Zdeněk Lodin

Constancy and variability in the content of DNA in cerebellar Purkinje cell nuclei

SummaryA cytophotometric study of DNA content in Purkinje cells of the cerebellum of rats, cats, chicken and humans (Feulgen staining) revealed that in a certain number of cells the amount of DNA ranged between the diploid and tetraploid level (H2C cells). The incidence of H2C Purkinje cells varied among the species studied. In rats, which were studied most thoroughly, these cells amounted on average to 3%. In some rats, as well as in some cats and chickens H2C Purkinje cells were entirely absent. In the group of animals possesing H2C Purkinje cells, great interindividual differences were observed. In rats for instance, the incidence of these cells varied from 1 to 23 per cent. Topographic analyses carried out in rat and human cerebellum revealed that H2C Purkinje cells occurred more frequently in the hemispheres than in the vermis. No significant differences were found in the number of H2C Purkinje cells in healthy and Kilham-DNA-virus infected rats.Densitometric analysis of the distribution of nuclear chromatin showed that H2C Purkinje cells were richer in condensed chromatin, especially in the region of the nucleolus, which apparently contains the hyperploid surplus of DNA. It is proposed that the phenomenon of DNA hyperdiploidy arises as a result of either incomplete S-phase in some immature Purkinje cell precursors or the amplification of some DNA sequences particularly those localized in the nucleolar region.

Immunological tolerance and tumour allografts in the brain

THE brain has been considered to be an immunologically privileged site with regard to tissue transplantation. In addition, a loss of ‘tumour surveillance’ due to a weakening of the immune system in the brain has been postulated. The data on the brain as a privileged site are not unequivocal, however. The evidence for and against this thesis has been discussed by Woodruff1 and Lance2. Barker and Billingham3 also speculated that “reports that the brain can prevent implanted homografts from inciting sensitivity because it lacks a lymphatic drainage, thus having no afferent pathway of the immunological reflex, are more equivocal”. The need for a critical re-evaluation of the brain as a privileged site has been stressed. We report here a study of the possible action of immunological tolerance in the brain and of the sensitivity of normal rats and tolerant rats to inoculation of allogeneic tumour cells into the brain. We show that transplantation tolerance is involved even in the brain; thus demonstrating the presence of specific immunity following inoculation of tumour cells into the brain. This finding suggests that previously held views of the brain as a privileged site may not be entirely valid and that specific immune processes are accomplished in the central nervous system.

In vitro effects of polyvinylpyrrolidone and sucrose on the acetylcholinesterase, succinate dehydrogenase and lactate dehydrogenase activities in the brain

SummaryThe supernatant prepared from the brain tissue homogenate incubated in vitro in the presence of PVP or sucrose exhibits a decrease of AChE, SDH as well as of LDH activity. A 0.75% PVP solution inhibits AChE activity by 30%, LDH activity is inhibited by 35% and SDH activity by 40%. A two hours lasting effect of a 7.5% PVP solution at 3° C on enzymatic preparations induces in AChE 20% inhibition of its activity, in LDH an inhibition of 44% and in SDH the inhibition of its activity amounts to 74%. 1 M Sucrose inhibits AChE activity by 34%, LDH activity by 41% and SDH activity is inhibited by 31%. After two hours lasting effect of 1.4 M sucrose at 3° C on the supernatant the AChE activity is inhibited by 22% and that of LDH by 30%. The SDH activity was after a two hours lasting effect of 1 M sucrose at 3° C inhibited by 34%. The inhibition of activity of the above mentioned enzymes localized in brain cortex preparations was compared with the inhibition of activity of the isolated serum cholinesterase. 0.25 M Sucrose inhibited the activity of this enzyme by 25% and 0.75% PVP by 45%. A two hours lasting effect of 7.5% PVP or 1 M sucrose at 3° C on the cholinesterase induced a 40% and 22% inhibition respectively. After double washing of the brain cortical minced tissue, prepared in a 7.5% PVP containing solution, AChE activity was constant. By triple washing of the brain cortical crude mitochondrial fraction, exposed for two hours at 3° C to the effect of 1 M sucrose, SDH activity was also constant.

