Group Of Intact Animals Research Articles

Значення порушень прооксидантно- антиоксидантних процесів у патогенезі розвитку експериментальної пневмонії та адреналінового пошкодження міокарда

Today, pneumonia is one of the most common diseases of the respiratory system, accounting for 35-45%. No less common is coronary heart disease (CHD) among the pathologies of the cardiovascular system. In the medical world, the problem of comorbid pathology is acute, which can reduce adaptive capabilities, reduce the effectiveness of treatment and increase the development of various complications. Pathogeny of development of pneumonia and CHD is in this time to the end not studied. Research aim: to find out the features of changes and role of lipoperoxidation processes and state of the antioxidant system in pathogenesis of development of experimental pneumonia of connected with the adrenalin damage of myocardium. Materials and research methods. There were undertaken studies on 82 guinea-pigs (males), that distributed on three groups: the first group is intact animals – control (10); the second (experience) group is animals with experimental pneumonia (ЕP) accordingly on 1th, 3th, 6th and 14th twenty-four hours of experiment (36); the third (experience) group is animals with the united pathology (ЕP and adrenaline myocardium damage – AMD) accordingly on 1th, 3th, 6th and 14th twenty-four hours of experiment (36). The fixed twenty-four (1th, 3th, 6th and 14th twenty-four hours) hours took into account, because they answer the classic stages of the abrupt used for setting fire reply (incubations, development of illness, height of illness, convalescence) and motion. ЕP recreated by the method of Shlapnikov V. N., Solodova T. L. et al. by the way of intranasal infection of animals of Staphylococcus of aureus. AMD designed by non-permanent intramuscular introduction of 0,18% solution adrenaline hydrotartrate («Darnytsia», Ukraine), in a dose 0,5 mg/kg. Assessment of lipid peroxidation was carried out by measuring levels of diene conjugates (DC) and malonyldialdehyde (MDA), and activity of antioxidant system was evalution by catalase (CAT) and superoxide dismutase (SОD). Euthanasia by decapitation of laboratory animals was conducted under act of chloroform anaesthesia. Determinations of DC in fabrics of kidney was carred by Gavrilov's method, content of МDА – by the method of Y. N. Korobeynikov, activity SОD – by the method of R. Fried, CAТ – by the method of B. Holmes and C. Masters. The statistical working of the obtained data was carried out by Student's t-test. Results and discussions. ЕP and AMD (1th, 3th, 6th, 14th twenty-four hours) is accompanied by the increase of content of DC in fabrics of kidneys accordingly on 62,5 %, 71,5 %, 79,5 %, 82,9 % (р < 0,05), MDA – on 55,0 %, 62,1 %, 72,7 %, 85,7 % (р < 0,05) on a background the decline of activity of SОD accordingly on 25,2 %, 27,2 %, 30,6 %, 37,2 % (р < 0,05), CAT – on 31,2 %, 34,7 %, 40,3 % 45,5 % (р < 0,05) in relation to the control group of animals, that testified to development of oxidative stress, that strengthened motion of pneumonia and AMD. Conclusion: the results of researches are Got by us allow to assert that comorbidity (EP and AMD) predetermines violation of balance between prooxidant and by an antioxidant the systems, strengthening of lipoperoxidation processes in the conditions of depression of the antioxidant securing for all stages of their development with advantage on 6th and 14th twenty-four hours of experiment against the intact group of animals and specifies on the important role of one of molecular (lipid) mechanisms of damage of cages in pathogenesis of formation of these models of illnesses.

