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Resident macrophages of different organs have structural and functional features, which can complicate their identification and analysis. A promising candidate for the role of a universal immunohistochemical marker of resident macrophages is the calcium-binding protein Iba-1, a well-known marker of brain microglia. The purpose of this work was to study the possibility of using one variant of antibodies to the Iba-1 protein for the immunohistochemical detection of resident macrophages in the liver, myocardium, lung, and choroid plexus of the rat brain. The study was performed on male Wistar rats (n = 15). It was shown that the use of rabbit monoclonal antibodies against Iba-1 allows highly effective detection of Kupffer cells in the liver, resident macrophages in the myocardium, alveolar and interstitial macrophages in the lung, and Kolmer cells in the choroid plexus of the rat brain. In all cases, the reaction is characterized by a high specificity and the absence of background staining. In contrast to the classical marker of macrophages, the CD68 molecule, the Iba-1 protein is evenly distributed in the cytoplasm of cell bodies and processes. This makes it possible to more fully identify cells using immunostaining for Iba-1, carry out their three-dimensional reconstructions, and study their structural and functional organization. Immunohistochemical reaction against Iba-1 can be successfully used as a universal alternative to other common methods for identifying resident macrophages., (© 2024. Pleiades Publishing, Ltd.)

Background: Chronic brain dyscirculation is one of the frequent type 2 diabetes mellitus (DM) complications and leads to patients' disability. Sodium-glucose co-transporter type 2 inhibitors (SGLT-2i) have been proven to have advantages for cardiovascular system, but their effect on the central nervous system (CNS) has not been studied enough., Aim: To study empagliflozin effect on CNS damage functional and laboratory parameters in patients with type 2 DM and, under experimental conditions, to investigate the mechanisms of the drug neurotropic effect., Materials and Methods: The clinical part of the study included patients with type 2 DM on metformin monotherapy (n=39). Patients with a target glycated hemoglobin level formed the "MET" group (n=19), in patients with a non-target glycated hemoglobin level empagliflozin was co-administered for the following 6 months (the "MET+EMPA" group, n=20). Healthy volunteers comprised the control group (n=16). The cognitive status and neuron-specific enolase (NSE) and neurofilament light chains (NLC) concentration were studied. DM was modeled in rats, thereafter the rats were treated with empagliflozin for 8 weeks. Microglia activation was assessed using anti-Iba-1 antibodies and morphological changes in neurons when stained by the Nissl method., Results: Both in the "MET+EMPA" and the "MET" groups cognitive deficits were observed, according to the Montreal Cognitive Assessment (MOCA) (24.0 (23.0; 27.0) and 25.0 (21.0; 27.0) points) and the Mini-Mental State Examination (MMSE) (23.75 (23.0; 27.0) and 25.0 (21.0; 27.0) points). Empagliflozin therapy led to the cognitive status normalization after 6 months (26.5 (24.0; 27.0) points according to the MOCA scale and 27.5 (24.0; 28.0) points according to the MMSE). Initially, all patients had a significant increase of NSE (3.60 (2.66; 3.76) ng/ml in the "MET" group, 3.22 (2.94; 3.54) ng/ml in the "MET+EMPA» group, 2.72 (2.13; 2.72) ng/ml in the «Control» group) and NLC (4.50 (3.31; 5.56) ng/ml in the «MET» group, 5, 25 (3.75; 6.25) ng/ml in the «MET+EMPA» group comparing with 3.50 (2.25; 3.50) ng/ml in the «Control» group). Empagliflozin therapy led to a significant decrease in NLC already after 3 months (3.80 (3.25; 3.87) ng/ml), without significant influence on the NSE level. In the experiment, DM was characterized by an increased number of activated microgliocytes and destructured neurons and a decreased number of neurons with a normal structure. Empagliflozin therapy was accompanied by a decrease in the number of immunopositive microgliocytes in the CA1 zone of the hippocampus and an increase in the number of structured neurons., Conclusion: Type 2 diabetes mellitus is characterized by functional and biochemical changes in the central nervous system even under satisfactory glycemic control. Therapy with empagliflozin has a neuroprotective effect, manifested in an improvement in cognitive status and a decrease in NLC level. Empagliflozin reduces neuronal damage and abnormal microglial activation.

