Effect of partial hepatectomy on the glycogen level in hepatocytes in the portal and central lobule zones in cirrhotically-changed rat liver

Using cytophotometric method, the content of glycogen was studied in hepatocytes of the portal and central zones of a liver lobule in norm, in cirrhosis, and 1, 3, and 6 months after a partial hepatectomy of the normal and cirrhotic rat liver. As we showed earlier, glycogen content in cirrhotic liver hepatocytes rose 2-3-fold, along with obvious impairment of glycogen metabolic heterogeneity in these. In cirrhotic liver glycogen dominates in the central zone, whereas in norm more glycogen is observed in the portal one. The objective of this study was to find out to what degree a partial hepatectomy of cirrhotic liver may promote recovery of the metabolic glycogen heterogeneity in hepatocytes. Glycogen was determined in hepatocytes, using a quantitative variant of PAS-reaction on sections of the material obtained from serial supravital punctate liver biopsies. Glycogen amount in hepatocytes of different liver lobule zones was determined by an image analyzer technique that allows to bring together the cytophotometric analysis of the substance with its localization in a particular liver lobule. Results of these studies have shown that a partial hepatectomy of cirrhotic liver promotes restoration of the hepatocyte metabolic heterogeneity in the liver lobule.     

Metabolic heterogeneity of glycogen in hepatocytes of patients with liver cirrhosis

Concentrations of the total glycogen (TG) and of its labile and stable fractions (LF and SF, respectively) were determined in hepatocytes of portal and central zones of the normal human liver and in the liver of patients with cirrhosis of viral and alcohol etiology. Using the PAS reaction, TG and its LF and SF were revealed in histological sections of the material obtained by liver punction biopsies. Concentrations of TG and its fractions were measured by television cytophotometry. In liver cirrhosis, concentrations of TG, LF, and SF in both zones of the hepatic lobule were much higher than in the normal liver. The ratio between hepatocyte TG concentration in the portal zone and that in the central zone (P/C ratio), both in norm and in viral cirrhosis, exceeds 1.0 to reach, respectively, 1.26 +/- 0.02 and 1.03 +/- 0.01. The glycogen fraction composition in cells of both liver lobule zones in viral cirrhosis does not significantly differ from that in norm. On the contrary, in the liver of patients with alcoholic cirrhosis, the P/C ratio falls to 0.82 +/- 0.02 to be accompanied by qualitative changes in glycogen composition.     

The antidepressant-like effect of Ocimum basilicum in an animal model of depression

We investigated the efficacy of Ocimum basilicum (OB) essential oils for treating depression related behavioral, biochemical and histopathological changes caused by exposure to chronic unpredictable mild stress (CUMS) in mice and to explore the mechanism underlying the pathology. Male albino mice were divided into four groups: controls; CUMS; CUMS plus fluoxetine, the antidepressant administered for pharmacological validation of OB; and CUMS plus OB. Behavioral tests included the forced swim test (FST), elevated plus-maze (EPM) and the open ?eld test (OFT); these tests were performed at the end of the experiment. We assessed serum corticosterone level, protein, gene and immunoexpression of brain-derived neurotropic factor (BDNF) and glucocorticoid receptors (GRs) as well as immunoexpression of glial fibrillary acidic protein (GFAP), Ki67, caspase-3 in the hippocampus. CUMS caused depression in the mice as evidenced by prolonged immobility in the FST, prolonged time spent in the open arms during the EPM test and reduction of open field activity in the OFT. OB ameliorated the CUMS induced depressive status. OB significantly reduced the corticosterone level and up-regulated protein and gene expressions of BDNF and GR. OB reduced CUMS induced hippocampal neuron atrophy and apoptosis, and increased the number of the astrocytes and new nerve cells. OB significantly increased GFAP-positive cells as well as BDNF and GR immunoexpression in the hippocampus.     

Analysis of glycogen structure in rat hepatocytes using cytochemical and FRET methods

Using cytochemical and FRET (Forster, Resonance Energy Transfer) methods, the glycogen structure in rat hepatocytes was investigated during fasting and at different time intervals after per os glucose administration to animals. Hepatocytes on slides were stained with fluorescent PAS-reaction. Staining the slides with ethidium bromide-SO2 (EtBr-SO2) for 40 min revealed a labile glycogen fraction (LE), and the subsequent staining the same samples with auramine-SO2 (Au-SO2) for 50 min showed a stable glycogen fraction (SF) in the cells. The total glycogen content (LF and SF) in the hepatocytes at different stages of refeeding was determined by means of cytofluorimetry, and then efficiency of FRET was measured in the same cells. Registration of FRET in several areas of the cells was carried out on a laser scanning confocal microscope Leica TCS SP5 with application of FRET AB (Acceptor Photobleaching) procedure. In this procedure, auramine served as a donor (D) and ethidium bromide was an acceptor (A). It was shown that the efficiency of FRET varied from 10 to 14 % during refeeding, while the glycogen structure had a marked influence on the value of this parameter. FRET efficiency was shown to correlate with the ratio A/D in the cells of hungry rats and at the early stages after glucose administration to animals, which reflected the degree of filling of the external tiers of glycogen molecules of glucose residues. At later stages, this correlation was either less pronounced or absent. It was found that the FRET efficiency can vary by 3-4 times at the same value of A/D. Since the probability of energy transfer from D to A is proportional to 1/R6, where R is a distance between D and A, such variations of the FRET efficiency indicate that the glycogen molecules possess a labile structure in which the chain of glucose residues can deviate from its axis by a distance of about half their diameter.     

