Sinusoidal profiles of lactate dehydrogenase activity in rat liver

Lactate dehydrogenase activities were measured along two sinusoidal paths (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the livers of male Wistar rats, using a Lowry technique. The established profiles of enzyme activity provide further support of functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed. Within the hepatocytes a pronounced heterogeneity in enzyme activity was recorded surrounding small portal tracts and central veins. The lowest values of activity were determined in those cells located in close proximity to the vessels, which emphasizes their exceptional morphological and functional position.     

The distribution pattern of NADP+-dependent isocitrate dehydrogenase in rat liver

Activities of the NADP+-dependent isocitrate dehydrogenase were measured along the entire sinusoidal path (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the liver of male Wistar rats using a Lowry technique. The measured activities show a slight increase from the periportal to the perivenous end, whereas no such septal-) perivenous gradient could be established. These profiles of enzyme activity give further support to previous studies, suggesting functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed.     

Sinusoidal profiles of lactate dehydrogenase activity in rat liver

Summary Lactate dehydrogenase activities were measured along two sinusoidal paths (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the livers of male Wistar rats, using a Lowry technique. The established profiles of enzyme activity provide further support of functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed. Within the hepatocytes a pronounced heterogeneity in enzyme activity was recorded surrounding small portal tracts and central veins. The lowest values of activity were determined in those cells located in close proximity to the vessels, which emphasizes their exceptional morphological and functional position.Supported by grants of the Forschungsförderung des Landes Nordrhein-Westfalen, No. 40002585 and the Verein der Freunde und Förderer der Universität KölnDedicated to Professor Dr. Dietrich Eichner on the occasion of his 65th birthday     

Distribution pattern of alanine aminotransferase activity in rat liver

Summary Activities of the alanine aminotransferase were measured along the entire sinusoidal paths (1) between small portal tracts and central veins and (2) between regions of adjoining septal branches and central veins in the livers of male Wistar rats using a Lowry technique. The established profiles of enzyme activity give support to previous studies, suggesting functional heterogeneity of liver sinusoids and their abutting hepatocytes related to morphological differences of the sinusoidal bed.Dedicated to Professor Dr. T.H. Schiebler on occasion of his 65th birthdaySupported by grants of the Forschungsförderung des Landes Nordrhein-Westfalen, No. 40002585 and the Verein der Förderer und Freunded der Universität Köln     

Expression pattern of glutamine synthetase marks transition from collecting into conducting hepatic veins.

The expression of glutamine synthetase (GS) is confined to a rim of hepatocytes surrounding the efferent hepatic veins in all mammalian species investigated. In rat liver, a two- to three-cell thick layer of GS-positive (GS(+)) hepatocytes uniformly surrounds the two to four terminal branching generations of the hepatic vein that collect blood from sinusoids (central veins). With increasing diameter of the efferent vessel, this multilayered rim of GS(+) hepatocytes becomes confined to patches surrounding the decreasing number of central vein outlets. The remaining part of the wall of these sublobar hepatic veins is bordered by a one-cell thick layer of GS(+) hepatocytes. Around still larger veins, this single-cell layer of GS(+) hepatocytes gradually disappears. The expression pattern of GS is therefore a convenient biological parameter to delimit sinusoidal draining ("collecting") from nondraining ("conducting") surfaces in the wall of the efferent hepatic vessels. The hepatocytes surrounding a single tree of central veins together form a compound liver lobule. (J Histochem Cytochem 47:1507-1511, 1999)     

Transient expression of laminin alpha1 chain in regenerating murine liver: restricted localization of laminin chains and nidogen-1

Most interstitia between epithelial and endothelial cells contain basal laminae (BLs), as defined by electron microscopy. However, in liver, the sinusoidal interstitium (called space of Disse) between hepatocytes and sinusoidal endothelial cells (SECs) lacks BLs. Because laminins are major components of BLs throughout the body, whether laminins exist in sinusoids has been a controversial issue. Despite recent advances, the distribution and expression of laminin chains have not been well defined in mammalian liver. Here, using a panel of antibodies, we examined laminins in normal and regenerating mouse livers. Of alpha chains, alpha5 was widely observed in all BLs except for sinusoids, while the other alpha chains were variously expressed in Glisson's sheath and central veins. Laminin gamma1 was also distributed to all BLs except for sinusoids. Although the beta2 chain was observed in all BLs and sinusoids, the expression of beta1 chain was restricted to Glisson's sheath. Detailed analysis of regenerating liver revealed that alpha1 and gamma1 chains appeared in sinusoids and were produced by stellate cells. The staining of alpha1 and gamma1 chains reached its maximum intensity at 6 days after two-thirds partial hepatectomy (PHx). Moreover, in vitro studies showed that alpha1-containing laminin promoted spreading of sinusoidal endothelial cells (SECs) isolated from normal liver, but not other hepatic cells. In addition, SECs isolated from regenerating liver elongated pseudopodia on alpha1-containing laminin more so than did cells from normal liver. The transient expression of laminin alpha1 may promote formation of sinusoids after PHx.     

