Ultrastructural study of Kupffer cells in teleost liver under normal and experimental conditions

Summary The Kupffer cells in the liver of the teleost fish, Pimelodus maculatus, are attached by desmosomes to the endothelial cells lining the sinusoids. These provide a strong attachment allowing them to resist the passage of blood. Following perfusion with India ink, both endothelial and Kupffer cells ingest India ink particles by pinocytosis and micropinocytosis. It is suggested that both cell types may represent two different functional states of the same cell.     

The organization of the sarcoplasmic reticulum in teleost ventricular myocardial cells

Summary The morphology of the sarcoplasmic reticulum in myocardial cells of the ventricle of the trout heart is described as the result of an investigation with the electron microscope. The sarcoplasmic reticulum is sparse in distribution compared to that of birds or mammals but shows a fundamentally similar organization. A very loose network of fine tubules is in intimate contact with the myofibrils but with no local modification with respect to the arrangement of myofilaments within the sarcomeres. There is no special association of the sarcoplasmic reticulum with the Z-bands. Some tubules pass to the cell periphery where they expand to form subsarcolemmal cisternae in which electron-dense matter is often seen. The occurrence of the subsarcolemmal cisternae (peripheral couplings) is random and they are not observed in the vicinity of intercalated discs. The sarcoplasmic reticulum is discussed in relation to excitation-contraction coupling in teleost myocardial cells, and in comparison with that of other vertebrates.I am grateful to Professor J. D. Lever for making certain facilities available, and to Mr. P. F. Hire for photographic assistance.     

Characterization of sinusoidal endothelial cells of the liver and bone marrow using an intravital lectin injection method

The vascular endothelia express a variety of structural and biological phenotypes. We used an intravital injection method of plant derived lectins (Lycopersicon esculentum lectin (LEL), Ricinus communis Agglutinin-I (RCA-I), Ulex europaeus Agglutinin-I (UEA-I) and Concanavalin A (ConA)) to elucidate the morphology and function of the sinusoidal endothelium of the liver and bone marrow. All four lectins stained the sinusoidal endothelia of the liver and bone marrow in a patchy granular pattern which differed from the uniform and smooth staining pattern of non-sinusoidal vessels in other organs. By transmission electron microscopy, the granular pattern was identified as internalization of these lectins and subsequent accumulation within the endothelial cells, suggesting their active endocytosis. The endocytosis of these lectins emphasizes the fact that sinusoidal endothelial cells of the liver and bone marrow belong to the reticuloendothelial system (RES), a cell system characterized by internalization of foreign material. We introduce this intravital lectin injection as a useful technique to discriminate sinusoidal endothelial of the liver and bone marrow from other vascular endothelia.     

Three-dimensional structure of endothelial cells in hepatic sinusoids of the rat as revealed by the Golgi method

Summary The three-dimensional structure of endothelial cells in the hepatic sinusoids of the rat was studied by application of light- and electron microscopy on Golgi-impregnated specimens. A number of endothelial cells could thus be individually delineated throughout the hepatic lobules. The cytoplasm, showing heavy silver deposits, consists of two distinct areas, a thick and thin portion. The thick portion, issuing from the region of the perikaryon, branches and tapers toward the cell periphery. The thin portion, occupying the remainder of the cytoplasm, consists largely of highly fenestrated sieve plates. Some intralobular variation can be noted; the thick portion of the endothelial cells is well developed in the periportal zone, while the cells in the centrilobular zone are relatively rich in thin portions. In addition, the area of distribution of an individual endothelial cell is larger in the centrilobular sinusoids than in the periportal zone. Some endothelial cells also possess unique cytoplasmic processes projecting into the intercellular space between hepatocytes and connecting the sinusoidal walls of neighboring sinusoids. These processes may anchor the endothelial cells to the hepatic plates.     

