Protection Of Vertebrate Animals Research Articles

Indicators of leukocyte blood formula of horses of different breeds

The work highlights the research results of some indicators of the leukocyte blood formula of Hutsul horses and their comparative characteristics with the same indicators of horses of other breeds, of different performance areas, as well as with normative indicators of blood in horse breeding. The research object was directly healthy adult Hutsul horses of the Zakarpattia region, in which blood leukocytes, eosinophils, neutrophils (rod-nuclear, segmental-nuclear), lymphocytes, and monocytes were determined. For comparison, the same average blood values of purebred riding horses, Russian trotters, and New-Alexandrian weight-carrying horses were taken. For research, blood was taken from the horses' jugular vein, and 10 ml of blood was taken. Laboratory studies were conducted according to generally accepted methods. When performing experimental research, all bioethical norms concerning animals were observed, which meet the requirements of the Law of Ukraine No. 3447-4 “On the Protection of Animals from Cruelty Treatment”, the provisions of the European Convention on the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 1986) and the regulation on the use of vertebrate animals for research and other scientific purposes at the Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv. The number of leukocytes, lymphocytes, eosinophils, rod-nuclear, and segment-nuclear neutrophils has clearly expressed characteristics in horses of different performance areas. All these indicators are within the limits of the norm determined for horses. The obtained data allow us to assert the existence of a connection between the number of leukocytes, the blood leukogram of horses with their work performance, which is related to the intensity of oxidation-reduction processes, as well as the natural resistance of the body of Hutsul horses to diseases. The conducted research provides a basis for the organization of the selection process for the reconstruction of the gene pool of the Hutsul breed with the restoration of its inherent qualities.

THE STATE OF THE NITROGEN OXIDE SYSTEM AND SOME INDICATORS OF ANTIOXIDANT PROTECTION OF THE CEREBRAL CORTEX DURING THE INTRODUCTION OF CARBACETAM TO RATS WITH EXPERIMENTAL NEURODEGENERATION

One of the most important medical and social problems of today is the search fornew pathogenetic areas of drug prevention and treatment of neurodegenerativediseases of the central nervous system. The pathogenesis of these processes has notbeen definitively studied, at the same time a number of scientific works testify to thesignificant role of the imbalance of the prooxidant- antioxidant system provoked by thehyperproduction of nitric oxide. Recent studies have also demonstrated the effect of nitricoxide on inhibiting GABA-ergic synaptic transmission. In addition, abnormal expressionof neuronal NO synthase has been found to be an early marker of Alzheimer’s disease andrelated cognitive impairment.Objective – to study the effect of carbacetam on the performance of nitric oxide andantioxidant enzymes in the cerebral cortex of rats under conditions of scopolamineinduced neurodegeneration.Materials and methods. The experiments were performed on nonlinear laboratory whitemale rats with scopolamine- induced neurodegeneration. The studies were performedin compliance with the Council of Europe Convention on the Protection of VertebrateAnimals. Carbacetam was administered intraperitoneally at a dose of 5 mg / kg (14 days).Antioxidant protection was assessed by the activity of superoxide dismutase (SOD) and catalase. The state of the NO system was determined by the content of stablemetabolites of nitric oxide – nitrite anions (NO2), and the activity of NO synthase.Results. In the group of rats with model pathology after 14 days of carbacetam therewas a tendency to decrease the content of NO2 and NO-synthase activity in the cerebralcortex in relation to untreated animals. At the same time, the activity of SOD and catalasedecreased in rats with neurodegeneration. After 14 days of carbacetam, catalase activityincreased and only a tendency to increase the activity of SOD was observed.Conclusion. Therefore, the obtained results allow judging the corrective effectof carbacetam on the nitric oxide system and the activation of antioxidant protection inthe cerebral cortex of rats with scopolamine- induced neurodegeneration.

PATHOMORPHOLOGICAL CHANGES IN THE RETINAL LAYERS AT THE END OF THE SEVENTH DAY OF EXPERIMENTAL OPIOID EXPOSURE

The aim of the work was to clarify the fundamental issues regarding the dynamics of early changes in the retinal layers under experimental exposure of opioids. Research was done on 15 adult 4-months-old mature male mongrel rats, having 100.0-160.0 g weight. Opioid eff ects were modelled by daily intramuscular injections of nalbuphine 1 time per day in one period of time (10-11 am) for 7 days. The initial dose of nalbuphine was 0.212 mg ̸kg. Histological slides were prepared according to generally accepted methods using the dyes, as hematoxylin, eosin and azan according to the Heidenhain method.All animals were kept in the vivarium and the work was carried out according to the European Convention for t he Protection of Vertebrate Animals used for experimental and other scientifi c purposes [Strasbourg, 1985]. During the experimental opioid administration after one week of research the changes in the structure of the retinal layers were established. In the early stages of low-dose opioid analgesia administration, the fi rst pathomorphological changes were de tected during the fi rst week, in the form of initial manifestations of vacuole dystrophy of the pigmental epithelium, signs of microcystic degeneration of the outer retinal and neurofi brous layers with the vacuole dystrophy of retinal ganglion cells appearance.

