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PubMed1991

Properties of heart fibroblasts of adult rats in culture.

In the heart of the adult rat, fibroblasts are mainly responsible for the synthesis and deposition of the collagenous matrix. Because these cells in vitro may serve as an important model system for studies of collagen metabolism in heart tissue, we have cultured and characterized rat-heart fibroblasts from young adult and old animals. Conditions included use of media of different compositions with and without addition of ascorbate. Cells used were either cultured directly from fresh tissues or thawed previously frozen cells. Cultured cells were studied with respect to growth properties, morphology and ultrastructure and patterns of collagen. Heart fibroblasts generally resembled fibroblasts cultured from other tissues, but were more like skeletal muscle fibroblasts in that they deposited, in addition to type I collagen, type IV collagen and laminin. The fibroblasts showed a typical appearance in phase-contrast microscopy and electron microscopy. In the case of cells grown with added ascorbate, aligned collagen fibrils in the extracellular matrix showed a periodicity typical of type I collagen. The deposition of type I collagen occurred only in medium supplemented with ascorbate, and in that circumstance increased as a function of time past confluence; this was independent of the age of the animal from which the cells were obtained or of other changes of medium composition studied. Immunofluorescence studies with specific antibodies revealed that the cells deposited types I and IV collagens, laminin and fibronectin. In contrast to the case of type I collagen, the deposition of type IV collagen occurred in cells grown either with or without ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1983

In vitro cultivation of xenotransplanted human breast-cancer cells.

Xenotransplanted human breast-cancer tissue growing in the second to the ninth passage in nu/nu mice and normal tissue from nu/nu mice was cultivated in vitro. Thirty-two of fifty-two (62%) cultures grew successfully and have been analyzed. The cells were observed using phase-contrast microscopy and May-Grünwald-Giemsa staining. The following cell types could be distinguished: normal fibroblast-like cells (FC), normal epithelial-like cells (EP I), abnormal epithelial-like cells (EP II), and degenerated or dying cells (DC). After staining, the nuclear area of all cell types was measured. The results were: FC = 280 micrometers 2, EP I = 320 micrometers 2, and EP II = 800 micrometers 2. They were compared with the nuclear area of human breast-cancer cells obtained by aspiration biopsy.

Animals↗

PubMed1994

Effect of sulfur dioxide on mucociliary activity and ciliary beat frequency in guinea pig trachea.

The effects of 30 min exposure to sulfur dioxide on mucociliary activity (MCA) and ciliary beat frequency (CBF) were studied in 31 guinea pig tracheas. MCA was measured by recording the light reflected from ciliated mucous membranes using an infrared bar code reader. CBF of single ciliated cells obtained by brushing was measured with phase-contrast microscopy. Each tracheal sample was exposed to SO2 at concentrations ranging from 2.5 to 12.5 ppm, or to air for control purposes. MCA and CBF were measured before and immediately after gas exposure. A reduction in mean MCA of 63% (P = 0.0007) and statistically insignificant changes in CBF (P > 0.05) were recorded at concentrations of 2.5 ppm SO2. Higher SO2 concentrations caused a further impairment of MCA as well as a dose-dependent decrease in CBF (P = 0.002). A concentration of 12.5 ppm SO2 induced a decrease from baseline values of approximately 80% in mean MCA and of roughly 70% in mean CBF. This study demonstrates a dose-dependent SO2-induced decrease in MCA of guinea pig tracheas. The decrease in MCA was associated with an impairment of CBF only at SO2 concentrations higher than 5.0 ppm.

Animals↗

PubMed1980

Direct studies of dikaryotization in Schizophyllum commune. II. Behavior and fate of multikaryotic hyphase.