Changes in GM1 ganglioside content and localization in cholestatic rat liver

(Glyco)sphingolipids (GSL) are believed to protect the cell against harmful environmental factors by increasing the rigidity of plasma membrane. Marked decrease of membrane fluidity in cholestatic hepatocytes was described but the role of GSL therein has not been investigated so far. In this study, localization in hepatocytes of a representative of GSL, the GM1 ganglioside, was compared between of rats with cholestasis induced by 17α-ethinylestradiol (EE) and vehicle propanediol treated or untreated animals. GM1 was monitored by histochemical reaction employing cholera toxin B-subunit. Our findings in normal rat liver tissue showed that GM1 was localized in sinusoidal and canalicular hepatocyte membranes in both peripheral and intermediate zones of the hepatic lobules, and was nearly absent in central zones. On the contrary, in EE-treated animals GM1 was also expressed in central lobular zones. Moreover, detailed densitometry analysis at high magnification showed greater difference of GM1 expression between sinusoidal surface areas and areas of adjacent cytoplasm, caused as well by increased sinusoidal staining in central lobular zone as by decreased staining in cytoplasm in peripheral zone. These differences correlated with serum bile acids as documented by linear regression analyses. Both GM1 content and mRNA corresponding to GM1-synthase remained unchanged in livers; the enhanced expression of GM1 at sinusoidal membrane thus seems to be due to re-distribution of cellular GM1 at limited biosynthesis and could be responsible for protection of hepatocytes against harmful effects of bile acids accumulated during cholestasis.     

Immunogold localization of procollagen III,fibronectin and heparan sulfate proteoglycan on ultrathin frozen sections of the normal rat liver

Summary In the present study, we have localized by immunocytochemistry at the LM and EM level, procollagen type III (PIIIP), fibronectin (FN) and heparan sulfate proteoglycan (HSPG). Intracellularly, PIIIP was observed in both parenchymal and endothelial cells. In parenchymal cells, PIIIP was found in Golgi derived vesicles. This observation suggests that PIIIP synthesis is a normal function liver parenchymal cells. In endothelial cells, vesicles, which could not be identified, were seen to contain PIIIP. This result does not allow to conclude, whether sinusoidal endothelial cells secrete or take up PIIIP. Extracellularly, PIIIP was present around portal and central veins, in the space of Disse and between adjacent parenchymal cells. In the space of Disse, almost all interstitial collagen fibrils reacted with the anti PIIIP antibodies. This observation leads to the conclusion that most fibrils of the space of Disse contain type III in addition to type I collagen molecules.By immunofluorescence, FN was seen mainly along the sinusoids in discrete dots. By EM, FN was found to be present in diffuse material closely associated with the sinusoidal membrane of the parenchymal cells and in strands connecting adjacent parenchymal cells, parenchymal and endothelial cells or parenchymal cells and collagen fibrils. FN was also present in vascular and ductular basal laminae.Strong HSPG reaction was observed around bile ducts. Moderate reaction was seen around blood vessels and in the space of Disse. In the latter location, the ultrastructural distribution of HSPG resembles that of FN, i.e. HSPG is present in diffuse material and in strands.In honour of Prof. P. van Duijn     

Immunogold localization of procollagen III, fibronectin and heparan sulfate proteoglycan on ultrathin frozen sections of the normal rat liver

In the present study, we have localized by immunocytochemistry at the LM and EM level, procollagen type III (PIIIP), fibronectin (FN) and heparan sulfate proteoglycan (HSPG). Intracellularly, PIIIP was observed in both parenchymal and endothelial cells. In parenchymal cells, PIIIP was found in Golgi derived vesicles. This observation suggests that PIIIP synthesis is a normal function of liver parenchymal cells. In endothelial cells, vesicles, which could not be identified, were seen to contain PIIIP. This result does not allow to conclude, whether sinusoidal endothelial cells secrete or take up PIIIP. Extracellularly, PIIIP was present around portal and central veins, in the space of Disse and between adjacent parenchymal cells. In the space of Disse, almost all interstitial collagen fibrils reacted with the anti PIIIP antibodies. This observation leads to the conclusion that most fibrils of the space of Disse contain type III in addition to type I collagen molecules. By immunofluorescence, FN was seen mainly along the sinusoids in discrete dots. By EM, FN was found to be present in diffuse material closely associated with the sinusoidal membrane of the parenchymal cells and in strands connecting adjacent parenchymal cells, parenchymal and endothelial cells or parenchymal cells and collagen fibrils. FN was also present in vascular and ductular basal laminae. Strong HSPG reaction was observed around bile ducts. Moderate reaction was seen around blood vessels and in the space of Disse. In the latter location, the ultrastructural distribution of HSPG resembles that of FN, i.e. HSPG is present in diffuse material and in strands.     