Cyclic AMP and growth regulation in rat glioma cells in tissue culture

Abstract C6 glioma cells grown in vitro for 3 to 4 days changed their morphology and showed a decreased [ 3 H]thymidine labeling index after adding dibutyryl cAMP, theophylline, isoprenaline, noradrenaline, or adrenaline to the culture medium. Changes in morphology appeared earlier (≦1 h) than those in the labeling index and/or cell proliferation (≦20 h). Dibutyryl cAMP and theophylline were found to be more effective in decreasing the rate of cell division than catecholamines. Specific blocking of adrenergic receptors suggests that catecholamines may affect cells via stimulation of both α- and β-adrenergic receptors.

Regional distribution of membrane-bound γ-glutamyl transpeptidase activity in mouse brain

Activity of membrane-bound γ-glutamyl transpeptidase (γ-GTP) was examined in various regions of mouse brain, in capillaries of the cerebral cortex and in telencephalic choroid plexuses. The level of activity in the capillaries was double and that of the choroid plexus nine times that of the γ-GTP activity found in the brain, septum, hippocampus, hypothalamus, thalamus, cerebellum, frontal cortex, pons, medulla oblongata, and amygdala. Histochemically the γ-GTP activity was demonstrated in the surface membranes of choroidal cells and in the endothelium of small capillaries.The activities of γ-GTP of cerebral cortex, choroid plexus, and capillaries from rabbit were 5–17 times greater than those from corresponding areas of mouse brain. While 30 mM methionine stimulated (in vitro) the enzyme from mouse brain, no such effect was observed with the enzyme activity from rabbit brain. The γ-GTP activity from the capillaries of cerebral cortex of both mouse and rabbit was not effected by the presence of methionine.These findings suggest existence of differences in the specificity of γ-GTP activity in these two species.

The metabolism of glucose of nerve cells cultivated under different conditions.

1. The consumption of glucose and formation of lactate was studied in medium of long-term cultivated nerve tissue. Fragments of chicken brain embryo dissociated and reaggregated brain cells were cultivated in Rose chambers, Falcon plastic dishes and Erlenmayer flasks. 2. Dissociated cells were cultivated in Petri dishes in media containing 100 mg/100 ml glucose. Consumption of glucose and formation of lactate increases until the 9th day. Glucose is completely exhausted in the medium up to the 2nd week of cultivation. 3. The time curve of both glucose consumption and lactate production is similar in cultures cultivated in Rose chambers, Petri dishes and Falcon plastic dishes. Cultures in Rose chambers utilize glucose at later stages anaerobically, whereas in Petri dishes and Falcon plastic dishes approximately 25% is utilized by aerobic glycolysis. 4. Cells dissociated by trypsinization and sieving are metabolically more active than cells separated mechanically (sieving only). During later stages of cultivation of enzymatically dissociated cells in 100 mg/100 ml glucose, lactate is utilized like a substrate, because of concentration of glucose in the medium is not being sufficient. 5. The concentration of glucose is essential for utilization by the aerobic pathway. In dissociated cells, cultivated in media enriched by 400 mg/100 ml of glucose in Falcon plastic dishes 75% of aerobic glycolysis is found during first 10 days and 50% in later stages. In the same system, cultivated in 100 mg/100 ml of glucose, glucose is exhausted up to the 12th day and lactate is utilized as a substrate. 6. In the close system of cultivation, i.e. in Rose chambers, 50% of glucose is utilized by the aerobic pathway if the medium contains 400 mg/100 ml of glucose. Early cultivation period of dissociated cells in Falcon plastic dishes is slowed, because cells adhere slowly to the plastic ground. 7. Structural development of cultures and differentiation of cells was studied during the cultivation period. Cells cultivated in elevated glucose concentration exhibit signs of better differentiation.

Structure and ultrastructure of cultivated glial cells from corpus callosum

1. Explants and dissociated cells from Corpus callosum (c. c.) of rats and rabbits were cultivated in Petri dishes and Rose chambers. 2. Different types of glial cells were found in the cultivated Corpus callosum (c. c.) explanted from 12 days old rats: a) adendritic glial cells, typical for migrating oligodendroglial cells, b)-migrating large, nondifferentiated astrocytes with pronounced phagocytosing activity, c) macro- and microglial cells which differentiated during cultivation. 3. The population of differentiated glial cells is mostly composed of oligodendroglia, less of astrocytes and microglial cells are rare. 4. Differentiation of dissociated cells from c. c. in homogenous and mixed population was studied. The appearance of first processes of macroglial cells is postponed to 6 to 10 days of cultivation. No substantial difference was observed between homogenous and mixed population. A higher incidence of macrophages was observed in the later. 5. Glial cells differentiate surrounded by degenerated nerve fibers and myelin, exhibiting phagocytoses and cleaning reaction.

