Vladislav Mareš

Uptake and transport of lectins from the cerebrospinal fluid by cells of the immature mouse brain.

40 mice (C57BL/6J) 2, 3, 6, and 10 d old were injected intraventricularly with 2% wheat germ agglutinin (WGA) or soybean agglutinin (SBA). The transport of lectins from the ventricular cerebrospinal fluid (CSF) into brain parenchyma was studied by an unlabelled antibody technique ( BORGES and SIDMAN 1982). The granule product of the immunohistochemical reaction was found in the cytoplasm of ventricular ependymal cells, the choroid plexus and meninges in all age groups studied. Penetration of lectins into brain parenchyma was higher in younger animals and at more immature-cell-surrounded parts of lateral and the IVth ventricle. The stain product was dispersed throughout the neuropil excepting cell nuclei. Higher concentration of lectins appeared in nerve cell cytoplasm of some regions of the brain of younger animals (P2 to P3); in 10 d old mice granules of stain appeared in the cytoplasm of PURKINJE cells and pyramidal neurons of the hippocampus. A fiber-like staining, apparent especially in large commissures adjacent to brain ventricles (corpus callosum, fornix hippocampi) in all age groups, suggests that lectins are transported within axons, even at the earliest postnatal ages. Unlike WGA, densely stained glial cells appeared in parenchyma of SBA injected animals, especially of the younger age group (P3 to P6). The results of this study confirm earlier findings that macromolecules within the ventricular CSF can penetrate into brain parenchyma but suggest that the penetration is higher in less mature animals and at the ventricle regions surrounded by less differentiated parenchyma.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of different light regimes on DNA synthesis and cell division in the rat visual centres

Abstract BD III strain rats were reared either in a natural day - night light cycle or in complete darkness from birth. At age 4 and 11 weeks the animals were injected with H-3 thymidine and killed after 2 hrs. The percentage of non - neuronal cells synthesizing DNA /LI%/ in the dorsal part of lateral geniculate body /dLGB/ of 4-week-old control and dark-reared animals did not differ. Towards the 11th postnatal week the LI% decreased by 59% in controls and by 80% in dark-reared rats. The difference in LI% between both groups of 11-week-old animals amounted in the dLGB to 66% /p

N-Acetyl-l-aspartyl-l-glutamate changes functional and structural properties of rat blood–brain barrier

Intracerebroventricular administration of N-acetyl-L-aspartyl-L-glutamate (NAAG), an agonist at group II metabotropic and NR1/NR2D-containing N-methyl-D-aspartate (NMDA) ionotropic glutamate receptors, increased the permeability of the blood-brain barrier (BBB) to serum albumin in the striatum, but produced no similar effects in the entorhinal cortex or in the hippocampal formation. Electron microscopy showed that NAAG, but not its hydrolytic products L-glutamate and N-acetyl-L-aspartate, increased the number of transport vesicles in the hippocampal endothelial cells. Furthermore, immunocytochemistry detected NR2D subunits on hippocampal capillaries. Consequently, NAAG may have influenced the vesicular transport via NMDA receptors. There was, however, no correlation with the regional pattern of BBB changes (increased permeability in the striatum) that, in turn, could not be directly related to the NAAG-induced neurodegeneration described previously in the hippocampus where no significant changes in BBB permeability were detected.

Influence ofcis-dichlorodiamineplatinum on glioma cell morphology and cell cycle kinetics in tissue culture

SummaryC6 glioma cells (CCL 107) were cultured for three days and then treated withcis-dichlorodiamineplatinum (cis-DDP) at doses of 0.2–10 µg/ml medium. Changes in DNA synthesis and DNA content, as well as morphology of cells and chromatin distribution, were examined from the first post-treatment day onwards. The number of cells labelled with [3H]thymidine, detected autoradiographically, decreased after treatment with 0.2–10 µg/ml by approximately one half on post-treatment day 1 and diminished further by the third day after treatment. The labelled cells were entirely absent only after treatment with 10µg/ml, 7 days post-treatment. Mitoses decreased from 1.4–0.6% by post-treatment day 1 and completely disappeared by day 3 (1 µg/ml). Feulgen cytophotometry and propidium iodide cytofluorimetry revealed accumulation of cells in the S-phase, especially the latter part (0.5 and 1.0 µg/ml, post-treatment day 1) and subsequently also in G2 phase (post-treatment day 3). Incomplete cyto- and karyokinesis in some cycling cells was indicated by an increased number of binucleate cells and nuclei of higher ploidy classes. Labelled cells with intermediate DNA values were, on average, labelled less intensively, as was revealed by simultaneous measurements of DNA content and [3H]thymidine incorporation. Some cells displayed reduction in grain density over heterochromatin clumps. This would be in agreement with the late S-phase block of DNA replication. After post-treatment day 3 the density of cells in cultures was substantially lower. This was due to slowed transversing through the cell cycle and cell death occurring after post-treatment day 1 with higher doses or after day 2 with lower doses (up to 1 µg/ml). The size of the nuclei of surviving cells enlarged initially (post-treatment day 1) and later (day 7) giant cells with long, branched fibres similar to those of reactive astrocytes occurred. Texture analysis of Feulgen-stained nuclei revealed that the chromatin of cells treated withcis-DDP became less evenly distributed. This might be due either to the direct influence ofcis-DDP on the DNA molecule, or mediated by changes in cytoskeleton and cAMP levels described earlier.

