Vol 19, No 4 (2019)

Immunohistochemical markers for neurobiology
Korzhevskii D.E., Grigor’ev I.P., Gusel’nikova V.V., Kolos E.A., Petrova E.S., Kirik O.V., Sufieva D.A., Razenkova V.A., Antipova M.V., Chernysh M.V.

In neurobiological studies, crucial is the selection of most appropriate and informative experimental methods, one of which is immunohistochemistry. This review briefly summarizes the experience of adaptation of immunohistochemical methods to nervous system studies accumulated over years the Laboratory of Functional Morphology of the Central and Peripheral Nervous System (Institute of Experimental Medicine). The aim of this work was to determine the most effective and reliable immunomarkers for neurobiological studies. The article contains theoretical basis and practical recommendations for use of key cytospecific and functional markers used in studies of structural and functional organization of brain and spinal cord of mammalian animals and human. In particular, the results of immunohistochemical reactions to neural markers (NeuN, neurofilament proteins, alpha-tubulin, alpha-synuclein), neurotransmitter synthesizing enzymes (tyrosine hydroxylase, glutamate decarboxylase, choline acetyltransferase, NO synthase) and glial markers (GFAP, glutamine synthetase, Iba-1, vimentin) are demonstrated. The presented methodology is useful for experimental neurobiology and clinical morphological diagnostics.

Medical academic journal. 2019;19(4):7-24
Molecular diagnostics in oncology: new trends
Imyanitov E.N.

Molecular diagnostics is a mandatory component of modern clinical oncology. The most known examples of molecular diagnostic procedures include the detection of hereditary cancer syndromes and the analysis of somatic drug-sensitizing mutations in protein kinases. Advances in cancer research as well as the development of new technologies led to emergence of new trends in this area of medicine. The invention of next generation sequencing (NGS) has a potential to dramatically change the landscape of molecular diagnostics. NGS allows to significantly improve the efficiency and availability of genetic testing for hereditary cancers as well as to undertake comprehensive tumor mutation profiling to guide the therapy choice. Tumors usually change their properties during therapeutic intervention. Monitoring of these properties is important for proper selection of further treatment options. So-called “liquid biopsy” is essential for this purpose, as it allows to detect key molecular features of the tumors by a non-invasive approach. There is an increasing popularity of ex vivo tumor models, which allow to cultivate tumor cells and to select the therapy based on the results of drug sensitivity tests.

Medical academic journal. 2019;19(4):25-32
SAS Enterprise Guide 6.1 for physicians: multiple comparisons
Bunenkov N.S., Bunenkova G.F., Komok V.V., Grinenko O.A., Nemkov A.S.

Objective: to develop algorithm of multiple comparisons data of prospective non-randomized clinical trial AMIRI – CABG (ClinicalTrials.gov Identifier: NCT03050489) using SAS Enterprise Guide 6.1.

Materials and methods. Data collection was performed according prospective non-randomized clinical trial AMIRI — CABG in Pavlov First St. Petersburg State Medical University, Saint Petersburg, Russia between 2016–2019 years with 336 patients. There is database with clinical, laboratory and instrumental data. Multiple comparisons test was performed with SAS Enterprise Guide 6.1.

Results. There was developed algorithm of multiple comparisons data of prospective non-randomized clinical trial AMIRI — CABG for independent group. This algorithm could be useful for physicians and researchers for data analysis.

Conclusion. Presented algorithm of data analysis could make easier and improve efficient data analysis. SAS Enterprise Guide 6.1 allows fast and accurate process data

Medical academic journal. 2019;19(4):33-40
Basic studies
Antidepressant-like action of hypoxic postconditioning is accompanied by the up-regulation of hippocampal HIF-1α and erythropoietin
Zenko M.Y., Rybnikova E.A.

Objective. The aim of the study was to reveal the role of HIF-1α in the effects of hypoxic postconditioning in the rat experimental model of depression “learned helplessness”.

Materials and methods. The studies were performed in the “learned helplessness” paradigm which represents a reliable experimental model of depression in rats. The development of the pathology was evaluated in the behavioral open field test and by the baseline level of plasma corticosterone. Correction of behavioral deficit was performed by three episodes of hypoxic postconditioning (360 mmHg, 2 h). Changes in the immunopositivity of HIF-1α and erythropoietin in the hippocampus of rats were evaluated. An inhibitor of HIF-1α subunit translation topotecan (1 mg/kg, i.p., Santa Cruz, USA) was used on the 4th day after the footshock stress. On the 9th day, animals were tested in the open field test to assess the level of depressive-like behavior.

