The Pattern of Granuloma Formation in the Liver of Rats as a Reflection of the Mechanism of Internalization of Submicron Silicon Particles.

A morphological analysis of the liver of Wistar rats was performed 2 months after a single intravenous injection of porous silicon particles of different sizes (60-80, 250-300, and 500-600 nm; 2 mg/ml, 1 ml). Histological, immunohistochemical, and electron microscopic methods showed the development of CD68+ granulomas in all experimental groups. Injection of 60-80-nm porous silicon particles led to the formation of single large granulomas (>2000 µm2), while 500-600-nm nanoparticles caused the formation of numerous smaller granulomas. The mechanism of involution of granulomas by apoptosis of Kupffer cells and the absence of subsequent connective tissue remodeling of the organ tissue is shown.

Morphological Analysis of the Respiratory Tract of Rats after Parenteral Administration of Silicon Dioxide Nanoparticles.

Morphological analysis of the respiratory tract of Wistar rats was performed after a single parenteral administration of 12-nm silicon dioxide nanoparticles (1 ml, 2 mg/ml, intravenously) was performed. On day 21 and in 2, 4, and 6 months after the administration of nanoparticles, the development of macrophage infiltration in the interstitium of the respiratory tract was demonstrated by histological and immunohistochemical methods. The pool of alveolar macrophages increased in 4 months after administration (p=0.004) and returned to the control values in 6 months. The number of mast cells did not significantly change at all stages of the experiment. Connective tissue remodeling in the interstitium of the respiratory tract was not observed throughout the observation period.

[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.

The Spatial Organization of the Intranuclear Structures of Human Brain Dopaminergic Neurons.

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.

[Immunohistochemical characteristics of the substantia nigra neurons of the human]. / Immunogistokhimicheskaia kharakteristika neironov chernogo veshchestva golovnogo mozga cheloveka.

AIM: To determine the cytochemical characteristics of unchanged neurons of the human substantia nigra using a wide range of immunocytochemical markers some of which (glutamate decarboxylase-65, PGP 9.5, non-phosphorylated neurofilament proteins, alpa-tubulin) have never been used for study of human dopaminergic neurons. MATERIAL AND METHODS: Fragments of human midbrain (17 men and women, aged from 28 to 78 years) from the archives of the Department of General and Specific Morphology of the Institute of Experimental Medicine were used. The study was performed using classical neurohistological techniques and immunocytochemistry using antibodies to 15 different proteins. RESULTS: Most neurons in substantia nigra exhibited a reduced expression of common neuronal markers such as neuronal nuclear protein NeuN, PGP 9.5 protein, and neuron-specific enolase. GABAergic (GAD65-immunopositive) neurons were not found in the substantia nigra. Single cholinergic neurons without neuromelanin were identified in the dorsal part of the substantia nigra. Calcium-binding proteins calbindin and calretinin were not found in the majority of nigral cells although calbindin was rarely seen in some neurons of the dorsal part and calretinin in the ventral one. Nitric oxide synthase (NOS) was present in the substantia nigra both in neuropil and neuronal bodies. CONCLUSION: The results suggest the unique cytochemical properties of the nigral neurons, which may be related to their increased susceptibility to lesion and degeneration.

Immunohistochemical demonstration of specific antigens in the human brain fixed in zinc-ethanol-formaldehyde.

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.

[GAP-43 and its proteolytic fragment in spinal cord cells of rats with experimental autoimmune encephalomyelitis].

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.

[Glial fibrillary acidic protein: the component of intermediate filaments in the vertebrate brain astrocytes].

Glial fibrillary acidic protein (GFAP) refers to the type III intermediate filament proteins and is the essential component of the cytoskeleton in astrocytes of all vertebrates. This review presents current data on the molecular organization of GFAP in a comparative aspect. The results of most relevant studies using immunocytochemical labeling of the protein are summarized. The data on the changes in expression of GFAP in Alexander disease caused by the primary pathology of astrocytes are presented.

[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.

[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.

[Method for simultaneous visualization of mast cells and nerve terminals in the rodent thymus].

The purpose of this study was to develop the method for the simultaneous visualization of mast cells (MCs) and nerve terminals, based on generally accepted techniques of histochemical identification of MCs with alcian blue and immunohistochemical detection of synaptophysin. The protocol presented allows simultaneous identification of mast cells and nerve terminals in the sections of paraffin-embedded thymus of laboratory mammals with high selectivity and good reproducibility. The method can be used for both visualization of spatial relationship between MCs and nerve terminals and independent research of the innervation of mammalian internal organs. Zinc-ethanol-formaldehyde is recommended as an optimal fixative.

[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.

[Catecholaminergic neurons of mammalian brain and neuromelanin].

Brain catecholaminergic neurons belong to the most extensively studied populations of nerve cells. Presence of a pigment neuromelanin in their cytoplasm is a specific morphological feature of these neurons in many mammalian species. Elucidation of the role of neuromelanin is of importance for comparative neurobiology, as it is absent in neurons of another neurotransmitter systems and, moreover, even in catecholaminergic neurons of some laboratory animals, which limits the possibility of experimental verification of existing hypotheses of its functions under physiological and pathological conditions. For recent years, neuromelanin is an object of particular interest in the scientific community involved in research of neurotoxicity and modeling the Parkinson's disease. The present review summarizes and analyzes new data on the structure and functions of neuromelanin and its probable role in pathogenesis of Parkinson's disease is discussed.

[NeuN nuclear protein in neurons of human brain substantia nigra].

The expression of NeuN protein was examined immunocytochemically in the neurons of human substantia nigra (n=14, age: 27-78 years). Some of substantia nigra neurons demonstrated weak NeuN immunopositive reaction, while the others were NeuN-immunonegative. In general, NeuN immunocytochemical reaction in neurons of human substantia nigra was expressed much weaker than in the nucleus rubrum neurons located in the same sections.

[Advantages and disadvantages of zink-ethanol-formaldehyde as a fixative for immunocytochemistry and confocal laser microscopy].

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.

[Diagnostic potential of the histochemical methods used in histological studies of the heart].

The authors proposed a number of immunohistochemical markers to be used in histological diagnostic studies of the heart and protocols for their identification that extend the possibilities for the estimation of the heart function in case of pathological changes and sudden cardiac death.

[Method of immunocytochemical demonstration of cholinergic neurons in the central nervous system of laboratory animals].

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