Vibrational spectroscopy-based quantification of liver steatosis.

The liver plays a central role in lipid metabolism, and abnormal lipid accumulation in the liver is a key feature of Non-Alcoholic Fatty Liver Disease. In experimental studies, quantification of liver steatosis is commonly based on lipids staining or biochemical analysis. Here, we present a spectroscopic approach for quantitative analysis of the lipid content in the freeze-dried liver. The method is based on vibrational spectroscopy (Raman and infrared) measurements applied for Partial Least Squares (PLS) regression modeling. The obtained PLS models show a good correlation of the spectroscopic data with the reference histological evaluation of steatosis based on Oil Red O (ORO)-stained images of liver cross sections. Vibrational spectroscopy with PLS-based modeling described here represents a useful approach for the fast assessment of the liver steatosis in a small sample of freeze-dried liver tissue. In conclusion, our work demonstrates the easy-to-use method that can be applied in laboratory routine as a beneficial alternative to the established ORO staining.

Alterations in NO- and PGI2- dependent function in aorta in the orthotopic murine model of metastatic 4T1 breast cancer: relationship with pulmonary endothelial dysfunction and systemic inflammation.

BACKGROUND: Patients with cancer develop endothelial dysfunction and subsequently display a higher risk of cardiovascular events. The aim of the present work was to examine changes in nitric oxide (NO)- and prostacyclin (PGI2)-dependent endothelial function in the systemic conduit artery (aorta), in relation to the formation of lung metastases and to local and systemic inflammation in a murine orthotopic model of metastatic breast cancer. METHODS: BALB/c female mice were orthotopically inoculated with 4T1 breast cancer cells. Development of lung metastases, lung inflammation, changes in blood count, systemic inflammatory response (e.g. SAA, SAP and IL-6), as well as changes in NO- and PGI2-dependent endothelial function in the aorta, were examined 2, 4, 5 and 6 weeks following cancer cell transplantation. RESULTS: As early as 2 weeks following transplantation of breast cancer cells, in the early metastatic stage, lungs displayed histopathological signs of inflammation, NO production was impaired and nitrosylhemoglobin concentration in plasma was decreased. After 4 to 6 weeks, along with metastatic development, progressive leukocytosis and systemic inflammation (as seen through increased SAA, SAP, haptoglobin and IL-6 plasma concentrations) were observed. Six weeks following cancer cell inoculation, but not earlier, endothelial dysfunction in aorta was detected; this involved a decrease in basal NO production and a decrease in NO-dependent vasodilatation, that was associated with a compensatory increase in cyclooxygenase-2 (COX-2)- derived PGI2 production. CONCLUSIONS: In 4 T1 metastatic breast cancer in mice early pulmonary metastasis was correlated with lung inflammation, with an early decrease in pulmonary as well as systemic NO availability. Late metastasis was associated with robust, cancer-related, systemic inflammation and impairment of NO-dependent endothelial function in the aorta that was associated with compensatory upregulation of the COX-2-derived PGI2 pathway.

Atomic Force Microscopy Reveals the Dynamic Morphology of Fenestrations in Live Liver Sinusoidal Endothelial Cells.

Here, we report an atomic force microscopy (AFM)-based imaging method for resolving the fine nanostructures (e.g., fenestrations) in the membranes of live primary murine liver sinusoidal endothelial cells (LSECs). From data on topographical and nanomechanical properties of the selected cell areas collected within 1 min, we traced the dynamic rearrangement of the cell actin cytoskeleton connected with the formation or closing of cell fenestrations, both in non-stimulated LSECs as well as in response to cytochalasin B and antimycin A. In conclusion, AFM-based imaging permitted the near real-time measurements of dynamic changes in fenestrations in live LSECs.

Morphology and force probing of primary murine liver sinusoidal endothelial cells.

Liver sinusoidal endothelial cells (LSECs) represent unique type of endothelial cells featured by their characteristic morphology, ie, lack of a basement membrane and presence of fenestrations-transmembrane pores acting as a dynamic filter between the vascular space and the liver parenchyma. Delicate structure of LSECs membrane combined with a submicron size of fenestrations hinders their visualization in live cells. In this work, we apply atomic force microscopy contact mode to characterize fenestrations in LSECs. We reveal the structure of fenestrations in live LSECs. Moreover, we show that the high-resolution imaging of fenestrations is possible for the glutaraldehyde-fixed LSECs. Finally, thorough information about the morphology of LSECs including great contrast in visualization of sieve plates and fenestrations is provided using Force Modulation mode. We show also the ability to precisely localize the cell nuclei in fixed LSECs. It can be helpful for more precise description of nanomechanical properties of cell nuclei using atomic force microscopy. Presented methodology combining high-quality imaging of fixed cells with an additional nanomechanical information of both live and fixed LSECs provides a unique approach to study LSECs morphology and nanomechanics that could foster understanding of the role of LSECs in maintaining liver homeostasis.

Label-free spectroscopic characterization of live liver sinusoidal endothelial cells (LSECs) isolated from the murine liver.

