[CELL CONTACT PROTEIN BETA-CATENIN IN EPENDYMAL AND EPITHELIAL CELLS OF THE CHOROID PLEXUS OF THE CEREBRAL LATERAL VENTRICLES].

The purpose of this study was to examine the distribution pattern of cellular contacts protein beta-catenin in the choroid plexus and ependyma of lateral ventricles of the brain. The study was conducted on frontal sections of the brain of Wistar rats (n = 10) using polyclonal antibodies against beta-catenin. The obtained preparations were analyzed by microscopy in transmitted light and using confocal laser microscopy. To study the distribution of beta-catenin in different projections, three-dimensional reconstruction was performed. The study demonstrated different distribution patterns of this protein in ependyma and choroid plexus. Unlike ependyma, in the cells of the choroid plexus beta-catenin was distributed in the same way as in simple epithelial tissues (on the basal and lateral borders of the cells). This may indicate different tissue attribution of the ependyma and the choroid plexus epithelium, despite their common origin.

[CURRENT HISTOCHEMICAL METHODS OF TISSUE IRON DEMONSTRATION BASED ON PERLS' REACTION].

The article presents the information on the modern modifications of Perls' reaction including diaminobenzidine enhancement according to R. Meguro (for various tissues). M.A. Smith (for nervous tissue), S.M. Levine (for detection of iron in oligodendrocytes and myelinated nervous fibers), and our own modification for visualization of the nucleolar iron. The analysis is performed of the advantages and disadvantages of these modifications of histochemical demonstration of iron. It is shown that the use of new methodological approaches significantly increases the sensitivity of Perls' reaction. Control procedures allow to to eliminate the possibility of artifacts.

[INTRANUCLEAR ACCUMULATION OF Iba-1 PROTEIN IN MICROGLIOCYTES OF THE HUMAN BRAIN].

Iba-1 protein which is a recognized marker of the microglial cells, was previously detected by the authors in the nucleus of microgliocytes. The present study was aimed to define more exactly these data using the methods of immunohistochemistry and confocal laser microscopy. The study was performed on the fragments of the human brain (n=18, age 25­78 years). The areas examined included cortex, striatum, substantia nigra, and nucleus rubrum. The Iba-1 protein was shown to accumulate in one or several parts of microgliocyte nucleus not identical to the nucleolus or the heterochromatin granules. The reasons for this fact are unclear. It may be speculated that Iba-1 protein besides its major function (involvement in phagocytosis) can perform the role of a transcriptional factor.

[RAPID METHOD OF THE AMYLOID STAINING WITH CONGO RED FOR LIGHT AND FLUORESCENCE MICROSCOPY].

The aim of the present study was to optimize the histochemical method of amyloid staining using Congo red. The study was performed on specimens of the myocardium of left ventricle of the heart obtained at autopsy from the patients with amyloidosis of myocardium diagnosed postmortem. It was shown that a positive impact on the quality of the staining of amyloid is provided by a procedure of pre-heating the slides in the liquid, especially at an acidic pH. The staining protocol was developed allowing to obtain preparations characterized by high-contrast staining of amyloid at light microscopic level and by high intensity of its fluorescence. The advantage of the protocol presented is also a significant reduction in the total duration of staining, an increase in stability of the specific staining of amyloid and the absence of nonspecific background staining.

[RAPID METHOD OF THE AMYLOID STAINING WITH CONGO RED FOR LIGHT AND FLUORESCENCE MICROSCOPY].

The aim of the present study was to optimize the histochemical method of amyloid staining using Congo red. The study was performed on specimens of the myocardium of left ventricle of the heart obtained at autopsy from the patients with amyloidosis of myocardium diagnosed postmortem. It was shown that a positive impact on the quality of the staining of amyloid is provided by a procedure of pre-heating the slides in the liquid, especially at an acidic pH. The staining protocol was developed allowing to obtain preparations characterized by high-contrast staining of amyloid at light microscopic level and by high intensity of its fluorescence. The advantage of the protocol presented is also a significant reduction in the total duration of staining, an increase in stability of the specific staining of amyloid and the absence of nonspecific background staining.