Morphological and morphometrical characteristics of cattle heart structure

The paper presents the results of macro- and microscopic structure of cattle heart on the organ-, tissue- and cell levels. The samples of the selected material (n = 5) were preserved in a 10–12 % water solution of neutral formaline with its further charging into wax. Histologic sections not more than 10 mkm were made from wax blocks by using a sliding microtome MC-2. Hematoxilin- and eosin staining of histological sections by Heydengine technique was used for studying cell morphology, carrying out morphometrical studies and for receiving review samples. Histoarchitecture of the structural parts of the heart (ventricle and auricle) and their morphometrical parameters were studied on the histological preparations using the light microscopy technique. The experimantal part of the scientific research was carried out in compliance with the requirements of “European Convention for the Protection of Vertebrate Animals used for Experimantal and other Scientific Purposes” (Strussburg, 1986). The cattle heart is located in a thorax between two lungs, in front of a diaphragm and shifted left. In the 3rd–4th rib region the heart adjacents to a thoracic wall. The heart apex is in the 5th rib region. The absolute weight of a cattle heart equals 2143.27 ± 38.76 g, the relative weight equals – 0.43 ± 0.006 %. The net weight of the heart without the epicardial fat equals 1926.12 ± 31.12 g. Herewith, the weight of the aortic ventricle equals 978.54 ± 19.52 g, the weight of the pulmonic ventricle equals 554.17 ± 14.21 g, the weight of both ventricles equals 1539.08 ± 49.74 g. The auricles weight was the least and equaled 397.18 ± 11.21 g. The ratio of the ventricle weight of the heart to its net weight equals 1:0.2, and the ratio of the weight of the auricle myocard to the weight of the ventricle myocard equals 1:0.26. The heart height equaled 23.08 ± 0.11 сm, width – 13.9 ± 0.18 cm and the circumference – 38.08 ± 0.9 cm. According to the analysis of such liniar measurements, the cattle heart in the animals of the experimental group is characterized as that of an enlarged- and short form. The heart wall consists of three sacs – endocardium, myocard and epicardium. The dominating weight of the heart wall is in a muscular layer (myocard), which consists of transversus stripe muscular fibers which are formed on the basis of mononuclear cells – cardiomyocytes which are interlinked into muscular fibers. According to the cytometric analysis of cardiomyocytes, their largest volume – (11225.73 ± 824.42 mkm3) is observed in the aortic ventricle, smaller – (7963.60 ± 627.09 mkm3) – in a pulmonic ventricle and the smallest – (5361.60 ± 583.91 mkm3) in the auricle cardiomyocytes. Herewith, the volumes of cardiomyocytes nuclei in an aortic ventricle (124.55 ± 7.99 mkm3 and in a pulmonuc ventricle (121.67 ± 7.02 mkm3) are nearly the same, and in the auricle cardiomyocytes the nuclei volume is significantly smaller and it equals 101.05 ± 6.04 mkm3 respectively. Such morphometrical parameters of cardiomyocytes and their nuclei are evidenced in their nuclei-cytoplasmatic ratio, which is the smallest in the cardiomyocytes of an aortic ventricle – 0.0113 ± 0.0068, somewhat larger in a pulmonic ventricle – 0.0156 ± 0.0054 and the largest – 0.0234± 0.0058 in the auricle cardiomyocytes, that is connected with the special properties of the muscular tissue of a myocard which is capable of spontaneous rythmic beatings depending on their morphofunctional load: the ventricles pump the blood from the heart to the body performing the gratest load (the aortic ventricle acts a s a pump, and the pulmonic ventricle acts as a container), the auricles receive the blood which returns to the heart from the animal body, performing somewhat smaller load.

Immunohistochemical Characteristics of Ischemic Process in Hippocampal Formation in Posterity of Female Rats after Labor Induction

The response to perinatal hypoxia, developing in stimulating generic activity, is the activation of microglia, which induces the development of local inflammation of the brain and leads to the death of neurons. The formation of the hippocampus supports important physiological and behavioral functions, including spatial learning and memory, and is part of the brain, which is especially vulnerable to changes in blood glucose and oxygen. Thus, the study of the features of the development of hippocampal formation in the postnatal period after stimulating generic activity is relevant. The purpose of the study was to study the features of Glial fibrillary acidic protein and NeuN expression in the hippocampal formation in posterity of female rats after PgE2 injection for labor induction. Materials and methods. Pregnant females of the experimental group on the twenty-second day of pregnancy were injected intravaginally with PGE2 in the form of a gel to stimulate the generic activity. Birth occurred on the twenty-third day after conception. Birth in the intact group of rats occurred on the 23-24th day after conception. The large hemispheres of the rat brain were fixed in a 10% neutral formal solution, dehydrated in an ascending alcohol battery. For immunohistochemical studies, paraffin sections were used with 3 microns with a thickness of 3 microns. Glial fibrillary acidic protein Mouse Monoclonal Antibody (Santa Cruz Biotechnology, Inc.) is used to detect astrocytes (Santa Cruz Biotechnology, Inc.). NeuN Mouse Monoclonal Antibody (Santa Cruz Biotechnology, Inc.) was used to identify neurons on the 1st, 7th, 14th, 45th days after birth in histological cuts of hippocampus and gear, using a program for analyzing and processing images Image J, studied the relative area occupied by Glial fibrillary acidic protein + NeuN + cells. Data is processed by variation statistics. The results are reliable at p <0.05. All animal experiments were performed according to international principles of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes” (Strasbourg, 18.03.86) and the Law of Ukraine No. 1759-VI (15.12.2009) On the Protection of Animals from Cruelty. Results and discussion. We identified that by the changes in the hippocampus and the toothed gyrus of the rats after induction of labor, increased Glial fibrillary acidic protein expression on the first day after birth and reducing the NeuN expression on the 14th and 45th day of life in experimental animals compared to the control group were included. Conclusion. The density of the distribution of Glial fibrillary acidic protein + cells in the hippocampus and the dentate gyrus of rats changes wavily throughout the first month and a half of postnatal life. In the offspring of rats, after stimulation of labor on the first day of life, a reliable increase in the relative area occupied by Glial fibrillary acidic protein + astrocytes is determined compared to the control (49.3±2.6% and 36.8±5.9%, respectively). In the hippocampus and the dentate gyrus of rats after stimulation of labor during the first 45 days after birth, the gradual decrease in the relative area occupied by NeuN + neurons is determined, in contrast to the control animals, in which this index practically does not change. On the 14th and 45th days, the index of the relative area occupied by NeuN + neurons in experimental rats is reliably lower than in control (p <0.05).