Liver sinusoidal endothelial cells (LSECs) represent a highly specialized and unique type of endothelial cell in terms of their morphology and function. The biochemical and functional characterization of LSECs in vitro is restrained by the rapid change of LSECs' phenotype upon culturing under classical experimental conditions. In this work, we present a novel approach to characterize the biochemical content of murine LSECs, freshly isolated from the liver, with the use of microspectroscopic analysis. For comparison, hepatocytes and Hepatic Stellate Cells (HSCs) were analyzed. Our approach, based on label-free confocal Raman imaging of live cells combined with chemometric analysis, provided insight into the biochemical content of freshly isolated LSECs on a subcellular level. LSECs were featured by a distinct biochemical signature in comparison with other major cell types of the liver. Based on our work we claim that the non-invasive and non-destructive confocal Raman imaging may assist in obtaining chemical information spatially distributed within the cells that characterize the phenotype of primary LSECs as well as other types of liver cells. Furthermore, our approach provides a unique insight into LSECs' morphology and chemical composition that may help to understand their functions.

Haematological parameters, serum lipid profile, liver function and fatty acid profile of broiler chickens fed on diets supplemented with pomegranate seed oil and linseed oil.

The objective of the present study was to determine effect of pomegranate seed oil (PSO) and linseed oil (LO) on haematological parameters, serum lipid profile and liver enzymes as well as fatty acids profile of adipose tissue in broilers. Broilers (n = 400) were fed on diets containing graded PSO levels (0.0%, 0.5%, 1.0%, 1.5%) with or without 2% LO. After 6 weeks of feeding, 6 male broilers from each group were slaughtered and abdominal fat, liver and blood samples were collected. Mixtures of pomegranate seed oil (0.5%, 1%) with linseed oil increased white blood cell level in broilers. Total cholesterol was elevated after LO supplementation whereas administration of PSO (1.5%) significantly decreased this parameter. PSO administration caused c9,t11 conjugated linoleic acid (CLA) concentration-dependent deposition in adipose tissue. By LO addition α-linolenic acid (ALA) content was enhanced, decreasing the n-6/n-3 ratio. PSO and ALA also affected oleic acid proportion in adipose tissue. Neither pomegranate seed oil nor linseed oil had any effect on liver parameters. Pomegranate seed oil had no negative effects on broiler health status and can be considered as a functional poultry meat component.

Giant coronary sinus and absent right superior vena cava.

Although persistent left superior vena cava (PLSVC) is rare, it is the most common thoracic venous anomaly. The prevalence of PLSVC is 0.3 % in a general population and 10 % in patients with congenital heart disease. Diagnosis of PLSVC is usually incidental during cardiovascular imaging or surgery. Although PLSVC is usually not associated with any negative hemodynamic effect, it is important to be aware of its existence, since it may cause problems in central venous catheterization, pacemaker implantation and cardiopulmonary bypass. The overall proportion of absent right superior vena cava in patients with PLSVC is approximately 20 %. If PLSVC is associated with absence of the right superior vena cava, the coronary sinus may become gigantic due to excessive inflow. Herein, we report a case of PLSVC complicated by the absence of a right superior vena cava resulting in a giant coronary sinus.

The influence of estrogens, progesterone and their antagonists on the collagen content in the pregnant rat uterus.

The influence of progesterone, estrogens and their antagonists: mifepristone and tamoxifen on connective tissue metabolism in pregnant rat uterus near term was evaluated. Total collagen and its soluble fraction as well as water concentration was measured in a population of 65 pregnant rats; treated with progesterone (n = 10), mifepristone (n = 13), estradiol (n = 17) and tamoxifen (n = 12). The results suggest the important influence of sex steroids on the uterine connective tissue metabolism during pregnancy.

Prostaglandin-induced changes and the influence of cyclo-and lipoxygenase inhibitors on the uterine collagen concentration in late pregnancy in rats.

During late pregnancy, the influence of PGF2 alpha and PGE2 on the collagen concentration in a rat's uterus was analysed. In order to evaluate the influence of endogenous prostaglandins and leukotrienes on connective tissue metabolism, a cyclooxygenase inhibitor (Diclofenac) and a dual cyclo-and 5-lipoxygenase inhibitor (BW755C) were administered prior to the collection of samples. Total collagen and its soluble fraction as well as water content were determined in the uteri in a population of 58 pregnant rats treated with PGF2 alpha (n = 16), PGE2 (n = 11), Diclofenac (n = 11), BW755C (n = 7) and controls (n = 13). The most evident changes in tissue hydration and collagen concentration were observed in the PGF2 alpha and Diclofenac treated groups. The results clearly show the affinity of PGF2 alpha to the uterus and the role of endogenous PGs in the maintenance of pregnancy by influencing connective tissue metabolism.

The effect of platelet activating factor antagonist (BN 52021) on pregnancy duration and collagen content in the pregnant rat uterus and cervix.

In order to study the role of Platelet Activating Factor (PAF) in the mechanism of parturition and connective tissue metabolism in the pregnant rat uterus, we investigated the influence of the Platelet Activating Factor Receptor Antagonist (BN 52021) on gestation duration and collagen concentration in pregnant rat uterus and cervix at term. Total and soluble collagen as well as dry tissue/wet tissue ratio were measured in the BN 52021 treated group (n = 9) and in the control group (n = 13). Gestation duration was evaluated in a group receiving BN 52021 (n = 10) and in a control group (n = 19) receiving pure solvent. There were no significant differences in water content. The total and soluble collagen concentration was also similar to the control in both the uterus and the cervix. The only significant difference was observed in the soluble collagen/total collagen ratio in the uterus. BN 52021 administration did not alter the gestational period. Basing on this experiment we suggest that not all the phospholipid metabolites activated by phospholipase A2 significantly influence the duration of pregnancy and connective tissue metabolism in the pregnant rat uterus.