Modern Imaging Technologies of Mast Cells for Biology and Medicine (Review).

Mast cells play an important role in the body defense against allergens, pathogens, and parasites by participating in inflammation development. However, there is evidence for their contributing to the pathogenesis of a number of atopic, autoimmune, as well as cardiovascular, oncologic, neurologic, and other diseases (allergy, asthma, eczema, rhinitis, anaphylaxis, mastocytosis, multiple sclerosis, rheumatoid arthritis, inflammatory gastrointestinal and pulmonary diseases, migraine, etc.). The diagnosis of many diseases and the study of mast cell functions in health and disease require their identification; so, the knowledge on adequate imaging techniques for mast cells in humans and different species of animals is of particular importance. The present review summarizes the data on major methods of mast cell imaging: enzyme histochemistry, immunohistochemistry, as well as histochemistry using histological stains. The main histological stains bind to heparin and other acidic mucopolysaccharides contained in mast cells and stain them metachromatically. Among these are toluidine blue, methylene blue (including that contained in May-Grünwald-Giemsa stain), thionin, pinacyanol, and others. Safranin and fluorescent dyes: berberine and avidin - also bind to heparin. Longer staining with histological dyes or alcian blue staining is needed to label mucosal and immature mast cells. Advanced techniques - enzyme histochemistry and especially immunohistochemistry - enable to detect mast cells high-selectively using a reaction to tryptases and chymases (specific proteases of these cells). In the immunohistochemical study of tryptases and chymases, species-specific differences in the distribution of the proteases in mast cells of humans and animals should be taken into account for their adequate detection. The immunohistochemical reaction to immunoglobulin E receptor (FcεRI) and c-kit receptor is not specific to mast cells, although the latter is important to demonstrate their proliferation in normal and malignant growth. Correct fixation of biological material is also discussed in the review as it is of great significance for histochemical and immunohistochemical mast cell detection. Fluorescent methods of immunohistochemistry and a multimarker analysis in combination with confocal microscopy are reported to be new technological approaches currently used to study various mast cell populations.

[Mast cells of the human pineal gland.]

The purpose of this study was to assess the possibilities of identifying mast cells using different histochemical and immunohistochemical methods and elucidating the features of their localization in the human pineal gland. The undertaken study showed that mast cells are an essential component of the human pineal gland, regardless of age. The data obtained indicate an increase in the number of mast cells in the pineal gland with age. Mast cells are mostly located in the pineal stroma and their preferred location has not been related to concrements, cysts or melanin accumulations. Mast cells in the pineal gland are predominantly non-degranulating, which indicates their inactive state. The detectability of mast cells in the pineal gland depended significantly on the applied method of staining of the preparations. The largest number of mast cells was revealed by tryptase immunohistochemistry, which should be used to accurately determine the population of mast cells of the pineal gland.

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 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.

[MORPHOLOGICAL BASES OF REORGANIZATION OF THE RAT CEREBELLAR CORTEX IN SENESCENCE].

A histological and immunohistochemical study of the cerebellar cortex of senescent Wistar rats (36-month-old) was performed in comparison to young rats (36-month-old).The cerebellar cortex of senescent animals typically showed degeneration of the Purkinje cells accompanied by immunochemically determined loss of the calcium-binding protein calbindin. These results suggest that presence of calbindin in the Purkinje cells is a criterion of functional activity of these neurons. The Purkinje cells degeneration is accompanied by lesion of the synaptophysin-containing basket cell networks, which is an indication of impaired function of inhibitory (GABAergic) axo-axonal synapses. During senescence significant rear-rangement is observed in the glomeruli of the granular layer of the cerebellum, which are the structures where primary afferent animals disintegrate suggesting of alteration in the transmission of information from the cerebrum to the cerebellum.