BMP-2 and IL-1ß as Markers of Nasal Mucosa Inflammation in Rhinosinusitis with Nasal Polyps.

Rhinosinusitis with nasal polyps is characterized by chronic inflammation and hyperplasia of the nasal mucosa. The key mechanism for polyp formation is the expression of molecules that regulate proliferation and inflammation. We studied immunolocalization of bone morphogenetic protein-2 (BMP-2) and IL-1ß in the nasal mucosa in patients aged 35-70 years (n=70, mean age 57.4±1.52 years). The typology of polyps was determined depending on the distribution of inflammatory cells, subepithelial edema, the presence of fibrosis and cysts. The immunolocalization of BMP-2 and IL-1ß had the same pattern in edematous, fibrous, and eosinophilic (allergic) polyps. Goblet and connective tissue cells, microvessels and terminal sections of the glands were positively stained. BMP-2+ and IL-1ß+ cells predominated in polyps of the eosinophilic type. BMP-2/IL-1ß can be considered as a specific marker of inflammatory remodeling of the nasal mucosa in refractory rhinosinusitis with nasal polyps.

Evaluation of the Effectiveness of Bone Grafting in Alveolar Ridge Augmentation Using the Two-Stage Splitting Technique.

Stimulation of neoosteogenesis is the main mechanism of osseointegration during installation of dental implants, bone tissue recession, and alveolar process augmentation in adentia. In experiments on miniature pigs, we used the technology of two-stage splitting of the ridge of the alveolar process of the mandible in combination with a xenograft that was placed between the fragments of the split bone plate. The morphology of the reparative process and the distribution of osteogenic differentiation markers in the compact and trabecular bone of the alveolar crest were studied. Signs of reparative osteogenesis were observed in the bone regenerate that had a lamellar structure, formed osteons, and foci of woven tissue. It was found that the xenograft was replaced by newly formed trabecular bone tissue. These sites were characterized by increased expression of osteocalcin and CD44. Augmentation technology through two-stage splitting provides trophic relationship of osteoprogenitor cells and is an effective method of osteogenesis stimulation in the alveolar process.

Localization of VEGF, TGF-ß1, BMP-2, and Apoptosis Factors in Hypertrophic Nonunion of Human Tubular Bones.

We studied localization of VEGF, TGF-ß1, BMP-2, caspase-3, Bcl-2, and TNFα in the callus samples obtained from 5 patients (4 women and 1 man) aged 41-53 years during planned surgery for nonunion and pseudarthrosis of the clavicle (n=1), ulna (n=1), femur (n=1), and tibia (n=2) bones. Two control groups included material of hypertrophied callus (n=3) with consolidated fractures of long bones and samples of intact bones (n=3) obtained by postmortem autopsy of subjects without pathology of the musculoskeletal system. A nonuniform distribution of the studied markers was revealed. Active expression of VEGF was observed in fibroblast-like cells of the fibrous tissue, osteoblasts of the periosteum and osteons. Osteoblasts expressing BMP-2 were localized in the periosteum and the loose connective tissue of the Haversian canals. The number of immunopositive cells expressing TGF-ß1 and TNFα in the callus exceeded that in the control and correlated with the expression of caspase-3 in fibroblast-like cells, osteoblasts, chondroblasts, and microvascular endotheliocytes. The results allow considering fracture nonunion as a result of overproduction of cytotoxic and proapoptotic factors in chronic inflammation and dysfunction of the expression of morphogenetic proteins. The morphochemical patterns of the studied markers open up prospects for the development of new methods of pharmacological correction of fracture repair.

On-Bone Fixation of Free Gingival Graft Induces an Osteoinductive Effect in Human Alveolar Bone.

We examined alveolar bone samples in the area of on-bone fixation of a free gingival graft performed during surgery in patients aged 37-55 years with a diagnosis of secondary partial adentia of the upper and lower jaws. Six months after fixation of the graft in the alveolar bone, foci of neoosteogenesis were found in the contact zone. They were characterized by the appearance of appositional lines, cords of basophilic osteoblasts, and growing osteons. An immunohistochemical study revealed an increase in the number of CD44+, CD29+, and osteocalcin+ cells in the layer of the outer circumferential lamellae, primary osteons, and the lining of the Haversian canals. TGF-ß1+ cells were located in the intertrabecular reticular tissue and wall of microvessels. The results indicate activation of mesenchymal stem cells in the area of localization of the graft and differentiating osteoblasts. The observed osteoinductive effect of free gingival graft is associated with its participation in reorganization in MSC and induction of morphogenetic molecules.

