الفهرس 
Macroanatomy of the liver
Nonalcoholic Fatty LiverOnly 14 pages are availabe for public view
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Abstract
As a result of increasing rates of obesity, non-alcoholic fatty liver disease (NAFLD) is now considered the most common liver disease. NAFLD is present when more than 5% of hepatocytes are steatotic in absence of excessive alcohol consumption and other causes of steatosis. It ranges from simple steatosis to nonalcoholic steatohepatitis (NASH) which can progress to cirrhosis and hepatocellular carcinoma. There is an increasing evidence indicating the therapeutic benefit of bone marrow derived mesenchymal stem cells (BM-MSCs) transplantation in liver diseases. Mobilizing endogenous stem cells using StemEnhance (SE) is a recent method to induce regeneration and repair of damaged liver. Aim of the work: The present study aimed to compare between the therapeutic effect of intraportal transplantation of BM-MSCs versus mobilization of endogenous stem cells using SE on experimentally induced NAFLD in adult male albino rats using histological, immunohistochemical and biochemical methods with statistical analysis of the results. Material and methods: Seventy male adult albino rats were used in this study. Ten rats were used for extraction and isolation of BM-MSCs. The remaining sixty rats were further divided into control and experimental groups as follows: Control group (group I): Consisted of 20 rats and was further subdivided into two subgroups (10 rats / each subgroup) as follows: Subgroup Ia: The rats of this subgroup were fed standard rat diet for 10 weeks and received no treatment. Subgroup Ib: The rats of this subgroup were fed standard rat diet for 10 weeks and received SE orally from the starting of the 7th week till the end of 10th week. Experimental group (group II): This group consisted of 40 rats. They were fed high fat diet (HFD) for 6 weeks. Then the rats were randomly separated into 4 subgroups (10 rats / each subgroup) as follows: Subgroup IIa (10 weeks HFD): The rats were fed high fat diet for further 4 weeks then sacrificed. Subgroup IIb (HFD withdrawal): High fat diet was stopped and the rats were fed standard rat diet for another 4 weeks then sacrificed. Subgroup IIc (SE treated): The rats continued to be fed high fat for another 4 weeks, while they received SE orally at a dose of 270 mg/kg per day once daily from the start of 7th week till the end of 10th week then rats were sacrificed. Subgroup IId (BM-MSCs treated): The rats received BM-derived MSCs (3×106 BM-MSCs suspended in 0.5 ml phosphate buffer solution) at the start of the 7th week as a single dose intra-hepatic via the portal vein. These rats continued on fatty diet for further 4 weeks then sacrificed. At the end of experiment, rats were weighed then anaesthetized and sacrificed. Blood samples were taken for measurement of serum lipids, liver enzymes and flow cytometry studies for percentage of circulating CD34 and CD90 positive cells. The liver was extracted for gross examination then prepared for Hx.&E., Mallory trichrome and Oil Red O staining. Tumor necrosis factor-alpha (TNF-α) and Glial fibrillary acidic protein (GFAP) were used for immunohistochemical studies. Results: Gross examination of liver and body weight: Liver of subgroup IIa was enlarged and yellowish in color. Livers of subgroups IIb, IIc and IId were red-brownish color with smooth surfaces as compared to subgroup IIa. A highly significant increase of the mean body weight was observed in subgroup IIa. Subgroup IIb showed a highly significant reduction of the mean body weight, while it was significantly decreased in subgroups IIc and IId as compared to subgroup IIa. Biochemical results: Subgroup IIa showed a highly significant increase in the mean serum levels of TC, TG and liver enzymes (ALT & AST) and a highly significant decrease in the mean serum levels of HDL as compared to control group. On stoppage of HFD in subgroup IIb, there was a highly significant reduction in the mean serum levels of TC & TG, a non-significant increase in the mean serum level of HDL and significant reduction in the mean serum level of liver enzymes as compared to subgroup IIa. On the other hand, subgroup IIc showed a significant reduction in the mean serum levels of TC & TG and a significant increase in the mean serum level of HDL. As regards liver enzymes, there was a significant reduction in the mean serum levels of ALT and a highly significant reduction of AST as compared to subgroup IIa. Subgroup IId showed a significant reduction in the mean serum level TC and TG in