Histochemical analyses of hepatic architecture of the hagfish with special attention to periportal biliary structures

The hagfish liver was histochemically examined with special attention to biliary structures around the portal veins. Hepatocytes were organized into tubular structures surrounded by sinusoids. Biliary ductule structures, which resemble the ductal plates transiently appearing in mammalian liver development, were observed around the portal veins, but they did not appear around central veins. Thus, the hagfish liver demonstrates the same basic structure as the mammalian liver; that is, a vascular system from portal to central veins via sinusoids, and portal triad structures consisting of portal veins, hepatic arteries, and intrahepatic bile ducts. The epithelial cells of the ductal platelike structures strongly expressed cytokeratin, had some lectin-binding sites, and were delineated by the basal lamina, which was reactive for periodic acid-Schiff (PAS) staining and Iectin histochemistry. The lumina of the ductal plate-like structures were comparatively small and heterogeneous in diameter around the portal veins, suggesting that the biliary structures may not be efficient for bile secretion. The epithelial cells of the gall bladder had a simple columnar shape and were a PAS-positive cytoplasm. Those of bile ducts near the hilus, including extrahepatic and hepatic ducts, were simple columnar or cuboidal cells, and had large lumina. The cytoplasm in these cells was PAS-positive. These phenotypes with the expression of lectin-binding sites were clearly different from those of the ductal plate-like structures in the liver proper, suggesting that the extrahepatic and intrahepatic biliary structures may have different developmental origins.     

The liver: a model of organ-specific lymphocyte recruitment

The liver is constantly exposed to gut-derived antigens that enter via the portal vein, and it must modulate immune responses so that harmful pathogens are cleared but necessary food antigens are ignored. The liver contains a large resident and migratory population of lymphocytes and macrophages that provide immune surveillance against foreign antigen. This population of cells can be rapidly expanded in response to infection or injury by recruiting leukocytes from the circulation, a process that is dependent on the ability of lymphocytes to recognise, bind to and migrate across the endothelial cells that line the vasculature. Lymphocytes can enter the liver at several sites: the vascular endothelium in the portal tracts (comprising the hepatic artery, portal vein and bile ductule), the sinusoids (through which the blood percolates past the hepatocytes) or the central hepatic veins (through which the blood exits). The requirements and physical conditions at each site vary and there is evidence that different combinations of adhesion proteins are involved at these different sites. This article discusses the expression and function of adhesion molecules within the liver and demonstrates how specific populations of effector lymphocytes can be selectively recruited to the liver.     

Distribution of the major histocompatibility complex antigens on different cellular components of human liver

Monoclonal mouse antibodies to the "framework" determinants of the class I and II molecules of the major histocompatibility complex (MHC) were used to demonstrate the presence of the MHC antigens in human liver. First, the localization of these antigens was demonstrated from frozen section histology with indirect FITC immunofluorescence and the cell component(s) binding the mouse antibody were identified by rabbit marker antisera and indirect TRITC immunofluorescence. Second, the antigen expression on the cell surface was analyzed by the Staphylococcus aureus rosette method from cytological cell smears. All antibodies reacted with cells in the liver sinusoids, both with the Kupffer cells and at least partially with the sinusoidal endothelial cells. The same antisera reacted also with the bile duct cells, though weaker, and with some stromal cells in close proximity of the blood vessels. The vascular endothelial cells of hepatic artery, hepatic vein, and portal vein displayed no reaction. Thus human liver differs strikingly from, e.g., human kidney, where the vascular endothelial cells contain large amounts of MHC antigens on the cell surface. This difference may be one explanation to why liver allografts are less promptly rejected than renal allografts in man.     

