[Rat brain cells containing ezrin (cytovillin)].

Ezrin (cytovillin or p81 protein) is an actin-binding protein, a member of ERM (ezrin, radixin and moesin) family, which species contribute to stabilization of the plasma membrane-formed structures. The aim of the present study was to demonstrate the ezrin-containing cells in the rat brain and to describe their topography and morphological features. The most pronounced immunohistochemical reaction to ezrin was found in the epithelium of the choroid plexus, cells of the subcommissural organ and ventricular ependyma. Moreover, ezrin staining was also detected in the unidentifiable cells in the subventricular zone, rostral migration pathway and astrocytes in various brain areas. Preferential ezrin localization in the brain cells contributing to formation of barrier structures suggests its involvement in transport processes in the CNS.

[Application of semi-conducting nanocrystals (quantum dots) in immunocytochemistry].

Semi-conducting nanocrystals represent a new class of fluorescent inorganic objects which have a promising perspective for the application in biology and medicine. The aim of the current work was a determination of advantages and shortcimingss of quantum dots (QD) application in immunocytochemistry. It was shown that streptavidin-QD conjugates have more advantages (i.e. high intensity of fluorescence, photostability, wide excitation range, short and symmetric emission range) as compared to streptavidin conjugates with organic fluorochromes. This allows to recommend the use of QD in immunocytochemical studies. However, there are several disadvantages (like lower stability during long-term storage as compared to that one of organic fluorochrome conjugates, poor safety of aliquots, impossibility of long-term preservation of fluorescence of stained sections during their storage, incompatibility with several commercial mounting media) that limit the widespread application of nanocrystals in immunocytochemical studies.

[Comparative analysis of methodological approaches applied for the simultaneous demonstration of several antigens in immunohistochemical studies].

In immunomorphological studies, it is often necessary to visualize several antigens simultaneously within the same histological sections. However in practice, the application of double staining is limited due to methodological complexity. The aim of the current study was to reproduce different methods recommended for multiple immunohistochemical staining of one section and to estimate their advantages and drawbacks. Six different methodological approaches for detection of several antigens in one slide were examined. It was found that the method providing the widest resources for analysis was the fluorescent detection, the most convenient was the application of ready-to-use standardized reagents, and the cheapest method was two-color reaction with DAB-type chromogen.

[The study of cerebral astrocyte spatial organization using confocal laser microscopy].

Three-dimensional reconstruction of cytoarchitectonic relations of the main components of the nervous tissue appears to be an important, yet an extremely laborious task of neuromorphology. The aims of the present study were: to develop the method permitting accurate visualization of astrocytes in rat brain slices thicker then 30 microm, to create an adequate algorithm for the study of the slides received using a confocal microscope and to perform the three-dimensional reconstructions of astrocytes from Z-series of confocal images. Different variants of slide preparation (various clearing reagents, fluorochromes, mounting media) were investigated, together with the operating modes of confocal microscope, methods of digital image processing etc. The methodical approaches are recommended that may be successfully applied for three-dimensional reconstruction and detection of spatial relations of astrocytes in mammalian brain.

[Change of composition of intermediate filaments in cells of rat telencephalon at early period of postnatal ontogenesis].

The goal of the present work was to study composition and spatial-temporal distribution of cells containing various proteins of intermediate filaments (nestin, vimentin, GFAP) in various brain parts at the early postnatal period of rat ontogenesis. By using methods of immunochemical determination of proteins of intermediate filaments it has been established that at they early period of ontogenesis, in the course of maturation of the nervous tissue, in cells of cortex, hippocampus, and subventricular area there occurs a change ofimmunochemical profile of intermediate filaments: the nestin+/vimentin+/GFAP- -cells become the nestin-/vimentin-/GFAP- ones.

[Immunocytochemical demonstration of tissue antigens after a long-term storage of samples in methylsalicylate].

The aim of the present study was to test the possibility of different antigens detection in the biological objects after their storage in methylsalicylate for two or more years before embedding in paraffin, using routine immunocytochemical methods. Using immunocytochemical reactions demostrating intermediate filaments nestin, vimentin, and GFAP, as well as neuronal markers NeuN, neuron-specific enolase, and doublecortin, as the examples, it was shown that long-term storage in methylsalicylate does not prevent immunocytochemical detection of these antigens.

[Morphological manifestations of astrocyte local functional activation produced by a short-term total brain ischemia].