addition to a highly significant increase in the mean serum level of HDL with a highly significant reduction in the mean serum levels of ALT and AST as compared to subgroup IIa. Flow cytometry results: Flow cytometry for circulating peripheral CD34 +ve and CD90 +ve cells revealed that subgroups Ib and IIc showed a highly significant increase in the mean percentage of circulating peripheral CD34+ve as compared to all other subgroups. Also, there was a highly significant increase in the mean percentage of circulating peripheral CD90+ve in subgroup IId as compared to all other subgroups. Histological results: Control group (subgroups Ia & Ib): Hx. & E. sections of this group showed classic hepatic architecture formed of hexagonal hepatic lobules with polyhedral hepatocytes having rounded vesicular nuclei and granular eosinophilic cytoplasm separated by sinusoids which were lined with endothelial cells and Kupffer cells. Mallory trichrome stain showed faint collagen fibers around the central vein, portal tract and between hepatocytes. This group showed no TNF-α positive cells while some hepatic stellate cells appeared as brown stained star-shaped cells in GFAP immunostaining. Oil Red O (ORO) section revealed tiny red stained fat droplets within in the cytoplasm of the hepatocytes. Experimental group (group II): Subgroup IIa (10 weeks HFD): Hx. & E. staining showed loss of normal hepatic architecture with congested central vein and compressed hepatic sinusoids by multiple small fat droplets (microvesicular) and large lipid vacuoles (macrovesicular) present within the cytoplasm of hepatocytes of all zones with loss of the vesicular appearance of hepatocyte nuclei which appeared small and dark. Inflammatory cells appeared around portal tract while apoptotic Mallory Denk bodies were seen within the cytoplasm of some ballooned hepatocytes. Extensive increase of collagen fibers was detected in Mallory trichrome stain. Moreover, marked increase in the mean number of TNF-α positive cells and GFAP positive hepatic stellate cells was detected. Multiple small and large sized fat droplets were extensively distributed in ORO staining. Subgroup IIb (HFD withdrawal): Hx. & E. staining showed partial improvement of liver architecture, while some apparently normal hepatocytes with granular eosinophilic cytoplasm and rounded vesicular nuclei were observed. However, congested central veins, microvesicular and macrovesicular steatotic cells were still seen. As compared to subgroup IIa; collagen fibers, TNF-α positive cells and GFAP positive hepatic stellate cells were reduced. Also the number of ORO stained fat droplets was apparently decreased. Subgroup IIc (SE treated): Hx. & E. liver sections showed moderate restoration of the hepatic architecture with cords of hepatocytes radiating from central vein and separated by sinusoids which were lined with Kupffer cells, but simple steatosis was still seen. Moreover, decreased collagen fibers were present around central vein, portal tract and between hepatocytes in Mallory trichrome stained sections. There were few TNF-α positive cells, reduced GFAP positive hepatic stellate cells and few ORO stained fat droplets as compared to subgroup IIa. Subgroup IId (BM-MSCs treated): Hx. & E. sections showed obvious improvement in the hepatic architecture, where regular cords of hepatocytes were radiating from the central vein and separated by sinusoids which were lined with Kupffer cell. Hepatocytes appeared as polyhedral cells with granular eosinophilic cytoplasm and rounded vesicular nuclei, but microvesicular steatosis was within the cytoplasm of some hepatocytes around central vein. Mallory trichrome stain showed minimal collagen fibers around central vein, portal tract and between hepatocytes. Very few TNF-α positive cells, few GFAP positive hepatic stellate cells and few ORO red stained fat droplets were observed as compared to subgroup IIa. Morphometric results : a) Mean number of steatotic cells in Hx. & E. sections: The image analysis, using the Image J software, revealed that subgroups IIb and IIc and IId showed a highly significant decrease in the mean number of steatotic as compared to subgroup IIa. However, subgroup IId showed a highly significant decrease in the mean number of steatotic cells as compared to subgroup IIc. b) Mean number of TNF-α positive cells: Subgroups IIb, IIc and IId showed a highly significant decrease in the mean number of TNF-α positive cells as compared to subgroup IIa. However, subgroup IId showed a highly significant decrease in the mean number of TNF-α positive cells as compared to subgroup IIc.