The work aimed to investigate the biocompatibility and biological activity of the water-soluble fullerene adduct C 60 -Arg. It was found that the material is haemocompatible, is not cyto- and genotoxic, possesses pronounced antioxidant activity. Additionally, this paper outlines the direction of application of water-soluble fullerene adducts in the creation of neuroprotectors. It has been suggested that a putative mechanism of the protective action of the C 60 -Arg adduct is associated with its antioxidant properties, the ability to penetrate the blood-brain barrier, and release nitrogen monoxide as a result of the catabolism of L-arginine residues, which promote vascular relaxation. The action of the C 60 -Arg adduct was compared with the action of such an antioxidant as Edaravone, which is approved in Japan for the treatment of ischemic and haemorrhagic strokes., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2023. Published by Elsevier Inc.)

NeuN is a neuron-specific nuclear protein expressed in most mature neuronal cell types, with some exceptions. These exceptions are known mainly for the brain but not for the spinal cord or the spinal visceral networks for which only scarce information is available. One of the most defined visceral structures in the spinal cord is the sympathetic intermediolateral nucleus located within the thoracolumbar segments. We investigated the NeuN staining in the intermediolateral nucleus and compared it with the staining for two neurochemical markers of visceral neurons: nitric oxide synthase and calcium-binding protein calretinin in adult cats and in kittens aged 0, 14, and 35 days. A clear NeuN-immunonegativity was obtained for intermediolateral neurons labeled for nitric oxide synthase for both adult cats and kittens. In contrast, a matched immunopositivity for the NeuN and calretinin was obtained, showing an age-dependent degree of this colocalization, which was high in newborn kittens, decreased on postnatal 14 and 35 days and persisted at a moderate level up to adulthood. Perhaps our data displayed a heterogeneity of the intermediolateral neurons., (© 2022 American Association for Anatomy.)

Objective: Immunohistochemical investigation of archival histological material is a serious problem, since long-term storage of biological tissues, most often in formalin, leads to a loss of antigenic properties. However, the biological material can also be stored in the clearing agent methyl salicylate. The aim of this study was to assess the antigenicity of the human choroid plexus after extra long-term storage in methyl salicylate., Material and Method: The study was performed on samples of fixed human choroid plexus (occasionally with attached neighboring pineal gland) stored in either methyl salicylate or paraffin blocks for 25 years. Chromogenic and fluorescence immunohistochemistry of vimentin, GFAP, type IV collagen, β-catenin, α-smooth muscle actin, von Willebrand factor, CD68, mast cell tryptase, TMEM119, and synaptophysin was carried out., Results: The storage of human choroid plexus in methyl salicylate for 25 years does not impair its histomorphology and preserves the properties of all the antigens assessed, which makes their immunohistochemical visualization possible using both light and fluorescence microscopy. Additionally, we found that long-term storage of human choroid plexus in methyl salicylate does not cause an increase in autofluorescence., Conclusion: Methyl salicylate can be recommended as a medium for long-term storage of biological tissue, as it provides excellent brain tissue preservation and retains its antigenic properties for up to 25 years.