The goal of this work was to study changes structural and cytochemical organization of rat hippocampus activated astrocytes after a brief total brain ischemia. By methods of immunocytochemical determination of proteins of intermediate filaments, in was established that 7 days after the ischemia the functional activation of dorsal hippocampus astrocytes is morphologically manifested both as changes of size and shape of the cells and their processes and as accumulation of the intermediate filament proteins -GFAP and nestin. Two populations of the activated astrocytes are formed - more dispersed GFAP-positive astrocytes and nestin-positive astrocytes located predominantly in the area of massive death of nerve cells. The obtained data allow suggesting that the postischemic activation of astrocytes is accompanied by their acquistition of properties characteristic of immature cells of the nervous tissue; however, the absence of morphological signs of dedifferentiation does not permit these cells to be considered responsible for reparational neurogenesis in hippocampus.

[Perturbations of the vesicle cycle in reticulospinal synapses of axons in lamprey after presynaptic microinjections of GTPgammaS].

The synaptic vesicle cycle sustains neurotransmission and keeps pace between exo- and endocytosis in synapses. GTP-binding proteins function as key regulators of this cycle. The large GTPase dynamin is implicated in fission of clathrin-coated vesicles from the presynaptic membrane during endocytosis. The present study addresses the effect of the non-hydrolysable GTP analog, GTPgammaS, on the assembly of the dynamin fission complex in situ. Intraaxonal microinjections of GTPgammaS induced distinct ultrastructural changes in synapses: the number of synaptic vesicles at active zones was reduces, and the number of docked vesicles was increased; at the same time the number of clathrin-coated intermediates at the synaptic endocytic zone was increased, indicating that synaptic vesicle recycling was inhibited. Clathrin-coated intermediates with unusual shape were found. At low concentrations of GTPgammaS they were represented by long tubules decorated by spirals containing dynamin and clathrin-coated vesicles on the top. At high concentrations of GTPgammaS the tubulular structures were shorted and branched. The pitch of the spiral and tubule's diameter were significantly reduced (23.1 +/- 0.4 and 19.0 +/- 0.5 nm, respectively, as compared to those at low concentration of GTPgammaS, 26.6 +/- 0.4 and 23.3 +/- 0.4 nm; P < 0.001). We suggest that these structural changes correspond to distinct steps in the fission reaction. A model is proposed. It implies that the fast GTP hydrolysis leads to an increase in length of the spiral due to the straightening of the dynamin dimmers, composing the spiral. This leads to a fast increase both in the pitch and the diameter of the helix. The shift in diameter breaks the local hydrophobic interactions between the inner and the outer leaflets of the lipid membrane at the sites of dynamin binding. Stretching of the spiral leads to an expansion of the neck in the longitudinal direction and promotes severing of the membrane that subsequently results in the release of the clathrin-coated vesicle.

[Demonstration of nuclear protein neun in the human brain corpora amylacea].

The purpose of the present study was to examine the corpora amylacea (amyloid bodies, polyglucosan bodies) in human brain. To test the hypothesis on their neuronal origin, distribution of the selective neuronal marker NeuN was studied immunocytochemically in the brain areas containing amyloid bodies. The studied neuronal marker NeuN was detected in most of the amyloid bodies. The data obtained confirm the neuronal origin of the amyloid bodies.

[Ischemic damage-induced nestin synthesis in the rat brain cells].

Nestin, an intermediate filament protein, is known to be expressed in the proliferating and provisional cells of the developing mammalian brain and is lost during differentiation. The aim of the present study was to determine the morphological type and localization areas of the rat brain cells exhibiting the ability to synthesize nestin after a short-term global ischemia of the brain. Induction of nestin synthesis after a short-term ischemia was found within the injured brain areas in astrocytes, which exhibited structural features atypical for these cells in mature brain and maintained them for a long time, and in the morphologically undifferentiated cells of subventricular zone, which were able to proliferate. However, acquisition by astrocytes of some phenotypic properties of immature glial cell does not by itself support the view that they may transform into neural stem cells.

[Nestin in the cells of the central nervous system].

This review describes the most recent data on an intermediate filament protein nestin, which is regarded by most of the authors as a possible neural stem/progenitor cells marker. Several structure-functional characteristics of nestin, its occurrence in different cell types at various stages of ontogenesis in physiological and pathological conditions are reviewed.

[Optimization of the immunocytochemical detection of nestin in paraffin sections of the rat brain].

The aim of this study was to develop an optimal and easy protocol for immunocytochemical demonstration of nestin (an intermediate filament protein which is believed to be a neuronal stem cell marker) in paraffin sections of rat brain with target retrieval procedure being omitted for better preservation of the neural tissue structure. The influence of fixation, character of blocking of nonspecific antibody binding, temperature and time of incubation on the results of immunocytochemical reaction was studied. On the basis of the results obtained, we propose a processing protocol allowing distinct demonstration of nestin in rat brain cells together with the optimal preservation of the nervous tissue structure.