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.

[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.

[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.

[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.

[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.

[PROSPECTS OF THE NUCLEAR PROTEIN NeuN APPLICATION AS AN INDEX OF FUNCTIONAL STATE OF THE VERTEBRATE NERVE CELLS].

Assessment of safety, viability, and functional state of nerve cells is the major problem in studies of experimental effects on various structures of vertebrate brain and in search for correlation between structural abnormalities and changes in physiological parameters. Such an assessment is possible with applying an immunocytochemical reaction to neuronal nuclear antigen NeuN discovered in 1992. Numerous studies of the protein showed its neural specificity and its amino acid consequence was found to have high interspecies concervatism. This review summarizes and analyzes the available data about the function of the NeuN protein in nerve cells and the results of using this marker for assessment of functional state and viability of CNS neurons in experiments. Particular attention is paid to. the critical analysis of the basic data used for the conclusions on the properties and functions of NeuN. It is stated that there are no satisfactory explanation for lacking the constitutive expression of this marker in some neuronal populations of the mammalian brain and spinal cord. The analysis of our own and literature data presented in the review suggests the future prospects of NeuN labeling for investigation of nerve cell responses to damage including comparative interspecies studies. Key words: brain, neuron, immunocytochemistry, neuronal protein NeuN, vertebrates.

[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.

[Thymic mast cell localization at different stages of the ontogenesis in the mouse].

The aim of this research was to characterize the population of thymic mast cells (MC) which were demonstrated by histochemical methods, at different stages of mouse ontogenesis. First MCs appeared on day 19 of intrauterine life. MCs were localized to the thymic medulla during the whole embryonic period. Individual MCs could also be identified within the cortex. Single MCs were interspersed among other thymic medullary cells. In the newborn, young and mature animals, MCs were observed in the connective tissue of the capsule and in the interlobular septa, but some MCs were also found inside the thymic lobules. In the last-mentioned case, MCs were located in the subcapsular zone. The possible functional significance of MCs localization is discussed.

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.

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.