The activity of the enzyme link of the antioxidant defense system of cows organism in experimental fasciolosis

The antioxidant defense system of the organism controls and inhibits all stages of free radical reactions, starting from their initiation and ending to the formation of hydroperoxides and TBA-active products. The main mechanism of control of these reactions is associated with the chain of reversible redox reactions of metal ions, glutathione, ascorbate, tocopherol and other substances, the value of which is especially important for the preservation of long-standing macromolecules of nucleic acids and proteins, some components of membranes. The purpose of the research was to investigate the activity of the enzyme link of the antioxidant defense system of cows organism in experimental fasciolosis. Twelve cows of 4–5 years of age, Black-Spotted breed, were selected for the experiments, from which 2 groups were formed, six animals in each. Animals in control group (C) were clinically healthy. Animals of experimental group (E) were experimentally infected with adolescents. During the research, the rules of compulsory experiments were followed – selection and keeping of analogue animals in groups. The cows' ration was balanced in terms of nutrients and minerals. All animal manipulations were carried out in accordance with the European Convention for the Protection of Vertebrate Animals, which are used for experimental and scientific purposes. According to the results of our research, in cows affected by fasciolosis invasion, catalase activity in their blood decreased by 21.5 %, and superoxide dismutase activity by 30 %. In the research of the glutathione link of the antioxidant defense system, it was found that the activity of both glutathione peroxidase and glutathione reductase is also reduced in experimental fasciolosis. Thus, on the 25th day of the experiment, a decrease in these enzymes by 26.6 and 20.5 % is set. Thus, with the development of fasciolosis in ruminants, the balance in the complex “Antioxidant system – Lipid peroxidation” is disturbed, which in turn leads to complications of the disease. In the future it is planned to investigate the state of the non-enzymatic part of the antioxidant defense system of cows organism with experimental fasciolosis.

Morphological and morphometric changes on the background of cell cardiomyoplasty in experimental myocardial infarction

The aim: to study the morphological and morphometric changes in the myocardium against the background of cellular cardiomyoplasty in experimental myocardial infarction.Materials and methods: the experiment was carried out on 142 Wistar-Kyoto rats weighing 200-220 g, which were kept in the vivarium of the Department of Experimental Surgery of the State Institution “Institute of Emergency and Reconstructive Surgery named after V. K. Gusak of the National Academy of Medical Sciences of Ukraine” in the period from 2012 to 2013. The Wistar-Kyoto breed was used because it is inbred, which minimizes the rejection reaction, due to its genetic homogeneity. The animals were kept in a vivarium under conditions of 12-hour daylight hours, room temperature and access to water and food at libitum at an air temperature of +20 – + 22 °C, humidity no more than 50 %, in a light mode - day-night. The use of animals in the experiment was carried out in accordance with the rules regulated by the “European Convention for the Supervision and Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes” (Strasbourg, 1986), Directives of the Council of the European Union of November 24, 1986 and the order of the Ministry of Health of Ukraine No. 32 dated 02.22.88. The induction of myocardial infarction (MI) was carried out according to the technique developed by us under general anesthesia. A separate group consisted of 20 males, whom we used as donors of mesenchymal stem cells (MSC) for further research on the Y chromosome of cell homing in the body. Cultivation of MSCs was carried out in a mixture of nutrient media DMEM / F12, 1:1, (Sigma, USA). The material for morphological studies was the sections of the myocardium of laboratory animals. To assess the morphometric parameters, histochemical methods were performed according to the recipes, which are given in the instructions for histochemistry. Immunohistochemical study was performed on paraffin sections with a thickness of 5-6 μm by the indirect Koons method according to the Brosman method (1979).Results: it was found that cellular cardiomyoplasty significantly improves the structure of the postinfarction heart, manifests itself in a decrease in the scar area and connective tissue, respectively, in an increase in the number of vessels and the percentage of preserved muscle fibers. The best results were achieved with intramyocardial injection, which requires confirmation of this fact in a clinical study.Conclusions: cellular cardiomyoplasty with any method of introducing a cell graft has a positive effect both on the morphological substrate of the heart in the form of a decrease in the size of the scar during postinfarction remodeling, an increase in the number of newly formed vessels and an increase in the percentage of preserved cardiomyocytes. This occurs due to the homing of MSCs into the ischemic zone and the commonality of two mechanisms – direct differentiation into endothelial cells of the heart vessels, as well as due to the paracrine effect

The pharmacological safety of a combined pharmaceutical composition for the correction of placental dysfunction in pregnant women

