On the Application and Possibilities of In Vivo Microscopy in Liver Research

In vivo microscopy (IVM) provides a valuable method for studying the histophysiology of the living liver. The method allows observation of living cells in the intact organ of an anesthetized animal with an undisturbed microcirculation, at a magnification and a resolution comparable to normal light microscopy of sectioned material. Due to the absence of preparative procedures, the image differs substantially from histological sections, but it has the advantage of providing us with a reference preparation free of artifacts. In the case of the liver, we have the opportunity to observe directly such details as bile capillaries, intracellular fat droplets, lysosomes, nucleoli and different types of sinusoidal cells and blood cells. By using epifluorescence, it is possible to visualize the phagocytosis of 0.8 μm fluorescent latex (or other) particles by Kupffer cells, to observe fluorescing substances such as FITC labeled asialofetuin during the process of endocytosis and intracellular transport in parenchymal cells, and to study the behavior of specific cell types such as white blood cells which stain specifically with acridine orange. It might be expected that in the very near future, the application of modern techniques based on processing TV images, such as image intensifying, averaging, filtering, integrating, gating and others will tremendously improve the possibilities of IVM and bring it to the level of observing and following single molecules, such as FITC labelled peptide hormones and other probes, at the cellular level

Normal versus abnormal structure: considerations in morphologic responses of teleosts to pollutants.

Consideration of newer more quantitative morphologic approaches to the study of aquatic pollutants can provide opportunity for collaborative/integrated studies with other subdisciplines in toxicology. Current commonly employed morphologic approaches result largely in subjective findings difficult to analyze statistically and often are directed at levels of structural organization inconsistent with biochemical and physiological approaches. We review some of the methods and approaches available for correlated structure/function studies and present examples from normal and altered skin, gill, and liver of teleosts

Effects of acetaminophen on hepatic microcirculation in mice

Proceeding

A deep UVBRI CCD photometric study of open clusters Tr 1 and Be 11

We present deep $UBVRI$ CCD photometry for the young open star clusters Tr 1 and Be 11. The CCD data for Be 11 is obtained for the first time. The sample consists of $\sim$ 1500 stars reaching down to $V$ $\sim$ 21 mag. Analysis of the radial distribution of stellar surface density indicates that radius values for Tr 1 and Be 11 are 2.3 and 1.5 pc respectively. The interstellar extinction across the face of the imaged clusters region seems to be non-uniform with a mean value of $E(B-V)$ = 0.60$\pm$0.05 and 0.95$\pm$0.05 mag for Tr 1 and Be 11 respectively. A random positional variation of $E(B-V)$ is present in both the clusters. In the cluster Be 11, the reason of random positional variation may be apparent association of the HII region (S 213). The 2MASS $JHK$ data in combination with the optical data in the cluster Be 11 yields $E(J-K)$ = 0.40$\pm$0.20 mag and $E(V-K)$ = 2.20$\pm$0.20 mag. Colour excess diagrams indicate a normal interstellar extinction law in the direction of cluster Be 11. The distances of Tr 1 and Be 11 are estimated as 2.6$\pm$0.10 and 2.2$\pm$0.10 Kpc respectively, while the theoretical stellar evolutionary isochrones fitted to the bright cluster members indicate that the cluster Tr 1 and Be 11 are 40$\pm$10 and 110$\pm$10 Myr old. The mass functions corrected for both field star contamination and data incompleteness are derived for both the clusters. The slopes $1.50\pm0.40$ and $1.22\pm0.24$ for Tr 1 and Be 11 respectively are in agreement with the Salpeter's value. Observed mass segregations in both clusters may be due to the result of dynamical evolutions or imprint of star formation processes or both.Comment: 15 pages, 13 figures. Accepted for publication in MNRA

Sinusoidal Endothelial Dysfunction Precedes Inflammation and Fibrosis in a Model of NAFLD

Non-alcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome. Most morbidity associated with the metabolic syndrome is related to vascular complications, in which endothelial dysfunction is a major pathogenic factor. However, whether NAFLD is associated with endothelial dysfunction within the hepatic vasculature is unknown. The aims of this study were to explore, in a model of diet-induced overweight that expresses most features of the metabolic syndrome, whether early NAFLD is associated with liver endothelial dysfunction. Wistar Kyoto rats were fed a cafeteria diet (CafD; 65% of fat, mostly saturated) or a control diet (CD) for 1 month. CafD rats developed features of the metabolic syndrome (overweight, arterial hypertension, hypertryglyceridemia, hyperglucemia and insulin resistance) and liver steatosis without inflammation or fibrosis. CafD rats had a significantly higher in vivo hepatic vascular resistance than CD. In liver perfusion livers from CafD rats had an increased portal perfusion pressure and decreased endothelium-dependent vasodilation. This was associated with a decreased Akt-dependent eNOS phosphorylation and NOS activity. In summary, we demonstrate in a rat model of the metabolic syndrome that shows features of NAFLD, that liver endothelial dysfunction occurs before the development of fibrosis or inflammation

Flickering in AGB stars: probing the nature of accreting companions

Binary companions to asymptotic giant branch (AGB) stars are an important aspect of their evolution. Few AGB companions have been detected, and in most cases it is difficult to distinguish between main-sequence and white dwarf companions. Detection of photometric flickering, a tracer of compact accretion discs around white dwarfs, can help identify the nature of these companions. In this work, we searched for flickering in four AGB stars suggested to have likely accreting companions. We found no signs for flickering in two targets: R Aqr and V1016 Cyg. Flickering was detected in the other two stars: Mira and Y Gem. We investigated the true nature of Mira’s companion using three different approaches. Our results for Mira strongly suggest that its companion is a white dwarf