Blood-flow route from the hepatic artery and portal vein to the sinusoid in normal human liver observed by confocal laser scanning microscopy

OBJECTIVE: To observe the microvasculature in normal human liver. STUDY DESIGN: Four autopsy livers cut into 50-micron-thick sections were observed by confocal laser scanning microscopy. Immunofluorescence was performed using anti-alpha smooth muscle actin (alpha-SMA) antibody. In addition, double immunofluorescence was performed on the other sections using antilysozyme antibody. The routes from the portal vein branches and hepatic artery branches to the sinusoids were defined as follows: portal venule, septal branch, inlet venule, hepatic arteriole and terminal hepatic arteriole. RESULTS: The reactivity of the walls of septal branches and inlet venule was positive for alpha-SMA. Lysozyme-positive cells (Kupffer cells) were dense in the sinusoids but were sparse in the septal branches and absent from the inlet venules. Terminal hepatic arterioles were observed along the septal branch, and the anastomoses between them were observed at the peripheral portion. No routes opening directly from the terminal hepatic arteriole into the sinusoids or arterioportal anastomoses in the portal tract were observed on alpha-SMA-stained sections. CONCLUSION: Regulation of the microcirculation in human liver may be performed by the smooth muscle layer of both peripheral portal and hepatic arterial routes.     

Scanning electron microscopy of normal rat liver: the surface structure of its cells and tissue components.

Scanning electron microscopy (SEM) allows the surface ultrastructure of intrahepatic cells and other tissue components of liver to be delineated. Excellent depth of focus of the SEM makes it possible to visualize surfaces of intact cells in their native configurations. This report details the surface characteristics and inter-relationships of hepatocytes and hepatic plates, sinusoidal endothelial cells and sinusoids, presumed Kupffer cells, vessels, bile ducts, connective tissue, and the capsule of rat liver. Hepatocytes present three structurally distinctive faces--the intercellular face containing flat surfaces and bile canaliculus, the sinusoidal face, and the connective tissue face which abuts portal tracts and hepatic veins. Sinusoidal endothelium is penetrated by large (1 to 3 mum) and small (0.1 mum) fenestrae, the latter occurring in clusters of up to 50. The width of bile canaliculi and distribution of large fenestrae vary proximodistally along hepatic plate or sinusoid. The cells of portal bile ductules contain microvilli located in linear rows and sparse cilia. Endothelium of hepatic artery and of portal vein is sparsely fenestrated.     

Vascularization and related distribution of leucocytes in carp, Cyprinus carpio L., head kidney

The distribution of lymphoid cells in the carp head kidney was investigated in relation to the vascular system. Blood vessels in the head kidney were histologically identified into arteries, sinusoids and two types of veins: renal veins and portal, which were distinguished by India ink injection into the caudal vein. By histological and histoplanimetrical observations it was found that the head kidney contained a number of lymphoid cells, which mainly aggregate around the connections between the portal veins and sinusoids, and that the cellular density of the aggregations was higher than in the thymus.
Pigment-containing cell clusters were also observed around these connections. This arrangement of the blood vessels suggests that it is one of the structures able to trap foreign materials, and the occurrence of the lymphoid clusters around the portal veins is a phylogenetic sign of the morphological division between granulopoietic and lymphatic tissues in the carp head kidney.     

Adenoviral delivery of human connexin37 induces endothelial cell death through apoptosis

Gap junction channels formed of connexins directly link the cytoplasm of adjacent cells and have been implicated in intercellular signaling that may regulate the functions of vascular cells. To facilitate connexin manipulation and analysis of their roles in adult endothelial cells, we developed adenoviruses containing the vascular connexins (Cx37, Cx40, and Cx43). We infected cultured human umbilical vein endothelial cells with control or connexin adenoviruses. Connexin expression was verified by immunoblotting and immunofluorescence. Infection with the Cx37 adenovirus (but not control or other connexin adenoviruses) led to a dose-dependent death of the endothelial cells that was partially antagonized by the gap junction blocker alpha-glycyrrhetinic acid and altered the intercellular transfer of Lucifer yellow and neurobiotin. Cell morphology, Annexin V and TUNEL staining, and caspase 3 assays all implicated apoptosis in the cell death. These data suggest that connexin-specific alterations of intercellular communication may modulate endothelial cell growth and death.     

Effect of the ligation of hepatic artery on the microcirculation in the cirrhotic liver in the rat.

The significance of the hepatic arterial supply in the intrahepatic microcirculation in normal and carbon tetrachloride-induced cirrhotic livers was studied by dye injection method and by ligation of the hepatic artery. The in vivo distribution of dye injected into the hepatic artery evidenced the presence of arterio-venous shunts in the cirrhotic liver. When the hepatic artery of the cirrhotic liver was ligated, the elevated portal venous pressure dropped significantly, and the fast-flowing population of microvessels and sinusoids in the bimodal frequency distribution plot disappeared. The fast-flowing microvessel and sinusoids appeared to be the "arterial" microvessels and sinusoids, and they were converted into the slow-flowing venous channels after hepatic arterial ligation. The transmission of arterial pressure via the A-V shunts may be of greater significance in the pathophysiology of portal hypertension than previously believed.     