Ultrastructure of the liver of the fingerling rainbow trout Salmo gairdneri Richardson

Portions of the livers of fingerling rainbow trout were studied by light and electron microscopy. The histology, cytology and ultrastructure of mesothelial cells, serosal fibroblasts, hepatocytes, sinusoidal endothelial cells, endothelial cells of central veins and blood cells were described. Mesothelial cells and fibroblasts constituted a very thin capsule. Hepatocytes contained extensive areas of rough surfaced endoplasmic reticulum, consisting mainly of parallel cisternae and pools of glycogen. One or two nuclei and numerous mitochondria occurred in the areas of endoplasmic reticulum, but never in the pools of glycogen. Hepatocyte surface possibilities included hepatocyte to hepatocyte, hepatocyte to bile canaliculus, hepatocyte to space of Disse and hepatocyte to serosa. The trout liver was compared compared to channel catfish liver and to rat liver. Functional implications of the structural features were discussed.     

Morphology and vascular anatomy of the accessory respiratory organs of the air-breathing climbing perch, Anabas testudineus (Bloch)

The vascular organization and endothelial cell specialization of the air-breathing organs of Anabas testudineus were examined by light and scanning electron microscopy of fixed tissue and vascular corrosion replicas. The vessels supplying blood to the lining of paired suprabranchial chambers and the plicated labyrinthine organs within the chambers are tripartite, having a median artery and paired, lateral veins. Hundreds of respiratory islets, the functional units of gas exchange, cover the surfaces of both the chamber and labyrinthine organ. A median islet artery supplies the central aspect of each islet and gives rise to numerous short arterioles from which the transverse channels are formed. Transverse channels are parallel capillary-sized vessels that extend in two rows away from the medial arterioles and drain laterally into one of two lateral islet veins. Basally situated single rows of endothelial cells lining the transverse channels form thick, evaginated, tongue-like cytoplasmic processes that project freely into the lumen from the tissue side of the channel. Other thin, septate, cytoplasmic extensions of the same cells form valve-like septa that extend across the channel. Both the septa and tongue-like processes appear to direct the red blood cells to the epithelial side of the channel and thus decrease the diffusion distance between the air and red cell. A large sinusoidal space lies under the transverse channels and may support the channels and even elevate them during increased oxygen demand. The epithelium covering the transverse channels is smooth, which enhances air convection and minimizes unstirred layer effects. The epithelium between the channels contains microvilli that may serve to trap bacteria or particulates and to humidify the air chambers.     

A quantitative histochemical study of 5′-nucleotidase activity in rat liver using the lead salt method and polyvinyl alcohol

Summary 5-Nucleotidase (EC 3.1.3.5) activity was demonstrated in cryostat sections of rat liver using the Wachstein—Meisel medium and polyvinyl alcohol as tissue stabilizer. Optimum activity was obtained using an incubation medium containing 5mm AMP, 10mm magnesium chloride, 7.2mm lead nitrate, 0.1m Tris—maleate buffer, pH 7.2, and 17% (w/v) polyvinyl alcohol (Sigma, type III). The activity was localized at the bile canalicular and sinusoidal side of the plasma membranes of liver parenchymal cells as well as in the plasma membranes of endothelial cells of central veins and in fibroblasts surrounding portal tracts. The reaction was specific for 5-nucleotidase because it was inhibited by ADP. Alkaline phosphatase did not interfere in the reaction. Cytophotometric analysis revealed a linear relationship between the formation of the final reaction product and incubation times up to 20 min and section thicknesses up to 8m. The activity in pericentral zones was 1.35 times the activity in periportal zones. The Michaelis constant for AMP was 1.4mm in pericentral zones and 0.8mm in periportal zones, suggesting that the bile canalicular and sinusoidal enzymes differ in their kinetic characteristics.     

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.     

Microvascular changes of the liver preserved in UW solution. Pathological and immunohistochemical examination.

