Том 160, № 4 (2016)

Gradual cooling of homeothermic organisms is followed by slowing and arrest of breathing and heart contractions. During deep cooling, even relatively slight artificial ventilation decreases the lower temperature limit of life (by 4.5-5^°C) and provides minimum oxygen supply to the heart and whole body. This allows us to restore cardiovascular function and prevent animal death after lethal cooling without external warming.

We developed and tested a novel hardware-and-software system for recording the amplitude of the acoustic startle response in rodents. In our experiments, the baseline indexes of acoustic startle response in laboratory rats and pre-stimulation inhibition under the standard delivery of acoustic stimulation were similar to those evaluated by other investigators on foreign devices. The proposed system is relatively cheap and provides the possibility of performing experiments on freely moving specimens. It should be emphasized that the results of studies can be processed with free-access software.

We studied the effect of delta sleep-inducing peptide (40, 120, and 360 μg/kg intraperitoneally, 1 h before the experiment) on free radical oxidation in the liver, aminotransferase activity, and total serum protein content in male Wistar rats during restraint stress. Treatment with the peptide in a dose of 40 μg/kg increased catalase and superoxide dismutase (SOD) activities and malonic dialdehyde (MDA) concentration in the liver homogenate of animals subjected to acute stress. No significant changes were found after administration of this peptide in other doses. Under conditions of chronic stress, the peptide in a dose of 40 μg/kg caused the most pronounced effect. Catalase and SOD activities and MDA concentration decreased, while aminotransferase activity and protein content remained unchanged under these conditions. Administration of the peptide in a dose of 120 μg/kg was accompanied by a decrease in SOD activity and MDA concentration, increase in total protein content, and reduction of AST activity. Increasing the peptide dose to 360 μg/kg abolished its effects.

We studied anticonvulsant effects of combined treatment with citicoline, a nootropic substance with neuroregenerative and neuroprotective activities, and valproate, an antiepileptic agent widely used in the treatment of epilepsy, on the model of pentylenetetrazole-induced (75 mg/kg, intraperitoneally) acute generalized convulsions in male Wistar rats. Combined treatment with citicoline and valproate in minimum effective doses (70 and 300 mg/kg, respectively) potentiated the anticonvulsant properties of both agents.

In vitro experiments showed that heparin adsorbed on activated charcoal can bind antibodies raised against native and single-stranded DNA in a diluted sera pool with a high level of these DNA. Thus, heparin used as anticoagulant during hemosorption procedure can demonstrate supplementary therapeutic activity resulting from its interaction with various agents involved in acute and chronic inflammatory reactions such as DNA- and RNA-binding substances, proinflammatory cytokines, complement components, growth factors, etc. Research and development of heparin-containing carbonic adsorbents for the therapy of numerous inflammatory and autoimmune diseases seems to be a promising avenue in hematology.

Expression of genes encoding the individual subunits of ionotropic GABA_A receptor was assessed after acute and chronic intoxication of rats with ethanol. The chronic 1-month-long exposure to ethanol signifi cantly decreased (by 38%) expression of Gabrb1 gene in the hippocampus. Acute exposure to ethanol elevated expression of genes Gabrb1 (by 1.7 times), Gabra1 (by 3.8 times), and Gabra4 (by 6.5 times), although it diminished expression of Gabra2 gene by 1.4 times. In preliminarily alcoholized rats, acute intoxication with ethanol enhanced expression of genes Gabrb1 and Gabra5 by 1.7 and 8.7 times, respectively. There was neither acute nor chronic effect of ethanol on expression of gene Gabra3.

Lithium chloride (85, 255, or 255+127 μg/kg) or dexamethasone (0.2 or 2 mg/kg) were subcutaneously injected to 3-day-old rat pups, whose excretory system did not yet attain functional maturity. Both agents retarded the growth of rat pups and delayed the appearance of negative geotaxis. LD₅₀ and therapeutic index of lithium chloride were 255 μg/kg and TI≤3, respectively. Thus, lithium salts even in low doses can be highly toxic for the developing organism.

GABA analogs containing phenyl (phenibut) or para-chlorophenyl (baclofen) substituents demonstrated nootropic activity in a dose of 20 mg/kg: they improved passive avoidance conditioning, decelerated its natural extinction, and exerted antiamnestic effect on the models of amnesia provoked by scopolamine or electroshock. Tolyl-containing GABA analog (tolibut, 20 mg/kg) exhibited antiamnestic activity only on the model of electroshock-induced amnesia. Baclofen and, to a lesser extent, tolibut alleviated seizures provoked by electroshock, i.e. both agents exerted anticonvulsant effect. All examined GABA aryl derivatives demonstrated neuroprotective properties on the maximum electroshock model: they shortened the duration of coma and shortened the period of spontaneous motor activity recovery. In addition, these agents decreased the severity of passive avoidance amnesia and behavioral deficit in the open field test in rats exposed to electroshock. The greatest neuroprotective properties were exhibited by phenyl-containing GABA analog phenibut.