Sequential changes in redox status and nitric oxide synthases expression in the liver after bile duct ligation

Bile duct ligation (BDL) in rats induces portal fibrosis. This process has been linked to changes in the oxidative state of the hepatic cells and in the production of nitric oxide. Our objective was to find possible temporal connections between hepatic redox state, NO synthesis and liver injury. In this work we have characterized hepatic lesions 17 and 31 days after BDL and determined changes in hepatic function, oxidative state, and NO production. We have also analyzed the expression and localization of inducible NO synthase (NOS2) and constitutive NO synthase (NOS3). After 17 and 31 days from ligature, lipid peroxidation is increased and both plasma concentration and biliary excretion of nitrite+nitrate are rised. 17 days after BDL both NOS2 and NOS3 are expressed intensely and in the same regions. 31 days after BDL, the expression of NOS2 remains elevated and is localized mostly in preserved hepatocytes in portal areas and in neighborhoods of centrolobulillar vein. NOS3 is localized in vascular regions of portal spaces and centrolobulillar veins and in preserved sinusoids and although its expression is greater than in control animals (34%), it is clearly lower (50%) than 17 days after BDL. The time after BDL is crucial in the study of NO production, intrahepatic localization of NOS isoforms expression, and cell type involved, since all these parameters change with time. BDL-induced, peroxidation and fibrosis are not ligated by a cause-effect relationship, but rather they both seem to be the consequence of common inductors.     

Laminin expression during bone marrow mononuclear cell transplantation in hepatectomized rats

The adult bone marrow retains two populations of stem cells with emerging importance for the treatment of diverse liver diseases: hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the mechanisms that control liver regeneration after bone marrow cell transplantation are still controversial. Liver regeneration after partial hepatectomy is a complex process that requires the proliferation of all hepatic cells. Growth factors, cytokines and extracellular matrix molecules are key elements in this process. Laminins are a family of extracellular matrix proteins with adhesive and chemotactic functions, expressed in the portal and centrolobular veins of the normal liver. The aim of this study was to investigate laminin expression during liver regeneration induced by partial hepatectomy followed by bone marrow mononuclear cell (BMMNC) transplantation. Rat BMMNCs were isolated by Ficoll-gradient centrifugation, stained with DAPI and injected into recently hepatectomyzed rats via the portal vein. Liver sections obtained 15 min, 1 day and 3 days after the surgery were immunolabeled with anti-rat CD34 and/or laminin primary antibodies and observed under a laser scanning confocal microscope. Results showed that 15 min after partial hepatectomy, a transplanted CD34+ HSC was found in contact with laminin, which was localized in the portal and centrolobular veins of rat livers. Furthermore, 1 and 3 days after hepatectomy, transplanted BMMNCs were found in the hepatic sinusoids expressing laminin. These results strongly suggest that laminin might be an important extracellular matrix component for bone marrow cell attachment and migration in the injured liver.     

Simultaneous sampling of portal, hepatic and systemic blood during intragastric loading and tracer infusion in conscious pigs

A surgical model for catheterization at multiple sites has been developed for use in long-term metabolic studies. For blood sampling, catheters were inserted into the portal and hepatic veins and the common carotid artery. The hepatic vein catheter was inserted from the margin of a liver lobe and led through the venous system, until the tip was close to the bifurcation with the inferior vena cava. A new technique was developed to ensure correct placement of the hepatic vein catheter using the specific extraction of indocyanin-green over the liver during surgery. Gastrostomy was performed using a Pezzer catheter. Catheters in the artery and hepatic and portal veins were patent for blood withdrawal for up to 4 weeks, and thus allowed repeated metabolic studies. Studies were performed in conscious animals familiar with the experimental situation.     