Nuclear pore complexes in cells of the developing mouse cerebral cortex.

The nuclear pore complexes of cells of the superficial layers of the cerebral cortex of mice were studied by freeze-etch technique. The nuclear membrane was found to be randomly penetrated by typical octagonal pore complexes in all age groups studied. The density of pores (per micron2) amounted to 7.8, 14.0, 17.0, 18.1 and 14.1 on the 18th to 20th embryonic and the 8th, 15th, 50th and 180th postnatal day respectively. The total number of pores per nucleus increases 5.2 times from the 18th to 20th prenatal to the 15th postnatal day and then decreases toward the 180th postnatal day (1257, 6582 and 3385 pores per nucleus respectively). The density of pores in cells of brain cortex, found in young adult mice is relatively high, if compared with other cell types.

Experimental allergic encephalomyelitis: changes in growth and proliferation within the draining lymph node following injection of basic encephalitogenic protein in Freund's complete adjuvant.

Abstract Changes in wet weight, dry mass, and DNA synthesis of draining lymph nodes from rats injected with encephalitogenic basic protein in Freunds complete adjuvant (FCA) were studied. Lymph nodes of rats injected with encephalitogenic basic protein in FCA show accelerated enlargement from the second up to the fourth day after injection, as compared to lymph nodes of rats injected with FCA alone, or with nonencephalitogenic basic protein in FCA. The greatest difference in lymph node weight was found on the fourth day. At this time cell division is higher in the group injected with encephalitogenic protein in FCA than in the group injected with FCA alone. However, the increased division of cells in situ cannot account, in and by itself, for the enlargement of the lymph node which was observed. It is concluded that migration of lymphocytes into the lymph node makes a substantial contribution to the hyperplasia of the lymph node. The results suggest that accelerated lymph node enlargement may be specific, at least in part, for induction of experimental allergic encephalomyelitis.

Activation of phagocytotic activity of glial cells in corpus callosum in tissue cultures

Summary Explants of the corpus callosum (c. c.) prepared from 12-day-old rats were cultivated in Rose chambers for 1 to 67 days. After 1, 7 and 8 days of cultivation indian ink (i. i.) suspension was added to the cultivation medium for 4 to 60 h . Phagocytosing cells were found already 4 h after administration of i. i. They appeared more frequently however only 2 to 3 days after application. Initially the incidence of phagocytes was relatively higher at the periphery of the fragment, especially where calls migrating later to the outgrowth zone were located. During the subsequent cultivation the relative number of phagocytes in the outgrowth zone increased. Toward the 10th day of cultivation 1/3 to 1/5 of the cell population of this zone contained i. i. particles. Cells with ingested i. i. particles were found even 67 days after being phagocytosed. When i. i. administered to 7- or 8-day-old cultures phagocytes were relatively more frequent in the explants with delayed migratory activity. A significantly lower number of phagocytosing cells was observed in explants prepared from older donor (30-day-old rats). Phagocytosed i. i. particles were found in mesenchymal and microglial-like cells as well as in the immature calls of microglial type; in the differentiated microglial cells phagocytosis was observed only rarely. The 3 H-thymidine-light-microscope autoradiograpby revealed that phagocytosing cells synthesized DNA only exceptionally and vice versa i. e. cells close to the S-phase phagocytosed i. i. very rarely. Electron microscopic observations (performed on cultures without the i. i. in the medium) showed numerous vacuoles and increased number of lysosomes in microglial- and mesenchymal-like macrophages and immature microglial cells. The vacuoles contained osmiophilic material and remnants of membranes. The lipoidic characater of the vacuoles is indicated also by Sudan staining. They were present both in the cell body cytoplasm and cell processes. In older cultures a part of these vacuoles could however originate from the intracellular autodigesting processes. Present results revealed o a) a high capacity of cells of the cultivated corpus callosum to phagocytosis which represent a part of the “cleaning reaction” in the tissue cultures and b) decrease in activation of explanted white matter to phagocytosis with the age of donors.