We studied the effects of SNK-411, a new 5-pyrimidinol derivative, on serum cytokine profile of C57Bl/6 mice with Lewis lung carcinoma. The compound was injected intraperitoneally in doses of 25 and 50 mg/kg. A significant (by 3.5 times) increase in serum IL-4 content was detected in mice with tumors on day 9 of tumor development. In mice receiving SNK-411 in doses of 25 and 50 mg/kg, IL-4 content significantly decreased (by 4.0 and 3.6 times) on days 2–8 of carcinoma development; IL-2 content decreased by 1.4 and 1.2 times and IL-6 content decreased by 2.7 and 1.6 times, respectively, in comparison with control mice with tumors. Injections of SNK-411 in the same doses on days 8–15 of carcinoma development led to a significant decrease in IL-4 levels (by 2.2 and 4.5 times, respectively, in comparison with control mice with tumors) and did not affect serum levels of other cytokines.

Three parkinsonism models using neurotoxin 6-OHDA and pesticides rotenone and paraquat were reproduced in Wistar rats and parameters of astrocyte processes in the striatum (axon number and length, area occupied by them, and axon branching pattern) detected by immunohistochemical reaction for acid glial fibrillary protein were studied by computer morphometry. By these parameters, three morphological types of astrocytes were distinguished. Two variants of changes were found in the used parkinsonism models: 1) more intense branching and even elongation of all axons and 2) reduction of small and elongation of the main remaining stems, which manifested in polarization of glial cell. Type 1 reaction was obviously associated with compensatory increase in astrocyte interaction with neurons, while type 2 reflected astrocyte response to injury and impaired glioneuronal interactions.

Helicobacter pylori persistence in patients with chronic gastritis is associated with a complex of nonspecific structural reactions, the type of these reactions correlates with the severity of infection: catarrhal fibrotic changes in the gastric mucosa predominate in cases with manifest colonization, while the absence of H. pylori is associated with predominance of fibrotic process. Analysis of the incidence of some pathomorphological phenomena (degeneration, atrophy, metaplasia, and dysplasia of the surface epithelium) shows no relationship between the presence of H. pylori and colonization intensity. In all patients with chronic hepatitis, the gastric mucosa is involved in the pathological process; fibrosis (gastropathy) was the most common process. No appreciable correlations between the structural changes and hepatitis activity and the presence of H. pylori were detected.

Umbilical cord stroma is an easily available, convenient, and promising source of multipotent mesenchymal stromal cells for regenerative medicine. Cryogenic storage of umbilical cord tissue provides more possibilities for further isolation of multipotent mesenchymal stromal cells for autologous transplantation or scientific purposes. Here we developed a protocol for preparation of the whole umbilical cord tissue for cryogenic storage that in combination with the previously described modified method of isolation of multipotent mesenchymal stromal cells allowed us to isolate cells with high proliferative potential, typical phenotype, and preserved differentiation potencies.

We studied safety and clinical efficacy of transplantation of autologous bone marrow cell in complex therapy of 158 patients with chronic hepatitis and cirrhosis of the liver. The efficiency of cell therapy was assessed in 12 months after single injection of the cells. The positive response (alleviation of liver cirrhosis or stabilization of the pathological process) was observed in 70% cases. The efficacy of therapy correlated with the severity and etiology of the disease and was maximum in patients with Child–Pugh class A (in 82.5% cases) and class B liver cirrhosis (in 79% cases); in patients with class C liver cirrhosis, the positive response was achieved in 42.5% cases. In 39 patients, ultrasonic examination performed in 3 years after transplantation revealed no focal lesions or ectopic ossification foci.

We studied the mechanisms of adaptation of human breast cancer cells MCF-7 to hypoxia and analyzed the role of AMPK/mTOR signaling pathway in the maintenance of cell proliferation under hypoxic conditions. It was found that long-term culturing (30 days or more) of MCF-7 cells under hypoxic conditions induced their partial adaptation to hypoxia. Cell adaptation to hypoxia was associated with attenuation of hypoxia-dependent AMPK induction with simultaneous constitutive activation of mTOR and Akt. These findings suggest that these proteins can be promising targets for targeted therapy of tumors developing under hypoxic conditions.

The possibility of formation of lymphatic vessels after introduction of autologous bone marrow-derived multipotent mesenchymal stromal cells transfected with GFP gene into thrombosed femoral vein was studied by fluorescent microscopy. Vascular thrombosis caused by ligation of the great vein with subsequent injection of thrombin solution was accompanied by blockade of regional lymph flow. The cells injected into thrombosed vein directly participate in the formation of new lymphatic vessels in the paravasal tissue surrounding the vein, its tissue region, and around regional lymph nodes. This is seen from bright specific fluorescence of individual cells in the walls of lymphatic vessels and all vascular layers and valves in UV light.