Визначення білків гострої фази в крові щурів після імплантації поліпропіленових хірургічних сіток із покриттям на основі танталу та його похідних

At present, more than 30 types of polypropylene surgical meshes are available in the world, but the statistics regarding the secondary inflammatory process after implantation still remain disappointing. Since polypropylene surgical meshes do not decompose well in the body, they can stimulate the surrounding tissuesto develop an inflammatory process that causes adhesions. For the past 50 years, tantalum has been successfully used to produce biocompatible medical implants in surgery, orthopedics, and dentistry. Its excellent anti-inflammatory and antibacterial properties have been repeatedly noted, indicating the possibility of its use as a coating for mesh implants. In order to better predict the outcome after implantation of biomedical materials, it is important to determine the content of acute phase proteins, namely C-reactive protein, haptoglobin and ceruloplasmin. The aim of the study is to determine the content of acute phase proteins, namely C-reactive protein, haptoglobin and ceruloplasmin in the blood of rats after implantation of surgical meshes coated on the basis of tantalum, tantalum oxide and tantalum nitride. Materials and methods. The experimental group included 40 male rats of the WAG population. With the help of surgical intervention, a polypropylene surgical mesh measuring 15x15 mm was implanted between the abdominal wall and various sections of the colon. 28 days after surgery, the experimental animals were decapitated by cervical dislocation. Blood samples were used to determine the content of C-reactive protein, the content of haptoglobin and the content of ceruloplasmin. Results and discussion. The content of C-reactive protein in the blood of rats was statistically 260 % higher in the experimental group implanted with uncoated surgical mesh and 228.8 % higher in the experimental group implanted with polypropylene surgical mesh with tantalum nitride coating in compared with the results in the group of intact animals. Haptoglobin content was statistically 110.5 % higher and ceruloplasmin was statistically 52.6 % higher in the uncoated surgical mesh group compared to the results of the intact animal group. We obtained similar results in the experimental group, which was implanted with a polypropylene surgical mesh with a coating based on tantalum nitride. The content of haptoglobin was 130 % higher and the content of ceruloplasmin was 50.6 % higher statistically compared to the results of the group of intact animals. The C-reactive protein content was 113.3 % and 95.5 % higher, respectively, in the groups implanted with polypropylene surgical meshes with a coating based on tantalum and tantalum oxide compared to the results in the group of intact animals. The content of haptoglobin in the tantalum-based coating group was 83.8 % higher and the content of ceruloplasmin was 32.6 % higher compared to the results obtained in the group of intact animals. In the tantalum oxide- based group, the haptoglobin content was 60.1 % higher and the ceruloplasmin content was 29.3 % higher compared to the results obtained in the intact animal group. Conclusion. Based on the results of the study, it was established that polypropylene surgical meshes coated with tantalum and tantalum oxide significantly reduce the inflammatory reaction compared to uncoated meshes and meshes coated with tantalum nitride. This is supported by lower levels of key inflammatory markers, indicating improved biocompatibility and anti-inflammatory effects of tantalum and tantalum oxide coatings.

Pharmacologic activity of mGLUR4 agonist in a model of primary open-angle glaucoma: A preclinical experimental study

