Detection of Tissue Macrophages in Different Organs Using Antibodies to the Microglial Marker Iba-1.

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.

[Pathological changes in the nervous structures of the aortic wall tissue adjacent to an unstable atherosclerotic plaque]. / Patologicheskie izmeneniya nervnykh struktur v tkanyakh stenki aorty, prilezhashchikh k nestabil'noi ateroskleroticheskoi blyashke.

OBJECTIVE: To study of nerve structures in the aortic wall in atherosclerosis using a complex of immunohistochemical markers. MATERIAL AND METHODS: The objects of the study were excised fragments of the wall of the thoracic and abdominal aorta along with visually determined unstable atherosclerotic plaques. To study nerve structures on paraffin sections, immunohistochemical reactions were performed for the PGP 9.5 protein, tyrosine hydroxylase, and synaptophysin. RESULTS: It has been established that pronounced pathological changes are observed in the nervous structures of the aortic wall near unstable atherosclerotic plaques. Reactive, dystrophic, and severe degenerative changes in neurocytes, nerve fibers, and glial cells are described in the elements of the nervous apparatus of the adventitia (microganglia, nerve trunks, and nerve plexuses). It was found that only sympathetic neurons and their postganglionic fibers remain in the intramural ganglia, while the structures of the parasympathetic nervous apparatus undergo degeneration. Destruction of perivascular nerve plexuses and vasa vasorum in the adventitia, as well as degeneration of varicose axons of the main terminal synaptic plexus at the border of adventitia and superficial smooth muscle layer of the media were demonstrated. CONCLUSION: It is assumed that the presence of inflammatory infiltrates in the adventitia and intima, denervation and death of vasa vasorum can serve as factors determining the development of the atherosclerotic process.

Proliferative Markers and Neural Stem Cells Markers in Tanycytes of the Third Cerebral Ventricle in Rats.

The proliferative properties of tanycyte subpopulations during postnatal development and aging were studied. Using immunohistochemical markers, we described the distribution of proliferative markers and markers of neural stem cells (NSC) in 4 tanycyte subpopulations (α1-, α2-, ß1-, and ß2-tanycytes). During the first postnatal week, all tanycyte subpopulations exhibit proliferative activity. During aging, ß-tanycytes lose their proliferative activity and retain a limited set of NSC markers, whereas α-tanycytes maintain both the ability to proliferate and the properties of NSC throughout the entire postnatal development including aging. The data obtained significantly improve modern understanding of the proliferative potential of tanycytes and their subpopulation differences in early postnatal period and during aging.

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.

[Transthyretin amyloid cardiomyopathy. Features of histological diagnosis: study design].

AIM: To compare efficiency and specific features of transthyretin amyloid staining by different histological dyes and thus to assess their suitability for diagnostic purposes. MATERIALS AND METHODS: Samples of left and right heart ventricles were taken from patients over 70 years-old of both genders (n=10) with immunohistochemically verified transthyretin amyloidosis (ATTR). All samples were stained with Congo red, Alcian blue, toluidine blue and methylene violet. RESULTS: Specificity and sensitivity of Congo red staining was comparable to those of immunohistochemical staining. For verification of amyloid presence after Congo red staining one could use fluorescent microscopy instead of polarization microscopy. It allows a more accurate diagnosis of amyloidosis. Confocal microscopy with spectral unmixing improves detection sensitivity of amyloid by elimination of background fluorescence of muscle tissue and autofluorescence of lipofuscin. Alcian blue staining gives the same result as Congo red. In addition, its less labor-intensive and free of false-positive and false-negative results caused by final processing of slide preparation. Toluidine blue and methylene violet develop metachromatic staining upon binding to transthyretin fibrils, likely due to specific biochemical features of these fibrils. CONCLUSION: The most reliable method for histochemical diagnosis of ATTR is the Congo red staining with subsequent analysis using fluorescence or confocal microscopy. For diagnostic screening, the use of Sodium sulphate-Alcian blue staining method is highly promising. Metachromatic stains are less effective for ATTR diagnosis.

[Features of innervation of epicardial adipose tissue in rats during aging (immunohistochemical study).]