We studied the reaction of rat hippocampal microgliocytes to hyperbaric oxygen at a pressure of 5 ata (absolute atmosphere). Immunohistochemical analysis with selective macrophage marker CD68 (ED1) and microglial marker Iba-1 allowed separate analysis of these two cell populations. It was shown that macrophages do not significantly contribute to reactive changes in the total pool of Iba-1 + hippocampal cells induced by hyperbaric oxygen., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Pulmonary embolism is a life-threatening condition, which can result in respiratory insufficiency and death. Blood clots occluding branches of the pulmonary artery (PA) are traditionally considered to originate from thrombi in deep veins (usually in legs). However, growing evidence suggests that occlusion of the vessels in the lungs can develop without preceding deep vein thrombosis (DVT). In this work, we used an inferior vena cava (IVC) complete ligation model of DVT in Wistar rats to explore the possibility and mechanisms of PA thrombosis under the conditions where all routes of thrombotic mass migration from peripheral veins are blocked. We demonstrate that rats both with normal and reduced neutrophil counts developed thrombi in the IVC, although, neutropenia caused a substantial decrease in thrombus size and a shift from fresh fibrin toward mature fibrin and connective tissue inside the thrombus. Massive fibrin deposition was found in the PA branches in the majority of DVT rats with normal neutrophil counts, but in none of the neutropenic animals. Neutrophil ablation also abolished macroscopic signs of lung damage. Altogether, the results demonstrate that thrombi in the lung vasculature can form in situ by mechanisms that require local neutrophil recruitment taking place in the DVT setting.

Neuromelanin (NM) is a dark polymer pigment produced in certain populations of catecholaminergic neurons in the brain. It is present in various areas of the human brain, most often in the substantia nigra (SN) pars compacta and the locus coeruleus, the main centers of dopaminergic and noradrenergic innervation, respectively. Interest in NM has revived in recent years due to the alleged link between NM and the particular vulnerability of neuromelanin-containing neurons to neurodegeneration. The aim of this work was to study the structural, cytochemical, and localization features of cytoplasmic and extracellular neuromelanin in the human SN pars compacta during normal aging. Sections of human SN from young/middle-aged adults (25 to 51 years old, n=7) and older adults (60 to 78 years old, n=5), all of which had no neurological disorders, were stained histochemically for metals (Perls' reaction, Mayer's hematoxylin) and immunohistochemically for tyrosine hydroxylase (TH) and Iba-1. It was shown that dopaminergic neurons in SN pars compacta differ in the amount of neuromelanin and the intensity of TH-immunoreactivity. The number of neuromelanin-containing neurons with decreased TH-immunoreactivity positively correlates with age. Extracellular NM is present in SN pars compacta in both young/middle-aged and older adults. The number of extracellular NM accumulations increases with aging. Cytoplasmic and extracellular NM are predominantly not stained using histochemical methods for detecting metals in people of all ages. We did not detect the appearance of amoeboid microglia in human SN pars compacta with aging, but we found an age-related increase in microglial phagocytic activity. The absence of pronounced microgliosis, as well as a pronounced loss of neuromelanin-containing neurons, indicate the absence of neuroinflammation in human SN pars compacta during normal aging.

We studied spatial organization and structural characteristics of striatal glial cells in spontaneously hypertensive rats (SHR) in 48 h after 30-min focal ischemia. Immunocytochemical analysis of nestin and glial fibrillar acidic protein (GFAP) revealed 3 types of activated astrocytes: expressing only nestin, only GFAP, or both markers. There were no nestin-immunopositive astrocytes in the striatum of sham-operated rats. In cells expressing nestin and GFAP, localization of these markers did not completely coincide, which can be explained by different functions of these proteins or formation of heterodimers of nestin with other intermediate filament proteins.

