Effects of fasting and refeeding on jejunal morphology and cellular activity in rats in relation to depletion of body stores.

BACKGROUND: Intestinal mucosa atrophy following a period of starvation characterized by the mobilization of fat stores for energy expenditure (phase II) worsen after a long fast marked by an increase in protein catabolism (phase III). However, the morphology of the jejunum is completely restored after 3 days of refeeding. The aim of this study was to determine the mechanisms involved in the rapid jejunal restoration following the critical phase III. METHODS: Jejunal structure was observed through conventional and environmental scanning electron microscopy, whilst cellular dynamics were studied using classical optic microscopy tools and immunohistochemistry. RESULTS: Mucosal structural atrophy during fasting proved to worsen over the two phases. During phase II, apoptosis is still present at the tip of the villi, the number of mitosis in crypts showed a 30% decrease and a transient drop in cell migration is observed. During phase III, however, an 85% rise in mitosis was noticed along with an increase in cell migration and the disappearance of apoptotic cells at the villus tips. This increased cell renewal continues after food ingestion. CONCLUSIONS: Starved rats appeared to be in a phase of energy sparing in phase II, with depressed cellular events in the intestinal mucosa. In phase III, however, the preservation of functional cells and the early increase in crypt cell proliferation should prepare the mucosa to refeeding and could explain why jejunal repairs are complete after 3 days of refeeding following either phase II or phase III.

Intralipid 10%: physicochemical characterization.

OBJECTIVES: Parenteral fat emulsions contain two populations of particles: artificial chylomicrons rich in triacylglycerols (TAG), and liposomes (bilayer of phospholipids [PL] enveloping an aqueous phase). Centrifugation permits isolating the liposomes in the infranatant called mesophase. The aim of the present work was to better characterize this mesophase chemically and to view the particles it contains by electron microscopy. METHODS: Electron microscopy (Philips 410) was performed after cryofracture on native 10% Intralipid, mesophase (centrifugation for 1 h at 27 000 g), and a liposome-enriched fraction (ring of density 1.010-1.030 g/l obtained after centrifuging mesophase in a KBr density gradient at 100 000 g for 24 h). The TAG and protein content of the mesophase was analyzed and the proteins partially characterized by immunodetection (Western-blot). RESULTS: This electron microscope study of 10% Intralipid gives evidence for the coexistence of artificial chylomicrons (mean diameter, 260 nm) and liposomes (43 nm), the latter being smaller than expected and containing 8% w/w TAG after purification. The solubilization of TAG in PL bilayers (reported to be < or = 3.1% w/w) might have been increased in parenteral emulsions by the manufacturing process or/and the high TAG/PL ratio. Minute amounts of proteins have also been detected and partially characterized using a specific antibody raised against the human 7 kDa Anionic Polypeptide Factor (APF), known to strongly interact with PL in bile. CONCLUSIONS: This work has shown that the size (mean diameter, 43 nm) of the liposomes present in 10% Intralipid is smaller than that usually assumed. Traces of hydrophobic proteins in the emulsion may account for certain allergic reactions sometimes observed in infused patients.

Restoration of the jejunal mucosa in rats refed after prolonged fasting.

To investigate the importance of body fuel depletion on gut rehabilitation after food deprivation, we compared the kinetics of jejunal mucosa alteration and restoration in rats that were refed after reaching different stages in body fuel depletion. Rats (P2) were refed while still in the so-called phase II, where body protein utilization is minimized, whereas rats (P3) were refed when they had reached the stage of increasing protein utilization (phase III). There was a significant decrease in total mass of intestine (P2, -30%; P3, -40%) and jejunal mucosa (P2, -52%; P3, -60%), as well in the size of the crypts (P2, -15%; P3, -36%) and villi (P2, -37%; P3, -55%). Structural changes of the mucosa included disappearance of some villi and a reduction in the size and number of crypts. Despite the larger morphological alterations in P3, the restoration of mucosa was as fast and complete after only 3 days of refeeding for both P2 and P3 rats. The respective roles of the mitosis pressure and of the lamina propria dynamics were studied. The rapid reversibility of the gut mucosal alterations due to fasting might constitute an integrative process.

Na(+)/K(+)-ATPase activity and immunocytochemical labeling in podobranchial filament and lamina of the freshwater crayfish Astacus leptodactylus Eschscholtz: evidence for the existence of sodium transport in the filaments.