The growth, duplication and fate of multikaryotic hyphae bearing true clamp connections, as derived from compatible matings of Schizophyllum commune, were studied by phase contrast microscopy. The nuclei (N) of multikaryotic apices maintained a near central position during hyphal growth. True clamp connection formation occurred with near synchronous mitosis followed by septal synthesis across the clamp neck and main hyphal axis. Nuclear progeny after mitosis in a hexakaryon included 6 N in the apex, 1 N in the clamp and 5 N in the penultimate cell; the solitary nucleus in the clamp later entered the penultimate cell. Similar events occurred for clamp connection formation and mitosis in the trikaryon, quadrikaryon or pentakaryon, whether in the apex or primary branches. Nuclear content of the multikaryotic apex (2 N through 10 N) had no apparent effect on the rate of individual hyphal growth. Reduction of the nuclear number in a trikaryon occurred by long-term entrapment of a solitary nucleus in the clamp and subsequent outgrowth of the dikaryotic penultimate cell. Occasionally, more than one nucleus became entrapped in the clamp cell. The ephemeral nature of the multikaryon was indicated by the fact that older cultures appeared to be exclusively dikaryotic hyphae at the colony periphery.

Agaricales↗

PubMed1980

Preliminary observations on human trabecular meshwork cells in vitro.

This report presents our preliminary observations on the trabecular meshwork from human eyes up to 5 days post-mortem in tissue culture. Satisfactory primary cultures were obtained from about 20% of the 423 explants which were investigated. The period prior to growth was from 4 days to 4 weeks and from the appearance of the initial outgrowth it took 25 to 30 days to reach maximum cellular spread within the culture chambers. The progress of the explant and the spreading of the trabecular meshwork cells was monitored by phase-contrast microscopy, time-lapse cinephotomicrography, light microscopy, transmission electron microscopy, scanning electron microscopy and autoradiography (using tritiated thymidine). On the basis of their ultrastructural appearance the cultured meshwork cells seems to be metabolically active. Their cytoplasm contained abundant rough endoplasmic reticulum, many mitochondria, a well developed Golgi apparatus and many coated and uncoated micropinosomes. However, even in short-term culture the trabecular meshwork cells had adapted to the artificial environment of our system and no longer resembled "normal" trabecular meshwork cells as seen in vivo. Since trabecular meshwork cells can quickly adapt their morphology in a culture environment and because the adult human meshwork contains a significant population of non-trabecular cells, the value of long term culture as a means of investigating the cellular activity of the normal and glaucomatous outflow system must be open to question.

Culture Techniques↗

PubMed1976

Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha.

The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes D-aminoacid oxidase and L-alpha-hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.

Alcohol Oxidoreductases↗

PubMed1981

Cochlear vascular histology in animals exposed to noise.

The regularly occurring cochlear vessels in the external wall and spiral lamina were studied in the guinea pig and chinchilla following exposure to various types and durations of noise. A soft-surface specimen technique with or without injection of a contrast medium into the vascular system was used, and the occurrence of specified vascular parameters was assessed using phase-contrast microscopy. Noise does not seem to result in any extraordinary vascular pathology, but a slight, overall decreased blood supply to the cochlea and localized changes depending on cochlear turn are suggested.

Animals↗

PubMed1977

Histology of the cochlear vessels in the guinea pig.

Normal adult guinea pigs were studied in phase contrast microscopy. Perivascular spaces were clearly more common in vessels facing perilymph than in vessels facing endolymph. The so-called "avascular channels" which have been suggested to signify a degenerative phenomenon were found in some inner ear vessels in the normal animal. These sections of vessels, lacking blood cells, may constitute a normal vessel, subjected to plasma skimming. Small openings were found on some of these "avascular channels". Both perivascular spaces and "avascular channels" appear to be a part of the fluid transport system in the cochlea.

Animals↗

PubMed1987

Light microscopical study of Blastocystis spp. in monkeys and fowls.