Among the properties of lactoferrin (LF) are bactericidal, antianemic, immunomodulatory, antitumour, antiphlogistic effects. Previously we demonstrated its capacity to stabilize in vivo HIF-1-alpha and HIF-2-alpha, which are redox-sensitive multiaimed transcription factors. Various tissues of animals receiving recombinant human LF (rhLF) responded by expressing the HIF-1-alpha target genes, hence such proteins as erythropoietin (EPO), ceruloplasmin, etc. were synthesized in noticeable amounts. Among organs in which EPO synthesis occurred were brain, heart, spleen, liver, kidneys and lungs. Other researchers showed that EPO can act as a protectant against severe brain injury and status epilepticus in rats. Therefore, we tried rhLF as a protector against the severe neurologic disorders developed in rats, such as the rotenone-induced model of Parkinson's disease and experimental autoimmune encephalomyelitis as a model of multiple sclerosis, and observed its capacity to mitigate the grave symptoms. Moreover, an intraperitoneal injection of rhLF into mice 1 h after occlusion of the medial cerebral artery significantly diminished the necrosis area measured on the third day in the ischaemic brain. During this period EPO was synthesized in various murine tissues. It was known that EPO induces nuclear translocation of Nrf2, which, like HIF-1-alpha, is a transcription factor. In view that under conditions of hypoxia both factors demonstrate a synergistic protective effect, we suggested that LF activates the Keap1/Nrf2 signaling pathway, an important link in proliferation and differentiation of normal and malignant cells. J774 macrophages were cultured for 3 days without or in the presence of ferric and ferrous ions (RPMI-1640 and DMEM/F12, respectively). Then cells were incubated with rhLF or Deferiprone. Confocal microscopy revealed nuclear translocation of Nrf2 (the key event in Keap1/Nrf2 signaling) induced by apo-rhLF (iron-free, RPMI-1640). The reference compound Deferiprone (iron chelator) had the similar effect. Upon iron binding (in DMEM/F12) rhLF did not activate the Keap1/Nrf2 pathway. Added to J774, apo-rhLF enhanced transcription of Nrf2-dependent genes coding for glutathione S-transferase P and heme oxygenase-1. Western blotting revealed presence of Nrf2 in mice brain after 6 days of oral administration of apo-rhLF, but not Fe-rhLF or equivalent amount of PBS. Hence, apo-LF, but not holo-LF, induces the translocation of Nrf2 from cytoplasm to the nucleus, probably due to its capacity to induce EPO synthesis.

We studied the intranuclear localization of protein nucleophosmin (B23) and ubiquitin in the dopaminergic neurons of human substantia nigra (n = 6, age of 25-87 years) using immunohistochemistry and confocal laser microscopy. Intranuclear ubiquitin-immunopositive bodies that morphologically correspond to Marinesco bodies were found to be present in substantia nigra dopaminergic (tyrosine hydroxylase-immunopositive) neurons but absent in non-dopaminergic neurons. The number of bodies varied from 0 to 6 per cell nucleus. Nucleophosmin (B23) was found in the neuronal nucleolus, with the nucleolus size being constant in the nigral neurons of each individual brain. All the observed neurons had only one large nucleolus with intense nucleophosmin immunoreactivity and a lightly stained region (1-2 μm in diameter) that apparently represents the giant fibrillar center (GFC). An intensely immunostained nucleophosmin-containing granule was often observed at the GFC periphery. Double labeling demonstrated that nucleophosmin-immunoreactive nucleolus and ubiquitin-immunoreactive Marinesco bodies can occur both closely to and remotely from each other. Three-dimensional reconstruction indicates that rounded Marinesco bodies are polymorphic and often have a complex shape, with some flattening and concavities, which may be associated with contact not only with the nucleolus, but also, presumably, with other intranuclear structures free of ubiquitin or nucleophosmin. Ubiquitin-immunoreactive structures with a relatively small size (up to 1 μm in length) and various clastosome-like shapes (Lafarga et al., 2002) often occur near Marinesco bodies. There were no cases of detection of ubiquitin in the nucleoli of dopaminergic neurons and nucleophosmin/B23 in typical Marinesco bodies. The obtained information may be helpful in unraveling the molecular mechanisms of the selective vulnerability of substantia nigra dopaminergic neurons to damaging factors.

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.

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.

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 25–78 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.

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.

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.

Tissue fixation is critical for immunohistochemistry. Recently, we developed a zinc-ethanol-formalin fixative (ZEF), and the present study was aimed to assess the applicability of the ZEF for the human brain histology and immunohistochemistry and to evaluate the detectability of different antigens in the human brain fixed with ZEF. In total, 11 antigens were tested, including NeuN, neuron-specific enolase, GFAP, Iba-1, calbindin, calretinin, choline acetyltransferase, glutamic acid decarboxylase (GAD65), tyrosine hydroxylase, synaptophysin, and α-tubulin. The obtained data show that: i) the ZEF has potential for use in general histological practice, where detailed characterization of human brain morphology is needed; ii) the antigens tested are well-preserved in the human brain specimens fixed in the ZEF.