Results. It was shown that postconditioning by three episodes of mild hypobaric hypoxia resulted in the correction of behavioral deficit produced by the “learned helplessness” aversive stress, and the levels of corticosterone did not differ from the baseline in these animals. These behavioral and hormonal effects were accompanied by the increased level of immunopositive HIF-1α and its transcriptional downstream target erythropoietin in the dorsal and ventral hippocampus. Using of HIF-1 inhibitor topotecan dramatically worsen the severity of the depressive-like symptoms.

Conclusion. The findings suggest that HIF-1 appears to have the antidepressant-like activities and that hypoxic postconditioning-induced stimulation of HIF-1α and erythropoietin level might contribute to the endogenous mechanisms which compensate for the pathogenic effects of stressors, particularly for the development of stress-induced depression.

Medical academic journal. 2019;19(4):41-46
Kinetic features of the dopamine release and uptake in the dorsal and ventral striatum of rats
Mukhin V.N., Borovets I.R., Sizov V.V., Pavlov K.I., Klimenko V.M.

Kinetics of the evoked dopamine release and subsequent uptake in the parts of the rat striatum has not been studied sufficiently.

The aim of this study is to fill this gap and to investigate kinetics of dopamine release and uptake in vivo so that comparison can be made between the dorsal and the parts of the ventral striatum and with taking into account the overlapping electrochemical factors during the subsequent analysis of voltammetry recordings.

Materials and methods. The evoked wave of dopamine release and uptake in the dorsal striatum, core, and shell of the nucleus accumbens in the different groups of rats was recorded by the fast-scan cyclic voltammetry. Voltammetry recordings were subjected to principal component analysis and only the components associated with dopamine were taken for further analysis. The values of the parameters of the curves of dopamine release and uptake were defined. Then factor and variance analyses of the parameters were carried out.

Results. Factor analysis showed that the set of parameters of the dopamine wave can be reduced to the 4 factors that are comparable with the variables of the known from the literature mathematical model that describes the dopamine wave based on the Michaelis–Menten equation. Two of the factors and the corresponding parameters of the dopamine curve differ within the dorsal and ventral striatum. Factor 1 is associated with the parameters HL, T80_20, T20_0, slope_T20T0, which are significantly larger in the core of the nucleus accumbens. Factor 3 is associated with the parameters T50_2, AUC, FWHH, T100_80 which are significantly less in the dorsal striatum.

Conclusions. The parameters of the curve of dopamine release and uptake are determined by 4 factors. Among the dopamine curve parameters, the best measures of the factors are T50_1, DAC, T100_80 и T20_0. The kinetics of stimulated dopamine release and uptake varies within the dorsal and ventral striatum. The final phase of dopamine uptake is slowed in the core of the nucleus accumbens in comparison to the shell, and the dorsal striatum. The slope of initial phase of dopamine uptake in the dorsal striatum is steeper.

Medical academic journal. 2019;19(4):47-54
Influence of individual psychological features on the functional state of aviation specialists in conditions of hypobaric hypoxia
Blaginin А.A., Kaltygin M.V., Kotov O.V., Sinelnikov S.N., Dzankisov R.A.

The study is devoted to the study of the influence of individual psychological characteristics of the personality of aviation specialists on the tolerability of moderate degrees of hypobaric hypoxia, simulated in a pressure chamber. The study included the assessment of physiological parameters (blood pressure, heart rate, arterial blood saturation, Roufier index) and psychophysiological indicators (critical frequency of light flickering fusion, complex sensorimotor response, indicators of subjective assessment of the state (SAN test)) depending on the level of neuroticism, extroversion and personal anxiety level.

As a result of the study, it was found that the functional state of the body of subjects with high levels of neuroticism in hypobaric hypoxia changes significantly than in subjects with low levels of neuroticism, which is confirmed by a more pronounced decrease in health, activity and mood by 17.5% (p < 0.01), 16.9% (p < 0.05) and 9% (p < 0.01), respectively, an increase in the time of complex sensorimotor response by 12.6% (p < 0.01), as well as more pronounced functional changes in the cardiovascular system.

It was revealed that changes in some physiological parameters in extroverts had a large negative dynamics in hypoxia, so the increase in the Roufier index in the group of introverts at 30, 60 minutes and after the rise by 97.3%, 98.6% and 15.1%, respectively, against an increase of 118.2%, 123.6% and 16.4% in the group of extroverts was revealed. In the group of extroverts, an increase in the time of complex sensorimotor response after the end of the study was found by 8.0% (p < 0.05), an increase in critical frequency of light flickering fusion compared to the indicators obtained before the rise by 5.2% (p < 0.05).