Morphology and function of cultured hepatocytes isolated from rats with experimental toxic hepatitis

The goal of the study was to examine the morphology and function of primary hepatocytes isolated from rats with toxic hepatitis induced by a combination of CCl₄ and ethanol. Fluorescent immunocytochemical analysis demonstrated that normal and pathologic hepatocytes in culture formed actin cytoskeleton, cell-cell, and cell-matrix contacts. In this investigation, the morphology of mitochondria and their localization in hepatocytes was assayed with Rhodamine 123 staining. Glycogen and DNA contents in cultured hepatocytes were determined by fluorescent cytometry. It was found that the ploidy of hepatocytes isolated from normal and injured livers were different. Cells were maintained in culture for 5 days and no changes in ploidy distribution were observed. The glycogen content was 50% higher in the experimental group than the control one; it was decreased in control and cirrhotic hepatocytes treated with collagenase. Intact hepatocytes accumulated glycogen within 3 days; the glycogen level remained low in pathologic hepatocytes.     

Dynamics of glycogen content in the normal and cirrhotic liver following glucose administration to starving rats

Using biochemical, cytofluorimetric and television cytophotometric methods, glycogen contents were studied in normal and cirrhotic rat liver at various intervals after glucose administration to fasting animals. The obtained data indicate that after a 48 h fasting glycogen contents in normal and cirrhotic liver are equally poor. A marked rise of glycogen content in cirrhotic liver was observed only 20-30 min after glucose administration to rats. It has been established that at all intervals after glucose administration to rats hepatocytes of the portal lobule zone, both in normal and in cirrhotic liver, accumulate more glycogen than those of the central zone. Again, the intensity of glycogen accumulation in cirrhotically altered liver is significantly lower than in normal liver, due, presumably, to a lower rate of glycogen synthesis in pathologically changed liver.     

Features of the effect of bemethyl on glycogen metabolism in hepatocytes of pathological changed human liver

Effect of actoprotector bemithyl (2-ethylthiobenzimidazole hydrobromide) on glycogen metabolism in hepatocytes of patients with chronic hepatitis and liver cirrhosis was investigated. Using cytofluorimetric method, the content of glycogen and its fractions in isolated hepatocytes was measured. The treatment with bemithyl resulted in a decrease in glycogen levels in hepatocytes, and in a marked restoration of fractional glycogen composition as compared to the basic therapy. Besides, it was established that the degree of glycogen decrease in cells of patients with chronic hepatitis depended on the increase of glucose-6-phosphatase activity (r = 0.75, P < 0.05), and on the levels of glycogen in hepatocytes prior to bemitil treatment (r = = 0.87, P < 0.01). Positive changes in glycogen metabolism after bemithyl treatment are pronounced in patients with chronic hepatitis. These positive alterations take place simultaneously with the conservation of basic structural disturbances in the liver parenchyma. However, even in this case, the indices of glycogen metabolism do not reach the normal levels.     

Analysis of structure of glycogen in rat hepatocytes using cytochemical and FRET methods

Using cytochemical and Förster resonance energy transfer (FRET) methods, the structure of glycogen was studied in rat hepatocytes during starvation and in some time intervals after the peroral administration of glucose to the animals. Hepatocytes were stained with a fluorescent variant of PAS reaction on object glasses. The staining of preparations for 40 min with ethidium bromide-SO₂ (EtBr-SO₂) revealed the labile fraction (LF) of glycogen, while their subsequent staining with auramine-SO₂ (Au-SO₂) for 50 min revealed the stable fraction (SF) of glycogen in cells. The total glycogen content (LF + SF) in hepatocytes at various stages of rat refeeding was determined using a cytofluorimeter; then, in the same cells, the FRET efficiency was measured. Recording FRET at several sites of cells was performed using a Leica TCS SP5 laser scanning confocal microscope by using the FRET Acceptor Photobleaching (FRET AB) procedure. In this procedure, auramine was used as the donor (D), while ethidium bromide was used as the acceptor (A). The efficiency of FRET in the course of rat refeeding with glucose has been shown to change from 10 to 14%, and the glycogen structure markedly affects the value of this parameter. It is found that, in cells of starved rats and in early terms after the administration of glucose, the FRET efficiency correlates with the A/D ratio, which reflects the degree of filling of external tiers of glycogen molecules with glucose residues. At later terms of refeeding, this correlation is either less pronounced or completely absent. It has been established that, at the same A/D value, the FRET efficiency can change by three to four times. Since the probability of energy transduction from D to A is proportional to 1/R⁶, where R is the distance between D and A. These fluctuations of the FRET efficiency mean that the glycogen molecules have the labile structure, in which chains of glycoside residues can deviate from its axis at a distance of about a half of their diameter.     

The influence of hepatoprotector 2-ethylthiobenzimidazole hydrobromide (bemithyl) on the content of glycogen in cirrhotic rat liver hepatocytes located in various microenvironments

Using absorption and fluorescent cytophotometry methods, glycogen contents were studied in hepatocytes located in liver lobules and in hepatocytes, which make the general population of these cells in normal and cirrhotic rat liver. In cirrhosis, the content of glycogen in hepatocytes located in lobules obviously rises in comparison with the norm, but to a lesser degree, than in hepatocytes making the general population of these cells in cirrhotic liver. The content of glycogen in hepatocytes, located in lobules of pathologically changed liver in bemithyl treated rats, did not differ from the norm. At the same time, the glycogen content in hepatocytes, representing the general population of these cells in cirrhotically altered bemithyl injected rat liver, remained higher than in the norm. The data obtained indicate that distinctions in particular cell microinvironment, obviously present in cirrhotic liver, render essential influence on hepatocyte functional activity.