Optimal conditions for the measurement of lipid peroxidation products (lipofuscin) in individual cultivated human glial and glioma cells.

Age pigment (lipofuscin) has been quantified in individual cultivated human glial and glioma cells using fluorescent microscopic techniques and the effects of cell spreading after subcultivation, fixatives, mounting media and storage conditions on these measurements were investigated. The use of formalin fixation on cells that were permitted to spread at least 6 h after subcultivation, combined together with glycerol mounting media and subsequent storage at 4 degrees C provided the optimal versatility for lipofuscin quantification when multiparametric analysis of the cells was desired. Alternatively, Entellan was the mounting media of choice when long-term, non-multiparametrical studies are conducted and the cellular material cannot be stored below room temperature.

Identification and characterization of non-dividing cell populations in phase II cultures of human glial cells.

It has been reported that cultures of normal cells in phase II contain a non-multiplying cell population, the size of which increases with passage number. In phase II cultures of normal human glial cells we have found two subpopulations of non-proliferating cells, one of which has a characteristic morphology, and differs from the actively dividing cells in a number of respects: (1) they are larger although of various sizes and are well spread over a very large substratum area: (2) they contain a great number of granules showing acid phosphatase activity, being heavy metal positive and displaying the characteristic natural fluorescence of lipofuscin pigment; and (3) they frequently contained a central somewhat irregular nucleus with various numbers of darkly staining nucleolar-like structures. Cytophotometric nuclear DNA measurements of the described "large" cell population show a decreased proportion of diploid cells as compared to their smaller sister cells. Moreover, with increasing passage number, the DNA values for large cells shift towards higher ploidy levels resulting in a scattered aneuploid pattern in the oldest passage. This "large" cell subpopulation consists of between 2% and 3% of all passages and becomes greatly decreased following subcultivation. The other subpopulation of non-dividing cells is generally morphologically similar to the dividers, increases in size with passage number and is the more important in the phase III phenomenon.

Influence of oxygen tension, pro-oxidants and antioxidants on the formation of lipid peroxidation products (lipofuscin) in individual cultivated human glial cells.

Cultivated human glial cells, kept in a state of density-dependent inhibition of growth, accumulate age pigment (lipofuscin) within their lysosomal vacuomes which has the same characteristics as the age pigment observed in vivo. The rate of formation and accumulation of lipofuscin is greatly accelerated under the conditions of routine cell culture in comparison to the in vivo event. Lipofuscin is generally considered to be composed of products of lipid peroxidation and thus it would be reasonable to suggest that factors which influence lipid peroxidation would also alter the rate of lipofuscin formation and accumulation. Human glial cells were grown in the presence of various oxygen concentrations (5%, 10%, 20%, 40%) or exposed to pro-oxidants (vitamin C/Fe) or antioxidants (vitamin E/Se, dimethylsulfoxide, reduced glutathione) treatments in the presence of normal oxygen concentration (20%). It was found that these factors can modulate (accelerate or decrease) the rate of formation of lipofuscin. This study thus provides: (1) important supportive evidence for the lipid peroxidation origin of lipofuscin, (2) a useful model system for studying the effect of lipofuscin accumulation on lysosomal function and cell growth kinetics, (3) evidence that our culture conditions are far from ideal: oxygen concentration may drastically alter rates of lipofuscin formation and accumulation. Cell culture technique, as we know it today, may benefit from more closely controlled oxygen tensions, i.e. by reducing oxygen to levels that more closely approximate conditions in vivo.

The effect of reactive oxygen metabolites (ROM) on the attachment and ingestion phases of C3b- and IgG-mediated phagocytosis by macrophages.

The effects of enzymatically produced reactive oxygen metabolites (ROM) on the attachment and ingestion phases of C3b- and IgG-mediated phagocytosis by cultured mouse peritoneal macrophages (MPM) was investigated using a hypoxanthine-xanthine oxidase ROM-generating system. ROM-exposure at a dose which did not affect cell viability caused a slight decrease in the percentage of phagocytosing cells. The total number of cell-associated (attached and ingested) C3b- and IgG-coated particles initially decreased in relation to controls. After 120 min the number of attached and ingested C3b-particles had returned to the level of controls, while the corresponding value for IgG-particles lagged behind. The number of ingested particles decreased in both C3b- and IgG-groups at each time-point studied (30-150 min). A linear increase in the formation of lipid peroxidation products was measured during the period of observation, while transmission electron microscopical studies showed largely intact morphology. These results indicate that ROM species may induce membrane-related changes in inflammatory cells such as macrophages, probably due to lipid peroxidation; affecting the binding functions of the C3b- and Fc-receptors, without any obvious alteration in cellular fine structure.