An investigation of a Blastocystis species obtained from several species of monkeys and fowls was conducted to clarify the morphology of the organism, using light microscopical techniques including Giemsa, Heidenhain iron hematoxylin, Trichrome stains, iodine mount and phase-contrast microscopy. A comparison was made with Blastocystis hominis from humans. Blastocystis spp. were found in 15 out of 26 monkeys and in all of 12 fowls (10 chickens and 2 ostriches) examined. The behaviour of the parasites in the bowels was also examined, using paraffin-embedded sections. Microscope examination of the lumen contents at death revealed the organism in the caecum of monkeys and fowls. The organisms from faeces, cultures and the lumen contents of the caecum of humans, monkeys and fowls were similar, except for variations in the size and contents of the central vacuoles, which occupied the centre of the organisms.

Animals↗

PubMed1985

Immunocytological characterization of the outer acrosomal membrane (OAM) during acrosome reaction in boar.

In order to study the acrosome reaction in boar, spermatozoa were incubated in a calcium-containing medium in the presence of the calcium ionophore A23187. The time course of the acrosome reaction was assessed by phase-contrast microscopy and correlated with the movement characteristics of the spermatozoa determined by means of multiple-exposure photography (MEP). Different stages of the acrosome reaction could be observed by indirect immunofluorescence using an antibody fraction raised in rabbits against the isolated outer acrosomal membrane (OAM). At the start of the acrosome reaction, a bright fluorescence located exclusively at the acrosomal cap of the sperm head could be observed, whereas after 60-120 min, the fluorescence vanished, indicating the complete loss of the OAM. However, to gain more insight into the stages of the plasma membrane and OAM during the acrosome reaction, immunoelectron-microscopical studies were performed using anti-OAM antibodies detected by the protein-A gold method. Ultrathin sections and total preparations in combination with transmission electron microscopy (TEM) confirmed, that boar spermatozoa start their acrosome reaction by a vesiculation of the plasma membrane, thus exposing the heavily labelled OAM, which is then lost as sheets or large vesicles. The newly exposed inner acrosomal membrane did not show any labelling with gold, thereby indicating clear differences in the antigenicity of both acrosomal membranes.

Acrosome↗

PubMed1988

Normal splenic function in children with the nephrotic syndrome.

Children with the idiopathic nephrotic syndrome (NS) are known to be susceptible to bacterial infections. A recent report suggested that splenic hypofunction may be responsible for this immunological defect. We assessed splenic function by counting the circulating pocked red blood cells (PkRBCs) using interference phase contrast microscopy. PkRBCs are removed by the spleen, so that normal eusplenic individuals have less than 2% PkRBCs while asplenics have 15%-30%. Intermediate values are seen in hyposplenism. Thirty-three measurements of PkRBCs were made in 19 children with NS (mean age 7.5 +/- 0.8 years). PkRBCs were normal in all children tested (range 0-0.8%), including two patients with bacterial peritonitis associated with relapse. Thus we were unable to find evidence of hyposplenism in children with NS.

Adolescent↗

PubMed1993

In vitro encystment and experimental infections of Blastocystis hominis.

Cultures of Blastocystis hominis were induced to encyst using three encystation media: (a) an encystation medium (EM) comprising yeast extract in buffered saline containing 50% horse serum, (b) an encystation medium (CEM) comprising EM conditioned with bacterial soluble products and (c) an encystation medium (TEM) containing 0.5% trypticase in EM. Two isolates of B. hominis were studied, an axenized isolate C and a non-axenized isolate MS. In EM, isolate C did not encyst, whereas 6.1% of isolate MS had encysted by day 1. However, in CEM and TEM, 17.4% and 25.7% of isolate C, respectively, had encysted by day 5. In all three media, isolate MS encysted more readily than isolate C, with as much as 91.7% of the former encysting in TEM. As viewed by phase-contrast microscopy, cyst-like stages appeared highly refractile. Direct stool examination of juvenile Wistar rats infected with 10,000 cyst-like stages of both C and MS isolates showed Blastocystis at day 2 post-infection. At autopsy on day 7, large numbers of Blastocystis were seen in the cecum, with smaller numbers being observed in the large intestine. In contrast, rats fed with various inocula of the vacuolar stages of isolates C and MS did not become infected, indicating the importance of the encysted stages in the transmission of the parasite.