Dynamics of Renewal of Cell Populations of the Bone Tissue on the Surface of Titanium Implants with Bioactive Coating during Fracture Modeling in Rats.

Localization of PCNA, CD44, osteocalcin, Mdm2, p53, and caspase-3 on the surface of implant with calcium phosphate and hydroxyapatite coating was studied by immunocytochemical method in a model of femur fracture in rats. PCNA+, Ost+, CD44+, and Mdm2+ cells were found in the periosteum, in the layer of the outer surrounding plates, and in the connective tissue of the Haversian canals. Cell density increased on day 7 after fracture and then decreased by day 30. The number of p53+ and CASP3+ cells reached a maximum on day 14 (they were predominantly located in the periosteum and bone plates adjacent to it) and decreased by day 30. Calcium phosphate coating stimulated proliferative activity of cells at the early stages of the regeneration phase and apoptotic death at the later stages. Components of coating can be viewed as a positioning clue for differentiation of mesenchymal stromal cells. The effectiveness of reparative osteogenesis is determined by the balance of proliferative and destructive factors at the site of the fracture healing. This process can be optimized with various nanostructured materials with osteoinductive properties, in particular bioresorbable calcium phosphate coatings on titanium implants. However, the influence of these components on the state of cambial cells, their differentiation, and positioning in the repair zone is unknown.

Osteogenic and Regenerative Potential of Free Gingival Graft.

We studied immunolocalization of CD29, CD44, osteocalcin, and TGF-ß1 in the bone tissue of the mandible of miniature pigs with extra-bone fixation of a free gingival graft. Three months after surgery, neoosteogenesis foci with high expression of the studied markers were found in the contact area of the free gingival graft with the alveolar bone. The markers were localized in the layer of external circumferential lamellae, on the surface of concentric lamellae of the growing osteons, and in the connective tissue of the Haversian canals. TGF-ß1-immunopositive cells predominated in the connective tissue of the Haversian and Volkmann canals and in the adventitia and inner lining of the vascular wall. The established morphochemical patterns of osteogenous cells indicate significant reparative capabilities of a free gingival graft and allows considering it as an effective osteoinductive factor.

Brain-Derived Neurotrophic Factor (BDNF) As a Regulator of Apoptosis under Conditions of Focal Experimental Stroke.

The immunolocalization of apoptotic factors in rat neocortex was studied on the model of permanent occlusion of the middle cerebral artery with administration of exogenous BDNF. We revealed heterogeneous distribution of pro- and anti-apoptotic factors in the stroke area and in the surrounding penumbra, where caspase-3+ and p53+ cells were found. Their number was maximum on day 3 of ischemia. The number of neurons containing anti-apoptotic factors was relatively decreased. Injection of BDNF changed the distribution of apoptotic factors. In the penumbra area, BDNF enhanced the expression of Mdm2 primarily in the pyramid cells of layers V/VI and Bcl-2 in interneurons of layers II and III. Localization of p53 and caspase-3 varied at different terms of the ischemic period and showed an inverse dependence. Considering the selective neuroprotective effect of BDNF, various mechanisms of the formation of ischemic tolerance in neurons are proposed.

[Expression of pro- and anti-apoptotic molecules in the mucous membrane of the nasal cavity with polypous rhinosinusitis]. / Ekspressiya pro- i antiapoptoticheskikh molekul v slizistoi obolochke polosti nosa pri polipoznom rinosinusite.

OBJECTIVE: To study the expression of proapoptotic (p53, p21) and antiapoptotic (MDM2) factors, as well as the distribution of proliferating PCNA-immunoreactive cells in the nasal mucosa in various types of polyposis rhinosinusitis (PRS). MATERIAL AND METHODS: We studied the immunolocalization of proapoptotic (p53, p21) and antiapoptotic (MDM2) factors, as well as the distribution of proliferating PCNA immunoreactive cells in the mucous membrane of the nasal cavity for various types of polypous rhinosinusitis. RESULTS AND DISCUSSION: Comparing with the control group, increased expression of all factors is detected. The main portion of marked cells is located in the loose connective tissue of the lamina propria of mucous membrane, a relatively small number of cells are detected in the epithelial layer. The edematous, eosinophilic (allergic) type of PRS is characterized by the expression of p53 and PCNA and by the reduction of MDM2 immunoreactive cells, while the expression of p53 and p21 is reduced in fibroinflammatory (neutrophilic) type. CONCLUSION: PRS is accompanied by epithelial metaplasia, by formation of inflammatory cell infiltrates, fibrosis and edema. The results are discussed in connection with the data on the regulatory role of apoptosis in the pathomorphism of chronic productive inflammation.