It is established that the functional status of subjects with high levels of trait anxiety had a more pronounced negative trend in terms of the effects of hypobaric hypoxia, which is confirmed by a significant higher values of the Roufier index in groups with high levels of trait anxiety, with the background study, 30th, 60th minute and after rising; extending the time complex sensorimotor response to 45 minute 6.1% (p < 0.05), and after altitude study by 9.5% (p < 0.01); by the 40th minute of stay in the hyperbaric chamber the indicators of well-being decreased by 20.3% (p < 0.01), activity by 18.8% (p < 0.05) and mood by 13.7% (p < 0.01).

Thus, it was shown that in assessing the tolerability of moderate degrees of hypoxia, it is necessary to take into account the dependence of changes in the functional state of the organism on individual psychological characteristics.

Medical academic journal. 2019;19(4):55-66
Effect of long-term emotional-painful stress on the leukocyte composition of blood in rats with different levels of excitability of the nervous system
Shalaginova I.G., Sheremet V.V., Khlebaeva D.A., Vaido A.I., Dyuzhikova N.A.

Post-stress states in animals and humans are accompanied by the development of neuro- and peripheral inflammation. The mechanisms of such immune dysfunctions, their contribution to the pathogenesis of stress-related diseases, as well as the dependence of the intensity of poststress inflammation on genetically determined features of the nervous system, have not been clarified.

Aim: to assess the dynamics of the development of poststress inflammation depending on the genetically determined level of excitability of the nervous system in rats.

Materials and methods. The study was carried out on male rats of two lines, selected by the threshold of excitability of the nervous system-the line HT (high threshold of excitability) and LT (low threshold of excitability). As a model of chronic stress, the Protocol of long-term emotional and pain exposure according to Hecht was used. To investigate the dynamics of changes in the leukocyte formula, three time points were selected: 24 hours, 7 days and 24 days after the stressor. Morphological analysis of blood was carried out to determine the leukogram, for which the leukocytes were counted in a blood smear stained by Romanovsky-Gimza.

Results. Chronic stress leads to an increase in the leukocyte shift index only in the experimental group of highly excitable rats of the LT line compared with control animals of the same line. The significance of the differences is confirmed on day 7 after the end of the stressor. No interline differences in neutrophil/lymphocyte ratios were found in intact animals of LT and HT lines.

Conclusion. In rats with a genetically determined high level of excitability of the nervous system, post-stressor systemic inflammation appears 7 days after the end of the stressor. Animals with a low level of excitability of the nervous system had no signs of post-stress inflammation throughout the observations. The article discusses the possible mechanisms of detected immune dysfunctions in animals due to high excitability of the central nervous system.

Medical academic journal. 2019;19(4):67-74
Correction of acid base disorders in rats with acute ethylene glycol poisoning
Sivak K.V., Lyubishin M.M., Kalinina E.Y.

The aim of the article. The aim of this study was to evaluation of the effectiveness of standard antidote therapy and dimephosphon administration in rats with acute ethylene glycol poisoning. The tasks of the study included modeling acute ethylene glycol poisoning in rats, conducting experimental therapy with ethanol antidote in combination with sodium bicarbonate and dimephosphon therapy, comparing the effectiveness of drugs in relation to indicators of acid-base state impairment and renal function.

Materials and methods. Ethylene glycol (EG) was administered per os to Wistar male rats (190–210 g b.w.) at a single dose of 6 mL/kg b.w. through an atraumatic gastric tube. The animals were divided into 4 groups of 6 individuals each: intact (negative control), EG poisoning (positive control), EG + standard antidote therapy, EG + dimephosphon therapy. Experimental therapy was carried out for first 24 hours using standard antidote therapy: ethanol (30% solution 2 mL/kg b.w. i.p. after 1, 4, 6, 12, 18 hours) and sodium bicarbonate (4 % solution 6 mL/kg b.w. i.p. 3 times on the first day), as well as administration of dimephosphon (150 mg/kg i.p. 3 times on the first day, 450 mg/kg b.w. per day). Daily urine on day 3 after poisoning was collected in metabolic cages. Creatinine concentration in urine and blood serum samples were measured, and creatinine clearance was calculated. After 24 hours of therapy, the pH, level of sodium, potassium, calcium, magnesium, chlorides, bicarbonates, lactate, D-3-hydroxybutyrate, albumin, urea and creatinine (measured parameters) were determined in venous blood samples. Anion gap, ∆рН, ∆AG, ∆HCO3, ∆AG/∆HCO3 and ∆Gap were calculated. The mechanism of death was determined for the dead animals. Data processing was performed using GraphPad Prism 6.0.