Background. Worldwide, more than 67 million people suffer from glaucoma. In Russia, this number exceeds 1.08 million people. Annually, the number of primary cases increases by 3–4%. The increasing prevalence of glaucoma intensifies the search for neuroprotectants that can reduce the loss of retinal ganglion cells, thereby impeding the progression of the disease. Objective. To study of the pharmacological activity of mGLUR4 agonist, ZC64-0001 substance, on a model of primary open-angle glaucoma. Methods. The conducted preclinical study involved 60 sexually mature male Wistar rats, weighing 180–220 g. The observation period was 73 days. The animals were divided into 6 groups with 10 animals in each. Group 1 included intact animals (without any manipulations); Group 2 included negative control animals with the water injected in the anterior chamber of the eye; in Group 3–6, primary open-angle glaucoma was modelled by injecting 1% hyaluronic acid solution into the anterior chamber of the eye once every 7 days from day 1 to day 62 of the study. Animals in Group 4 were treated intragastrically with H-[(4-chlorophenyl)methyl]-1,6-dihydro-4-methoxy-1-(2-methylphenyl)-6-oxo-3-pyridazinecarboxamide under the laboratory code of ZC64-0001 at a dose of 10 mg/kg. Animals in Group 5 received Mexidol as a comparison drug intramuscularly at a dose of 25.7 mg/kg. Animals in Group 6 were treated with a Timolol instillation at a dose of 0.009 ml/kg. The studied compounds were administered from day 63 of the study once a day for 10 days. The evaluated indicators included the level of microcirculation in the retina, the amplitude of a-wave and b-wave of the electroretinogram, and the number of retinal ganglionic layer nuclei in the setting of the conducted treatment. Statistical processing of the data was performed using the Statistica 10.0 software (StatSoft, USA). Differences were determined at the significance level of p ˂ 0.05. Results. ZC64-0001 increased the level of microcirculation relative to the group with modelled glaucoma by 11.5%, with this indicator being statistically significantly different from that both in the group with modelled glaucoma and the intact group ( р < 0.05). In the group of animals receiving ZC64-0001, the amplitude of a-wave increased relative to the primary glaucoma group by 17.7%, with this index being statistically different from the groups of intact animals and negative control (р < 0.05). The b-wave amplitude increased by 34.4% relative to the group with modelled glaucoma, being statistically different from the intact group, negative control group, pathology modelled group, and comparison drug groups ( р < 0.05). Administration of ZC64-0001 increased the number of retinal ganglion cell nuclei relative to the group with modelled glaucoma by 41.0%, which had a statistically significant difference from all the studied groups ( р < 0.05). Conclusion. The ZC64-0001 compound demonstrated high neuroprotective properties in a model of primary open-angle glaucoma, leading to an improvement in retinal microcirculation, an increase in the wave amplitude according to the conducted electrophysiological study, and an increase in the number of ganglion cell nuclei.

EVALUATION OF THE EFFECT OF NEUROPEPTIDES OF THE MELANOCORTIN FAMILY ON STRESS-ASSOCIATED CHANGES IN THE LEVEL OF CORTICOTROPIN-RELEASING HORMONE AND CORTICOSTERONE

Introduction. To date, the problem of studying the neuropeptide system of regulation of adaptive processes is relevant, since the stress reaction is a trigger for the occurrence of various diseases, in particular depression. Corticoliberin is considered as the main regulator of the hypothalamic-pituitary-adrenal axis, released in response to stress from the paraventricular nucleus of the hypothalamus, modulating the synthesis of adrenal hormones and further the processes of integration of neuroendocrine, autonomic and behavioral responses to stress. The aim of the study was to evaluate the effect of neuropeptides on stress–induced changes in corticotropin-releasing hormone and corticosterone. Material and methods. The experiment was carried out on male rats in the amount of 70 individuals, which were divided into several groups: a group of intact animals receiving an equiobject of water solution for intraperitoneally injection for 20 days (10 individuals), a group of animals receiving an equiobject of water solution for intraperitoneally injection in an experimentally created model of a depressive-like state for 20 days (20 individuals); experimental groups of animals who received intraperitoneally studied neuropeptide compounds Met-Glu-His-Phe-Pro-Gly-Pro and His-Phe-Arg-Trp-Pro-Gly-Pro at a dose of 100 mcg/kg/day for 20 days from day 1 of an experimentally created model of a depressive-like state. A depressive-like state was modeled by daily intermale confrontations for 20 days, as a result of which submissive and aggressive types of behavior were formed in rats. Neuroendocrine status was assessed by determining the level of key hormones of the hypothalamic-pituitary-adrenal axis (corticotropin-releasing hormone (corticoliberin) and corticosterone) in blood serum using enzyme immunoassay (ELISA). Results. In conditions of stress-induced depression, a statistically significant increase in corticoliberin and corticosteroid in the blood of rats of both types of behavior was found, the difference in comparative aspect with the indicators of the control group, while exposure to neuropeptides against the background of stressful exposure contributed to a decrease in corticosterone levels by an average of 30% (p<0.05). Conclusion. Thus, it was found that stress-induced depression is accompanied by hyperactivation of the hypothalamic-pituitary-adrenal axis, presumably due to a violation of the reverse inhibitory effects of endogenous glucocorticoids due to dysfunction of glucocorticoid receptors, which contributes to the development of a complex of manifestations of the main reactions, in particular, a decrease in the level of neurotrophic factors and the development of neurogenic inflammation. Neuropeptides of the glyproline series, in turn, showed the ability to reduce the activity of the hypothalamic-pituitary-adrenal axis by inhibiting the level of corticoliberin and corticosterone in conditions of stress-induced depression.

