Actins ; analysis ; Animals ; Dimethylnitrosamine ; Immunohistochemistry ; Liver ; metabolism ; pathology ; Male ; Necrosis ; chemically induced ; pathology ; Rats ; Rats, Wistar ; Reticulin ; analysis ; Silver Staining

Animals ; Liver ; pathology ; Liver Cirrhosis ; pathology ; Male ; Rats ; Rats, Wistar ; Reproducibility of Results ; Staining and Labeling ; methods

OBJECTIVETo investigate the relationship between mast cell and hepatic fibrosis by histopathological method and semi-quantitative measurement.

METHODSSeventy-two Wistary male rats, the control group and the normal group of each only 16, experimental group of 40 rat liver fibrosis was induced by injection of DMN and was sampled at eight different time points. HE, histochemistry, immunohistochemistry (ABC method) and immunofluorescence were performed. The size of fibrosis and the number of mast cells were counted. The expression of MMP-2 and TIMP-2 was documented and electron microscopic examination was performed.

RESULTSAfter injection of DMN, the fibrosis was the most severe in the 2 week (3.72%) and the first month (3.73%, P = 0.2626), and then gradually diminished, although residual fibrosis was still present at 12 months (1.42%, P = 0.0003). The appearance of mast cells began at 2 weeks (1.73 per 200 power field in average by light microscope) after the injection and reached the peak at 4 months (3.06, P = 0.008). Residual amount of mast cells were present at 12 months (1.04, P = 0.045). However, the degree of fibrosis was not proportional or overlapping with the number of mast cells in this experiment model. Mast cells expressed MMP-2 but not TIMP-2.

CONCLUSIONSIn the DMN-induced rat liver fibrosis model, mast cell may be an integral player in the pathogenesis of liver fibrosis and may contribute to the degradation of fibrosis by synthesizing and secreting MMP-2.

Actins ; metabolism ; Animals ; Cell Count ; Dimethylnitrosamine ; Liver Cirrhosis ; chemically induced ; metabolism ; pathology ; Male ; Mast Cells ; metabolism ; pathology ; ultrastructure ; Matrix Metalloproteinase 2 ; metabolism ; Rats ; Rats, Wistar ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Tryptases ; metabolism

OBJECTIVETo investigate the morphological changes and regeneration mechanism of sinusoidal endothelial cell.

METHODSSixty male Wistar rats (bought from SLC company limited of Japan) were divided into three groups. Fifty of them belonged to experiment group, five rats belonged to untreated group, and the rest five ones belonged to normal saline treated group. The experiment group was then divided into ten subgroups. All the rats of the experiment group were killed under anaesthesia using aether at 12, 24, 36 hrs, and 2, 3, 5, 7, 8, 10 and 14 days subsequently after an one-off injection of dimethylnitrosamine (DMN) (50 mg/kg). The liver tissues, bone marrows and peripheral blood of the rats were taken out rapidly. All the tissues received with HE staining, immunohistochemistry staining and double immunofluorescence labelings, and they were observed under a light microscope and electron microscope. The livers, bone marrows and peripheral blood from the rats at 24 hrs to 14 days after an injection of DMN were examined by light microscopic, immunohistochemical, and ultrastructural methods.

RESULTSSmall focal necrosis of the liver tissues was found at 12 hrs after the DMN injection, and gradually becomes more obvious from the 24 hrs. The most obvious necrosis, with lots of ED-1 (monocyte/phagocyte marker of rats) positive cells infiltration, was observed at 36 hrs. On the 2nd day and 3rd day after injection, the necrotic fragments and red cells were phagocyted by ED-1 positive macrophages. On the 5th day, some of the ED-1-positive cells were transformed from round to spindle in shape. On the 7th day, these cells contacted with residual reticulin fibers and became positive for SE-1, a marker of hepatic sinusoidal endothelial cells and Tie-1, an endothelial cell-specific surface receptor, associated with frequent occurrence of ED-1/SE-1 and ED-1/Tie-1 double positive spindle cells. On the 8th day, the histomorphology of liver tissue was similar with that on day 7, except that the range of the lesions had become smaller. On the 10th day, the regeneration of liver tissue increased, filling in the necrosis. On the 14th day, the necrotic tissues were almost replaced by regenerated liver tissues and thin bundles of central-to-central bridging fibrosis. 12 hrs after the DMN injection, bone marrow studies showed an increase in the number of ED-1 positive mononuclear cells, some of which were both BrdU/ED-1 positive. The number of ED-1 positive mononuclear cells reach their highest level at 36 hrs. These cells are morphologically similar to round mononuclear cells in bone marrows and could be found in the peripheral blood from 24 hrs to the 10 days. They reached their highest level in peripheral blood at the same time as in the bone marrow. These cells morphologically resembled ED-1 positive cells in necrotic tissues of the liver.

CONCLUSIONSThese findings suggest that round mononuclear ED-1-positive cells proliferate first in the bone marrow after DMN treatment, reach necrotic areas of livers through circulation, and differentiate to sinusoidal endothelial cells. Namely, hepatic sinusoids in DMN-induced necrotic areas may partly be reorganized possibly by vasculogenesis.

Animals ; Chemical and Drug Induced Liver Injury ; pathology ; Dimethylnitrosamine ; Endothelial Cells ; pathology ; ultrastructure ; Liver ; blood supply ; metabolism ; pathology ; ultrastructure ; Liver Regeneration ; Male ; Necrosis ; chemically induced ; pathology ; Neovascularization, Physiologic ; Rats ; Rats, Wistar