In the crayfish Astacus leptodactylus, the podobranch, bearing respiratory and ion-transporting filaments, is attached to the lamina. The Na(+)/K(+)-ATPase activity is higher in filaments than in lamina. Using a mouse monoclonal antibody directed against the alpha subunit of Na(+)/K(+)-ATPase, and indirect immunofluorescence microscopy, we can observe that the immunoreactivity of the antibody is different for each kind of structure. The lamina presents an ion-transporting type epithelium, which is thick and presents a developed apical infolding system but no developed basolateral infolding system. The immunoreactivity of the antibody in the lamina is very weak. Respiratory filaments present a thin epithelium with a few small apical folds and scarce mitochondria. The immunoreactivity of the antibody in the respiratory filaments is very weak. In contrast, the ion-transporting filament epithelium is thick, presents a short apical infolding system, a well-developed basal infolding system, and numerous mitochondria. The immunoreactivity of the antibody in the ion-transporting filaments is strong. Ion-transporting filaments, which have a cuticle permeable to cations, seem particularly implicated in Na(+)regulation, by way of the sodium pump.

Transepithelial potential difference of a single gill filament isolated from the crayfish Astacus leptodactylus Esch.: a new method.

A new method is described that allows in vitro perfusion and transepithelial electrical potential measurements of a single filament (3-5 mm long; 200 microns in diameter) isolated from the podobranch of the crayfish Astacus leptodactylus. An electrophysiological study was carried out on the preparation to validate this technique. The good physiological quality of the isolated filament preparation has been established and results of continuous measurements of the potential difference under two perfusion conditions are reported. Filaments were perfused with Van Harreveld physiological saline inside and either with Van Harreveld saline or artificial fresh water outside. Large potential differences up to 150 mV between inside and outside of the filament were recorded. When filaments were symmetrically perfused, the behavior of the electrical potential difference allowed two populations of filaments to be distinguished.

[Morphological characterization of cell lines of human uveal melanoma]. / Caractérisation morphologique de lignées cellulaires de mélanome uvéal humain.

Several cell lines have been derived from an ocular melanoma obtained from an enucleated patient. Three cell types are observed during the time in culture of all the cell lines under study. Two of them have epithelial and spindle shape respectively. A third cell type, having a spheroidal shape, is formed from spindle cells and may be transformed into epithelial cells upon re-seeding. Further experiments showed that the same cell may change of shape following the cycle: spheroidal-->epithelial-->fusiform-->spheroidal. Scanning microscopy shows the coexistence of the three cell shapes in the same culture and the presence of several filaments and processes protruding at the surface of the cells. Transmission electron microscopy shows that the cell lines, in general, contain melanosomes empty or fairly pigmented and several filaments and microtubules. The presence of melanin may be stimulated by seeding of melanoma cells over a "feeder layer" of fibroblasts.

Gill epithelial cells kinetics in a freshwater teleost, Oncorhynchus mykiss during adaptation to ion-poor water and hormonal treatments.

The aim of this work was to determine the kinetics of the dramatic development of the gill chloride cells (CCs) during adaptation of the salmonid Oncorhynchus mykiss to an ion-poor environment.To monitor cell division, the incorporation in the mitotic cell DNA of bromo-deoxyuridine (BrdUrd) was visualized with a monoclonal antibody. The density of labelled nuclei was used as an index of cellular division (proliferation), concomitantly with morphometry of phenotypic changes monitored with SEM.In the filament epithelium, a phase of CC differentiation occurred within 12h after the transfer, followed by a delayed phase of cell proliferation (48h). In the lamellar epithelium, the present study demonstrates the absence of cell proliferation after ion-poor water transfer. The conclusion is that proliferation (mitosis) is important in the primary filament whereas differentiation and migration (from the filament) is the main mechanism for the appearance of CCs on the secondary lamellae.The present study suggests that cortisol promoted differentiation, but not division, of cells. CCs, presumably premature, were stained by anti-cortisol monoclonal antibody indicating the presence of cortisol. No mature CCs were stained.Growth hormone (oGH, ratGH) increased the rate of cell division both in lamellar and filament epithelium.

The neuroepithelial cells of the fish gill filament: indolamine-immunocytochemistry and innervation.

The neuroepithelial cells (NECs) of the fish gill filament share several morphofunctional features with the cells of the neuroepithelial bodies in the lungs of air-breathing vertebrates. In the present study, a detailed indolamine-immunocytochemical analysis of the branchial neuroepithelial cells and nerves was undertaken in non-teleost and teleost species, with particular emphasis on the latter. In the rainbow trout, Oncorhynchus mykiss, the chemical degeneration of either catecholaminergic (by 5- and 6-hydroxydopamines) or indolaminergic (by 5,6-dihydroxy-tryptamine) innervations associated with the NECs was studied using electron microscopy. In teleosts, the NECs are located primarily on the distal half of the filament. In the trout particularly, these cells are innervated mainly by non-indolaminergic nerves taking up sympathetic neurotoxins. The proximal half of the filament contains isolated NECs innervated additionally by intrinsic indolaminergic neurons. Serotonin-like immunoreactivity of the NECs is evident in the granular vesicles packed within the basal soma and processes which surround non-vascular and vascular smooth muscles in the filament. Apical processes from the neuroepithelial cells occasionally contact the water on the surface of the filament epithelium. The secretory function of the NECs is discussed with reference to the probable involvement of serotonin in the modulation of fish gill function. In addition, their connections with both central and branchial nervous systems suggest a possible chemoreceptor role.

