Ulf Brunk

The fixation, dehydration, drying and coating of cultured cells for SEM

Cultivated cells form a valuable model system for studies on the effects of various preparative protocols for scanning electron microscopy (SEM). The various effects of each preparative step can be followed in detail in the light microscope and no diffusion gradients complicate the fixation and other procedures as in the case of solid tissues.

Characterization of residual bodies formed in phase II cultivated human glia cells

Residual body-like structures have been shown to occur in small numbers in actively growing in vitro cultivated phase II human glia cells. In contact inhibited phase II glia cells they have been shown to accumulate. Whether such structures are analogous to lipofuscin pigment granules in post-mitotic cells in vivo has been uncertain. We have characterized such bodies in actively growing and contact inhibited cells as to their acid phosphatase content, heavy metal content, autofluorescent characteristics, staining characteristics and morphology. It has been shown that many of these bodies are acid phosphatase positive, contain heavy metals, show the natural and characteristic fluorescence of lipofuscin pigment, are PAS-positive, acid fast with Ziehl-Neelsen and are morphologically similar to lipofuscin. They are thus analogous to the lipofuscin granules found in post-mitotic cells in vivo. The number of such granules was found to increase following contact inhibition of the mitotic activity. We conclude that lipofuscin granules are found continuously in the culture and that the cellular content of these granules can be diluted by cell division.

Quantitation of residual bodies in cultured human glial cells during stationary and logarithmic growth phases.

Abstract Recent report have stated that lipofuscin granules, similar to those found in post-mitotic cells in vivo, accumulate in phase II glial cells during density dependent inhibition of growth. No stereological studies have been carried out to date to investigate the degree or rate of this accumulation. Parallel cultures of cells were studied during log growth and subsequent density dependent inhibition of growth for a period of up to four months. During this period some cultures were subcultivated and the effects of renewed log growth on cytoplasmic organelles and especially the content of residual bodies were studied. We found that the mean volume percentage of the cytoplasm taken up by residual bodies increased steadily from the point of density inhibition of growth. The 24 hours tritiated thymidine (3H-TdR) incorporation fell below 2% 21 days after subcultivation. In log phase the mean volume percentage of cytoplasm taken up by residual bodies was 9.6, seven days after subcultivation. Four weeks later, and two weeks after the almost complete cessation of cell multiplication, this figure had risen to 15.3. Four and six weeks after density dependent growth inhibition, the figures were 29.1 and 32.2 respectively. Longer periods in stationary phase did not give any further significant increase in the residual body volume percentage of the cytoplasm. During this period cell volume increased by a factor of 1.9, nuclear volume decreased slightly and the cytoplasm, excluding the secondary lysosomes of the residual body variety, increased by a factor of approximately 1.4. The average cell accumulation of residual body structures increased by a factor of 7 thus constituting the greater part of the cell volume increase. Following subcultivation the volume of residual body structures decreased rapidly and after only seven days of log growth had been lowered to 22.5 vol.% of the cytoplasm and after a further 14 days to 9.8 which approaches the starting level. During prolonged density inhibition of growth there was a gradual cell loss with a parallel enlargement of the remaining cells which kept the monolayer intact and confluent. These results show that phase II glial cells accumulate residual bodies during density dependent growth inhibition. The fact that the volume percentage decreases rapidly following subcultivation shows that cells can decrease their load of residual bodies rapidly by dilution during cell division. Cell volume increases during prolonged periods of density inhibition of growth are to a considerable degree the result of this residual body accumulation.

The effect of aging on lysosomal permeability in nerve cells of the central nervous system. An enzyme histochemical study in rat.

SummaryAging neurons accumulate lipofuscin pigment granules which appear to be secondary lysosomes of the residual body variety. The biological significance of the residual bodies is debated. They were here studied with the aim of testing a hypothesis that the membranes surrounding these granules might be more vulnerable than the membranes around “younger” types of lysosomes.For this purpose large motor neurons of young and old rats were compared with respect to lysosomal membrane latency, using a modified Bitensky lysosomal lability test. Utilizing successively increasing incubation times, the lysosomes of old neurons, in particular the residual bodies in polar aggregates of old neurons—presumed to represent lipofuscin pigment granules—were found to have a clearly reduced latency in comparison with lysosomes of young neurons.These findings support the notion that the residual bodies are more fragile than “younger” lysosomes.

Histochemical evidence for lysosomal uptake of lead in tissue cultured fibroblasts.

SummaryLysosomes of embryonic rat fibroblasts cultivated in vitro normally contain heavy metals, as shown with a modified sulfide-silver method (SSM). Cultures which received lead added to the cultivation medium showed an enhanced SSM-positivity. However, since the SSM demonstrates several different heavy metals the sulfides of which are weakly soluble in water, it was not possible to distinguish between naturally occurring heavy metals—largely iron—and added lead (supposed to have been taken up by the cells).By treating the cells with a 0.2 M TCA solution after an initial exposure to HS− it was possible to dissolve FeS without affecting the PbS to any noticeable extent. When subsequently the development process of the SSM was applied to the cells, no metals could be demonstrated in the control cells whereas those exposed to lead showed presence of granules, most of which were identical with lysosomes as visualized with a Gomori type reaction. A lysosomal uptake of lead could thus be demonstrated with the modified SSM when combined with a simple dissolving process.