Role of Vascular Endothelial Growth Factor and Transforming Growth Factor-ß2 in Rat Bone Tissue after Bone Fracture and Placement of Titanium Implants with Bioactive Bioresorbable Coatings.

The study established enhanced expression of vascular endothelial growth factor (VEGF) in the subpopulation of osteoblasts located in the regeneration region of femoral bone fracture near the titanium implants with bioactive calcium phosphate and hydroxyapatite coatings and suppressed activity of transforming growth factor-ß2 (TGF-ß2) in chondroblasts during the two weeks after surgery. In the delayed posttraumatic period, the distribution of TGF-ß2 inversely related to its maximal activity. The data revealed the up-regulating effect of bioresorbable coatings on expression of VEGF and TGF-ß2 and their implication in the control over various stages of reparative osteogenesis.

[The experimental strategies in the study of ischemic stroke]. / Éksperimental'nye strategii issledovaniia ishemicheskogo insul'ta.

Literature data and own experience in the studies of experimental stroke methodology are reviewed. Advantages and disadvantages of the common models of focal ischemia used in the laboratory practice are discussed in details. The advantages of the filament occlusion of the middle cerebral artery in rats as the most adequate model of human stroke are substantiated. The authors suggest a modification of this variant using an additional coagulation of the pterygopalatine artery that allows the exclusion of the retrograde and collateral blood flow into the inner carotid artery after ligation of the common and external carotid arteries.

Small molecules regulating apoptosis in the synovium in rheumatoid arthritis.

OBJECTIVES: To study the rate of apoptosis and expression of pro- and anti-apoptotic molecules in the synovial membrane in early and late rheumatoid arthritis (RA). METHODS: Samples of the synovium, cartilage, synovial fluid, and blood serum were obtained from patients with seropositive RA. The localization of Bcl-2-, p53- and TUNEL-immunoreactive cells in the synovial membrane was studied. The level of the soluble Fas (sFas) receptor was determined in the blood serum and synovial fluid using an immunoassay. RESULTS: In early RA, p53-immunoreactive synovial cells of type A were found to form rare aggregates in the intima. In late RA, on the contrary, these cells increased in number and occurred predominantly in the synovial stroma. Bcl-2-immunoreactive synovial cells were observed in lymphocytic infiltrates in the intima. They were found mainly in early RA. In late RA, their number decreased. The apoptotic index determined from the proportion of TUNEL-reactive synovial cell nuclei reached a maximum in late RA. The temporal differences in the rate of apoptosis were correlated with the humoral level of sFas, which increased significantly in late RA. On the contrary, in the synovial fluid, the sFas level decreased monotonically from early to late RA. CONCLUSION: In early RA, in the synovial membrane, the rate of apoptosis and p-53-immunostaining intensity were low, and Bcl-2-immunostaining intensity was high. The sFas level in synovial fluid was high. In late RA, the rate of apoptosis and p-53-immunostaining intensity increased, Bcl-2-immunostaining intensity decreased, as did the sFas level.

Morphological characteristics of apoptosis and its significance in neurogenesis.

Results from our own studies and published data are used to provide a critical analysis of current views of the role of apoptosis in regulating the quantitative and qualitative constancy of cells in the developing brain. Detailed descriptions of the morphological features and mechanisms of the different phases of apoptotic neuron death are given. Apoptosis affects the ordering of connections in neural networks and is involved in the pathogenesis of neurodegenerative diseases--epilepsy, schizophrenia, and Alzheimer's disease. The question of the interaction of the NO-ergic mechanism and the cell in executing apoptosis is discussed. The data allow NO to be regarded as a cytotoxic factor which induces apoptosis. The ultrastructural features of post-mitotic immature neurons suggests the operation of the apoptotic and necrosis-like (NO-dependent) pathways of natural cell death during the process of differentiation.

[Morphological characteristic of apoptosis and its significance in neurogenesis].