[STRUCTURAL AND SPATIAL ORGANIZATION OF MICROGLIA IN THE MOLECULAR LAYER OF RABBIT CEREBELLAR CORTEX].

Using the methods of immunocytochemistry and confocal laser microscopy, structural organization and spatial distribution of microgliocytes in the molecular layer of the cerebellar cortex were studied in 5 adult male rabbits. Reaction to microglial cell marker Iba-1 was highly specific, while until recently their selective detection was impossible in rabbits. In the molecular layer of the cerebellar cortex, two patterns of microgliocyte organization were observed. Most common were the cells with tortuous intricately ramified processes, the main branches of which often had radial direction. Perivascular sparsely-branched spindleshaped microgliocytes were also found. The peculiarities of the structural organization of these cells are related to the protective functions they perform at the level of the blood-brain barrier.

CHARACTERISTICS OF AMYLOID DEPOSITS DETECTED IN THE INTERNAL ORGANS OF mdx MICE.

The dystrophin-deficient mdx mouse is the most commonly used experimental model of Duchenne muscular dystrophy (DMD). Although the amyloid has been shown in the muscle biopsies of patients with different types of muscular dystrophies, there are no data on the amyloid accumulations in the biopsy of DMD patients or mdx mouse. Therefore, the aim of the present study was to testify the hypothesis of probable accumulation of amyloid in the visceral organs of mdx mouse. Specimens of myocardium, kidneys, and liver of male and female mdx mice aged from 2 months to 1.5 years (n = 9) were used in the study. The histochemical staining with Congo red demonstrated amyloid accumulations in the studied organs of the mdx mice. Morphology and localization of the found accumulations were described in details and analyzed. The mass-spectrometric study determined the vitronectin and apolipoprotein A-II as the most probable components of the amyloid accumulations in the mdx mouse.

[INTRANUCLEAR IRON DISTRIBUTION IN THE PURKINJE CELLS OF HUMAN CEREBELLUM].

The distribution of iron ions in the cerebellum of 15 human subjects aged 20-89-years was studied using highly-sensitive variant of Perls' histochemical technique. Increased iron content was found in the white matter and in Purkinje cells. In 10 out of 15 cases examined iron was detected in the nuclei of Purkinje cells, while in some cases iron was found in the nucleolus.

[STRUCTURAL ORGANIZATION OF THE PROCESSES OF EPENDYMO- CYTES LINING THE LATERAL VENTRICLES OF THE RAT BRAIN].

The aim of this study was to examine the structural organization of processes of ependymocytes lining the lateral ventricles of the rat brain using vimentin immunocytochemistry and confocal laser microscopy. The study was performed on adult male rats (n = 3). It was found that most typical ependymocytes had basal processes, while 1/3 of these cells had none. Some vimentin-immunopositive tanycyte-like cells with long processes appoaching blood vessels, were found inside the ependymal lining In some typical ependymocytes, cytroskeleton wa s formed by intermediate filaments of mixed type containing both vimentin and glial fibrillary acidic protein.

[CEREBRAL MICROGLIA AND MICROGLIAL MARKERS].

In recent years, there has been a steady increase of interest in various aspects of the organization and functioning of microglia. However, the information on modern immunocytochemical methods of its identification is ambiguous and requires systematization. In the present paper. the main attention is focused on microglial markers--the proteins (Iba-1, CD11b, CD68, HLA-DR, and some others) expressed by normal microgliocytes and those activated by the effects of damaging factors. Characterization of markers and methods of microglia immunocytochemical labeling is combined with an analysis of the data concerning the origin and structural organization of microgliocytes.

[THE DISTRIBUTION OF CHOLINERGIC AND NITROXIDERGIC NEURONS IN THE SPINAL CORD OF NEWBORN AND ADULT RATS].