All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells

BACKGROUND & AIMS: Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen. METHODS: Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity. RESULTS: Cell preparation yielded the following cell counts per gram of liver tissue: 2.0+/-0.4x107 hepatocytes, 1.8+/-0.5x106 Kupffer cells, 4.3+/-1.9x105 liver sinusoidal endothelial cells, and 3.2+/-0.5x105 stellate cells. Hepatocytes were identified by albumin (95.5+/-1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5+/-1.2%) and exhibited phagocytic activity, as determined with 1mum latex beads. Endothelial cells were CD146+ (97.8+/-1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of alpha-smooth muscle actin (97.1+/-1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence. CONCLUSIONS: Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease

Toll-like receptor-2 deficiency enhances non-alcoholic steatohepatitis

<p>Abstract</p> <p>Background</p> <p>Previously we reported that mice deficient in toll-like receptor 4 (TLR-4) signalling were protected from diet-induced non-alcoholic steatohepatitis (NASH). Another member of the toll-like receptor family, TLR-2, has been shown to play a role in lipid trafficking via uptake of diacylated lipoproteins. However, a role for TLR-2 in NASH has not been elucidated. The objectives of the current study were to examine the influence of dietary fat quality and TLR-2 on NASH pathogenesis.</p> <p>Methods</p> <p>Steatohepatitis was induced in male Db, C57BL/6 and TLR-2^-/-mice by feeding an L-amino acid-defined diet that was deficient in methionine and choline (MCDD). Mice fed the base diet supplemented with methionine and choline (control diet; CD) were used as controls. To determine the role of fat quality, MCDD was enriched with polyunsaturated corn oil (PUFA) or coconut oil that is comprised mostly of saturated fat (SAFA); the total amount of each fat was 112.9 g/kg of diet. After 8 weeks of feeding CD or MCDD, hepatic steatosis, inflammation and necrosis were evaluated in histological sections. Total RNA was extracted from frozen liver samples and mRNA expression of TNFα, collagen α1, IL-10, peroxisome proliferator-activated receptor-γ (PPAR-γ), TLR-4, and CD14, was analyzed via real-time PCR. Protein levels of TLR-2 were analyzed by western blot.</p> <p>Results</p> <p>Panlobular macrovessicular steatosis and diffuse leukocyte infiltration were noted in PUFA-fed Db mice. Histological scores demonstrated significantly less steatosis, inflammation and necrosis in SAFA-fed mice of all mouse strains. However, compared to wild type mice, hepatocellular damage was notably more severe in TLR-2^-/-mice. Consistent with histological findings, mRNA expression of TNFα was elevated by approximately 3-fold in TLR-2^-/-mice; PPAR-γ expression was blunted in this strain compared to wild type. Expression of the matrix protein collagen αI was also significantly higher in TLR-2^-/-mice, indicating a pro-fibrogenic state. Sensitivity to steatohepatitis due to dietary fat or TLR-2 deficiency correlated significantly with alterations in the expression of TLR-4 as well as the co-receptor CD-14.</p> <p>Conclusions</p> <p>Our findings suggest that dietary saturated fat plays a protective role against MCDD-induced steatohepatitis, whereas TLR-2 deficiency exacerbated NASH. The mechanism underlying the response to dietary fat and TLR-2 likely involves altered signalling via the TLR-4 pathway.</p

3D Hepatic Cultures Simultaneously Maintain Primary Hepatocyte and Liver Sinusoidal Endothelial Cell Phenotypes

Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro

Gu-4 Suppresses Affinity and Avidity Modulation of CD11b and Improves the Outcome of Mice with Endotoxemia and Sepsis

BACKGROUND: Systemic leukocyte activation and disseminated leukocyte adhesion will impair the microcirculation and cause severe decrements in tissue perfusion and organ function in the process of severe sepsis. Gu-4, a lactosyl derivative, could selectively target CD11b to exert therapeutic effect in a rat model of severe burn shock. Here, we addressed whether Gu-4 could render protective effects on septic animals. METHODOLOGY/PRINCIPAL FINDINGS: On a murine model of endotoxemia induced by lipopolysaccharide (LPS), we found that the median effective dose (ED50) of Gu-4 was 0.929 mg/kg. In vivo treatment of Gu-4 after LPS challenge prominently attenuated LPS-induced lung injury and decreased lactic acid level in lung tissue. Using the ED50 of Gu-4, we also demonstrated that Gu-4 treatment significantly improved the survival rate of animals underwent sepsis induced by cecal ligation and puncture. By adhesion and transwell migration assays, we found that Gu-4 treatment inhibited the adhesion and transendothelial migration of LPS-stimulated THP-1 cells. By flow cytometry and microscopy, we demonstrated that Gu-4 treatment inhibited the exposure of active I-domain and the cluster formation of CD11b on the LPS-stimulated polymorphonuclear leukocytes. Western blot analyses further revealed that Gu-4 treatment markedly inhibited the activation of spleen tyrosine kinase in LPS-stimulated THP-1 cells. CONCLUSIONS/SIGNIFICANCE: Gu-4 improves the survival of mice underwent endotoxemia and sepsis, our in vitro investigations indicate that the possible underlying mechanism might involve the modulations of the affinity and avidity of CD11b on the leukocyte. Our findings shed light on the potential use of Gu-4, an interacting compound to CD11b, in the treatment of sepsis and septic shock