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.

The regenerative capacity of the Central Nervous System (CNS) is a key factor implicated in the pathogenesis of neurodegenerative diseases. In the present study, the regenerative capacity of the CNS is considered using one of the markers of regeneration, Growth Associated Protein-43 (GAP-43) and its proteolytic fragment GAP-43-3 in the Experimental Autoimmune Encephalomyelitis (EAE) animal model of multiple sclerosis. The EAE on Wistar rats was characterized as an adequate model of multiple sclerosis, with typical clinical (pares and paralysis) and morphological (infiltration of spinal cord and deformation of motoneurons) disorders. Normally about 60% of GAP-43 is cleaved by m-calpain and stays in the form of GAP-43-3. During severe form of EAE up to 85% of GAP-43 can be found cleaved. We speculated that the cleavage of GAP-43 can play a crucial role for regenerative capacity of CNS during EAE development. Thus the distribution of GAP-43 and GAP-43-3 in the spinal cord was analyzed. The manifestation of clinical signs of EAE has been found to be in correlation with the levels of GAP-43 proteolysis both in the homogenate of the spinal cord and on the spinal cord slices. The immunoreactive staining enabled the observation of the accumulation of GAP-43-3 predominantly in microglial cells.

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.

Marinesco bodies were discovered in the human substantia nigra neurons in 1902. However, relationships these intranuclear inclusions with other cell nuclear structures remains obscured yet. The aim of the present study was to investigate morphological and cytochemical peculiarities of these ubiquitin-immunopositive intranuclear bodies in neurons of the human substantia nigra and the character of their relationships with the nucleolus using light microscopy, immunocytochemistry, and confocal laser microscopy. It has been established that up to 20 % of the neurons of the substantia nigra contain ubiquitin-immunopositive Marinesco bodies. Only a third of them were closely adjacent to the nucleolus. Using a method of silver impregnation of argentophilic proteins associated with nuclear organizer, the lack of the argentophilic proteins typical for the nucleolus has been shown in the Marinesco bodies. We have found some specific ubiquitin-positive structures in the nuclei of neurons in addition to Marinesco bodies. These structures having less than 1 μm in size are supposedly the initial forms of the Marinesco bodies. Confocal laser microscopy has revealed two types of the ubiquitin-immunopositive intranuclear bodies--with high and low immunofluorescence, while the latter shows heterogeneity in distribution of the immunopositive product. With the use of a fluorescent dye SYTOX Green, the presence of DNA has been revealed in the Marinesco bodies. The absence of the peripheral zone of heterochromatin and poor perception of toluidine blue in combination with the DNA presence and loss of argentophilic proteins strongly suggest significant structural and chemical differences between Marinesco bodies and nucleoli and argue against the view that the revealed bodies may be changed nucleoli.

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 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.

Irradiation of rats with γ-quanta at relatively low doses induces a sustainable dose-independent increase in the occurrence of lethal cytoplasmic disorders in the renal tubules epithelium together with sustainable and as well dose-independent subcelluar compensation and restorative processes. Over the period of research (6 months) these processes led to no population recovery. The detected alterations are referred to the category of non-targeted non-mutagenic effects and they are of interest because they address the issue of the sensitivity of low renewable tissues to radiation.

The aim of this investigation was to develop an integrated approach to spatial reconstruction of the cells lining the ventricles of the brain using confocal laser microscopy and immunocytochemical reaction to vimentin. The work was performed on paraffin sections of rat brain of different thickness (5 and 10 microm). To visualize the immunocytochemical reaction the fluorescent dyes in the visible range were selected: SYTOX Green selectively staining the nucleus and indocarbocyanin (Cy-3) conjugated with streptavidin. As a result of testing of various processing conditions, the protocol which allows to receive an intensive staining of the structures was developed. The set of fluorochromes proposed in confocal laser microscopy allows to separate easily the channels, to study the structures independently, if needed, and does not require the use of an expensive ultraviolet laser.