Effect of hypokinesia and the combined action of gravitational load and hypokinesia on the structure of the hepatic portal system]

General hypokinesia during 1--6 weeks resulted in dilatation of the interlobular veins. sinusoids and central veins. The sequence of alterations corresponded to terms of hypokinesia. After exposure to "gravitation stress--hypokinesia for 1--6 weeks" stagnation in the portal system of the liver was less than after exposure to hypokinesia alone, but unevenness of lumens in the interlobular veins and sinusoids was more pronounced. The foci of the vessel spasm were determined. The signs of stagnation in the system of the portal vein and unevenness of the width of all the links of the portal bed were most pronounced after combination "hypokinesia for 1--6 weeks-- gravitation stress".     

Transport of amino acids in the splanchnic bed by blood plasma and blood in the preruminant calf]

Three preruminant calves were fitted with catheters in portal and hepatic veins and in a mesenteric artery. Two electromagnetic flowmeter probes were clipped around the portal vein and the hepatic artery. The calves were fed either a diet with a low (L) or a high (R) abomasal emptying rate for dietary proteins. Blood flow and free amino acid levels in plasma (P) and blood (S) were determined before the morning meal and during the following 7 h. In the portal vein, for most amino acids P/S ratios were correlated to the net amino acid balance of the digestive tract measured in plasma. By contrast in the hepatic vein, these ratios were mainly correlated to hepatic balance measured in whole blood. Correlations between digestive tract and hepatic balance calculated using either plasma or whole blood pool were different for some amino acids. This suggests that amino acid exchange between plasma and blood cells is low and absorbed amino acids are mainly transported to the liver by plasma, whereas whole blood rather than plasma is concerned in amino acid exchanges in the liver.     

Cholinergic nerves in the human liver

Summary The cholinergic innervation of the human liver was studied. Slices (150–200m thick) of human liver and of the greater hepatic blood vessels (hepatic artery and vein, portal vein) were incubated in a solution of 6-hydroxydopamine (6-HDA) in order to obtain a selective degeneration of adrenergic nerves. Controls were prepared from samples incubated with buffer alone. The slices were cut on a cryostat into 15–20m thick sections and processed for the histochemical detection of cholinesterases.Cholinergic nerve fibres innervate the extra hepatic and the intrahepatic branches of the hepatic artery, the portal vein as well as the hepatic vein. Fewer cholinergic fibres innervate the hepatocytes and the hepatic sinusoids. The 6-HDA treatment does not seem to alter the pattern of the cholinergic innervation of the liver. The findings indicate the presence of a cholinergic parasympathetic innervation in the human liver.     

一雄性灰鹤胃的血液供应

用血管铸型法对一只因伤致死的雄性灰鹤胃的血供进行铸型观察,结果显示,灰鹤的胃动脉均由腹腔动脉分出,腺胃由腺胃背侧动脉和腺胃腹侧动脉供应营养,肌胃由胃左动脉、胃右动脉和肌胃背侧动脉供应营养。腺胃的静脉有腺胃腹侧静脉、胃凹腹侧静脉和腺胃背侧静脉,分别经左（腺胃腹侧静脉和胃凹腹侧静脉）、右（腺胃背侧静脉）肝门静脉回流;肌胃的静脉有胃左静脉、胃右静脉和胃背侧静脉,分别经左（胃左静脉）、右（胃右静脉和肌胃背侧静脉）肝门静脉回流。此外本文将灰鹤胃的血供与其它动物的进行了比较。     

环颈雉胃的血供

用血管铸型法和大体解剖学方法对环颈雉胃动脉的起源、分布及胃静脉的回流情况进行了解剖学研究。结果表明,环颈雉的胃动脉均由腹腔动脉分出;腺胃由腺胃背侧动脉和腺胃腹侧动脉营养,腺胃背侧动脉直接起自腹腔动态的左侧,腺胃腹侧动脉起自腹腔动脉左支。腺胃血液的静脉有腺胃前静脉和腺胃后静脉,分别汇入后腔静脉和左肝门静脉。肌胃由肌胃左动脉、肌胃右动脉和肌胃背侧动脉营养,肌胃左动脉起自腹腔动脉的左支;肌胃右动脉起自腹腔动脉的右支;肌胃背侧动脉从腺胃背动脉分支而来。回流肌胃血液的静脉有胃右静脉、胃左静脉和胃腹侧静脉;胃右静脉汇入右肝门静脉,胃左静脉和胃腹侧静脉汇入左肝门静脉。另外腺胃和肌胃的表面缺乏主干动脉间的吻合。     

Modifications in connexin expression in liver development and cancer

The establishment of an elaborate gap junctional intercellular communication network, especially between hepatocytes, is important for normal liver development. In fact, the production of the gap junction building blocks, the connexins, undergoes several well-defined changes throughout the hepatic differentiation process. This ultimately results in the acquisition of an adult connexin expression pattern which is critical for maintaining the fully differentiated hepatocyte-specific phenotype. Abnormalities of connexin production are observed in a number of pathological conditions, such as during liver cancer. This article provides an overview of these processes with emphasis on the underlying molecular mechanisms.     