Differentiation of vascular pseudointima under normal and disturbed blood flow conditions: Ultrastructural observations in the rat

Summary To study the effect of haemodynamic stress on the morphological differentiation of pseudointima, the ultrastructure of the cells lining normally shaped and aneurysmal polyurethane vascular prostheses implanted into the abdominal aorta of rats was examined. In the normally shaped vascular prostheses the pseudointima was composed of several layers of smooth muscle cells, which varied in differentiation from normal smooth muscle cells to myofibroblasts, and which were lined by a continuous sheet of endothelial cells. In the aneurysmal vascular prostheses, a pseudointima, composed of only layers of smooth muscle cells had developed. Those smooth muscle cells which lined the lumen had a typical morphology: they were polygonal, flat cells of unequal size, with a distinct organelle-free zone, containing myofilaments, at the luminal peripheral cytoplasmic side. The other smooth muscle cells varied in differentiation from normal smooth muscle cells to myofibroblasts. Under severe haemodynamic stresses, such as occur in the aneurysmal vascular prostheses, the regeneration of endothelial cells is impaired and smooth muscle cells undergo morphological changes to form a pseudoendothelial lining.     

Ultrastructure and lipid content of the liver of the zebrafish,Brachydanio rerio,related to vitellogenin synthesis

The female zebrafish is capable of producing mature eggs on the fifth day of each reproductive cycle. During this five-day period the ultrastructure of hepatocytes undergoes several changes. The number of nuclear pores increases rapidly during spawning, followed by a proliferation of RER within 24 h. Two days after spawning, glycogen has disappeared and the liver contains large amounts of lipids. The lipid droplets are closely surrounded by elongated mitochondria. Golgi complexes are abundant, secreting dense bodies. Four days after spawning the hepatocytes tend to regain their pre-spawning appearance. It is suggested that the changes in the hepatocytes, which coincide with special phases of ovarian activity, are related to vitellogenin synthesis. Steroids, especially estradiol-17beta, may trigger this process in the liver.     

Peroxidase-positive endothelial cells in sinusoids of the mouse liver

The cytochemical localization of endogenous peroxidase activity in sinus lining cells of mouse liver has been investigated. Kupffer cells, as identified by their exclusive ability to phagocytize large (0.8 micron) latex particles, exhibited strong peroxidase activity in nuclear envelope and endoplasmic reticulum. In addition, weak to moderate peroxidase activity was found in 57% of all endothelial cells. The enzyme in endothelial cells was also localized in nuclear envelope and endoplasmic reticulum, with a negative reaction in the Golgi apparatus. These observations indicate that peroxidase staining, as a marker for identification of Kupffer cells in mouse liver, is only of limited value and should be used in conjunction with other methods (e.g., latex phagocytosis).     

A light- and electron-microscopic study on the migration of primordial germ cells in the teleost,Oryzias latipes

Summary The primordial germ cells (PGCs) of Oryzias latipes in migration to the gonadal anlage have been investigated by light and electron microscopy. The ultrastructure of the PGCs, which occur in the subendodermal space on the syncytial periblast, differ conspicuously from that of the surrounding endodermal cells. After the PGCs move to the cavity between lateral plate and ectoderm, they are taken into the somatomesodermal layer and transferred to the dorsal mesentery where they form gonadal anlage with mesodermal cells. During their translocation to the dorsal mesentery through the somatic mesoderm, apparently without formation of pseudopods, the PGCs are completely surrounded by mesodermal cells. Since these conditions seem unfavorable to the active translocation of the PGCs to the dorsal mesentery, it is more likely that the PGCs are transferred passively by the morphogenic activity of the lateral-plate mesoderm.Counts of the number of the PGCs revealed that they are mitotically dormant during the migratory period. After the completion of the migration, they regain their proliferative activity. The PGCs in the female proliferate more actively than those in the male, which provides the first morphological indication of sex differentiation in this species of fish.     