The aim of this study was to examine the distribution of cholinergic and nitroxidergic neurons in the spinal cord (SC) of adult and newborn rats. Using immunohistochemical demonstration of choline acetyltransferase (ChAT) and nitric oxide synthase (NOS), cervical portions of SC were studied in newborn (n=5) and adult (n=5) Wistar rats. It was found that ChAT-positive neurons were localized in the anterior horns of the SC, while individual cells were located in of SC posterior horns, in the central gray matter and at the boundary of VI-VII Rexed laminae. Nitroxidergic neurons were located in the superficial layers of SC posterior horns of grey matter, in the central gray matter and in the area of VI-VII Rexed laminae. It is found that SC of newborn and adult rats contained cholinergic neurons expressing NOS. Detection of cells containing both enzymes already at postnatal Day 1, suggests that they were formed in rat SC during prenatal ontogenesis

[HISTOCHEMICAL AND IMMUNOHISTOCHEMICAL IDENTIFICATION OF HUMAN MYOCARDIAL MAST CELLS].

This paper compares the results of application of various methods of histochemical and immunohistochemical staining of mast cells (MC) in human myocardium after formalin fixation and paraffin embedding of the material. It was shown that the optimal methods for description of their structure were toluidine blue staining and and Giemsa stain, while alcian blue staining represented the most suitable histochemical method for MC counting. In combination with immunohistochemical detection of synaptophysin, it could be used for identification of co-localization of MC and nerve terminals in the myocardium. Combined staining of MC with alcian blue and safranin is not suitable for human formalin-fixed myocardial MC. Immunohistochemical techniques of MC tryptase and chymase demonstration appear to be more sensitive when compared with histochemical methods, and allow the most comprehensive quantitative description of human myocardial MC population.

NeuN As a Neuronal Nuclear Antigen and Neuron Differentiation Marker.

The NeuN protein is localized in nuclei and perinuclear cytoplasm of most of the neurons in the central nervous system of mammals. Monoclonal antibodies to the NeuN protein have been actively used in the immunohistochemical research of neuronal differentiation to assess the functional state of neurons in norm and pathology for more than 20 years. Recently, NeuN antibodies have begun to be applied in the differential morphological diagnosis of cancer. However, the structure of the protein, which can be revealed by antibodies to NeuN, remained unknown until recently, and the functions of the protein are still not fully clear. In the present mini-review, data on NeuN accumulated so far are summarized and analyzed. Data on the structure and properties of the protein, its isoforms, intracellular localization, and hypothesized functions are reported. The application field of immunocytochemical detection of NeuN in scientific and clinical studies, as well as the difficulties in the interpretation of the obtained experimental data and their possible causes, is described in details.

[Simultaneous demonstration of glutamate decarboxylase and synaptophysin in paraffin sections of rat cerebellum].

The article presents highly reproducible and inexpensive protocol for simultaneous demonstration of glutamate decarboxylase (GAD67), the key enzyme of gamma-aminobutyric acid (GABA) synthesis and synaptophysin (SYP), a marker protein of synaptic vesicles using confocal laser microscopy. In the cerebellar cortex, GAD labels Purkinje cells and pinceaux in their basal parts and is unevenly distributed in the neuropil of molecular and granular layers. SYP clearly marks the contours of large dendrites of Purkinje cells in molecular layer, while in the granular layers it labels parts of cerebellar glomeruli--the terminals of the mossy fibers. GAD-immunopositive structures (GABA-ergic axons of stellate cells--Golgi cells) are often located at periphery of the glomeruli. In the peripheral zone of the glomeruli, colocalization of GAD- and SYP-immunopositive structures was observed, suggesting the presence of GABA-ergic synapses in this zone.

[DISTRIBUTION OF THE MARINESCO BODIES IN THE NEURONS OF HUMAN BRAIN SUBSTANTIA NIGRA].

The aim of the study was to investigate the occurrence and intranuclear distribution of Marinesco bodies in substantia nigra neurons of the human brain. Marinesco bodies were identified in substantia nigra sections of 5 men aged 28 to 58 years old using Nissl staining and immunohistochemical detection of ubiquitin--the protein characteristic of this intranuclear inclusion. Marinesco bodies were found in 1-2% of the substantia nigra neurons, but not in adjacent brain areas. One neuron contained 1-4 Marinesco bodies sized up to 6.7x5.1 microm, which were located both near and at a distance from the nucleolus. Most Marinesco bodies exhibited ubiquitin expression. A trend was found for the increased incidence of Marinesco bodies in human substantia nigra neurons with age.

