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PubMed1979

Cell suspensions from rat olfactory neuroepithelium: biochemical and histochemical characterization.

Cell suspensions were generated from rat olfactory epithelium by digestion with collagenase and hyaluronidase followed by gentle mechanical disruption. These cell suspensions excluded nigrosin dye and synthesized RNA, protein and carnosine from radiolabeled precursors. Sustentacular cells, repiratory epithelial cells and olfactory neurons but not basal cells could be identified by phase-contrast microscopy. Sedimentation of these cell suspensions at unit gravity in discontinuous gradients of buffered bovine serum albumin resulted in partial separation of the various cell types as indicated by the distribution of several biochemical markers. Olfactory marker protein and carnosine synthetase activity were found in the upper gradient fractions, while carnosinase activity was present predominantly in the lower gradient fractions. Cellular localization of olfactory neuron marker protein and non-neuronal S-100 protein by immunoperoxidase staining of gradient-fractionated cells indicated that neuronal cells were only partially separated from non-neuronal cells by our fractionation techniques. Evaluation of gradient fractionated cells by histochemical staining for carbohydrates demonstrated that secretory Bowman's gland cells were quite efficiently separated from neurons. This study demonstrates the ease with which cell suspensions may be produced from the olfactory epithelium, and emphasizes the importance of utilizing both biochemical and histochemical approaches in studies of mixed populations of cells, particularly when the purity of the cell fractions is a consideration.

Animals↗

PubMed1984

Cell production and morphological pattern formation in primary brain cell cultures. I. Pattern formation within the basal layer(s).

Spontaneous pattern formation within the basal cellular layer of primary brain cell cultures were studied. Basal cells were found to be organized into a limited number of morphologically well distinguishable types of cell-arrangements. Phase contrast microscopy was used to characterize the different cell-assemblies morphologically. Under standard culture conditions the time of appearance and specific changes of relative frequency in time were also characteristic of different patterns. The distribution of mitotically active cells among the different morphological patterns within the basal layer was also investigated by recording [3H]thymidine uptake of cells. Reduction of mitotic activities of cells in the basal layer was found in the vicinity of overlying cells. In a given area of cultures the reduction of mitotic activity was proportional to the number of overlying cells. The influence of cell proliferation on the morphological pattern formation in primary cultures is discussed.

Animals↗

PubMed1985

Protein synthesis requirement for the formation of synaptic elements.

The formation of synapses in cell cultures of rat cerebellum was examined in the presence of the protein synthesis inhibitor cycloheximide. First, cell survival in the presence of 25 micrograms/ml cycloheximide was determined by phase contrast microscopy, trypan blue exclusion, total protein and uptake of [3H]gamma-aminobutyric acid (GABA). Neurons with 24 h incubation in cycloheximide appeared normal with little cell death, but by 48 h incubation the first signs of cell death were found. Some viable neurons were still found in cultures incubated continuously in cycloheximide for 72 h. Normally, the number of synapses seen in cerebellar cultures with the electron microscope shows an increase during the first several weeks in culture. However, the number of synapses in cultures treated with cycloheximide decreased, indicating that inhibition of protein synthesis at least partially inhibited synaptogenesis. Cycloheximide also inhibited the maintenance of synapses already formed as seen by the decrease in the number of synapses from the time the cycloheximide was added. To determine the sensitivity of the forming presynaptic element to cycloheximide, the development of apparent presynaptic elements was investigated. In cultures treated with polylysine-coated sepharose beads, neurites grew and formed apparent presynaptic elements with the bead taking the position of the postsynaptic element. Cultures pretreated with cycloheximide for 1 h followed by 24 h incubation with both cycloheximide and coated beads showed a normal number of apparent presynaptic elements. The first decrease in numbers was seen after 12 h preincubation and 12 h incubation with both cycloheximide and coated beads. Even after 72 h continuous incubation some apparent presynaptic elements could be formed although at reduced levels. Results presented here suggest that continuous protein synthesis is not necessary for the formation of the presynaptic element, but that active protein synthesis is required for neurons to form and maintain postsynaptic elements.

Animals↗

PubMed1986

Intrinsic neurones and associated cells of the guinea-pig heart in culture.