Topicality. Along with the pronounced pharmacological effectiveness, high requirements for the safety are applied to new medicines. To obtain the necessary information regarding the safety of a potential drug and predict the risk of further side effects, a preclinical study of possible toxic effects, primarily acute toxicity, is performed.Aim. To study the acute toxicity of a new pharmaceutical composition for the correction of placental dysfunction in pregnant women.Materials and methods. The study object was the pharmaceutical composition in the form of solid gelatinous capsules containing active pharmaceutical ingredients from basic therapeutic group of drugs used for fetoplacental dysfunction in pregnant women. The acute toxicity was determined according to the methodological guidelines “Preclinical studies of drugs” edited by A. Stefanov, in outbreed female rats weighing 200-250 g in compliance with the “European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes” (Strasburg, 1985).Results and discussion. The study of the pharmaceutical safety has been performed by the acute toxicity criterion under the condition of short-term influence of a new pharmaceutical composition combining the required pharmaceutical ingredients in well-balanced doses affecting different chains of pathogenesis of placental dysfunction. Under the condition of the acute experiment a new combined composition (intraperitoneal introduction in the dose of 5000 mg/kg of the body weight) does not cause changes in the rat’s organism. It has been determined that this pharmaceutical composition belongs to practically nontoxic substances – Toxicity Class V according to the generally accepted classification system.Conclusions. The data for the preclinical safety assessment of a new combined composition for the correction of placental dysfunction in pregnant women have been obtained.

PECULIARITIES OF MORPHOFUNCTIONAL CHANGES OF MASTICATORY MUSCLES IN IODINE-DEFICIENT CONDITIONS

The problem of iodine deficiency is becoming increasingly important in Ukraine. According to the United Nations Children’s Fund (UNICEF), each and all regions of Ukraine suffer from “hidden hunger”. The World Health Assembly has stated that the elimination of iodine deficiency will be as much a triumph for health care system as the eradication of smallpox and poliomyelitis. The goal of this study was to establish the peculiarities of structural organization of the masticatory muscles of rats in experimentally induced iodine deficiency. The research material involved masticatory muscle of 36 white outbred male rats, which were divided into groups: group І – intact animals; group ІІ – kept in iodine deficiency condition; group III – exposed to iodine deficiency with the addition of goitriferous food. All manipulations with test animals were performed in agreement with the regulations on the protection of vertebrate animals. The methods used in the study included: biochemical, histological, histochemical, submicroscopic, morphometric and statistical research methods. It has been established that under the conditions of iodine deficiency the lumen of arterial vessels in masticatory muscles becomes narrowed, the vascular vessels become thickened due to membrane oedema. The number of hemocapillaries per unit area of muscle fibre goes down in all the studied muscles. The number of muscle fibres with intermediate succinate dehydrogenase activity decreases with the simultaneous increase in muscle fibres with low succinate dehydrogenase activity. Furthermore, the oedematous changes are the most prominent in muscle fibres with low succinate dehydrogenase activity. In conditions of iodine deficiency with the addition of goitriferous products, we can observe the progression of luminal narrowing of the artery and wall thickening in masticatory muscles where oedematous-dystrophic changes are considerably marked. There is a tendency in the growth in haemocapillaries number per 1 μm2. Histostructurally, the loss of cross striation is quite noticeable. The cross-section diameter increases, especially in muscle fibres with low succinate dehydrogenase activity and muscle fibres with high succinate dehydrogenase activity in the masticatory muscle proper of immature animals. Ultrastructurally, the oedematous-dystrophic changes of myofibrils and mitochondria were revealed. Thus, under the conditions of iodine deficient diet the lumen of arterial vessels narrows in the masticatory muscles, their wall thickens due to oedema of the membranes, which progresses in iodine deficient diet with the addition of goitriferous products. The number of hemocapillaries per unit area of muscle fibre decreases in both study groups. Changes in vascular bed lead to the redistribution of the number of muscle fibres and their significant oedema.

Вплив L-глутамінової кислоти та L-цистеїну на показники гемопоезу та імунного статусу щурів за токсичної дії фосфорорганічних сполук

The rapidly increasing use of xenobiotics in our life stimulates to pay greater attention to the toxic effect of these compounds on human and animal organism. The purpose of our work was to study the changes in hematological and immunological parameters of rat blood under the action of chlorpyrifos (CPF) and additional administration of L-glutamic acid (L-Glu) and L-cysteine (L-Cys) both separately and in the complex. Material and methods. Studies were conducted on albino Wistar rats (males), weighing 200–220 g. They were housed in cages under standard laboratory conditions with a 12-h light/12-h dark cycle. All rats had free access to a standard rodent diet and water ad libitum. After 1 week of acclimatization, the rats were divided into five experimental groups. Animals from first (D1), second (D2), third (D3) and fourth (D4) experimental groups were intraperitoneally exposed to chlorpyrifos. After that rats from the second experimental group were treated with an aqueous solution of L-Glu rats of the third experimental group received L-Glu and L-Cys, rats of the fourth experimental group took L-Cys. Rats of the control group were administered to the appropriate amount of saline. Rats of the control group were administered to the appropriate amount of saline. After the end of exposure period, animals were euthanized. The total number of erythrocytes, leukocytes, white blood cell count, indicators of T- and B-cell immunity was determined in the blood. All procedures were conducted according to the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes (Strasbourg, 1986) and General Ethical Principles of Experiments using Animals (First National Congress of Bioethics, Kyiv, 2001), in accordance with current legislation on animal experimentation in Ukraine. Results and discussion. Our results showed that the chlorpyrifos group of animals reflected in decrease in the total number of erythrocytes, the number of lymphocytes on the background of neutrophil increasing, changes in the number of total T-lymphocytes with medium receptor density. Conclusion. Additional administration of amino acids had a positive effect on animals

