literammp-9 mRNA Expression and Bridging Fibrosis Progression in Toxic Liver Injury.

Developing liver disease treatments, in which fibrosis is a key pathogenetic link, still remains an urgent problem in hepatology. In the present study, the level of mmp-9 mRNA expression and the number of FAP+, α-SMA+, CD45+ cells were analyzed at nine time points of fibrosis and cirrhosis. It was found that in the case of liver fibrosis, the choice of the optimal reference gene depended on the stage of fibrogenesis. When studying the specific stages rather than the entire process in a long-term experiment, it was shown that choosing an optimal reference gene has to be done additionally. In this case, the mmp-9 mRNA expression level should be considered as a marker of liver fibrosis initiation and development but not as that of cirrhosis progression. In the liver, two morphologically heterogeneous populations of myofibroblasts were simultaneously identified as able to synthesize various types of immunohistochemical markers. It was found that the FAP+ cells were the main contributor to the development of portal fibrosis and the initial stages of bridging fibrosis. In the selected experimental model, fibrosis initiation and the development stages preceding parenchyma restructuring were accompanied by a low level of inflammation.

Changes in Macrophage Subpopulations in Rat Liver at Different Stages of Experimental Fibrosis.

The number, phenotypic composition, and functional properties of macrophages in the liver of Wistar rats change depending on the stages of fibrosis induced by thioacetamide. In the sinusoidal capillaries of the liver of control rats, CD68+ wing-shaped cells were mainly detected. The number of CD68+ cells at the stages of fibrosis before the process of its transformation into cirrhosis was 2-fold higher (p=0.0000) than in the control. At later terms of the experiment, no significant differences were found. Immunohistochemical method revealed two morphologically different groups of CD68+ cells differing in shape and localization. At all stages of the experiment, round and elongated CD206+ cells of were detected in the sinusoidal capillaries. At the stage of cirrhosis (13 weeks), the number of CD206+ cells was higher than during the third week of the experiment by 3.21 times (p=0.0000). Later, a decrease in the number of CD206+ cells was observed. At the same time, in the portal zones and connective tissue septa around the false hepatic lobules, round CX3CR1+ cells were noted. By the end of the experiment (17 weeks), their number exceeded that on the third week of the experiment by 5.66 times (p=0.0000).

Cellular and Molecular Mechanisms of Toxic Liver Fibrosis in Rats Depending on the Stages of Its Development.

The aim is to study the cellular and molecular features of toxic liver fibrosis in rats and its dependence on development stages of this pathological condition. Materials and Methods: Liver fibrogenesis in male Wistar rats was induced with the thioacetamide solution by introducing into the stomach with a probe at a dose of 200 mg/kg of animal body weight 2 times per week. The process dynamics was studied at 5 time points (control, week 3, week 5, week 7, and week 9). The mRNA levels of tweak, fn14, ang, vegfa, cxcl12, and mmp-9 genes in liver were detected by real-time polymerase chain reaction. Immunohistochemical study was performed on paraffin sections. The CD31, CD34, CK19, α-SMA, FAP, CD68, CD206, CX3CR1, and CD45 cells were used as markers. Fibrosis degree was determined in histological sections, stained in line with the Mallory technique, according to the Ishak's semi-quantitative scale. Results: Two simultaneously existing morphologically heterogeneous populations of myofibroblasts expressing different types of markers (FAP, α-SMA) were identified in rat liver. Prior to the onset of transformation of fibrosis into cirrhosis (F1-F4, weeks 3-7), FAP+ and SMA+ cells were localized in different places on histological specimens. All stages of liver fibrosis development were accompanied by an increase in the number (p=0.0000), a change in the phenotypic structure and functional properties of macrophages. The CK19+ cells of the portal areas differentiated into cholangiocytes that formed interlobular bile ducts and ductules, as well as hepatocytes that formed rudiments of new hepatic microlobules. Pathological venous angiogenesis and heterogeneity of endotheliocytes of the intrahepatic vascular bed were detected. Two options for changes in mRNA expression of the selected genes were identified. The level of the fn14 and mmp-9 mRNAs at all stages of fibrosis was higher (p=0.0000) than in control rats. For tweak, ang, vegfa, and cxcl12 mRNAs, the situation was the opposite - the level of genes decreased (p=0.0000). There were strong and moderate correlations between the studied target genes (p<0.05). Conclusion: It was established that the stages of toxic fibrosis had morphological and molecular genetic features. The FAP+ cells make the main contribution to development of portal and initial stage of bridging fibrosis. The stellate macrophages and infiltrating monocytes/ macrophages can potentially be used for development of new therapeutic strategies for liver pathology treatment. One should take into account the features of the markers' expression by endothelial cells during the study of the intrahepatic vascular bed. Joint study of genes is a necessary ad-hoc parameter in fundamental and preclinical research.

Effects of experimental increases and decreases in thyroxine levels on the extent of cataleptic freezing reactions in rats.

Numerous clinical observations have provided evidence for a tight connection between impairments in the functions of the hypothalamo-hypophyseal-thyroid system and nervous and mental disorders. The aim of the present work was to compare the effects of experimental decreases and increases in blood thyroxine levels on the extents of two types of pathological freezing reaction in male Wistar rats--spontaneous catalepsy and catalepsy evoked by pinches at the nape of the neck (pinch-induced catalepsy). Chronic administration of the thyroxine synthesis inhibitor propylthiouracil (5 mg/kg/day for 28 days) significantly decreased the blood hormone level and sharply increased the proportion of animals showing spontaneous catalepsy and the immobility time, but had no effect on the extent of pinch-induced catalepsy. At the same time, chronic administration of thyroxine (0.1 mg/kg/day for 28 days), which produced significant increases in blood hormone levels, had no effect on the extent of spontaneous catalepsy but significantly increased the proportion of animals showing pinch-induced catalepsy and the duration of this type of catalepsy. It is concluded that both insufficiency and excess of thyroid hormones have cataleptogenic actions, but enhance different types of catalepsy.

[Effect of experimental elevation and decrease of thyroxine level on catalepsy in rats]. / Vliianie éksperemental'nogo uvelicheniia i umen'sheniia urovnia tiroksina na vyrazhennost' reaktsii katalepticheskogo zamiraniia u krys.

Thyroid dysfunction is associated with mental disorders. The present study was aimed to reveal the effects of experimental decrease and increase of thyroxine level on expression of two types of extensive freezing: spontaneous and pinch-induced catalepsy, in Wistar rat males. Chronic administration of thyroxine synthesis inhibitor, propylthiouracil (5 mg/kg/day, 28 days), markedly decreased plasma hormone level and at the same time produced a significant increase in percentage of spontaneously cataleptic animals and immobility time, but had no effect on the expression of pinch-induced catalepsy. On the contrary, chronic thyroxin (0.1 mg/kg/day, 28 days) treatment produced no effect on spontaneous catalepsy expression, although it significantly increased percentage of cataleptic animals and immobility time of pinch-induced catalepsy. The results suggest that both the thyroid hormone deficit and excess provoke catalepsy in rats but enhance different forms of freezing reaction.