The structure of the coeliac ganglion of a teleost fish Myoxocephalus scorpius

Summary In order to compare the structure of a teleost sympathetic ganglion with those of other vertebrates, light, fluorescence histochemical and electron microscopy were carried out on the coeliac ganglion of the scorpion fish, Myoxocephalus scorpius. In common with studies on other vertebrates, fluorescence histochemistry distinguished two cell types: a) principal neurones which exhibited low levels of specific catecholamine fluorescence and comprise the majority of neurones in the ganglia, and b) smaller intensely fluorescent cells, some of which had processes tens of micrometers long.With the electron microscope, the principal cells were seen to make axodendritic and axosomatic synapses with axons containing mainly 30 nm agranular vesicles at the synaptic site while in other vertebrates usually only one or other synaptic association is present.Both the somata and the processes of intensely fluorescent cells contain 300–600 nm diameter vesicles many of which have electron dense cores. These cells are also innervated by axons containing 30 nm agranular vesicles.     

Fenestration patterns in endothelial cells of rat liver sinusoids

Endothelial fenestrae of both zone 1 and zone 3 acinar liver sinusoids have been studied in rats by an interactive analysis of scanning electron microscopical images. Two fenestration patterns have been recognized in the endothelial cells on the basis of local variation in size, distribution and clustering of pores in each acinar zone. Our data indicate that both the number of fenestrae per square micrometer of endothelial surface and the mean diameter of fenestrae are significantly larger in zone 3 than in zone 1. The number of sieve plates is about 1.74 times larger in zone 3 than in zone 1, and the number of fenestrae per plate in zone 3 is nearly twice that in zone 1. Two different classes of fenestrae have been considered: clustered pores, which prevail in zone 3 and have a mean diameter smaller than the other pores, and free pores, which prevail in zone 1 and are bigger.     

Structural changes produced in Kupffer cells in the rat liver by injection of lipopolysaccharide

Summary The fine structure of Kupffer cells has been studied at various times after an intravenous injection of lipopolysaccharide of Salmonella abortus equii. The most prominent effects were: an increase in the number and dimensions of phagocytic vacuoles (often containing ingested LPS and neutrophilic granulocytes); mitochondrial damage, including disintegration of the matrix and cristae; an increase in the amount of dilated, lucent rough endoplasmic reticulum; presence of fat droplets in the cytoplasm. Five days after injection of lipopolysaccharide, the Kupffer cells had resumed their normal ultrastructure.Several minutes after injection of lipopolysaccharide, platelets adhered to the Kupffer and endothelial cells. Between one and six hours, neutrophilic granulocytes accumulated in the liver sinusoids. The resulting obstruction of the hepatic microcirculation most probably affected cellular ultrastructure by ischaemia. At three days, the number of Kupffer cells was doubled, and increased further at later time intervals.     

Ultrastructure of endocrine cells in the stomach of two teleost fish Perca fluviatilis L. and Ameiurus nebulosus L.

Summary In the gastric mucosa of two teleost species, the perch (Perca fluviatilis) and the catfish (Ameiurus nebulosus) three endocrine cell types were found, located predominantly between the mucoid cells of the gastric mucosa. A fourth cell type is present in the gastric glands of catfish. Each cell type was defined by its characteristic secretory granules. Type-I cells were predominant in both fish. These cells contained round or oval granules with a pleomorphic core. The average diameter of granules was 400 nm for the perch and 270 nm for the catfish. Type-II cells of both species displayed small, highly osmiophilic granules about 100 nm in diameter. The secretory granules of type-III cells (260 nm in the perch and 190 nm in the catfish) were round or slightly oval in shape and were filled with a finely particulate electron-dense material. Type-IV cells of the catfish were found in the gastric glands only. Their cytoplasm was filled with homogeneous, moderately electron-dense granules averaging 340 nm in diameter. The physiological significance of these different morphological types of gastric endocrine cells requires further investigation.     