Features of Klotho Protein Expression in Rat Kidneys in Experimental Hyperglycemia Against the Background of Pharmacological Correction with GABA Derivatives

The aim is to identify the features of Klotho protein expression in the epithelium of the convoluted tubules of the kidney during prolonged experimental hyperglycemia and pharmacological correction with GABA derivatives. Material and methods. The study used 50 mongrel male rats (initial weight: 330,0–360,0 g) with streptozotocin diabetes lasting 6 months. 6 months after the injection of streptozotocin (60 mg/kg), animals with a glycemic level of ≥ 15 mmol/l were included in the study (after 4 hours of food deprivation), then GABA derivatives (aminalon, mefargine, succicard) were administered orally for 30 days. Proteinuria and serum creatinine concentrations were evaluated. After euthanasia, kidney tissue was fixed in buffered neutral formalin and examined using immunofluorescence microscopy. The intensity of the glow was evaluated based on a visual-analog scale. Results. In the group with chronic hyperglycemia without treatment, there was a significant increase in the protein content in daily urine and serum creatinine levels (p<0,05). In the group of animals with chronic hyperglycemia without treatment, there was a significant decrease in the expression of Klotho protein compared to the group of intact animals during immunofluorescence analysis. In the group treated with the GABA derivative succicard, there was an improvement in the functional state of the kidneys, accompanied by a significant increase (p≤0,0001) in the area of Klotho-positive material in the epithelium of the convoluted tubules of the kidney in relation to the group of animals with prolonged hyperglycemia without treatment. Conclusion. Prolonged hyperglycemia causes severe renal dysfunction in rats with streptozotocin diabetes: an increase the proteinuria and serum creatinine levels is accompanied by a decrease in the expression of Klotho protein in the kidneys. The GABA derivative succicard with course administration improves kidney function and this effect is accompanied by an increase in the expression of Klotho protein.

Studying the possibilities of pharmacological correction of hypoxia-induced pulmonary hypertension using a phenolic compound with a laboratory cypher KUD975

The aim of our work was to study a pharmacological activity of a selective arginase-2 and thrombin inhibitor from a phenolic compounds group with a laboratory cypher KUD975 on a model of arterial pulmonary hypertension induced by hypoxia.Materials and methods. To simulate pulmonary hypertension (РН), animals were placed in a normobaric hypoxic chamber and subjected to 5 weeks of hypoxia with an oxygen content of 10% in the air. After 3 weeks of hypoxia, the animals were administered with the test compound KUD975 (intragastrically, at a dose of 2 mg/kg once a day for 2 weeks). L-norvaline (intragastrically, 20 mg/kg) was used as a reference drug. To assess the development and correction of РН, measurements of cardiohemodynamics, analysis of blood gas composition, study of the number of circulating endothelial precursor cells (EPCs), quantitative PCR assessing the expression of mRNA VEGF-R2, SGF-1 (stromal growth factor-1) and MCP-1 (monocyte chemoattractant protein-1). Next, a histological examination of the lungs and heart was performed, the degree of pulmonary edema and the concentration of cardiotrophin-1 and atrial natriuretic peptide were assessed.Results. The administration of the studied phenolic compound with laboratory cypher KUD975, as well as the reference drug L-norvaline, led to a decrease in the right ventricular systolic pressure against the background of modeling РН. The present study shows a more than twice-fold decrease in the number of circulating (EPCs) in the animals group with modeling a hypoxia-induced circulatory РН (171.3±12.1) in comparison with the group of intact animals (296.1±31.7; p=0.0018). The recovery of EPCs was noted in the animals group administered with KUD-975 and L-norvaline, up to 247.5±34.2 (p=0.0009 compared with a pulmonary arterial hypertension (PAH) and 235.6±36.4 (p=0.008 compared to PAH), respectively. The studied compounds had a protective effect by statistically significantly increasing the expression of VEGF-R2 mRNA and decreasing the expression of SGF-1 mRNA, reducing the lung moisture coefficient and the concentrations of cardiotrophin-1 and atrial natriuretic peptide and preventing vascular remodeling caused by hypoxia.Conclusion. When studying the pharmacological activity, it was shown that the phenolic compound with the laboratory cypher KUD975 normalizes hemodynamic parameters, reduces the signs of remodeling of the heart and pulmonary vessels and has a pronounced endothelial protective effect on the model of hypoxia-induced РН, and is superior to the activity of the reference drug L-norvaline.