This paper describes a method for dissociation of intrinsic neurones from the atria and interatrial septum of newborn guinea-pig heart and their maintenance in culture. The appearance of the cultured intracardiac neurones, muscle and other non-neuronal cell types also present in the preparation has been observed by phase-contrast microscopy. Some of the neurochemical properties of the intracardiac neurones in culture have been investigated using histochemical methods. All the neurones studied were shown to contain acetylcholinesterase. No catecholamine-containing neurones were found. Using an indirect immunofluorescence technique, 20-50% of clearly identifiable neurones in culture contained neuropeptide Y-like immunoreactivity. Vasoactive intestinal polypeptide-like immunoreactive neurones were found in only one out of 15 culture preparations; no substance P-, neurotensin-, or enkephalin-like immunoreactivity was observed. These findings are consistent with those described for intracardiac neurones studied in situ, suggesting that the neurochemical differentiation of the intrinsic heart neurones is retained in culture. The culture preparation provides an opportunity to study the properties and role of intrinsic neurones of the heart. The characteristics of the intracardiac neurones may be distinguished from those of the extrinsic nerve fibres which degenerate in culture. Further, the intracardiac neurones are more accessible to experimental manipulation in culture than in situ.

Acetylcholinesterase↗

PubMed1987

Urinary bladder intramural neurones: an electrophysiological study utilizing a tissue culture preparation.

An enzymic dispersion technique was used to free the intramural ganglia from their usual close association with the other components of the urinary bladder wall. The isolated ganglia obtained were viable and could be kept in culture for several weeks. The development of the cultures was monitored by phase-contrast microscopy and their electrophysiological properties were investigated using intracellular recording techniques. Neurones could be visually identified after 2-3 days in culture; cell groups contained from 2-50 neurones. Three types of spontaneous activity were seen: small changes in membrane potential and action potentials, and slow oscillatory conductance changes. These events were not blocked by hexamethonium but were abolished by hyperpolarizing current. Most neurones spiked without adaptation to direct stimulation; in a few cells the train of spikes was damped out. No neurones generated long afterhyperpolarizations. Indirect stimulation produced responses in the ganglia which are consistent with synaptic activity. Summation of inputs was demonstrated. These results provide evidence for local intraganglionic circuits since the ganglia or neurone groups are unequivocally extrinsically denervated. It was concluded that the intramural ganglia have the capacity to integrate preganglionic input and the question of whether or not they might mediate reflex activity is raised.

Action Potentials↗

PubMed1989

Ketamine protects cultured astrocytes from glutamate-induced swelling.

Effects of ketamine on glutamate-induced swelling of astrocytes in primary cell culture were studied. Following the exposure to 1 mM glutamate (Glu) for 4 h the intracellular water space (as measured by 3-O-methyl-[14C]glucose uptake) of astrocytes was increased by two-fold concomitant with cell swelling and disappearance of cellular processes observed by phase-contrast microscopy. Ketamine, when co-incubated with Glu, reduced the astrocytic swelling in a dose-dependent manner. These data suggested that ketamine, in addition to its function as a non-competitive N-methyl-D-aspartate receptor antagonist, is also involved in protecting astrocytes from Glu-induced swelling.

Animals↗

PubMed1989

Aluminum salts induce the accumulation of neurofilaments in perikarya of NB2a/dl neuroblastoma.

NB2a/dl neuroblastoma cells were exposed to aluminum chloride or aluminum lactate (0.1-1 mM) for 3 and 6 days. Additional cultures were exposed to aluminum salts as the cells were stimulated to elaborate axonal neurites by dibutyryl cyclic AMP. By phase-contrast microscopy, aluminum salts had no effect on the morphology of undifferentiated (NB2a(-] or differentiated (NB2a(+] cells, or on neuritic elaboration and maintenance. Silver straining by the Bielschowsky method, however, demonstrated argyrophilic accumulations in perikarya of many NB2a(-) and NB2a(+) cells treated with aluminum salts. At the ultrastructural level, whorls of intermediate filaments were the most prominent abnormalities in neuronal perikarya. Although phosphorylated high-molecular weight neurofilament subunits (NF-H) are normally detected by immunocytochemical analyses only within axonal neurites of NB2a/dl cells, aluminum salt treatment caused the detection of phosphorylated epitopes of NF-H within perikaryal of NB2a(-) and NB2a(+) cytoskeletons, suggesting that the argyrophilic filamentous accumulations are composed at least partly of phosphorylated NF-H.

Aluminum↗

PubMed1990

Lack of excitotoxic cell death in serum-free cultures of rat cerebral cortex.

The excitotoxicity of glutamate, N-methyl-D-aspartate (NMDA), quisqualate and kainate were compared in rat cerebro-cortical cell cultures grown in different media. Excitotoxic cell death was examined by phase contrast microscopy and by the measurement of lactic dehydrogenase (LDH) activity released in the culture medium. Cells grown for 14 days in a serum-free, defined (N2) medium were resistant, while sister cultures maintained in the presence of serum responded to the excitotoxins by a significant degree of cell death. Cells cultured in serum-free medium for 7 days, and fed by a serum-containing one for an additional week exhibited vulnerability to the excitotoxins. It is concluded that the development of excitotoxicity in rat cortical cultures depends on one or more serum constituents.