Immunohistochemical demonstration of Angiopoietin-2 in lymphatic vascular development

The cellular expression of Angiopoietin-2 (Ang2) was studied during lymphatic development in mouse by immunohistochemistry and compared to that of lymphatic endothelial markers. At the earliest stage of lymphvasculogenesis, Prox1-identified lymphatic precursor cells of the cardinal vein displayed an intense immunoreaction for Ang2 in their cytoplasm, implying that Ang2 may adjust lymphatic specification and sprouting from the veins under the control of Prox1. Thereafter, Ang2 was constantly expressed in Prox1 and/or LYVE-1-immunopositive endothelial cells of lymphatic sacs and vessels, ranging from lymphatic capillaries to collectors, throughout embryonic and neonatal development, and the lymphatic endothelial cells simultaneously exhibited immunoreactivity to Tie2, a primary receptor for angiopoietins. These results suggest that lymphatic endothelial cells may regulate lymphatic development via their own Ang2-Tie2 signaling. Ang2 is further immunolocalized in the developing blood vessels including hepatic sinusoids, adrenal medullary vasculature and postnatal pulmonary vessels, thereby indicating that the blood vessels, which undergo vascular remodeling and sudden alteration of blood flow during the development, are also likely to express Ang2. The present study is first to demonstrate Ang2 expression in the lymphatic endothelial cells during development, and consequently Ang2 is regarded as a molecular profile of the developing lymphatic endothelial cells required for lymphatic vascular organization.     

Hepatic fibrosis and cirrhosis: the (myo)fibroblastic cell subpopulations involved

Fibrosis, defined as the excessive deposition of extracellular matrix in an organ, is the main complication of chronic liver damage. Its endpoint is cirrhosis, which is responsible for significant morbidity and mortality. The accumulation of extracellular matrix observed in fibrosis and cirrhosis is due to the activation of fibroblasts, which acquire a myofibroblastic phenotype. Myofibroblasts are absent from normal liver. They are produced by the activation of precursor cells, such as hepatic stellate cells and portal fibroblasts. These fibrogenic cells are distributed differently in the hepatic lobule: the hepatic stellate cells resemble pericytes and are located along the sinusoids, in the Disse space between the endothelium and the hepatocytes, whereas the portal fibroblasts are embedded in the portal tract connective tissue around portal structures (vessels and biliary structures). Differences have been reported between these two fibrogenic cell populations, in the mechanisms leading to myofibroblastic differentiation, activation and "deactivation", but confirmation is required. Second-layer cells surrounding centrolobular veins, fibroblasts present in the Glisson capsule surrounding the liver, and vascular smooth muscle cells may also express a myofibroblastic phenotype and may be involved in fibrogenesis. It is now widely accepted that the various types of lesion (e.g., lesions caused by alcohol abuse and viral hepatitis) leading to liver fibrosis involve specific fibrogenic cell subpopulations. The biological and biochemical characterisation of these cells is thus essential if we are to understand the mechanisms underlying the progressive development of excessive scarring in the liver. These cells also differ in proliferative and apoptotic capacity, at least in vitro. All this information is required for the development of treatments specifically and efficiently targeting the cells responsible for the development of fibrosis/cirrhosis.     

A lumped parameter mathematical model of the splanchnic circulation.

A lumped parameter mathematical model to describe the propulsion of blood in the splanchnic circulation was developed by integrating the principles of mechanics and physiology. A set of governing equations by derived by specifically considering the contractility of the portal vein, hepatic vein, liver sinusoids, and of the draining lymphatics. These equations were then simulated on a computer. The present simulation results substantiate previous experimental observations that hepatic venous pressure leads to portal hypertension and increased liver interstitial fluid volume.