The aim of the study was to develop the methodologic approaches for the demonstration of the complex synaptic groups (glomeruli) in human cerebellum using synaptophysin immunocytochemistry. The protocols presented in the paper allow to obtain of high qualify prepartaions for conventional light and confocal laser microscopy in which individual glomeruli and intraglomerular axonal terminals could be distinctly identified. The preparations obtained are suitable for a quantitative analysis.

It is known that during development of the brain, with the progress of ependendymocyte differentiation from radial gliocytes, the synthesis of nestin is stopped. However, it was shown that in the ependyma of the lateral brain ventricles nestin synthesis was resumed in response to ischemic injury. The aim of the present study was to test the hypothesis of possible re-expression of nestin in the ependyma in aging. The study was performed on male Wistar rats aged 4 (n = 4) and 28 months (n = 3). In older animals the expression of nestin was demonstrated in the ependyma of the lateral ventricles of the brain. It was also found that the area of the medial and upper walls of the lateral ventricle contained the regions of ependyma, in which all cells had intense cytoplasmic staining. The causes of the phenomenon described remain unclear.

The goal of the study was to identify the subependymal microglial cells of the III ventricle of the rat brain and to determine their structural characteristics. The sections of the brain of intact Wistar (n = 3) and Sprague-Dawley (n = 3) male rats were studied using the methods of immunocytochemistry and confocal laser microscopy. Subependymal microglia of the III ventricle was found to be a constantly present cell population. Two types of subependymal microgliocytes were identified--spindle-like and basket cells. Their processes penetrate the ependymal layer and reach its surface, thus contacting the cerebrospinal fluid (CSF), which suggests a possible participation of these cells in the structure of CSF-brain barrier.

Neuroglobin is a recently discovered heme-containing protein located predominantly in the mammalian brain. This paper for the first time presents the data on neuroglobin distribution in human cerbellum using immunohistochemistry. Neuroglobin immunoreactivity in the cerebellum was found in all the cases studied (n = 7), although its intensity varied. Distinct reaction was found in Purkinje cells and the areas of cerebellar glomeruli.

Ependymal cells of cerebral ventricles are the most probable candidate progenitor cells. We studied ependymal and subventricular zone cells expressing intermediate filament protein vimentin. The results suggest that the ventricular ependyma represent a homogenous cell population. Some ependymocytes have long processes contacting with blood vessels, which makes them similar to tanycytes of the third cerebral ventricle. New data on the structure of ependymocyte processes attest to their active involvement into creation of a special microenvironment for neural stem cells of the subventricular proliferative zone.

The paper presents an analysis of authors' results obtained using fixation of various tissues in zink-ethanol-formaldehyde (ZEF). It was found that fixation in ZEF, in comparison with other methods of fixation, allowed to achieve higher sensitivity of immunocytochemical reaction for a large number of antigens studied and, in many cases, to avoid heat unmasking of antigens. It also provided high resolution of the images obtained using the fluorescent and confocal laser microscopy. However, the studies of antigens with small molecular mass revealed the antigen diffusion from the site of original localization. The data obtained suggest that fixation of a material in ZEF fixative is promising for both immunocytochemical studies, including those using the fluorescent and confocal laser microscopy, and general histological practice.

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.

The distribution of alpha-tubulin, the major protein of the microtubules, was studied using immunohistochemistry in the rat forebrain structures. The differential distribution of alpha-tubulin immunoreactivity was detected: high immunoreactivity was found in cingulate and pyriform cortex, olfactory tubercles and optic chiasm, while the weakest immunohistochemical staining was observed in caudatoputamen, superficial layers of septum, cingulum and areas around the third cerebral ventricle. Immunoreactivity in the nervous cells was distributed along perikaryon periphery and in the apical dendrite. It is suggested that the intensity of alpha-tubulin immunohistochemical reaction could reflect the functional state of the neurons.