Amino Acids↗

PubMed1991

GABA accelerates excitotoxic cell death in cortical cultures: protection by blockers of GABA-gated chloride channels.

The effects of gamma-aminobutyric acid (GABA) and related agonists and antagonists on the excitatory cell death were examined in dispersed primary cultures of the rat cerebral cortex. The cytotoxic effects evoked by kainic acid, quisqualic acid and N-methyl-D-aspartic acid were evaluated by phase contrast microscopy and quantified by the measurement of lactic dehydrogenase release into the culture medium. GABA accelerated the cell death in a concentration-dependent fashion, but did not influence the amount of cells dying with 24 h after the treatment. This effect of GABA could be mimicked by GABAA rather than GABAB receptor agonists. Blockers acting at different sites of the GABAA receptor/chloride channel complex not only reduced the GABA-induced acceleration of cell death, but also showed a significant protection against the excitotoxic cell death in the absence of exogenous GABA. Blockers of chloride channels unrelated to GABA receptors produced similar protection. Our findings indicate that GABA-gated chloride channels may be effective in modulating excitotoxic vulnerability of cerebrocortical cells.

Animals↗

PubMed1994

Localization of vitronectin- and fibronectin-receptors on cultured human glioma cells.

Utilizing a human astrocyte-derived glioma cell line, we have demonstrated the presence of a vitronectin receptor, alpha v beta 3, and a fibronectin receptor, alpha 5 beta 1, on the surface of the cells spreading on the respective adhesion molecules by immunohistochemical analyses. By phase-contrast microscopy, these receptors were found to be expressed predominantly in the focal contact-like area, suggesting that they were involved in the spreading of the cells upon contact with these adhesion molecules. Interestingly, they appeared to have differential functions and roles as integrins as evidenced by different time-dependent distribution profiles on the cell surface in the serum-containing medium. Furthermore, both vitronectin and fibronectin seem to have chemotactic effects onto the glioma cells as observed in a Boyden chamber study. Although these receptors are not expected to be present on the surface of astrocytes under physiological conditions, they may be expressed thereon and involved in gliosis when the cerebral vasculature is traumatized and, thereby, blood proteins, including vitronectin and fibronectin, come into contact with the astrocytes.

Cell Adhesion↗

PubMed1994

Increased synthesis of specific proteins during glutamate-induced neuronal death in cerebellar culture.

We have previously shown that glutamate-induced neurotoxicity is mediated by a sodium-chloride component and a calcium component in our cerebellar granule cell culture. In order to further characterize these two different components, the time course of neuronal death induced by glutamate (100 microM) in basal solution and in low sodium-chloride solution was studied by morphological and biochemical criteria. As shown by phase-contrast microscopy, cerebellar granule cells exhibited clear neuronal degeneration within 4 h after exposure to this excitotoxin. These morphological changes correlated [35S]methionine incorporation into proteins which rapidly declined during the first hour of treatment. Qualitative change in [35S]methionine incorporation into proteins was further investigated by two-dimensional gel electrophoresis performed after glutamate exposure in basal solution and in low sodium-chloride solution. Most of the proteins showed a decreased labelling after glutamate exposure as expected, but some polypeptides showed an increased labelling or appeared to be newly synthesized. Furthermore, a different pattern of protein synthesis was observed when glutamate exposure was performed in basal solution or in low sodium-chloride solution. The identification of these polypeptides and their implication in the neuronal death are discussed.

Animals↗

PubMed1990

Laminin in lung development: effects of anti-laminin antibody in murine lung morphogenesis.

The pattern of deposition and the role of laminin, a major glycoprotein constituent of basement membranes, were investigated during lung morphogenesis in the fetal mouse. Lung primordia were removed from Day 13 embryos, right lower lobes were further dissected and placed in filter membrane assemblies. Explants were then cultured at the liquid-air interface for 3 days in the presence of anti-laminin, anti-thrombospondin (another extracellular matrix constituent), preimmune serum, laminin-neutralized anti-laminin, or medium alone. Cultures were monitored by (direct) phase-contrast microscopy, light microscopy, and immunofluorescence. We found that anti-laminin antibodies altered normal lung morphogenesis in a dose-dependent manner. The anti-laminin-treated explants presented a marked inhibition of branching morphogenesis and a distortion of the bronchial tree. A lower rate of growth was also observed in the explants exposed to this antibody. High concentrations of anti-thrombospondin antibodies, normal rabbit serum, or laminin-neutralized anti-laminin antibodies had no effect on lung morphogenesis. These results were not modified by culturing the explants in submersion culture or on Vitrogen 100-coated surfaces.