Results. Acute poisoning of rats with ethylene glycol leads to the development of toxic encephalopathy and nephropathy, acid-base abnormalities, high anion gap metabolic acidosis due to the presence of metabolites, as well as lactate-ketoacidosis due to depression of the central nervous system. 100% of the EG-treated (12 mL/kg b.w.) animals died within 3 days. Metabolic acidosis in combination with hypermagnesemia had provided a cardiodepressive effect, which with direct nephrotoxic and neurotoxic effects contributed to the development of a mixed variant of thanatogenesis and death. Death comes from toxic encephalopathy and nephropathy, high anion gap metabolic acidosis caused by direct nephrotoxic and neurotoxic effects of EG and its metabolites. The standard antidote therapy with ethanol in combination with sodium bicarbonate prevented a pH shift, lactic acidosis and ketoacidosis, an increase in urea, but did not affect the level of bicarbonate (p = 0,048), creatinine and its clearance (p = 0,037) and the anion gap (p = 0,033). The dimephosphon therapy prevented a decrease in creatinine clearance and blood bicarbonate level, limited the increase in lactate dehydrogenase activity, had a more pronounced effect on the AG and ∆AG (p = 0,042), but did not affect the hypocalcemia (p = 0,0076) and hypoalbuminemia (p = 0,021).

Conclusion. Acute ethylene glycol poisoning leads to the development of a mixed variant of thanatogenesis with damage to the central nervous and urinary systems, as well as the heart. Autopsy and histopathology confirmed the cause of animal death. In the model at a dose of 6 mL/kg of EG the dimephosphon therapy was more conducive to the correction of the main markers of high anion gap metabolic acidosis (HAGMA) than standard antidote therapy (both measured and calculated, p < 0,05). The dimephosphon therapy prevented a decrease in creatinine clearance. A comparative analysis of two methods for the correction of high anion gap metabolic acidosis in rats in acute poisoning with ethylene glycol showed that dimephosphon therapy vs. standard antidote therapy had a stronger effect on markers of metabolic acidosis and renal impairment.

Medical academic journal. 2019;19(4):75-86
Influence of PLL treatment on the long-term memory formation in Helix mollusk
Grinkevich L.N.

Relevance. The studies of the epigenetic mechanisms of long-term memory formation (LTM) has attracted the attention of many world leading laboratories since gained knowledge can be applied to correct cognitive impairments. miRNA dependent suppression of gene expression is the most complicated step in the epigenetic regulation, associated with a huge number of miRNAs (tens of thousands) and the diversity of their targets, thus the knowledge of miRNAs functions during LTM is still very fragmented.

Aim. The aim of this study was to investigate the involvement of miRNAs in the formation of long-term memory using the model of the food aversion conditioned reflex development in the mollusk Helix. Prevention of the formation of mature miRNAs via Poly-L-lysine hydrobromide (PLL) treatment — inhibitor of Dicer activity was used as the main approach.

Materials and methods. PLL was injected into animals during training, or 1, 3 or 5 hours after training. Success of the formation of conditioned reflexes was tested 72 hours after training.

Results. There was a significant deterioration in LTM in animals with injected PLL 1 and 3 hours after training procedure compared with trained animals that were not injected with PLL. The treatment with PLL during training, or 5 hours after training, had no effect on LTM.

Conclusion. Treatment with PLL, inhibitor of miRNA biogenesis disrupts formation of the food aversion reflex in Helix. Thus, miNRA’s are involved in the LTM formation on Helix. Impaired expression of miRNAs is critical for the long-term memory formation if occurs in the intervals of 1 to 3 hours after training. We can recommend PLL for the investigations in the area of the epigenetic mechanisms of long-term memory.

Medical academic journal. 2019;19(4):87-92
Analysis of concentration and activity of proteins involved in iron metabolism in rats with streptozotocin-induced hyperglycemia
Voynova I.V., Kostevich V.A., Elizarova A.Y., Karpenko M.N., Sokolov A.V.

Objective. We aimed to analyze the alterations of concentration and activity of iron metabolism proteins in samples obtained from rats with hyperglycemia induced by streptozotocin (STZ).