Animals↗

PubMed1991

Protective effect of tauroursodeoxycholate against chenodeoxycholate-induced damage to cultured rabbit gastric cells.

Ursodeoxycholate (UDC) and tauroursodeoxycholate (TUDC) have been reported to be protective against liver injury induced by other bile salts. UDC also has been shown to be effective against refluxed bile-induced gastritis after gastric surgery. However the mechanism of the therapeutic effect of UDC on gastric mucosa has not been known. In the present study, cytoprotective actions of UDC and TUDC against chenodeoxycholate (CDC)-induced gastric injury were investigated using rabbit gastric cell cultures without systemic factors. Rabbit gastric mucosal cells were cultured after the isolation of rabbit gastric cells with collagenase and ethylenediaminetetraacetic acid. Cytotoxicity was quantified by measuring 51Cr release from prelabeled cells and MTT assay. Prostaglandin (PG) E2 was assayed by radioimmunoassay. Concentrations of CDC greater than 0.5 mM or UDC greater than 5 mM caused cellular damage and increased 51Cr release in a dose-dependent and time-dependent fashion, while TUDC up to 10 mM did not. TUDC, but not UDC, showed a significant decrease of CDC (1.5 mM)-induced 51Cr release dose dependently. The protective effect of TUDC against CDC-induced damage was confirmed by MTT assay. On phase-contrast microscopy, disruption of monolayers induced by CDC (1.5 mM) was clearly protected by TUDC (10 mM). Free radical scavengers (500 units/ml of superoxide dismutase, 300 units/ml of catalase, and 100 mM of dimethyl sulfoxide) or a calcium blocker (10(-7)-10(-5) M verapamil) did not show significant protection against CDC-induced damage. Deprivation of Ca2+ in the media did not affect CDC-induced damage. Thus free radicals or Ca2+ might not be involved in the cell toxicity of CDC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1983

A monolayer culture of human gastric epithelial cells.

Our aim was to develop a fibroblast-free monolayer culture of human gastric mucosal cells, using the specimens obtained by routine endoscopic biopsy. Human gastric mucosa obtained from normal volunteers by endoscopic biopsy was dissociated from collagenase and hyaluronidase. Dissociated cells were cultured in supplemented Coon's modified Ham's F-12 medium. Within 24 hr of inoculation, the cells were attached to the culture dishes. This was followed by cellular outgrowth. On phase-contrast microscopy, all cells had epithelial characteristics and fibroblasts were not observed. Ninety percent of cells contained periodic acid Schiff reaction-positive mucous granules after diastase digestion consistent with mucous epithelial cells. Two percent of the cells gave a strong reaction for succinic dehydrogenase activity (parietal cells). Immunohistochemical staining for pepsinogen in cultured cells was negative. On EM, microvilli-like projections, junctional complexes, Golgi apparatus, and mucous granules were apparent in the majority of cells. Mitotic figures were observed by day 3 with Giemsa staining. Autoradiographically, these cells were able to incorporate [3H]TdR into the nuclei. Cells were capable of synthesizing DNA, and this function was inhibited by cycloheximide. Cells could be cultured for up to two weeks without fibroblast contamination. A method of primary monolayer culture of human gastric mucosa obtained by a routine endoscopic biopsy has been successfully developed.

Adult↗

PubMed1982

Herpes simplex virus infection of isolated autonomic neurons in culture: viral replication and spread in a neuronal network.