EXPERIMENTAL RESEARCH OF THE POSSIBILITIES OF INFLUENCE ON HEALING SMALL INTESTINAL ANASTOMOSIS

Abstract. A promising area of further research is the use of a combination of components of tissue growth factors in combination with low-intensity electromagnetic effects of light radiation.
 Objective. Investigation of various methods of influence on the healing of sutures of the small intestinal anastomosis in conditions of peritonitis.
 Materials and methods. The experiment was conducted on 20 white Wistar male rats weighing 180-250 g, the content, care and methods of experimental work with which corresponded to the observance of the International Principles of the European Convention on the Protection of Vertebrate Animals (Strasbourg, 1985)
 Results. The research work reflects the results of an experimental study, the purpose of which was to develop a method of sealing the small intestinal anastomosis under peritonitis. The experiment was performed on 20 white Wistar male rats weighing about 250 grams. The possibilities of using physical (light of a certain wavelength) and biological (tissue growth factors) methods in the prevention and treatment of acute inflammatory reactions and stimulation of reparative processes are shown. It is noted that a promising direction for further research on methods for the prevention of insolvency of small bowel anastomoses is the use of electromagnetic effects of low intensity of the light spectrum in combination with the application of tissue growth factors.
 Conclusion. A promising direction for further research is the use of a combination of sealing lines with the application of components of tissue growth factors in combination with a low-intensity electromagnetic effect of light radiation.

EVALUATION OF ANTITUMOR ACTIVITY OF TUMOR NECROSIS FACTOR ALPHA WITHIN THE ARTIFICIAL VIRUS-LIKE PARTICLE

Introduction. Tumor necrosis factor α (TNF-α) is a natural cytokine, characterized by pronounced antitumor properties. A wide range of side effects serves as an obstacle for the use of TNF-α in clinical practice. One of the ways to improve its therapeutic properties is to increase the tropism of the cytokine to the tumor tissue by incorporating it into the targeted delivery system.The aim of the study was to evaluate the antitumor activity of the preparation containing TNF-α as part of the artificial “virus-like particle” (VLP-TNF-α), developed in SRC VB “Vector” as a transport system for delivering proteins to target cells.Materials and methods. The antitumor effect of VLP-TNF-α preparation was evaluated in experimental B16F10 melanoma model by the change of dynamics of tumor growth (volume, mass) and its morphological structure (presence of necrotic processes, blood vessel destruction). The number of the effector immune cells (CD3+, CD11b+) in the tumor tissue was determined by immunohistochemical method.Results. It has been shown that VLP-TNF-α administered intravenously at the doses of 5 × 104 and 1 × 105 IU/mouse inhibits the growth of the primary tumor. The most pronounced and stable effect was observed with a five-fold administration at the dose of 1 × 105 IU/mouse every other day: tumor growth inhibition was 40 % on the 1st day, and 47 % on the 7 th day upon the treatment. Injections of the preparation resulted in the increase of necrosis number, destruction level of the tumor tissue, development of damage and destruction of the tumor blood vessels and its infiltration with immunocompetent cells.Conclusion. The obtained data indicates that TNF-α within the delivery system exerts antitumor activity, which suggests the possibility of its further use for the treatment of malignant neoplasms, in particular, melanoma.The study was performed in accordance with ethical principles adopted by the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes.

PRECLINICAL STUDY OF THE PHARMACOKINETICS OF BACTERIOSENS FOR THE PHOTODYNAMIC THERAPY OF MALIGNANT NEOPLASMS

Introduction. Special attention has been recently paid to photosensitizers that absorb and fluoresce in the near infrared region of the spectrum. One of the most promising photosensitizers is bacteriosens, a synthetic bacteriochlorin derivative. Objective. To conduct a preclinical study of the biodistribution and pharmacokinetics of bacteriosens in animals.Materials and methods. The active ingredient of bacteriosens is (meso-tetra(3-pyridyl)bacteriochlorin) with of λmax 747 nm). The biodistribution and pharmacokinetics of the agent were studied in mice and rabbits. It was administered intravenously once at three doses: 1.0; 2.5 and 6.25 mg/kg for the mice and 0.236; 0.59 and 1.475 mg/kg for the rabbits. Local fluorescence spectroscopy was used for the quantitative determination of the pharmacokinetic parameters of bacteriosens.Results. Bacteriosens was removed quickly from the mouse bloodstream at 1 and 4 days after using minimal (1.0 mg/kg) and maximal (6.25 mg/kg) doses, respectively. When given at doses of 6.25 mg/kg and 1.0 mg/kg, bacteriosens was recorded in the skin, muscle, and spleen for 4 days and 24 h, respectively. The agent most intensively accumulated and long persisted in the omentum, liver, and kidneys for more than 6 days (6.25 mg/kg) and 2 days (1.0 mg/kg). A similar pattern was observed in the rabbits. Bacteriosens was rapidly removed from the rabbit bloodstream at 1 and 3 days after using at doses of 0.236 and 1.475 mg/kg, respectively. The agent was recorded in the skin, muscle, and spleen up to 4 days (1.475 mg/kg) and 3 days (0.236 mg/kg). It most intensively accumulated and long persisted in the omentum, liver, and kidneys for more than 6 days (1.475 mg/kg) and 4 days (0.236 mg/kg).Conclusion. Bacteriosens was removed from the animal bloodstream within 3–4 days after administration of the maximum dose that was 2.5 times higher than therapeutic one. The half-life of bacteriosens for mice was directly proportional to the dose and increased from 8 to 24 min; the half-life for rabbits was 20 min, irrespective of the dose. The drug was recorded in the skin for no more than 4 days. The main routes of bacteriosens elimination from the body of animals were the kidneys and liver.The study was performed in accordance with ethical principles adopted by the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes.