Short-term cell culturing on basement membrane matrix is a common and very convenient in vitro model of angiogenesis. We studied the possibility of interaction of multipotent stromal cells from the umbilical cord and Ea.hy926 endothelial cells on this model at the early and late periods of the experiment. Multipotent stromal cells alone and in combination with endothelial cells formed an unstable tubular network. Clusters formed after its disassembling later became the sprouting centers in co-culture of the two cell types, but not in pure culture of multipotent stromal cells. Multipotent stromal cells with CD31+ phenotype constitute the structural basis of newly formed stable 3D capillary-like network. Prolongation of the time of culturing and combination of the two in vitro models of angiogenesis (tubulogenesis and sprouting) allowed more complete assessment of the angiogenic potential of multipotent stromal cells.

Culture of mesenchymal stem cells isolated from the bone marrow of primates by their characteristics met the requirements of stem cells. It was shown that transplantation of allogeneic mesenchymal stem cells (2 million cells per 1 kg body weight) immediately after ligation of the left anterior descending coronary artery between the middle and upper thirds led to neovascularization and capillarization of the ischemic myocardium.

We studied the dynamics of erythropoiesis in CBA mice during gestation against the background of treatment with iron-binding drug. The mechanisms of suppression of the bone marrow erythroid stem were evaluated. Administration of deferoxamine in a dose of 1 g/kg induced hypoplasia of the erythroid hemopoietic lineage. Suppression of bone marrow erythropoiesis manifested in a decrease of hemoglobin concentration and counts of reticulocytes, erythrocytes, and erythrokaryocytes. These changes were accompanied by a decrease in functional activity of erythropoietic precursors and secretion of erythropoietically active humoral factors by bone marrow myelokaryocytes. These data indicate that deferoxamine can be used for modeling of iron defi ciency anemia in pregnancy.

Induction of long-term potentiation in rat hippocampus was followed by short-term activation of transcription factor p53 and its subsequent degradation. We studied the effects of EX-527 (inhibitor of deacetylase Sirt1, a negative regulator of p53) and pifi thrin-β (inhibitor of p53-dependent transcription) on the levels of p53 protein and mRNA of its target gene S100B during long-term potentiation. Pifi thrin-β limited the increase in S100B mRNA content after tetanization, which confi rmed signifi cant contribution of p53 in the regulation of S100B during long-term potentiation. EX-527 completely prevented p53 degradation and increased S100B expression induced by tetanization. Thus, Str1 regulates stability of p53 and expression of its target gene S100B in rat hippocampus during long-term potentiation.

We studied the response of hematopoietic stem cells and progenitor cells to sympatholytic reserpine in intact C57Bl/6 mice and in animals with cyclophosphamide-induced leukopenia. The count of long-term hematopoietic stem cells (Lin^–Sca-1⁺c-kit⁺CD34^–) in the bone marrow of healthy mice increased in 7 min after reserpine injection and remained elevated in 2 h in parallel with elevated content of short-term stem (Lin^–Sca-1⁺c-kit⁺CD34⁺) and progenitor (Lin^–Sca-1⁺c-kit⁺) cells. Reserpine produced no effect on recruitment of hematopoietic stem and progenitor cells into the peripheral blood, but increased the serum level of granulocyte CSF and increased the count of metamyelocytes and neutrophilic granulocytes in the blood (in 2 h postinjection). Transplantation of bone marrow hematopoietic stem and progenitor cells from reserpine-treated donor C57Bl/6 mice to recipient CBA mice with 5-fluorouracil-induced leukopenia accelerated regeneration of the granulocytic lineage cells in leukemic mice. In cyclophosphamide-treated C57Bl/6 mice, reserpine reduced the level of short-term hematopoietic stem cells and increased the count of progenitor hematopoietic cells in the bone marrow in parallel with recruitment of the progenitors into the peripheral blood.

The model of streptozotocin-induced diabetes mellitus in C57Bl/6 mice was employed to study the role of precursors of insulin-producing β-cells, hematopoietic stem cells, and progenitor hematopoietic cells in inflammation. In addition to provoking hyperglycemia, streptozotocin elevated serum levels of IL-1β and hyaluronic acid, induced edema in the pancreatic insular tissue and its infiltration by inflammatory cells (neutrophils, lymphocytes, and macrophages) and fibroblasts. Inflammation in pancreatic islets was accompanied by necrotic processes and decreasing counts of multipotent progenitor β-cells (CD45^–, TER119^–, c-kit-1^–, and Flk-1^–), oligopotent progenitor β-cells (CD45^–, TER119^–, CD133⁺, and CD49f^low), and insulinproducing β-cells (Pdx1⁺). Pancreatic infl ammation was preceded by elevation of the number of short-term hematopoietic stem cells (Lin–Sca-1⁺c-kit⁺CD34⁺) relative to long-term cells (Lin^–Sca-1⁺c-kit⁺CD34^–) in the bone marrow as well as recruitment of hematopoietic stem and progenitor cells into circulation. Transplantation of bone marrow hematopoietic stem and progenitor cells from diabetic C57Bl/6 donor mice to recipient CBA mice with 5-fluorouracilinduced leukopenia accelerated regeneration of granulocytopoiesis in recipient mice.