Materials and methods. Concentration and activity of ceruloplasmin (Cp) and transferrin (Tf), concentration of glucose, fructosamine, hemoglobin, ferritin (Ft), and iron were measured in blood samples obtained from rats after injection of STZ or saline (control). To develop in-house ELISA, highly purified preparations of Cp, Tf, and Ft, as well as specific antibodies against these proteins, were obtained.

Results. The hyperglycemia in rats after STZ injection was confirmed by increase in glucose and fructosamine serum concentrations. Increase in Cp concentration and decrease in specific ferroxidase activity of Cp, concentration of Tf and Ft were observed in hyperglycemic rats. However, the absence of changes in iron concentration and total iron binding capacity of serum is indicative of compensatory response.

Conclusion. STZ-induced hyperglycemia in rats was characterized by alteration of activity and concentration of iron metabolism proteins, however, this alteration was negated by homeostatic response. The alterations of Cp and Tf concentrations observed in this study are similar to those in acute phase of inflammation.

Medical academic journal. 2019;19(4):93-102
The influence of new coumarin derivatives on survival rate of mice in model conditions of acute hypoxia
Rodionova O.M., Safonova A.F., Kashirin A.O., Polukeev V.A., Bychkov E.R., Lebedev A.A., Shabanov P.D.

Objective. The article is devoted to study the anti-hypoxemic properties of new coumarin derivatives in the models of hypoxemic hypoxia with hypercapnia, hemic hypoxia and histotoxic hypoxia.

Materials and methods. Нypoxemic hypoxia with hypercapnia was modeled as follows: mice were placed in hermetic 200 cm3 jars one in a jar. Hemic hypoxia was reproduced in mice by single subcutaneous introduction of sodium nitrite in a dose of 230 mg/kg. Histotoxic hypoxia was caused in mice by intraperitoneal introduction of sodium nitroprusside in a dose of 20 mg/kg. Coumarin derivatives under lab codes IEM-2266 and IEM-2267 were dissolved in distilled water with addition of twin-80, and then a single intraperitoneal infusion of them in doses 25 and 50 mg/kg was made 45 minutes before placing to the model conditions. Increased life time of an animal compared with the control served the criterion of anti-hypoxemic effect of the studied substances.

Results. In hypoxemic hypoxia with hypercapnia test compound under IEM-2267 in doses of 25 and 50 mg/kg increased mice life time by 26 and 34% respectively in comparison with control. In hemic hypoxia model, the positive effect was seen with IEM-2266 compound in a dose of 50 mg/kg which increased life time of animals by 45% in comparison with control. In histotoxic hypoxia model, at preventive introduction of IEM-2266 compound in a dose of 25 mg/kg and IEM-2267 in a dose of 50 mg/kg life time increased up to 117% and 123% respectively.

Conclusion. The coumarin derivatives IEM-2266 and IEM-2267 relieved the course of acute hypoxia and increased life time of animals in the models of hypoxemic hypoxia with hypercapnia, hemic hypoxia and histotoxic hypoxia.

Medical academic journal. 2019;19(4):103-108
Clinical research
Comparative immunohistochemical and morphometric research of interleukin-17 in various atherosclerotic lesions in human
Pigarevsky P.V., Snegova V.A., Maltseva S.V., Davydova N.G.

The aim of the artical — to investigate cellular and tissue localization of IL-17 in various atherosclerotic lesions of arteries of human and on the basis of the obtained data to make a hypothesis of a possible role of Th17 of cells in destabilization of an atherosclerotic plaque.

Material and methods. On autopsy material by means of histologic, immunohistochemical and morphometric research techniques aorta segments, the coronary arteries and a. basilaris with various types of atherosclerotic lesions (43 samples of tissue) were studied. In samples of tissue studied the endothelial and mononuclear cells expressing IL-17.

Results. It is shown that endothelial cells of an intima are capable to produce IL-17 in all types of atherosclerotic plaques. Increase in number of the mononuclear cells expressing IL-17 in an intima of arteries was at the same time revealed. It is shown that the maximum number of the cells expressing IL-17 was found in an intima of an unstable atherosclerotic plaque, it is especially frequent around a rupture of its cap. What can demonstrate pro-inflammatory action of Th17-cells and IL-17 expressed by them and significant effect them on formation of unstable atherosclerotic lesions.

Conclusion. On the basis of the obtained data for the first time it was succeeded to make a hypothesis of a possible role of Th-17 of cells in destabilization of an atherosclerotic plaque.

Medical academic journal. 2019;19(4):109-113

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