The purpose of the article is to study the structure, innervation and state of the epicardial adipose tissue of the aortic-pulmonary region of the heart of rats at the age of 3-4 months and 18-23 months using neuroimmunohistochemical markers. Using a complex of histological and immunohistochemical methods, various nervous apparatus (ganglia, clusters of chromaffin cells, nerve trunks, nerve fibers, nerve plexuses, synaptic endings) with different mediators were identified in the white and brown adipose tissue of the base of the rat heart. It was found that parasympathetic and sympathetic postganglionic nerve fibers are involved in the innervation of white and brown adipose tissue. They penetrate into adipose tissue as part of Remak's cords of varicose axons along arterial vessels, form terminal synaptic plexuses of the en passant type, and are involved in the innervation of adipocytes of both types of epicardial adipose tissue. It was found that PGP 9.5+ cholinergic terminal nerve fibers predominate over catecholaminergic ones. During aging of rats, neurodegenerative, involutive (desimatization), and destructive pathological changes in white adipocytes were noted in epicardial adipose tissue.

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.

Changes in the Thickness of Rat Nerve Sheaths after Single Subperineural Administration of Rat Bone Marrow Mesenchymal Stem Cells.

The sheaths of the damaged peripheral nerve of Wistar-Kyoto rats were studied after single subperineural administration of bromodeoxyuridine (BrdU)-labeled bone marrow mesenchymal stem cells (MSC) from the same rats. The sciatic nerve was damaged by ligation for 40 sec directly before MSC administration. BrdU+ MSC were identified in the recipient nerve within 1 week after transplantation and were detected not only in the endoneurium, but also in the epineurium and perineurium. It was found that single administration of MSC into the damaged nerve trunk led to an almost 2-fold increase in the thickness of its sheaths (perineurium and epineurium) in comparison with the control group (ligation). It can be hypothesized that MSC induce thickening of nerve sheaths through the production of factors that stimulate angiogenesis and adipogenesis.

[Morphological signs of neurogenic inflammation in the heart of rats during aging.]

Using histological methods of staining with toluidine blue, hematoxylin-eosin and immunohistochemical reactions for the PGP 9,5 protein, tyrosine hydroxylase (TH), Iba-1 protein, cellular changes in different parts of the heart of Wistar rats at the age of 18-23 months were studied. In the connective tissue of the heart base, focal inflammatory infiltrates were found, near which PGP 9.5+ and TH+ plexuses, consisting of parasympathetic and sympathetic nerve fibers, were detected. In the area of the valvular heart apparatus, at the border of the anneau fibreux and the myocardium of the right atrium, pathological changes in nerve structures were found: degeneration of nerve fibers and granular destruction varicose axons of the terminal plexus. A close relationship has been established between axons of the terminal nervous network and cells of inflammatory infiltrates and blood capillaries. The features of the localization of neurocellular inflammatory complexes consisting of nerve fibers, blood capillaries and cells participating in the local inflammatory process (mast cells, histiocytes, monocytes, fibroblasts, plasma cells) in various parts of the myocardium in old animals are described. The chronic nature of neurogenic inflammation in the heart during aging has been established.

Immunohistological Detection of Active Satellite Cellsin Rat Dorsal Root Ganglia after Parenteral Administration of Lipopolysaccharide and during Aging.

Immunohistochemical reaction to glial fibrillar acid protein (GFAP) is widely used for identification of activated satellite cells in sensory ganglia. We used this marker in studies of satellite cells activation in dorsal root ganglia during aging and under conditions of experimental systemic inflammation: in young (4 months) and aged (18-19 months) rats and animals with experimental LPS-induced systemic inflammation. The number of GFAP+ satellite cells increased significantly after parenteral injection of LPS and during aging, which can indicate similarity of mechanisms of reactive glial changes during aging and systemic inflammation.

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.

Spinal Cord Microglia in Health and Disease.

The review summarizes data of recent experimental studies on spinal microglia, the least explored cells of the spinal cord. It focuses on the origin and function of microglia in mammalian spinal cord embryogenesis. The main approaches to the classification of microgliocytes based on their structure, function, and immunophenotypic characteristics are analyzed. We discuss the results of studies conducted on experimental models of spinal cord diseases such as multiple sclerosis, amyotrophic lateral sclerosis, systemic inflammation, and some others, with special emphasis on the key role of microglia in the pathogenesis of these diseases. The review highlights the need to detect the new microglia-specific marker proteins expressed at all stages of ontogeny. New sensitive and selective microglial markers are necessary in order to improve identification of spinal cord microgliocytes in normal and pathological conditions. Possible morphometric methods to assess the functional activity of microglial cells are presented.

A Novel Method for Amyloid Detection in Human Tissue Load Using a Fluorescent Dye - Congo Red Analogue.