Histomorphological changes in the rat pancreas after methionine administration

The effectiveness of using various methionine preparations for activating pancreatic function is ambiguous; the reasons may include differences in dosage and duration of methionine administration. The question remains, in what extent the methionine application is efficacious for increasing functional activity of a healthy pancreas. The aim of our study was to investigate morphological changes in pancreas after prolonged administration of methionine. The experiments were carried out on 24 males of Wistar rats at the age of 15 months. During 21 days, the experimental animals received methionine at a daily dose of 250 mg/kg of body weight in addition to the standard diet. Histological preparations were made from pancreatic tissue according to standard method. Morphometry was performed using the computer program «Image J». The rats were taken out of the experiment under ether anesthesia. The studies were carried out in accordance with the provisions of the "European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes" (Strasbourg, 1986). Upon completion of the experiment, histomorphological sings of an increase in functional activity were registered in both exocrine (enlarged acini’s areas and their epithelium height, higher nuclear-cytoplasmic ratio of exocrinocytes, and higher number of nucleoli in cell nuclei) and endocrine (enlarged sizes of the Langerhans islets and increased number of endocrinocytes in the islets) parts of the rat pancreas. In the experimental rats, the relative area of ​​the connective tissue and the stromal-parenchyma index of the pancreas, as well as the width of the interlobular and interacinus layers of connective tissue decreased. A decrease in the mass of connective tissue in the pancreas can be considered as one of the signs of its function activation, an improvement in metabolism between acini, and an increase in regenerative capabilities. Thus, additional administration of prophylactic doses of methionine to healthy animals results in distinct morphological signs of increased pancreatic activity.

УЛЬТРАСТУКТУРНА ОРГАНІЗАЦІЯ ГЕМОМІКРОЦИРКУЛЯТОРНОГО РУСЛА ШКІРИ БІЛОГО ЩУРА НА РАННІХ ЕТАПАХ ПЕРЕБІГУ ЕКСПЕРИМЕНТАЛЬНОГО СТРЕПТОЗОТОЦИН- ІНДУКОВАНОГО ЦУКРОВОГО ДІАБЕТУ


 
 
 This article is presented information about electron microscopic characteristic the angioarchitectonics of hemomicrocirculatory white rat skin net in experimental streptozotocin-induced diabetes mellitus. Was used in the experiment 30 adult white male rats weighing 120-130 grams, which were maintained on a standard diet, had free access to food and water under normal conditions. The insulin-dependent form of type I diabetes mellitus was modeled by a single intraperitoneal injection of streptozotocin from Sigma at a rate of 7mg per 100g of body weight of the animal (prepared on 0.1M citrate buffer, pH = 4.5). The development of experimental diabetes mellitus during 4 weeks was monitored by observing an increase in blood glucose, which was measured by the glucose oxidase method. Studies were performed on rats with glucose levels of 12.00 mmol/l and above. The experiment was carried out in accordance with the provisions of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, 1986), Council of Europe Directive 86/609 / EEC (1986). Rats were removed from the experiment by an overdose of intraperitoneal anesthesia using sodium thiopental (calculated at 25 mg / kg body weight of the animal). Applying the method of electron microscopy (the study and photographing of the material was carried out on an electron microscope EM-100 AK at an accelerating voltage of 75 kV and magnification 6000-8000 times). As a material for this examination, the skin intact from the internal surface of the thigh and the back of the white rats was used. The study showed that after 2 weeks of diabetes occure gradually and are progressing alterations of the skin's hemomicrocirculatory bed of white rats, which characterised mainly by small spasm of some vessels. The wall of the most vessels still had normal structure but revealed a narrowing of the capillaries due to the protrusion of the nuclear zone of the endothelial cells. After 4 weeks of the experimental streptozotocin-induced diabetes mellitus were already observed reconstruction practicaly all skin's microvessels of white rats. The lumen of the capillaries became irregular. The contours of the nuclei of endotheliocytes were elongated with an uniform homogen chromatyn, which is concentrated in lumps, thickened bazal membrane, observed proliferation of endothelial cells, which leads to narrowing of the lumen of blood vessels. At the end of the experiment, we observe the inclusion of compensatory properties of the organism - the wall of blood vessels of the hemomicrocirculatory bed of the skin thickens, while the lumen of the microvessels become narrows. The results of investigation can be used in the practice medicine for diagnosis and treatment of skin's diseases in diabetes mellitus.
 
 