Cultures of isolated neurons, derived from the superior cervical ganglion (SCG) of the newborn rat and maintained in the absence of nonneuronal cells, were infected with herpes simplex virus (HSV) type 1. By phase-contrast microscopy, including time-lapse cinematography, cytopathologic changes appeared first in neuronal cell bodies and only approximately 24 hours later were axonal abnormalities detectable. Despite low yields of viral progeny, infection spread readily within the two-dimensional network of neurons and their processes. Immunoperoxidase staining for viral antigens confirmed the replication and spread of virus and revealed that antigen extended along axons during infection. Antiviral antibody added to the overlay medium slowed but did not prevent the spread of infection, indicating that virus passed from neuron to neuron over axonal pathways. Despite alteration of neuronal macromolecular synthesis early in infection, axonal transport is apparently preserved long enough to allow propagation of virus over interconnecting neural pathways.

Animals↗

PubMed1985

Effect of diuresis on urinary erythrocyte morphology in glomerulonephritis.

The differentiation between glomerular and non-glomerular haematuria by phase-contrast microscopy has proved to be a useful tool in the diagnosis of glomerulonephritis. In an attempt to evaluate the effect of marked diuresis on the altered red cell morphology in patients with biopsy proven glomerulonephritis, urinary sediments were examined following water or furosemide-induced diuresis. In both diuretic states urine flow increased, urine osmolality decreased and the percentage of glomerular erythrocytes was significantly reduced in the urinary sediment. These data demonstrate that the alteration in urinary red cells in glomerulonephritis is mainly caused by tubular forces. The diagnostic significance is reduced during increased diuresis and the evaluation of urinary red cell morphology should not be performed.

Adult↗

PubMed1986

Accumulated strain mechanism for length determination of thick filaments in skeletal muscle. I. Experimental bases.

The kinetics of dissociation of myosin from both ends of thick filaments in glycerinated skeletal muscle fibres and myofibrils was studied in the presence of MgATP by use of an optical diffraction method and phase-contrast microscopy. The dissociation velocity, v (identical to -dL/dt where L is the length of thick filaments at time t), increased with increasing KCl concentration (0.225 to 0.5 M), or increasing pH (6.5 to 8.0) but hardly changed with temperature (5 and 25 degrees C), micromolar concentrations of Ca2+ or sarcomere length (2.4 and 2.75 micron). Over a wide range of filament length, the dissociation velocity could be expressed by v0exp(alpha L), where v0 and alpha are positive constants depending upon the dissociation condition. When the effects of crossbridge formation are minimized it was thus shown that the structural stability of thick filaments in a muscle fibre and a myofibril gradually decreases from the central part to the tips of the filaments. On the basis of these results we propose that the length of thick filaments is largely regulated by an accumulated strain mechanism in which the free energy of association of myosin molecules increases with filament length.

Animals↗

PubMed1983

Effects of epidermal growth factor on osteoblastic cells in vitro.

The effect of epidermal growth factor (EGF) on clone MC3T3-El cells that have osteoblastic activity was examined by phase-contrast microscopy and electron microscopy; hydroxyproline content, collagen synthesis, collagen pattern, and alkaline phosphatase (ALP) activity were also determined. We found that EGF (0.4 ng/ml) transformed the cells from their normal polygonal shape to a spindle-like morphology by 8 h. This hormone also caused dose-related suppression of hydroxyproline content and ALP activity which was detectable 2 days and 1 day, respectively, after EGF addition. Indomethacin did not affect hydroxyproline content and ALP activity, suggesting that the effect of EGF on the cells may not be mediated by prostaglandins. Epidermal growth factor at concentrations of 2 to 50 ng/ml significantly decreased collagen synthesis in the cells, whereas protein synthesis was stimulated. Electron microscopy demonstrated that collagen fiber formation was also reduced by EGF; an immature type of fibril was observed compared with the typical cross-striated one in the controls. Moreover, the hormone treatment also resulted in the appearance of type III collagen in addition to the type I already present in the cells. These suppressive effects of EGF on MC3T3-El cells in vitro suggest that this hormone may be involved in bone remodelling in vivo as well.

Alkaline Phosphatase↗