Elaboration of the concept of neural stem cells at the end of the twentieth century resulted in the appearance of the multitude of new terms used for the designation of cells localized in the proliferative zones of the brain. Concurrently, a problem arose concerning the correlation of newly named cells, generally known elements of the nervous tissue, and classic views on its development. In the present study, an attempt is made on the basis of the literature data review and authors' own experience in the exploration of proliferative zones, to classify cell populations involved in the neurogenesis considering, primarily, the possibilities of their morphological and immunocytochemical in situ identification.

A protocol of immunocytochemical demonstration of choline acetyltransferase (ChAT), a key enzyme of acetylcholine synthesis, in paraffin sections of the brain of some laboratory animals, is presented. The method is simple, gives fairly reproducible results and allows for demonstration of ChAT in neurons, nerve fibers, and terminals in preparations of at least three species of laboratory animals including rat, rabbit, and cat. Different kinds of fixation (10% formalin, 4% paraformaldehyde, or zinc-ethanol-formaldehyde) were found suitable for immunocytochemical visualization of ChAT, however, optimal results were obtained with the application of zinc-ethanol-formaldehyde

One of the poorly studied problems of the pathogenesis of the brain ischemic damage is the early reorganization of blood vessels in the zone of the transitory ischemia. The aim of the present study was to investigate the structural organization and cytochemical characteristics of smooth muscle cells (SMC) in the wall of the intracerebral blood vessels in 16 Wistar rats during the early postischemic period (48 hrs after a 30 minute-long ischemia). Examination of the lesioned hemisphere revealed the stellate cells, demonstrating positive reaction to alpha-smooth muscle actin, which seem to be a special population of structurally modified SMC, uncharacteristic to the intact brain of control animals (n=5). The functional role of these cells remains to be elucidated.

Importance of study of astrocytes for fundamental biology and medicine is due their key role in formation of the brain barrier system. On taking into consideration the controversial data on structure of the mammalian neocortex superficial layers, of great actuality are the comparative studies of the structural and cytochemical organization of astrocytes in human and in the laboratory animals used in the experimental studies connected with modeling of brain diseases and traumas. The goal of the present work was to study structural organization of astrocytes in the human and rat neocortical layer I. The work was on the autopsy and experimental material from Wistar rats. Astrocytes were revealed immunocytochemically by using antibodies to GFAP, vimentin and nestin. The preparations were examined with aid of light and confocal laser microscopy. No significant difference in the sizes of perinuclear areas were established between the rat and human astrocytes. In the majority of cortex regions, the specter of proteins forming intermediate filaments in these cells was identical. However, there were essential differences revealed in organization of the superficial glial bordering lamina (SGBL). The human SGBL is formed by interlacing of thin processes in the layer I processes, whereas the rat SGBL is represented by specialized astrocytes spread along the cortical surface and connected with the wide-blade processes. The human layer I astrocytes have translaminar processes passing via several cortical layers, whereas in rats such processes are located within the limits of one layer. The revealed differences in the astrocyte structural organization should be taken into account when interpreting results of experimental studies carried out on rats and extrapolating these results to human.

The aim of the study was to evaluate, by using immunohistochemistry (demonstration of Iba-1 protein), the morphological features of the rat hippocampal microglia and to describe its cellular types during the response to a single total brain ischemia of 12 min duration. The results obtained suggest the existence of several morphologic types of microgliocytes in the intact hippocampus, while some more types appear in response to ischemic lesion of neurons. These changes in general population of the hippocampal microgliocytes is related to their functional activation. During the post-ischemic period (3, 7 and 14 days following the challenge), there appeared multiple micro-outgrowths of the microglial cell processes, which suggests the enhancement of their interaction with surrounding cellular elements and may be the manifestation of the process of the elimination of neuropil lesioned synaptic structures.