THE EFFECT OF MELATONIN ON THE ENERGY METABOLISM IN HEART OF DIABETIC RATS

The aim of this study was to determine the effect of melatonin on activity of pyruvate kinase (PK) and lactate dehydrogenase (LDH) in the heart tissue and on the activity of succinate dehydrogenase (SDG) and H+-ATPase in mitochondria of cardiomyocytes of rats with dexamethasone diabetes. Methods. The research was performed in compliance with the Rules of the work with test animals (1977) and the European Convention on the Protection of Vertebrate Animals used in experiments and other scientific purposes (18 March 1986). The experiments were carried out on 36 sexually mature male albino rats with the body mass 0.18 – 0.20 kg. The animals were divided into 3 groups:1) control group; 2) group with diabetes mellitus DM (BG level ≥ 8.0 mmol/l); 3) diabetic animals which were injected with melatonin. Melatonin (“Sigma”, USA) was injected intraperitoneally in a dose of 10 mg/kg of body weight daily over 13 days. Dexamethasone diabetes was evoked by daily subcutaneous administration of dexamethasone (solution for injection 4 mg / ml, KRKA, Slovenia) at a dose of 0.125 mg / kg body weight for 13 days (O.V. Stefanov, 2001). The mathematical model of the insulin-glucose correlation by the Homeostasis Model Assessment (HOMA) was used to evaluate the insulin resistance in the animals (D.R.Matthews et al., 1985). The blood was taken from the tail vein to evaluate the BG level using “OneTouchUltra” (“LifeScan”, USA). The rats were sacrificed on the 14th day of the experiments in accordance with the ethical treatment of animals. 5% homogenate was prepared from cold-isolated rat hearts on chilled 50 mM Tris-HCl buffer (pH = 7.4) with sucrose. Mitochondria were isolated by differential centrifugation. Enzyme activities in the mitochondrial fraction was determined by: succinate dehydrogenase (SDG) according to the method of Eshchenko N.D. et al. (1992), H + -ATP-ase - for Gabibiv M.M. (1986). Serum insulin content was determined using an automatic immunochemiluminescent analyzer (Snibe Co., Ltd, PRC) using a Maglumi test kit. The insulin resistance index (HOMA-IR) was calculated by the formula: HOMA-IR = (glucose (mmol / l) • insulin (mcU / ml)) / 22.5. To determine the enzymes activities by standart methods, heart muscle tissue was quickly removed, rinsed in saline, blotted, weighed and homogenized. Then the homogenate (5% in ice-cold 0.25 mM tris-HCl-buffer) was ultracentrifugated (10 min at 1500r/min) and the supernatant was used for measurements. Statistical analysis was performed using Statistica 10 (StatSoft Inc). Prior to analysis, Shapiro-Wilk test was used to assess the normality of the group data. According to the criterion, the samples distributions differed from normal distribution. Given these, use of the Mann-Whitney test was considered sufficient for valid conclusions to be made. Differences were considered to be statistically significant when P ≤ 0.05. Results. In the blood of all diabetic rats on the 14th day the glucose content exceeded 8.9 mmol / l, and the HOMA-IR index increased 7 times. According to results, we have obtained the activity of PK in the heart tissue of diabetic animals was 55% less than in control group. It can be explained by low uptake of glucose from the blood by heart muscle tissue in conditions of insulin resistance. Reduced glucose uptake by peripheral tissues, in turn, leads to a reduced rate of glucose metabolism. That means now substrate for next catabolic changes in glycolysis. The activity of LDH increases by 32% in the heart muscles of diabetic rats compared with the control values. We have found out that the activity of PK and LDH has become normalized by the introducing of melatonin in dose of 10 mg/kg of body weight. In the mitochondria of the animals with dexamethasone diabetes, the activity of SDG and H+ -ATPase is significantly lower than that in intact animals by 45 and 32%, respectively. Probably, melatonin enhances the uptake of glucose by heart muscles. The blood glucose content of diabetic rats, which were administered is not significantly different from that of the intact animals, and the HOMA-IR was 6.3 times lower than that of diabetic rats. The activity of SDG and H+-ATPase in the mitochondria of rats receiving both melatonin and dexamethasone does not differ from that of intact rats. Conclusion. A 14-day introduction of melatonin leads to a marked improvement of the state of carbohydrate metabolism as well as the energy metabolism in heart tissue that is accompanied by the normalization of the indices studied.

Zebrafish: Housing and husbandry recommendations.

This article provides recommendations for the care of laboratory zebrafish (Danio rerio) as part of the further implementation of Annex A to the European Convention on the protection of vertebrate animals used for experimental and other scientific purposes, EU Commission Recommendation 2007/526/EC and the fulfilment of Article 33 of EU Directive 2010/63, both concerning the housing and care of experimental animals. The recommendations provide guidance on best practices and ranges of husbandry parameters within which zebrafish welfare, as well as reproducibility of experimental procedures, are assured.Husbandry procedures found today in zebrafish facilities are numerous. While the vast majority of these practices are perfectly acceptable in terms of zebrafish physiology and welfare, the reproducibility of experimental results could be improved by further standardisation of husbandry procedures and exchange of husbandry information between laboratories. Standardisation protocols providing ranges of husbandry parameters are likely to be more successful and appropriate than the implementation of a set of fixed guidance values neglecting the empirically successful daily routines of many facilities and will better reflect the wide range of environmental parameters that characterise the natural habitats occupied by zebrafish.A joint working group on zebrafish housing and husbandry recommendations, with members of the European Society for Fish Models in Biology and Medicine (EUFishBioMed) and of the Federation of European Laboratory Animal Science Associations (FELASA) has been given a mandate to provide guidelines based on a FELASA list of parameters, ‘Terms of Reference’.

Condition of neuro-hormonal systems in traumatic brain injury and influence of Carbacetam