The aim of the study was to develop a new technology for the detection of amyloid in human tissues based on the fluorescent dye, disodium salt of 2,7-(1-amino-4-sulfo-2-naphthylazo)fluorene (DSNAF). MATERIALS AND METHODS: Synthesis of DSNAF was performed by diazotization of 2,7-diaminofluorene in a stream of argon followed by azo coupling with naphthionic acid. Identification of DSNAF was performed using MALDI mass spectrometry. Human myocardial samples from males and females aged from 85 to 98 years (n=11) were the material for the histochemical study. Myocardial paraffin sections were stained with a 0.1% aqueous solution of Congo red or with an aqueous solution (0.1 or 0.034%) of DSNAF under the same conditions. RESULTS: It has been demonstrated for the first time that a new fluorene-based analogue of Congo red, DSNAF, can be successfully used to identify amyloid deposits in histological sections of human myocardium. In terms of the specificity and intensity of amyloid staining, DSNAF is comparable to Congo red, which is the gold standard for detecting amyloid deposits. The fluorescence intensity of DSNAF when binding to amyloid fibrils is significantly higher than the intensity of Congo red fluorescence (with a lower intensity of background fluorescence of heart muscle tissue). This is especially useful for identifying small deposits of amyloid in the human tissues which is important when using small biopsies. CONCLUSION: The advantages of using DSNAF allow us to consider the developed technology for the detection of amyloid as a new promising method of identifying amyloid deposits in human tissues.

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.

[Transthyretin amyloidosis in a cohort of old and very old patients with chronic heart failure].

BACKGROUND: Life-time diagnostics of wild type transthyretin amyloidosis (ATTR(wt)-amyloidosis) is virtually absent, even though ATTR(wt)-amyloidosis is an underestimated cause for morbidity and mortality, particularly in the older age group. AIM: To study incidence, demographic characteristics, and morpho-functional features of ATTR(wt)-amyloidosis in patients with FC IV CHF and LV hypertrophy > 15 mm according to autopsy data. MATERIALS AND METHODS: Postmortem reports were retrospectively analyzed for patients (n=141; 19 % males, 81 % females) of cardiology departments aged ≥69 with the underlying CHF syndrome. From all formalin-fixed fragments of the myocardium embedded in paraffin were prepared 5-7 mkm cuts, which were stained with Congo red (Sigma, USA) and viewed under normal and polarized light. Immunohistochemical analysis was also performed using antibodies to AA-amyloid, transthyretin, kappa and lambda-light chains of immunoglobulins. RESULTS: deposits were found in both old and very old persons aged 91.25±9.67, mostly in women due to shorter life span of men. In different FCs associated with LV hypertrophy, according to autopsy data amyloid deposits were observed in virtually every fifth deceased (21 % of cases). The amount of myocardial amyloid deposits was generally small (56 % of cases had (+) and 27 % had (++) amyloid deposits); 17 % of cases had considerable amyloid deposits (7 % had (+++) and 10 % had (++++)). The presence of amyloid deposits did not influence indexes of myocardial hypertrophy, such as ventricular septum thickness, LV posterior wall thickness, and heart mass. In the presented cases we observed focal amyloid deposition in the myocardium typical for old age-related amyloidosis; in 97 % cases, amyloid was located in the interstitium, around cardiomyocytes and in 3 % of cases - exclusively around blood vessels. CONCLUSION: ATTR (wt)-amyloidosis was detected in every fifth patient in the old and very old cohort, primarily in women (83 %), and was not diagnosed during the life time. Characteristic morphological manifestations of ATTR(wt)-amyloidosis were focal amyloid deposits mostly in the myocardial interstitium.

Vascularization of the Damaged Nerve under the Effect of Experimental Cell Therapy.

Quantitative analysis of blood vessels in the distal segment of rat sciatic nerve after its ligation for 40 sec and subperineurial administration of mesenchymal stem cells or dissociated cells of rat embryonic spinal cord was carried our by immunohistochemical tracing of von Willebrand factor, a marker of endothelial cells of blood vessels. It was found that the number of blood vessels per unit area of the nerve trunk in 21 days after injury and administration of mesenchymal stem cells increased by more than 1.5 times in comparison with the control (damaged nerve). After administration of dissociated cells of the embryonic spinal cord, this effect was not observed. It is assumed that mesenchymal stem cells stimulate the growth of vessels of the damaged nerve via production of angiogenic factors.

[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.

[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.

Activation of Microglyocytes in the Anterior Horns of Rat Spinal Cord after Administration of Bacterial Lipopolysaccharide.

We studied the reaction of the microglia of the anterior horns of the rat spinal cord to intraperitoneal administration of bacterial LPS. Immunohistochemical analysis showed that acute systemic inflammation leads to activation of more than half of microglial cells as soon as in 24 h after LPS injection, while the total number of microglial cells does not change significantly. It was hypothesized that activated microglial cells are involved in the reorganization of synaptic connections, but do not have a neurotoxic effect.