Experimental study of the pattern of genes activated by multi-day intermittent hypoxia in the rat pancreas

Intermittent hypoxia affects a variety of pathological conditions in the body and is used in medicine to reproduce the sanogenic therapeutic effect. Intermittent hypoxia is used for clinical indications to improve lung function; increase the body’s adaptive capacity; in obstructive sleep apnoea syndrome; in anaemia, diabetes mellitus. In cancer radiotherapy, intermittent hypoxic training helps to increase the sensitivity of the tumour to treatment. Intermittent hypoxia is useful in rehabilitation medicine to improve the physical recovery of patients after surgery or injury to improve the body’s functional capabilities. However, to date, the activity of regulatory genes, that activate the molecular mechanisms of the above-mentioned sanogenic effects of intermittent hypoxia has not been sufficiently studied. The aim of the study: to determine the expression of hypoxia-related genes in pancreas of Wistar rats under intermittent hypoxia. Materials and methods. The CFX-96 Touch™ real-time reverse transcription polymerase chain reaction (PCR) (Bio-Rad, USA) and the RT2 Profiler™ PCR Array Rat Hypoxia Signalling Pathway kit (QIAGEN, Germany) were used to analyse gene expression in experimental animals, where 84 genes involved in the hypoxia signalling pathway identified in the pancreas were studied. Results. According to the results of PCR analysis of pancreatic samples from intact animals and animals exposed to hypoxic training, the activity of the gene panel can be distributed as follows: genes with high expression compared to the intact group of animals, genes with low expression compared to the intact group of animals and genes in which no significant changes were detected in the samples compared to the intact group of animals. We have found, that the genes Cops5, F10, Jmjd6, Lgals3, Rbpjl, Vegfa have high expression activity compared to the intact group of animals. Conclusions. The increase in the expression of Cops5 by 10.29 times, Lgals3 by 2.94 times and Rbpjl by 5.73 times as a result of intermittent hypoxia can be regarded as an activating factor for the increase in the proliferation of endocrine and exocrine pancreatic cells and the growth of their antiapoptotic potential. The 3.20-fold increase in Jmjd6 gene expression under intermittent hypoxia can be interpreted as a regulatory effect through protein dehydroxylation and promotion of antiapoptotic protein activity in endocrine and exocrine pancreatic cells. The 5.99-fold increased level of Vegfa expression may promote angiogenesis in the pancreas under intermittent hypoxia.

DYNAMICS OF CHANGES IN THE VASCULAR BED AND ORGANOMETRICAL INDICATORS OF WHITE RATS TESTICLES DURING EXPERIMENTAL SKIN THERMAL INJURY AND UNDER THE CORRECTION WITH A XENODERMAL SUBSTRATE

DYNAMICS OF CHANGES IN THE VASCULAR BED AND ORGANOMETRICAL INDICATORS OF WHITE RATS TESTICLES DURING EXPERIMENTAL SKIN THERMAL INJURY AND UNDER THE CORRECTION WITH A XENODERMAL SUBSTRATE