Damage and repair of the immature rat cerebellum after cis-dichlorodiammineplatinum II (cis-DDP) treatment. An ultrastructural study.

SummaryThe aim of this electron microscopy study was to further investigate the effects of cis-dichlorodiammineplatinum (cis-DDP) on the cerebellum of the immature rat. Ten-day-old animals were treated with cis-DDP subcutaneously and killed after 1, 7, 15 or 21 days. On postinjection day 1, cis-DDP effects were evident mainly in the external granular layer, with nuclear damage in many dividing cells, while their cytoplasm appeared to be less affected. Some binucleate cells were also present. On the contrary, in postmitotic or more differentiated cells, only cytoplasmic alterations were found. At later stages (postinjection day 7), the frequency of damaged cells in the external granular layer decreased, but there was a cellular deficit in the internal granular layer. Many postmitotic neurons underwent coagulative necrosis. Finally (postinjection days 15 and 21), the cellular deficit was partly compensated for by “reactive” structures, e.g., glial cell fibers, which underwent hypertrophy after initial edema. Moreover, packing densities of Bergmann astrocytes and oligodendrocytes were higher.

Changes in cell cycle and chromatin distribution in C6 glioma cells treated by dibutyryl cyclic AMP

Abstract. C6 glioma cells in culture were treated with 1 mm dibutyryl cyclic AMP (Db‐cAMP) for 5,8,24 and 72 h. The cells were labelled with [3H]‐thymidine before either the end, or the beginning, of the Db‐cAMP treatment. The cell cycle passage was monitored by the simultaneous determination of DNA content and DNA synthesis in propidium iodide stained autoradiograms. The data revealed an early (t ≤ 3–8 h) and moderate inhibitory effect of Db‐cAMP on all phases of the cell cycle except mitosis; some cells (2%) were completely blocked in the S phase. Later (8 < t < 24–72 h), the cycling of a substantial part of the population became inhibited in G1 phase. Microdensitometric texture analysis of Feulgen‐stained nuclei, performed 24 h after administration of Db‐cAMP, showed a higher inhomogeneity of the DNA distribution in cell nuclei, caused by the condensation of a part of the chromatin. This may reflect either changes in genome expression taking part in the process of cAMP induced differentiation or transit of some cells into quiescent G0 or S0 phases.

Quinolinic acid induces NMDA receptor-mediated lipid peroxidation in rat brain microvessels.

Abstract Quinolinic acid increased the generation of lipid peroxidation products by isolated rat brain microvessels in vitro. The effect was inhibited both by a specific NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid and by reduced glutathione (GSH). Furthermore, quinolinic acid displaced specific binding of [3H]-L-glutamate by cerebral microvessel membranes, particularly in the presence of NMDA receptor co-agonist (glycine) and modulator (spermidine). We conclude that quinolinic acid can cause potentially cytotoxic lipid peroxidation in brain microvessels via an NMDA receptor mediated mechanism.

An attempt to influence DNA content in postmitotic Purkinje cells of the cerebellum

Feulgen-DNA content has been measured cytophotometrically in granule and Purkinje cells of the rat and mouse cerebellum. The study has confirmed that under normal conditions a very small part of the P. cell population in rats (less than 3%) possesses a Feulgen-DNA surplus ranging from 2C to 4C. In mice, the hyperdiploid (H2C) P. cells are even more rare. The occurrence of H2C-P. cells in rats and probably also in mice has not been substantially changed in animals exposed to factors interfering with chromatin structure and/or its template activity; the H2C-P. cells seem to be slightly more frequent after injecting mice with corticoids (Urbason) or in animals suffering from ectromelia or hereditary Purkinje cell degeneration. The incidence of H2C-P. cells has neither been substantially affected by experimental conditions which are known to lead to functional and/or metabolic stimulation of the cerebellum. Functional changes in the number of H2C-P. cell nuclei may, however, be short-term or transient in character and therefore might have excaped detection in our models. The findings rule out an impact of some reasons suspected for the artefactual origin of Feulgen H2C DNA values as e.g. compactness of the chromatin.

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.

Inhibition of mitochondrial ATPase by dicarbopolyborate, a new enzyme inhibitor

Polyborate anions were found to inhibit mitochondrial ATPase. Mercapto and chloro derivatives of dicarbononaborates showed full inhibition of the enzyme activity at 0.5–0.8 mM. The inhibitory effect of dodecaborates was lower. The inhibition was of competitive type with respect to ATP. The inhibition of soluble F1-ATPase indicates a direct interaction of the polyborate anion with the catalytic part of the enzyme molecule.