Reaction of the Hippocampal Microglia to Hyperbaric Oxygen.

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.

Expression of Nestin and Glial Fibrillary Acidic Protein in the Marginal Ischemic Zone of the Brain in SHR Rats.

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.

Erythropoietin and Nrf2: key factors in the neuroprotection provided by apo-lactoferrin.

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.

[Experience with using cyanoacrylate glue in endovascular treatment of varicose veins]. / Opyt primeneniia tsianoakrilatnogo kleia pri éndovaskuliarnom lechenii varikoznoi bolezni.

The method of cyanoacrylate-mediated obliteration of subcutaneous veins is known to be an alternative to thermal endovascular obliteration and eliminates the need for tumescent anaesthesia. This technique is based on glue-induced damage to the venous intima, followed by immune response according to the delayed-type hypersensitivity principle. The authors report herein their first experience with using cyanoacrylate-mediated embolization in treatment of patients presenting with varicose veins. The operation was carried out using the VenaSeal closure system (Medtronic). Under ultrasonographic guidance, we performed cyanoacrylate-mediated obliteration of the trunk of the great saphenous vein, without tumescence. The procedure turned out to be well tolerated, with no pain in the zone of cyanoacrylate obliteration reported by the patients in the postoperative period. By means of ultrasonographic control carried out within 1-month of follow up we assessed obliteration of the vein, with the diameter of the obliterated portion amounting to 0.3-0.4 cm. No phlebitis, allergic reactions, nor evidence of deep vein thrombosis were observed. We also performed a morphological study of the removed suprafascial segment of the vein, containing the cyanoacrylate adhesive. The obtained findings demonstrated detachment and destruction of the intima, swelling and loosening of the media, as well active degranulation of mast cells, thus making it possible to suppose the presence of toxic damage to the venous wall induced by cyanoacrylate glue. Hence, the experience thus gained appears to be unequivocally suggestive of remarkable simplicity of performing cyanoacrylate-mediated embolization whose indisputable advantages include the painless nature of the procedure and no need for tumescent anaesthesia. In order to assess efficacy and safety of this technique, further studies are required.

[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.

[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.

[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.

[INTRANUCLEAR UBIQUITIN-IMMUNOPOSITIVE STRUCTURES OF THE HUMAN SUBSTANTIA NIGRA NEURONS].

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.

[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.

[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.

[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.

[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.

[On the issue of non-mutagenic non-targeted effects in low renewable tissues. Analysis of low dose radiation effects on the rat renal tubule epithelium].

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.

[Nestin expression in ependymal cells of lateral ventricles of the rat brain in aging].

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.

[Three-dimensional visualization of the brain ependyma and tanycytes].

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.

[Subependymal microgliocytes of the third ventricle of the brain].

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.

[Demonstration of the glomeruli in human cerebellum using synaptophysin immunohistochemistry and confocal laser microscopy].

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.

[Neuroglobin distribution in the human cerebellar cortex (an immunohistochemical study)].

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.