Malignant gliomas are most common and fatal primary brain tumors. In addition to neoplastic cells, the tumor tissue contains microglial cells and monocyte-derived macrophages. It is an established fact that monocyte recruiting promotes the tumor growth and dissemination. Monocyte chemotactic protein-1 (MCP-1) is the major attractant for monocytes. We have previously synthesized an MCP-1 antagonist ingramon, a synthetic peptide fragment (65–76) of this chemokine. In the present study, we demonstrated that glioma-conditioned medium contains MCP-1 and stimulates migration of blood monocytes. Ingramon inhibited the effect of glioma-conditioned medium on monocyte migration.

We fabricated bioresorbable microcarriers from water solution of Bombyx mori silk fi broin. The microcarriers are 3D structures with intricate surface and pores allowing penetration of culture medium, gas exchange, and cell adhesion. Fibroin molecules form hydrophobic structures and normally have a negative charge, which stimulates migration, but inhibits cell adhesion and makes it less effective. In order to improve adhesion efficiency and velocity, gelatin (hydrophilic biopolymer with integrin-recognizing RGD sequence) was added to the microcarrier composition. The resultant bioresorbable microcarriers support adhesion and proliferation of 3T3 murine fibroblasts.

Real-time quantitative PCR (Dentofl or kit) was used to detect DNA of periodontal pathogens in specimens from 92 patients with chronic periodontitis and from a control sample of 12 normal subjects. A bimodal distribution of patients by periodontium colonization with A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythensis, and T. denticola was demonstrated. A new approach to interpretation of the results of quantitative evaluation of periodontal pathogens, including the notion of pathological colonization level, led to classification of all cases with chronic generalized periodontitis into 3 groups: associated with A. actinomycetemcomitans, with T. forsythensis/T. denticola complex, and cases of uncertain genesis.

The study examined the effect of VEGF and erythropoietin on proliferative and migratory activities of skin fibroblasts and multipotent mesenchymal stromal cells of human adipose tissue. VEGF stimulated proliferation and migration of fi broblasts, but produced no significant effect on functional activity of multipotent mesenchymal stem cells. Erythropoietin stimulated proliferation of both cell types, but did not affect their migration.

Cytomorphological signs of bacterial infection agents were studied by atomic force microscopy. Analysis of the elastic mechanical characteristics of Staphylococcus spp. from the skin of patients with chronic dermatoses showed lower elasticity of S. aureus cell membrane in comparison with that of transitory flora representatives. Significant differences in characteristics of cell membrane relief and presence of fimA pathogenicity factor were detected in E. coli isolated from the reproductive tract mucosa of clinically healthy women and patients with inflammatory urogenital infections.

The tumor-suppressive effect of rat mesenchymal stem cells against low-differentiated rat C6 glioma cells during their direct and indirect co-culturing and during culturing of C6 glioma cells in the medium conditioned by mesenchymal stem cells was studied in an in vitro experiment. The most pronounced antitumor activity of mesenchymal stem cells was observed during direct co-culturing with C6 glioma cells. The number of live C6 glioma cells during indirect co-culturing and during culturing in conditioned medium was slightly higher than during direct co-culturing, but significantly differed from the control (C6 glioma cells cultured in medium conditioned by C6 glioma cells). The cytotoxic effect of medium conditioned by mesenchymal stem cells was not related to medium depletion by glioma cells during their growth. The medium conditioned by other “non-stem” cells (rat astrocytes and fibroblasts) produced no tumor-suppressive effect. Rat mesenchymal stem cells, similar to rat C6 glioma cells express connexin 43, the main astroglial gap junction protein. During co-culturing, mesenchymal stem cells and glioma C6 cells formed functionally active gap junctions. Gap junction blockade with connexon inhibitor carbenoxolone attenuated the antitumor effect observed during direct co-culturing of C6 glioma cells and mesenchymal stem cells to the level produced by conditioned medium. Cell–cell signaling mediated by gap junctions can be a mechanism of the tumor-suppressive effect of mesenchymal stem cells against C6 glioma cells. This phenomenon can be used for the development of new methods of cell therapy for high-grade malignant gliomas.