Rat livers preserved in University of Wisconsin (UW) solution for 24 h were compared with those preserved in Euro-Collins (EC) solution before and after liver transplantation using an immunohistochemical method. Tissue ATP and total tissue adenine nucleotide (TAN) were measured using HPLC. The levels of TAN in the UW group or the EC group were significantly low compared with the control group (no preservation) after 24-h storage. In the EC group, the levels of tissue adenine nucleotides (TAN) decreased 1 h after reperfusion and never reached control levels. In the UW group, the levels of TAN increased a little 1 h after reperfusion and increased more 3 h after reperfusion. After 24-h preservation, the expression of factor VIII-related antigen (FRA) in endothelial cells of central veins was weak in the EC group; in the UW group, FRA was clearly detected in these cells. After reperfusion, although severe endothelial cell damage to the central veins and numerous FRA-positive substances were observed in EC group, endothelial cells of central veins retained their normal structure and FRA-positive substances were rarely noted in the UW group. In both groups, no endothelial changes were detected in portal veins. From these results, it is concluded that UW solution prevents endothelial cell damage and microcirculatory injury in zone III during the preservation period resulting in prevention of initial graft nonfunction. Also, measurement of the TAN level after reperfusion is useful to predict the function of the graft.     

The superficial cerebral veins

Estimated were the number, the course, and the width of the superficial cerebral veins. The veins on the superolateral surface of the brain are the prefrontal superficial lateral superior, the precentral superficial lateral superior, the central superficial lateral superior, the parietal superficial lateral superior, and the occipital superficial lateral superior veins which drain to the superior sagittal sinus, to bridging veins, and to the falx cerebri. The veins which drain the lateral surface of the brain downwards are the middle superficial cerebral veins, the temporal inferior, occipital inferior, and anastomotic veins. The diameters of these veins were measured at the perforation of the arachnoid membrane and the diameters of the anastomotic veins on their narrowest area. On the medial side of the hemispheres, we divided in precentral superficial superior medial, central superficial medial, parietal superficial medial, occipital superficial medial dorsal veins of the corpus callosum, and internal occipital veins. On the basal surface of the hemispheres, we studied and described the uncal veins and the inferior hemispheric veins. Studied and discussed are also the bridging veins in the course of the inferior cerebral veins, the paracavernous sinuses, and the last course of the veins and their connections with the dura mater or the course inside the dura. Given are besides the numbers of these veins, the area of perforation of the arachnoid membrane, and their width and medical importance.     

Microscopy of vascular architecture and arteriovenous communications in the spleen of two odontocetes

The red pulp of the spleens of the short-finned pilot whale (Globicephala macrorhynchus) and the Pacific bottle-nosed dolphin (Tursiops truncatus gilli) (Odontoceti) were examined by light and electron microscopy and found to comprise two venous layers, an inner and an outer. The inner layer is homologous to the intermediate zone (IZ) of primitive-type mammalian spleens and contains sinusoids consisting of endothelial cells and a thin layer of extracellular deposits. Its vascular structure is unclear. The venous vessels of this layer eventually communicate with veins of the perivenous outer layer. The perivenous layer contains veins of various sizes, interstitial elements, and trabeculae. It is filled with blood cells, particularly plasma cells, but no myeloid cells. The perivenous layer (PVL) is homologous to the red pulp of common mammalian spleens but shows signs of involution. The white pulp gives origin to arterial terminals that end in the red pulp, where they communicate directly with the sinusoidal veins producing a closed circulation. The arterial terminals do not show ellipsoids. The presence of an IZ with a closed circulation and the involution of the red pulp makes the spleen of Odeontoceti another example of a mammalian spleen of the primitive type that has been altered by the evolutionary process. Vascular remodelling of the spleen of Odontoceti seems to follow the pattern noted in the spleens of nonmammalian vertebrates. © 1994 Wiley-Liss, Inc.     

The transmural migration and release of blood cells in acute myelogenous leukemia.

The transmural passage of malignant blood cells from the extravascular parenchyma into sinusoidal lumen has been studied in the bone marrow of rats with myelogenous leukemia. The Shay myelogenous leukemia was chosen as a model system because an increased bone marrow cellularity is, in this leukemia, usually accompanied by an increase in circulating myeloid cells. By means of light microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM) it was found that the sinusoidal endothelial lining of the bone marrow remains intact and continuous even in advanced stages of the disease. SEM shows that the malignant myeloblast-like cell enters the sinusoidal lumen by means of a temporary migration pore, which appears only during the transmural passage of the cell. Certain nondegenerative changes in the sinusoidal blood vessels are associated with the myelogenous leukemia. The normal radial alignment of sinusoids about the central sinusoid is changed into a tortuous pattern, and intraluminal cytoplasmic bridges which impede the blood flow are formed by the endothelial cells.     