Nestin and Musashil (Msi-1) proteins are most often used for labeling of neural stem cells and progenitor cells in vivo, however it remains unclear if these markers really label the same cells. As a result of the study of structural characteristics and localization of nestin- and Msil-expressing cells it was found that these proteins were detected in non-identical cell populations in the brain of intact 15 rats. We failed to find cell groups demonstrating a coexpression of nestin and Msi-1. However, after ischemic lesion of the brain, which was caused in 38 rats by an endovascular occlusion of the left medial cerebral artery for 30 min with the following reperfusion for 48 hours, both markers were detected in cells of subventricular zone and in ependymocytes. These results indicate the changes in cytochemical patterns of the candidate stem cells.

Microgliocytes are currently known as structurally labile cells, that, by changing the length of their processes, control the synaptic organization of the brain. The aim of this study was to investigate structural organization of microglia of the striatum and of the forebrain periventricular area in rat following transitory focal ischemia. In the absence of brain infarction, ischemic lesion was found to activate microglia, which contributed to rearrangement of striatal neuropil. As microgliocytes became activated, they changed their shape from characteristic dendritic one to oval and rounded, which are typical to amoeboid microglia. Microgliocytes were shown to be capable of proliferation.

Phosphorylated H3 histone is of interest for the study of proliferative activity of the cells in various tissues. Its detection is used in histopathological tumor diagnosis. The purpose of this study was to develop immunocytochemical protocols for the demonstration of H3 phosphohistone in the cells of the developing nervous system for classical light microscopy and confocal laser microscopy. The protocols presented allow to identify the dividing cells in the structures of developing brain with high selectivity and good reproducibility. Zinc-ethanol-formaldehyde is recommended as an optimal fixative for H3 phosphohistone.

Forebrain subventricular zone (SVZ)--the putative major source of neural stem cells in the brain of adult mammals--can hardly be visualized using routine histological staining. The present study was focused on the possibility of application of immunocytochemical approach for accurate delineation of the border between SVZ and striatum. It was shown that immunocytochemical reactions demonstrating tyrosine hydroxylase or synaptophysin were optimal for the determination of the border between SVZ and striatum in different mammals.

A study of structural and functional organization of the boundaries separating CNS compartments is a fundamental task of neurobiology. Taking into account the contradictory data on the structure of superficial layers of mammalian neocortex, it is pertinent to study structural and cytochemical organization of astrocytes--the main components of the brain barrier system in animals that are often used for experimental modeling of brain diseases and injuries. The aim of the present work was to study the structural organization of layer I astrocytes of rat neocortex. Astrocytes were demonstrated immunocytochemically using anti-GFAP, anti-vimentin and anti-nestin antibodies using light and confocal laser microscopy. The results of the study demonstrated that the superficial glial limiting membrane had significant structural differences in different cortical regions. Astrocytes in layer I of rat neocortex were different from typical protoplasmic astrocytes, common to gray matter The regional peculiar features of superficial glial limiting membrane organization that were found in this study, are probably determined by the differences in functional characteristics of CSF-encephalic barrier in the specific regions of the brain.

The aim of the study was to examine the distribution of cells expressing Msi-1 (Musashi-1) protein which is believed to be a marker of neural stem cells, in rat telencephalon. These cells were found to be concentrated in the subventricular proliferative zone and diffusely scattered in striatum and hippocampus. Moreover, neurons with extensive Msi-1 reaction were found in habenular nuclei. The data obtained are not in full agreement with current views on the localization of neural stem cells in the brain.

Subventricular proliferative zone is the brain area most intriguing and least understood in terms of cellular and spatial organization. The objective of this study was to focus on the structural and cytochemical organization of astrocytes of the subventricular zone of the rat brain. Astrocytes were detected immunocytochemically. The preparations were analyzed using conventional light microscopy and confocal laser microscopy. The study performed showed that the major cellular population of the subventricular zone was represented by a special kind of atypical fibrous astrocytes with the intermediate filaments containing only glial fibrillary acidic protein (GFAP). The processes of these cells formed a complex double-layer network beneath the ependyma. The described structural pattern of the glial skeleton of the subventricular zone seems to be determined by specific functions of the area and its location in the zone of CSF-brain barrier.