Animals↗

PubMed1991

Identification of laminin domains involved in branching morphogenesis: effects of anti-laminin monoclonal antibodies on mouse embryonic lung development.

We recently found that polyclonal antibodies to laminin, a basement membrane-related glycoprotein, inhibited murine lung morphogenesis when added to organ cultures of mouse embryonic lung. Using a series of monoclonal anti-laminin antibodies with previously characterized subunit specificity (termed AL-1, AL-2, AL-3, AL-4, and AL-5), the deposition and functional involvement of different laminin domains in the developing lung were investigated. By immunohistochemistry the antibodies' reactivity was largely localized to the basement membrane, but was also present diffusely in the extracellular matrix throughout the mesenchyme. Organ cultures of lung explants from Day 12 embryos were cultured for 3 days in the presence of 50-100 micrograms/ml of each antibody or in the presence of the same concentration of immunoglobulins G and M, laminin-neutralized antibody, or medium alone. Cultures were monitored by phase-contrast microscopy, light microscopy, and immunofluorescence. Although all antibodies penetrated the tissues in culture, only two of them inhibited branching activity. These two antibodies were AL-1, which binds on or near the cross region of laminin, and AL-5, which binds to the lateral short arms at the globular end regions of the B chain of laminin. Inhibition of branching with these two antibodies was dose-dependent and statistically significant for the two concentrations used. AL-2, AL-3, AL-4, laminin-neutralized antibodies and control immunoglobulins did not alter lung morphogenesis. The two domains of laminin that promote lung branching morphogenesis have been reported by others to promote the attachment of a variety of cells and/or bind heparin. These domains of laminin may promote branching morphogenesis by facilitating cell attachment and, consequently, cell proliferation.

Animals↗

PubMed1992

Differentiation of retinal precursor cells born in vitro.

It is not known whether the differentiated fate of retinal precursor cells is determined before, during, or after terminal mitosis. Previous studies from this laboratory led to the hypothesis that retinal precursor cells remain plastic after final mitosis and will follow a photoreceptor "default pathway" unless induced to develop as neurons by intraretinal factors. This hypothesis predicts that isolated precursors undergoing terminal mitosis and differentiation in cell culture, in the absence of the retinal microenvironment, should become photoreceptors, regardless of embryonic age. To test this prediction precursor cells were dissociated from 5- to 8-day chick embryo retinas and grown as single cells in vitro. Bromodeoxyuridine (BRDU)- and [3H]thymidine-labeling techniques, coupled with serial photography of precursor development in culture, showed that at all donor ages some of the isolated cells divided one or more times and became postmitotic in vitro. Analysis of cell phenotype by phase-contrast microscopy, sequential photography, autoradiography, and immunocytochemistry showed that the majority of precursors from all donor ages differentiated as photoreceptors. These observations support a prediction derived from the "photoreceptor default" hypothesis.

Animals↗

PubMed1983

Separation of VX-2 rabbit carcinoma-derived cells capable of releasing collagenase.

Primary and secondary cultures of VX-2 carcinoma produced high levels of collagenase activity in both active and latent forms in serum-free media. These cultures appeared morphologically heterogeneous in phase-contrast microscopy and revealed the presence of mainly three distinct forms: epithelial-like cells (E cells), fibroblast-like cells (F cells), and large rounded-flat cells which may represent a subclass of the F cells. Cell separation techniques such as brief dispase treatment, Percoll gradient centrifugation, thimerosal treatment, and rabbit serum were used to obtain predominantly one form or the other. The E cells never formed a monolayer but rather grew as limited size clusters of intimately associated cells with large nuclei and often appeared multinucleated. These cells were difficult to maintain in culture or serially passed more than a few times. The F cells, rare in early cultures but having the highest growth potential, appeared in various morphological forms ranging from spindle- to stellate-shaped cells. The cells in their third passage were capable of producing palpable tumors, similar in light and electron microscopic studies to the original tumor from which they were derived, when injected intramuscularly into recipient rabbits and produced specific collagenase activity in active and latent forms in serum-free media. Ultrastructural studies suggested that the E cells were of epithelial origin whereas the F cells were similar to stromal fibroblasts. Cytogenetic studies demonstrated that almost all of the E cells showed both numerical and structural chromosomal changes in a modal number of 54 chromosomes. On the other hand, the major cell population of the F cells resembled normal rabbit fibroblasts; both contained a normal diploid (2n = 44). However, few cells (4-6%) in the F-cell population were hyperdiploid with a modal chromosome number of 54. These cells may represent inadvertent contaminating E cells and account for the apparent limited turmorigenicity observed in early F-cell cultures. The data suggested that the E cells were of tumor origin whereas the majority of the F-cell population appeared to be of host origin. Furthermore, it is suggested that the E cells stimulate tumor-associated stromal cells to produce elevated levels of collagenolytic activity and contribute to collagen degradation during tumor invasion.