The characteristics of endothelial progenitor cells derived from mononuclear cells of rat bone marrow in different culture conditions

Endothelial progenitor cells (EPCs) derived from bone marrow are known to be heterogeneous. In this study, we tried to find favorable conditions that induce the differentiation of mononuclear cells (MNCs) from bone marrow into EPCs. The differentiation capacity of MNCs from rat bone marrow was investigated in different conditions, such as different media, different induction times and different culture surfaces. The cell morphology and endothelial biomarkers associated with differentiated MNCs were studied. Our results indicated that MNCs cultured in EGM-2MV (Endothelial cell basal medium-2, plus SingleQuots of growth supplements) developed a bursiform shape, a late EPC-like morphology, while MNCs cultured in complete medium (CM, M199 with 10% FBS, 20 ng/mL VEGF and 10 ng/mL bFGF) showed a spindle shape, an early EPC-like morphology. Cells of both morphologies were able to incorporate DiI-ac-LDL and bind lectin in vitro. MNCs cultured in EGM-2MV exhibited a higher proliferation rate and higher eNOS expression than MNCs cultured in CM. MNCs cultured in EGM-2MV had the ability to form tubes on Matrigel. Flow cytometry results indicated that CD133 expression was highest at day 12 and that the greatest number of cells positive for both FLK-1 and CD133 appeared at day 20 from cells cultured in dishes without fibronectin coating. In addition, the expression levels of CD133, CD31 and FLK-1/CD133 were not significantly different between cells of different shapes. Our experiments suggest that MNCs from bone marrow can be differentiated into late EP-like cells in EGM-2MV, which have the ability to rapidly proliferate. These MNCs can also be differentiated into early EP-like cells in CM. Additionally, fibronectin may not be necessary for the differentiation of EPCs to mature ECs after three generations. Differentiated MNCs from bone marrow in EGM-2MV have the characteristics of EPCs, although the expression levels of EPC markers were lower than previously reported.     

Sca-1+ endothelial cells (SPECs) reside in the portal area of the liver and contribute to rapid recovery from acute liver disease

The liver has a high potential to regenerate however, the relation between oval cells and endothelial cells in the portal area during liver regeneration has not been adequately described. We have focused on sca-1+ endothelial cells (SPEC: sca-1+CD31+CD45− cells) and analyzed their localization, growth potential, and the role of these cells in damaged liver. SPECs are localized in the portal area and comprise approximately 20-30% of CD31+CD45− cells. These cells have higher growth potential than sca-1− endothelial cells and grow aggressively when the liver is severely damaged on the lateral side of the oval cells. In an in vivo study we show that when the liver is severely damaged in the presence of a VEGF (vascular endothelial growth factor)-inhibitor, the frequency of SPECs decreased and the recovery of liver volume was also delayed. These results strongly suggest that SPECs play important roles in the recovery of severely damaged liver.     

The effect of water acidification on prolactin cells and pars intermedia PAS-positive cells in the teleost fish Oreochromis (formerly Sarotherodon) mossambicus and Carassius auratus

Summary Although exposure to acid water (pH 3.5) induces severe and prolonged reduction in plasma osmolarity and total plasma calcium concentration in tilapia (Oreochromis mossambicus) and goldfish (Carassius auratus), the responses of the hypophyseal cells are clearly different. In tilapia, the size of the rostral pars distalis of the pituitary gland is enlarged as a result of the increase in size and number of prolactin cells. The pars intermedia PAS-positive (PIPAS) cells are not noticeably changed. Conversely, in goldfish, prolactin cells are unaffected, whereas the number of enlarged PIPAS cells increases markedly. Stimulation of prolactin secretion may be responsible for the partial restoration of plasma osmolarity and calcium levels observed in tilapia after two weeks exposure to acid water. Prolactin cells apparently play a role in the adaptation to acid stress by counteracting osmoregulatory disturbances. Goldfish show no restoration of plasma osmolarity during the course of the experiment. Plasma calcium levels tend to increase. Although prolactin may have an osmoregulatory function in goldfish under steady state conditions, goldfish prolactin cells do not seem to participate in the physiological adaptation to environmental changes that disturb water and ion homeostasis. The function of PIPAS cells in tilapia remains unclear and is apparently unconnected with ion regulation. The observations on these cells in goldfish are consistent with the hypercalcemic activity suggested for them.