A biphasic model for sinusoidal liver perfusion remodeling after outflow obstruction

Liver resection can lead to focal outflow obstruction due to transection of hepatic veins. Outflow obstruction may cause additional damage to the small remnant liver. Drainage of the obstructed territories is reestablished via dilatation of sinusoids. Subsequently, sinusoidal canals are formed draining the blood from the obstructed territory to the neighboring unobstructed territories. We raised the phenomenological hypothesis that the blood pressure gradient is the main driving force for the formation of sinusoidal vascular canals. We generated a biphasic mechanical model to describe this vascular remodeling process in relation to the variable pressure gradient. Therefore, we introduced a transverse isotropic permeability relation as well as an evolutional optimization rule to describe the relationship between pressure gradient and the direction of the sinusoidal blood flow in the fluid phase. As a next step, we developed a framework for the calculation concept including the representation of the governing weak formulations. Then, we examined a representative numerical example with simulation of the blood flow under both conditions, the physiological situation as well as after outflow obstruction. Doing so, we were able to reproduce numerically the experimentally observed process of reestablishing hepatic venous drainage via redirection of blood flow and formation of new vascular structures in respect to the fluid flow. The calculated results support the hypothesis that the reorientation of blood flow mainly depends on the pressure gradient. Further investigations are needed to determine the micromechanical influences on the reorientation of the sinusoids.     

Dual origin of avian lymphatics

The earliest signs of the lymphatic vascular system are the lymph sacs, which develop adjacent to specific embryonic veins. It has been suggested that sprouts from the lymph sacs form the complete lymphatic vascular system. We have studied the origin of the jugular lymph sacs (JLS), the dermal lymphatics and the lymph hearts of avian embryos. In day 6.5 embryos, the JLS is an endothelial-lined sinusoidal structure. The lymphatic endothelial cells (LECs) stain (in the quail) positive for QH1 antibody and soybean agglutinin. As early as day 4, the anlagen of the JLS can be recognized by their Prox1 expression. Prox1 is found in the jugular section of the cardinal veins, and in scattered cells located in the dermatomes along the cranio-caudal axis and in the splanchnopleura. In the quail, such cells are positive for Prox1 and QH1. In the jugular region, the veins co-express the angiopoietin receptor Tie2. Quail-chick-chimera studies show that the peripheral parts of the JLS form by integration of cells from the paraxial mesoderm. Intra-venous application of DiI-conjugated acetylated low-density lipoprotein into day 4 embryos suggests a venous origin of the deep parts of the JLS. Superficial lymphatics are directly derived from the dermatomes, as shown by dermatome grafting. The lymph hearts in the lumbo-sacral region develop from a plexus of Prox1-positive lymphatic capillaries. Both LECs and muscle cells of the lymph hearts are of somitic origin. In sum, avian lymphatics are of dual origin. The deep parts of the lymph sacs are derived from adjacent veins, the superficial parts of the JLS and the dermal lymphatics from local lymphangioblasts.     

Culture and characterization of sinusoidal endothelial cells isolated from human liver

Summary Although most vascular models use large vessel endothelial cells from human umbilical veins, there is marked heterogeneity among endothelial cells from different vascular beds and organs. More accurate modeling of endothelial involvement in liver diseases, including metastasis, may result from the use of human hepatic sinusoidal endothelial cells. Liver resection specimens were sectioned, then treated with a 1.2 U/ml dispase solution. The tissue slurry was mechanically disaggregated and separated by centrifugation on a Percoll density gradient. Cells were then cultured in an endothelial-specific media with growth factors. These techniques resulted in a homogeneous monolayer consistent with endothelial cells by light microscopy. An endothelial origin was further confirmed by the expression of Factor VIII, binding of Ulex lectin, and uptake of acetylated low density lipoprotein. Electron microscopy showed transcellular fenestrations consistent with a sinusoidal origin. These human hepatic sinusoidal endothelial cells were then studied for expression of the adhesion molecules CD31/PECAM, CD34, E-selectin, ICAM-1, L-selectin, LFA-3, P-selectin, and VCAM-1 plus the binding of wheat germ agglutinin lectin. The patterns of adhesion molecule expression and lectin binding by these cells are characteristic of hepatic sinusoidal endothelia. In this paper, we have described a method for isolation and culture of human cells with the morphologic and phenotypic characteristics of hepatic sinusoidal endothelia.