Animals↗

PubMed1985

Interactions of human ovarian tumor cells with human mesothelial cells grown on extracellular matrix. An in vitro model system for studying tumor cell adhesion and invasion.

Human ovarian tumors metastasize by direct extension into the peritoneal cavity leading to tumor cell implantation onto peritoneal surfaces. Successful formation of peritoneal implants is dependent on the ability of ascitic tumor cells to infiltrate the mesothelium, and become firmly adherent to the underlying extracellular matrix (ECM). In order to investigate this process in more detail, an in vitro model system was developed employing human mesothelial cells grown on ECM-coated culture dishes. The ability of human ovarian carcinoma cells derived from ascitic fluid to attach to the mesothelial cell monolayer grown on ECM, ECM alone or plastic was quantitated with the use of 51Cr radio-labelled tumor cells. Tumor cells exhibited a more rapid and firmer attachment to ECM than to the mesothelial cells or to plastic. Using agitation to stimulate peritoneal fluid dynamics and shear forces in vivo, tumor cell arrest was found to be limited to the ECM, but it occurred at a slower rate than it did without agitation. Tumor cell attachment was also restricted to areas of exposed ECM in wounded mesothelium as assessed by phase-contrast microscopy. Morphologic alterations of the mesothelium induced by tumor cells were observed with the use of scanning electron microscopy (SEM) and immunohistochemical staining which included disruption of intercellular junctions leading to retraction of mesothelial cells, exposure of underlying ECM, subsequent attachment and proliferation on ECM. This model system would appear to be useful for elucidating mechanisms of ovarian tumor cell adhesion and proliferation, and for assessing various therapeutic modalities for their ability to block tumor cell implantation, invasion and growth on peritoneal surfaces.

Animals↗

PubMed1987

Endothelial cell plasma membrane obtained by chemically induced vesiculation.

A method using low concentrations of formaldehyde and dithiothreitol was applied to obtain 'right-side out' luminal plasmalemma-derived vesicles from bovine aortic endothelial cells (EC) in culture, and from human umbilical vein and bovine or porcine aortas perfused ex vivo with the vesiculation solution. Vesicle formation and shedding were examined by phase-contrast microscopy and by transmission (TEM) and scanning electron microscopy (SEM). Vesicles showed the characteristic trilaminar pattern of the unit membrane and did not contain cellular organelles. As detected in freeze-fracture preparations, vesicle membrane displayed intramembrane particles and filipin-detectable cholesterol. Like EC plasmalemma, vesicle surface was heavily stained by Ruthenium Red and bound under a normal pattern cationized ferritin and ferritin hydrazide. As indicated by lectin agglutination assays and by ultrastructural cytochemistry, vesicles maintained on their ectodomains glycoconjugates bearing monosaccharides such as N-acetyl-neuraminic acid, beta-N-acetylglucosamine and beta-D-galactose, and expressed 5'-nucleotidase activity. The electrophoretic profiles of externally disposed 125I-labelled polypeptides of vesicles were found to be similar to those of intact EC. Chemically-induced vesiculation appears as a suitable method to obtain EC plasmalemma for studying its composition and functions in various vascular beds.

Animals↗

PubMed1987

Viability and recovery from degranulation of isolated rat peritoneal mast cells.

Using a culture system that allows prolonged maintenance of purified populations of peritoneal mast cells, we have examined them following stimulation by non-immunologic or immunologic agents. Employing phase-contrast microscopy of living cells and various pharmacological manipulations, we have noted that the recovery process includes a reduction in cell size, the probable sealing of exocytotic cavities, a pronounced displacement of the cell nucleus and a resynthesis of histamine. During recovery, mast cells can entrap molecules from the extracellular fluid and later release these substances by a Ca-dependent mechanism. Our results suggest that microfilaments, calmodulin, Ca, and metabolic energy are necessary for recovery.

Animals↗