Active urea transport independent of H+ and Na+ transport in frog skin epithelium.

We investigated the relationship between H+ secretion (JH), Na+ absorption (JNa), and urea transport (Ju) in skin of frogs (Rana esculenta) adapted to running tap water, NaCl (100 mM), and KCl (100 mM). In addition, cell morphological changes, particularly in the mitochondria-rich cells (MRC), were followed. NaCl adaptation stimulated an active Ju, reduced JNa and JH, and caused a decrease in the apical surface of MRC. After KCl adaptation, JNa and JH were increased and highly correlated, with a twofold increase in Ju, whereas the numerous MRC developed infoldings on their apical membranes. No correlation was found between JH and Ju. Clamping the skins in a range of +/- 50 mV or changing the external pH from 7.4 to 5.4 (at high cellular buffering power) had no effect on Ju. Depolarization of the basolateral membranes (serosal KCl-Ringer) had no effect on Ju. Ju was reversibly blocked by acidification of the cells by oxygen-free solution and sulfhydryl reagents (Hg2+, p-chloromercuribenzenesulfonic acid, and N-ethylmaleimide). Diethylstilbestrol, a proton transport blocker, had no effect on Ju. Apical addition of amiloride and derivatives (phenamil and ethylisopropyl amiloride) reversibly blocked Ju, whereas ouabain had no effect. We conclude that a cation (Na+ or H+)-dependent process is unlikely to exist in R. esculenta skin. A primary active transport in a two-step process is the simplest hypothesis to account for the energy-dependent Ju that develops in NaCl-adapted frogs.

A study of in vitro and in vivo morphological changes of ependymal cells induced by galactocerebrosides.

Ependymal cells in culture and in vivo were treated with mixture of galactocerebrosides. Galactocerebroside is the major glycolipid of myelin and in demyelinating diseases is found in cerebrospinal fluid. Morphological changes induced by this treatment were examined by microscopy at both optical and ultrastructural levels. In vitro, cilia, microvilli, and junctions between the cells disappeared, processes containing intermediate filaments developed, and the cells lost characteristics typical of ependymal cells and became more astrocyte-like. As shown by vital staining with a fluorescent compound and by nuclear incorporation of bromodeoxyuridine, cells did not proliferate during the period of galactocerebroside treatment and the morphological transformation was restricted to the ependymal cells. In contrast, asialoganglioside-GM1 and sulfatides had no effect on ependymal cell morphology. Some of the in vitro observations could be reproduced in vivo. Junctions between ependymal cells disappeared and intercellular spaces appeared between these cells and the cerebral parenchyma at the basolateral side of the ependymal layer. At the apical side, morphological modifications of junctions and cilia were less evident. As these experimental conditions resemble those existing during demyelination the morphological changes described may account for perturbations of the physiological functions of the ependymal cell.

Neuroepithelial cells in fish gill primary lamellae.

Formaldehyde-induced fluorescence reveals numerous serotonin-containing cells within the primary epithelium of the fish gill. These cells are isolated or clustered and are supported by the epithelial basal lamina. They never reach the external medium and are found on the internal side of the primary lamellae, facing the respiratory water flow. With the electron microscope, these cells are found to contain dense-cored vesicles (DCV) of 80-100 nm. Nerve profiles are consistently found close to DCV cells. After having crossed the basal lamina, nerve fibers form endings on DCV-containing cells. These endings display both small clear vesicles and DCV and are in direct contact with DCV cells. Specific membrane alterations are suggestive of efferent synapses. These cells are considered neuroepithelial cells, similar to those found within the wall of lung airways in mammals and submammalian vertebrates. Structure and localization are suggestive of a tissue O2 sensor.

Ultrastructure of marine teleost gill epithelia: SEM and TEM study of the chloride cell apical membrane.

This paper deals with the structure of gill epithelia in the sole, Solea solea, as revealed by transmission and scanning electron microscopy. In this marine teleost the chloride cell and its accessory cell form a cellular complex. Apically the plasma membranes of these cells are loosely juxtaposed, thus forming a leaky epithelium covering a large part of the gill.