On the basis of the literature data and the results obtained by the authors, modern concepts on the role of apoptosis in the regulation of a quantitative and qualitative constancy of the cells in the developing brain, is critically analyzed. The detailed description of morphological features and mechanisms of various phases of apoptotic death of neurons is given. Apoptosis affects the ordering of connections in the neural circuits and is involved in the pathogenesis of the neurodegenerative disorders - epilepsy, schizophrenia and Alzheimer's disease. The problem of the NO-ergic mechanism interaction with a cell in the apoptosis realization is examined. The data obtained support the view that NO should be considered as a cytotoxic factor inducing apoptosis. On the basis of ultrastructural features of postmitotic immature neurons, the presence of the apoptotic and necrosis-like (NO-dependent) ways of the natural cell loss during the differentiation process, is proposed.

The role of nitric oxide in the apoptosis of neurons in the retina of the human fetal eye.

The locations of NADPH-diaphorase (NADPH-d), inducible NO synthase (iNOS), and TUNEL-immunoreactive neurons in the retina of human fetuses collected during the first to third trimesters of pregnancy were studied. High levels of NADPH-d activity were seen in the inner segments of light-sensitive cells, amacrine cells, and ganglion cells. The population of NADPH-d-positive amacrine cells included three types of neuron. Type 1 neurons were large and had sparse dendritic fields occupying the inner nuclear and outer retinal layers. Small type 2 neurons were located in the inner retinal layer. Ectopic amacrine cells, type 3, were located in the outer part of the ganglion layer. A high density of NADPH-d-positive neurons was seen in the central part of the retina, surrounding the central fovea and optic disk area. NADPH-d activity increased progressively during ontogenesis and correlated with the appearance of immunoreactive iNOS in neurons. iNOS labeled a subpopulation of amacrine and ganglion cells, which appeared at 20-21 weeks of development and reached a peak of immunoreactivity by the end of the third trimester. TUNEL-immunopositive neuron nuclei with signs of apoptotic destruction were seen at 30-31 weeks of pregnancy. The greatest apoptotic index was seen in the ganglion and amacrine cell populations. These data identify NO as a factor mediating apoptosis of neurons during the critical period of differentiation of interneuronal connections in the human retina.

[Neurogliaform cells: neurochemistry, spatial arrangement, and their role in the neocortical inhibitory system].

The neurogliaform cells (NGFC) localized in area 4 of the human motor cortex were found to express choline acetyl transferase (ChAT), GABA, and calbindin. ChAT-positive neurons were located in cortical layer II. Their dendrites lay in a close proximity to those of pyramid cells, neighbouring neurogliaform cells, and bodies and dendrites of other cortical neurons. The NGFCs revealed by Golgi staining fell into two groups. Cells of the first group had locally (within cortical layer II) spreading axons, while those of the second group had axons extending into the adjacent layers. Neurochemical heterogeneity of NGFCs is discussed in the context of information processing in cortical modules and interaction of excitatory and inhibitory interneurons.

Neurochemical diversity of neurogliaform cells in the human primary motor cortex.

Neurogliaform cells of area 4 of the human motor cortex were found to express choline acetyl transferase (ChAT), gamma-aminobutyric acid, and calbindin. GABA- and calbindin-positive NGCs were mainly localized in layers II and VI and were relatively rare in layer I of the cortex. ChAT-positive NGCs were observed in the upper and middle thirds of layer II, occurring occasionally in layer I and the upper portion of layer III. Their numbers were low compared to those of GABA- and calbindin-positive NGCs in layers II/III. The dendrites of ChAT-positive NGCs were short and few in number. Axonal arborizations of neighboring ChAT-positive cells interpenetrated considerably so that each ChAT-positive cell body was normally surrounded by axonal trees of the parent and a few other ChAT-positive NGCs. NGC axon collaterals surrounded small neuropil areas containing perikarya presumptive pyramidal neurons. The findings are discussed in the context of information processing in cortical modules and interaction of excitatory and inhibitory interneurons.

[Role of nitric oxide in apoptosis of retinal neurons in human fetuses].

The localization of NADPH-diaphorase (NADPH-d), inducible NO-synthase (iNOS) and TUNEL-immunoreactive neurons was studied in the retina of human fetuses in the I-III trimesters of pregnancy. High NADPH-d activity was found in internal segments of photosensory cells, amacrine and ganglion cells. The population of NADPH-d-positive amacrine cells included three types of neurons. Neurons of the 1st type had large size and scarce dendritic field, occupying the inner nuclear and outer plexiform layers. Small neurons of the 2nd type were located in the inner plexiform layer. Ectopic amacrine cells of 3rd type could be found in the outer part of the ganglion cell layer. High density of the NADPH-d-positive neurons was detected in the central portion of retina surrounding fovea centralis and the optic disk area. The activity of NADPH-d was found to grow progressively in ontogenesis and to correlate with the appearance of immunoreactive iNOS in neurons. Immunoreactive iNOS marked a subpopulation of amacrine and ganglion cells which appeared in weeks 20-21 of gestation and attained maximal immunoreactivity by the end of the III trimester. TUNEL-immunoreactive nuclei of the neurons with the signs of the apoptotic destruction were found in weeks 30-31 of gestation. The highest apoptotic index was found in the population of ganglion cells. The data obtained strongly suggest that NO is a factor, mediating the neuronal apoptosis during the critical period of a differentiation of interneuronal connections in the human retina.

