12 (sdf), and mmp-9 genes in the liver was detected by real-time polymerase chain reaction. Immunohistochemical study was performed on paraffin sections. α-SMA, FAP, CD68, CD206, CX3CR1, CD45 were used as markers. The area of interlobular veins and interlobular arteries was measured (µm²). The number of sinusoidal capillaries and interlobular veins was counted.

RESULTS: Based on the results obtained, it is possible to establish the transition point of fibrosis to cirrhosis as an independent separate stage of fibrogenesis. This transition was identified at stage F5, and the process itself — from F4/F5 to F6.

With the growth of fibrous tissue and nodular restructuring of the liver parenchyma, no progression of dystrophic processes and an increase in the zones of necrosis and necrobiosis of hepatocytes were noted. The number of α-SMA⁺ and FAP⁺ cells in the period F4–F5 did not change (p=0.2073 and p=0.3775, respectively). At the same time, significant F6 cirrhosis was accompanied by an increase in their number by 1.5 times (p <0.00001). Differences in the number of CD68⁺ cells were revealed only at the F4/F5 stage (2.0 times higher than the control, p <0.00001). The number of CD206⁺, CX3CR1⁺ and CD45⁺ cells remained the same. An increase in the number of interlobular veins (p <0.00001) and a decrease in sinusoidal capillaries (p <0.00001) were found compared to the control.

The transition to cirrhosis was characterized by changes in the expression levels of tweak, fn14, ang, vegfa, cxcl12, and mmp-9 mRNAs, as well as the presence and strength of the relationship between them. Significant correlations were revealed between the target genes (r=0.5–0.84; p <0.01).

Keywords

rats / liver fibrosis / liver cirrhosis / gene expression / mRNA level / immunohistochemistry / cells / vessels / correlations

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