Use of GABA-ergic modulators in the traumatic brain injury (TBI) is actively investigated; their positive influence on brain circulation, inhibition of glutamate excitotoxicity, decrease of free radical damage, improvement of metabolism and power supply of neurons is determined. However, influence on functioning of neuro-hormonal systems in TBI isn't studied. The goal of given study was to investigate the Carbacetam effects on the condition of neuro-hormonal system in dynamics of experimental TBI. Methods. Research was carried out on white rat-males, age of six months, weighing 180–220 g. TBI was modeled by V. N. Elskyy and S. V. Ziablitsev model (2008) with the observance of Declaration of Helsinki adopted by the World Medical Association General Assembly, and general norms and principles of European Convention for the Protection of Vertebrate Animals. In comparison group 1 ml of normal saline solution was injected intraperitoneally. In animals of experimental groups 1 and 2 Carbacetam and Actovegin were injected intraperitoneally in doses of 5 mg/kg and 16 mg/kg of body weight, respectively, within 10 days after trauma. In blood the content of adrenocorticotropic hormone (ACTH), somatotropic hormone (STH), corticosterone and vasopressin were determined. Results and discussion . Administration of Carbacetam within 10 days after TBI prevented primary hyperactivation and contributed to the preservation of functioning of hypothalamic-neurohypophysis system and hypothalamic-pituitary-adrenal axis. In comparison with Actovegin, renewal of secretion of ACTH, corticosterone and vasopressin against the background of Carbacetam administration happened faster (from the 3 rd to the 7 th days). Administration of Carbacetam promoted prevention of the hyposecretion of STH significantly more than Actovegin. Identified modulating effects of Carbacetam could create conditions for more effective metabolism renewal and reparation processes in TBI. Conclusions . Administration of Carbacetam in the posttraumatic period renews functioning of neuro-hormonal systems more effectively than Actovegin, by prevention of their hyperactivation and exhaustion or excessive inhibition. Such an effect can be considered modulating with a positive restoring effect.

Білковий профіль крові свиней за згодовування їм ГМ-сої

Дослідження проводились на поголів’ї свиней-аналогів з живою масою та породністю у кількості 24 голови (8 свинок та 4 кнурця в групі). В раціон свиней входило за масою 10 % повножирової екструдованої сої , в контрольній групі сорту «Ворскла» без генетично-модифікованих організмів (ГМО), а в дослідній – за наявності ГМО (RR, GTS 40.3.2). За умов тривалого згодовування ГМ-сої спостерігаються вірогідні зміни концентрації білкових фракцій у крові 8-місячних свиней дослідної групи: вміст α 2 глобулінів знизився на 21,54 % (р = 0,001), а кількість γ глобулінів підвищився на 24,33 % (р = 0,0005) порівняно з контролем, що опосередковано свідчить про зниження рівня естрогенів в їх крові. Зміни окремих показників, що характеризують резистентність, можуть у подальшому негативно позначитись на функціональній діяльності репродуктивної системи організму свині. Отже, за умов використання генетично-модифікованої сої в раціонах свиней, призначених для відтворення, необхідно вести контроль за біохімічним складом крові, зокрема її білковим профілем.

Modeling of chronic ovary inflammation

In our country preservation of the population reproductive health is a high-priority direction of modern medicine. In many cases, the cause of reproductive disorders in women is a chronic infectious inflammation of the small pelvis, the frequency of which in recent years had no tendency to decrease. The choice of inactivated vaccine of Staphylococcus aureus as a phlogogen was due to the fact that the etiological role of the aerobic infection remains the leading one in gynecological pathology. The aim of research was studying of the ability to use the inactivated vaccine of Staphylococcus aureus strain 209 for modeling of chronic inflammation of the ovaries in laboratory mice. Materials and methods. 25 mature outbred white female mice weighing 18-20 g were used as experimental animals, which formed next groups: 1) control (n=5) – animals without any interventions and 2) experimental (n=20) – animals with one-fold intraperitoneal injection of inactivated Staphylococcus aureus strain 209 vaccine in the dose of 50х10 6 microbial bodies in 0,3 ml of physiological solution. Efficiency of the modeling pathology was performed by histomorphometric and hematological methods on the 7th, 14th, 21st and 31st days. All the manipulations with animals were carried out in accordance to the requirements of bioethics and the international principles of the European Convention for the protection of vertebrate animals. For statistical study ANOVA and t-Student tests were used with application of Microsoft Excel Program. Results. In the group of control animals the form and histological structure of ovaries were regular for mature mice without signs of inflammatory changes. The leukocyte infiltration, hemodynamic disorders and minor dystrophic changes of granulosa cells were determined on the 7th day in the ovaries of experimental animals. The increasing of observation period up to 14 days on the background of hemodynamic disorders resulted in the appearance of degenerative follicles, atresia of which proceeded by the type of the productive process with the formation of multilayer granulose tunica. The increase in the atretic follicles number, the migration of macrophages in their cavity and disorganization of follicular epithelium cells were determined on the 21st day after the vaccine injection. The proliferation of connective tissue was noted under the basement membrane of the follicles, between generative elements and ovarian medulla. To the 31st day of modeling granulosa cell layers lost ordered structure, reflected in the increasing of intercellular space and accumulation of fluid between the cells. During the first 14 days after the vaccine the leukocyte number in peripheral blood increased by 3,2 times. However, on the 21st day investigated index was 1,75 times lower in comparison with the previous period (P<0,05). Further, the leukocyte level was not statistically changed (P<0,05), but did not reach values of intact animals (P<0,05), indicating the reduction of the inflammatory process severity. At the same time there was a statistically significant (P<0,05) reduction in the number of follicles that was probably due to the intensification of processes of atresia. Due to their progression on the 31st day after vaccine injection, the number of follicles in the studied preparations decreased by 1,7 times (10,55±2,48) relatively to the index of control animals (18,25±4,52). The conclusions. Intraperitoneal injection of inactivated Staphylococcus aureus strain 209 vaccine to the 21st day leads to the formation of chronic inflammation of the ovaries in laboratory mice, which was characterized by the prevalence of atresia processes over follicle growth with the development of sclerotic changes and decreasing of their total number. At the initial time leukocytosis was observed in the blood of experimental animals, and starting from the 21st day inflammation acquired signs of chronic process, which manifested in reducing of number of cells without the normalization of this index until the end of the experiment.