Induction of NO synthase and glial acidic fibrillary protein in astrocytes in the temporal cortex of the rat with audiogenic epileptiform reactions.

The localizations of NADPH-diaphorase (NADPH-d), inducible NO synthase (iNOS), and glial acid fibrillary protein (GFAP) in astrocytes of the temporal cortex were studied in Krushinskii-Molodkina rats, which are genetically predisposed to audiogenic convulsive seizures. Convulsive reactions were induced in rats by three exposures to acoustic stimuli. Controls consisted of Wistar rats and Krushinskii-Molodkina rats not subjected to acoustic stimulation, these not developing convulsive reactions. The neocortex of animals with audiogenic convulsions consistently showed foci of brain tissue damage. Foci, of diameter 300-400 microm, were located in layers III-V and were groupings of NADPH-d-positive astrocytes; these were seen in both hemispheres. Astrocytes in foci of damage expressed iNOS and had elevated GFAP levels. The numbers of GFAP-immunopositive cells were increased by 25-37% in damage foci as compared with levels in controls and undamaged areas of the cortex. The induction of NO synthase and GFAP in astrocytes seen here indicates the involvement of glia in compensatory NO-dependent mechanisms formed in damage foci in response to audiogenic convulsive seizures.

Unipolar brush cells--a new type of excitatory interneuron in the cerebellar cortex and cochlear nuclei of the brainstem.

Published data and the authors' own studies on the morphology, neurochemical specialization, and spatial organization of unipolar brush neurons (UBN) in the cerebellar cortex and cochlear nuclei of the brainstem are reviewed. UBN represent an exclusive category of excitatory interneurons, with a single dendrite which forms a compact branching with a shape reminiscent of that of a brush in its terminal segment. These cells are characterized by an uneven distribution in the granular layer of the cerebellum, being located mainly in its vestibular zones. UBN synthesize glutamate, calretinin, and metabotropic and ionotropic glutamate receptors. The dendritic brush of UBN form giant synapses with the rosettes of glutamatergic and cholinergic mossy afferent fibers. UBN axons form an intracortical system of mossy fibers which, forming rosettes and glomeruli, make contact with the dendrites of other UBN and granule cells. In the circuits of interneuronal communications, UBN can be regarded as an intermediate component, amplifying the excitatory effects of mossy afferent fibers on granule cells in the cerebellar cortex and cochlear nuclei of the brainstem.

[Induction of NO-synthase and glial fibrillary acidic protein in astrocytes of the temporal cortex in rats with audiogenic epileptiform reaction]. / Induktsiia NO-sintazy i glial'nogo kislogo fibrilliarnogo belka v astrotsitakh visochnoi kory krys s audiogennoi épileptiformnoi reaktsiei.

The localization of NADPH-diaphorase (NADPH-d), inducible NO-synthase (iNOS) and glial fibrillary acidic protein (GFAP) was studied in the astrocytes of the temporal cortex in rats of Krushinsky-Molodkina strain which are genetically prone to audiogenic seizures. The seizure was evoked by thrice-repeated acoustic stimulation. Wistar rats and acoustically untreated seizure-free Krushinsky-Molodkina rats were used as a control. The foci of brain damage were consistently found in the neocortex of the animals with audiogenic seizures. Epileptic foci, 300-400 microm in diameter, were localized in layers III-V; they were found to consist of the clusters of NADPH-d-positive astrocytes and to be present in both hemispheres. In the foci of cortical damage astrocytes expressed iNOS and an elevated level of GFAP. The number of GFAP-immunopositive astrocytes in the foci of damage was increased by 25-37% compared to the control and to undamaged areas of the cortex. Astrocyte NOS and GFAP induction found in this work, suggests the participation of glia in compensatory NO-dependent mechanisms, that are formed in the damage foci of neocortex during the audiogenic seizures.