The measurement of lipid peroxidation products (lipofuscin) in individual cultivated human glial cells.

The natural fluorescence (autofluorescence) of the age pigment lipofuscin has been quantitated and related to the dry mass and DNA content (after Feulgen staining) of individual cultivated human glial cells which were density-dependent growth-inhibited for 3 and 18 months, subcultivated and kept in log growth phase for a period of up to 3 weeks. Stereological measurements at the ultrastructural level were performed in parallel to determine the volume density of secondary lysosomes of the residual body type (containing lipofuscin). The autofluorescence/dry mass/cell and the volume density of residual bodies showed a positive correlation. Cells in S-phase following subcultivation demonstrated similar autofluorescence values to cells in G1-phase. These results demonstrate that (1) multiparametric measurements of this type can be utilized to correlate many parameters which may be indicative of differing cell functions in individual cells, (2) there is a positive correlation between the ultrastructural and the autofluorescence techniques to measure lipofuscin, and (3) despite the limited data, even high cellular lipofuscin content does not appear to be detrimental to the ability of cells surviving subcultivation to re-enter the cell cycle.

A versatile localization system for microscopic multiparametric analysis of cells.

A new, simple and relatively inexpensive electronic digital position readout (DPRO) system which can be applied to the rapid localization and recovery of microscopic material is described. It is based upon a commercially available digital position readout system which is routinely utilized by industry for small machine tools and measuring equipment. This has been mounted onto the stage of various microscopic instrumentation to provide X and Y coordinates relative to an arbitrary reference point. The integration of small computers interfaced to scanning interferometric, microdensitometric and fluorescence microscopes were used to demonstrate the reliability, versatility and ease of application of this system to problems of multiparametric measurements and analysis of cultured cells. The system may be expanded and applied to clinical material to obtain automatized, multiparametric measurements of cells in haematology and clinical cytology.

An approach to the assessment of membrane stability of cultured cells.

A simple method for assessing the combined stability of the plasma and lysosomal membranes of cultured cells is described. Monolayers of normal, human glial cells were incubated in situ in an isotonic, buffered sucrose solution (pH 5.0) containing the acid phosphatase (AP) enzyme substrate p-nitrophenyl phosphate (PNPP). The rate of appearance, in the solution, of the reaction product p-nitrophenol (PNP) was measured spectrophotometrically, curves then plotted, and fitted by computer. "Lag time" (LT) was calculated, and an index of membrane lability constructed, termed "fragility index" (FI). Transmission electron microscopy (TEM), "vital" staining of the cells with fluorescein diacetate (FDA) and Evans Blue (EB), and use of a Gomori-type cytochemical technique, indicate that the data reflects the combined stability of lysosomal and plasma membranes. The latter playing the more critical role. Cell cultures pre-incubated with various membrane labilizing or stabilizing agents were compared. Control, 0.3 M sucrose, and normal saline treated cells demonstrated similar stability. Distilled water decreased AP latency (increased fragility), and the magnitude of this effect was time dependent. Cells fixed in glutaraldehyde (GA) retained much of their osmotic reactivity, as confirmed by distilled water treatment. Oxygen derived free radicals caused pronounced fragility, while dexamethasone, a membrane stabilizing agent, decreased membrane fragility. Triton X-100 abolished latency completely, and total AP activity was very rapidly recovered outside the cells in the surrounding incubation medium. These results suggest this technique yields a measure of membrane stability which is sensitive enough to differentiate between known stabilizers and labilizers of membranes. Hence, this may prove an easy and useful aid for the assessment of how various substances and environments modulate the lysosomal and plasma membrane stability of cultured cells.

Free radicals as mediators of tissue injury.

Polymorphonuclear leukocytes and other inflammatory cells on exposure to appropriate stimuli release superoxide anion radical into the extracellular space. This results in the further generation of other activated oxygen species such as hydrogen peroxide, hydroxyl radical and singlet oxygen. We have assessed the influence of activated oxygen species, generated by substrate-xanthine oxidase systems, on the degradation of hyaluronic acid, human glial cells in culture and the microcirculation of the hamster cheek pouch. Hyaluronic acid degradation was observed and morphological changes, culminating in cell death, were seen in glial cells. Increased microvascular permeability and increased polymorphonuclear leukocyte-endothelial adhesion were also observed. The results suggest that a variety of alterations to macromolecular and cellular elements of tissues can be mediated by specific free radical species. Similar mechanisms may play a role in the structural and cellular injury occurring in the microcirculation during inflammation and other disease processes.