[CYTOPLASMIC LOCALIZATION OF NEUN/Fox-3 NUCLEAR PROTEIN IN THE PANCREATIC CELLS].

The study of the sections of Wistar rat pancreas (n=4) using the methods of immunocytochemistry and confocal laser microscopy has demonstrated previously unknown fact of the presence of NeuN protein in the secretory epithelial cells of the exocrine part of this organ. The protein was located in the apical part of the cytoplasm slightly below the zone of zymogen granules. Until now the nuclear protein NeuN was considered to be the panneuronal differentiation marker and was used in the studies of neurogenesis.

[CHANGES IN THE PANCREATIC ISLETS AND NERVOUS ELEMENTS IN RATS DURING AGING (AN IMMUNOHISTOCHEMICAL STUDY)].

The neural apparatus and the endocrine part of the pancreas was studied in Wistar rats aged 3-4 and 19 months (n = 24) using the immunohistochemical reactions for synaptophysin (Syn), tyrosine hydroxylase (TH) and protein gene product 9.5 (PGP 9.5). Since Syn and PGP 9.5 are highly selective in detection of pancreatic islet (PI) endocrinocytes, it was possible to examine their topography and density in all parts of the pancreas. It was found that in rats aged 19 months, the total number of PI was decreased as compared to that in young animals. The study of PI size distribution has shown that the number of large islets decreased with age. Young animals showed rich innervation of the pancreas which was represented by three nerve plexuses: the first was a broadly-looped one, formed by small nerve trunks and bundles of unmyelinated and myelinated nerve fibers, the second consisted of thin bundles of postganglionic axons and microganglia, and the third (main terminal plexus) was formed by axons with varicosities and synapses of "en passant" type. In aged rats, marked degenerative changes in the neurons of intramural ganglia, nerve trunks and bundles were noted together with the reduction or complete absence of Syn- and TH-positive efferent parasympathetic and sympathetic terminals around blood vessels, excretory ducts, denervation of the exocrine and endocrine parts of the pancreas. Innervation disturbances in some lobules were accompanied by small inflammatory perivascular infiltrates.

[Extraependymal ependymocytes in the rat brain].

The aim of the study was to test the hypothesis on the presence, in intact rat brain, of the cells located outside the ependymal layer, but possessing the structural organization and cytochemical characteristics similar to those of ependymocytes. The study was carried out on Wistar rats (n=10). Ependymocytes were identified using immunocytochemical reactions to ezrin and vimentin and were visualized with light and confocal microscopy. Cells, structurally and cytochemically similar to typical ependymocytes, were found outside the layer of ependymocytes in the nervous tissue of intact rat brain. It is suggested that extraependymal ependymocytes may have a function of reserve population of the neural stem cells in the brain.

[Vimentin and S100 protein in the developing rat dorsal root ganglion cells].

The aim of this research was to study the expression of vimentin, one of the intermediate filament proteins, and S100 protein in the cells of developing dorsal root ganglion in prenatal ontogenesis in Wistar rat embryos (n=22) on 12-19 days of development and in newborn rats (n=6). Immunohistochemical staining methods were used to study the formation of rat dorsal root ganglion glial cells. Vimentin-immunopositive neural and glial progenitor cells were present in dorsal root ganglion primordia in rat embryos at 12-17 prenatal days. At the later stages (day 19 of prenatal life and day 1 of postnatal life) vimentin was found to be synthesized only by the differentiating glial cells. It was shown that at the same time, S100 protein was first detected in the glial cells of rat dorsal root ganglion. On postnatal day 1, the tendency was noted for the accumulation of S100 protein in the larger neurons, that was characteristic to mature dorsal root ganglia.

[Application of the immunohistochemical detection of PGP 9.5 protein for the study of rat and human heart innervation].

In this paper we describe the modern immunohistochemical method detecting the neuronal marker PGP 9.5. The application of this method for the study of the innervation of rat and human heart, the detailed examination of the topography of the cardiac nervous apparatus, as well as their changes in pathological states, is demonstrated. Structural organization and the nature of the terminal branches of nerve apparatus suggest that they have afferent function. Protocol of the reaction demonstrating PGP 9.5 on paraffin sections is presented.