Heavy metal localization and age related accumulation in the rat nervous system

SummaryA modified sulfide-silver method was used to demonstrate tissue bound heavy metals in the rat brain at various ages. An accumulation of sulfide-silver positive material was found to accompany aging, indicating heavy metal accumulation. This was verified by quantitative analysis using atomic absorption spectrophotometry. Iron appears to be the most important heavy metal. Besides differences between various ages, regional variations in heavy metal contents could constantly be shown. The heavy metals appear, at least in part, to be located in lysosomes. A heavy metal influence on the lysosomal membrane permeability is discussed.

CYTOPLASMIC EFFECTS OF X-IRRADIATION ON CULTURED CELLS IN A NON-DIVIDING STAGE

Cultured human glial cells were blocked in interphase (G1) by 24 h of serum starvation and subsequently subjected to 200 Gy, 8 MV X-radiation. Immediately following irradiation the cultures were transferred to serum-containig medium. Time-lapse cinemicrography performed during the next 48 h showed a profoundly disturbed motility with decreased ability for polarization and locomotion of the irradiated cells. Specimens fixed 24 and 48 h after irradiation and investigated by immunofluorescence with tubulin-antibodies and DNase/DNase-antibodies, and by whole cell transmission electron microscopy showed derangements in the distribution of microfilament bundles related to the cytoplasmic ramification of the irradiated cells, but otherwise no detectable alterations in structure or distribution of either microtubules or microfilaments. It is suggested that the alteration in the arrangement of filament bundles is of importance to the impaired locomotion of the irradiated cells.

Lability of human decidual cells. In vitro effects of autolysis and osmotic stress.

Abstract Decidual cells have been found previously to be damaged by hypertonic saline injected intra-amniotically as well as extra-amniotically, while the trophoblast appeared mainly unaltered. To investigate the apparent differences in stability between the two types of cells, they were compared regarding their vulnerability during osmotic stress and autolysis in vitro. The decidual cells were found to be exceedingly fragile and contained lysosomes of low resistance. Since the decidual cells are rich in prostaglandins—or their precursors—the stability of the decidual cell membranes should be of importance for the maintenance of pregnancy. It is suggested that damage to the easily disrupted decidual cells with consequent liberation of prostaglandins is the mechanism by which hypertonic saline induces abortion. Factors influencing the stability of the lysosomal and plasma membranes could be important for the prevention of abortion and premature labor, or for the initiation of abortion and normal labor at term.

Effects of clofibrate administration to rats on their hepatocytes

Abstract Changes in the hepatocytes of rats treated with the hypolipidemic agent clofibrate for a 1 week period were investigated. During this time liver weight increased while, in contrast to peroxisomes, mitochondrial and microsomal protein were not changed significantly. While there was no effect on cytochrome oxidase activity carnitineacetyl transferase activity increased more than 20-fold. The urate oxidase activity of the homogenate decreased about 30% and palmitoyl-CoA oxidation increased more than 10 times. The ratio of protein to phospholipid in microsomes was not affected, nor were the rates of incorporation of [ 3 H]leucine, [ 3 H]glycerol and [ 3 H]mevalonate into total protein, phospholipid and cholesterol, respectively. Injection of labeled glucosamine, mannose and galactose into the animals revealed an increased incorporation of these substances into lipid intermediates and endogenous luminal and membranous protein acceptors after clofibrate treatment. The significant stimulation of enzymes participating in hydroxylation reactions may be the main reason for the complex cellular changes brought about by clofibrate.

LABILITY OF HUMAN DECIDUAL CELLS. IN VIVO EFFECTS OF HYPERTONIC SALINE

Abstract. Recently, decidual cells, in contrast to trophoblastic cells, were found to be exceedingly fragile in vitro. In the present investigation the stability, in vivo, of decidual cells was studied by histological and cytochemical methods. The material was obtained either by vacuum aspiration or hysterotomy at various intervals following intrauterine injection of 20 % saline solution. Fifteen minutes following the injection, decidual cells in part of the decidua showed distinct degenerative changes and signs of leakage into the cell cytoplasm of the lysosomal “marker” enzyme acid phosphatase. The extent of these changes and the number of cells involved increased in parallel with the interval between the injection and the evacuation. The alterations in the decidual cells were of the same type and magnitude whether the hypertonic solution was injected intra‐amniotically or extra‐amniotically. The present findings further support the hypothesis that the mechanism by which hypertonic saline provokes abortion involves damage of the decidua and subsequent liberation of prostaglandins.