INFLUENCE OF COPPER COMPLEXES [CU(PTA)4]BF4 AND [СU(ACAC)2] ON RATS IRRADIATED WITH RADIOISOTOPE TECHNETIUM

One of the priority tasks of modern radiobiology is the search for new, effective radioprotective compounds. In this area, metal complexes with high antioxidant activity are of particular interest. In order to study the possible favorable radioprotective effect of two copper complexes in different oxidation states, [Cu(PTA)4]BF4 and [Сu(acac)2], on the irradiated organism, we studied cytogenetic parameters in 4 groups of experimental animals. Group I included intact animals; Group II consisted of animals exposed to technetium (Tc) radioisotope, which were intraperitoneally injected with an isotope with an activity of 4.8 mCi in a volume of 2 mL - "pure irradiation"; Group III consisted of animals that were intraperitoneally injected with the copper(I) complex [Cu(PTA)4]BF4 at a dose of 50 mg/kg in a volume of 2 mL one hour before the administration of the Tc isotope (“irradiation + [Cu(PTA)4]BF4”). Group IV included animals that received the copper(II) complex [Сu(acac)2] before irradiation. We studied survival and cytogenetic parameters, determined the mitotic index, chromosomal aberrations and the percentage of polyploid cells. The survival rate of group II was 40%. In the group of intact animals, as well as in groups III and IV the survival rate was 100%. The dynamics of survival was described by regression equations, which make it possible, by means of extrapolation, to determine the change in the percentage of survival in the long term of the experiment and to predict the further outcome of the experiment. Investigating cytogenetic indicators, for all 3 indicators, a significant difference was found between intact and irradiated animals, i.e. these indicators can be considered as markers of technetium isotope exposure. In terms of proliferative activity, a significant difference was found in irradiated animals compared with groups III and IV, which indicates the radioprotective property of both the copper compounds. As for the [Cu(acac)2], when it was used in group IV and compared with others, we obtained a pronounced significant difference from the group with “pure irradiation” in all studied cytogenetic parameters. It was found that the copper complexes [Cu(PTA)4]BF4 and [Cu(acac)2] have the ability to prevent or mitigate the effect of ionizing radiation on an animal's body. Based on cytogenetic parameters, it can be concluded that these compounds promote reparative processes in the bone marrow cells of irradiated animals. Multiregression analysis of cytogenetic parameters confirmed the highest efficiency of the copper (II) complex [Cu(acac)2]. The results of the research indicate the need to continue work in the direction of searching for agents that have a therapeutic effect in radiation injuries.

MORPHOMETRIC FEATURES OF CHANGES IN THE MUSCLES OF THE ANTERIOR ABDOMINAL WALL AFTER IMPLANTATION OF THE CAPROMESH MESH COMBINED WITH PRP IN THE EXPERIMENT

Summary. Despite the use of meshes and other improvements, currently popular methods of surgical treatment of diastasis recti of the abdominal muscles and umbilical hernia have certain disadvantages and risks, especially in the group of patients with morbid obesity. The priority surgical intervention for diastasis and umbilical hernia under the conditions of morbid obesity is the performance of E-Milos video assisted operation. However, this type of procedure does not involve suture or stapled fixation of the mesh in the retromuscular space, which can lead to a number of complications. The analysis of the obtained results determines the relevance of this study.
 Purpose. Carry out a quantitative morphometric assessment of the changes in the arteriomuscular-aponeurotic layer and muscles of the abdominal wall under the conditions of the E-Milos operation using meshes based on e-caprolactone treated with PRP (plasma enriched with growth factors) in the experiment.
 Materials and Methods. The morphometric study involved the analysis of the muscles of the anterior abdominal wall after the implantation of the Capromesh based on e-caprolactone treated with PRP into the retromuscular space under the light-optical examination of micronutrients. Experiments were conducted on 16 sexually mature male Vietnamese pigs, which were divided into 4 groups.
 Results. Measurements made in a group of intact animals showed that the outer diameter of small-caliber arteries was equal to (35.40±0.39) μm,, the inner diameter was (16.95±0.15) μm, the media thickness was (6.36±0 .06) μm, the Kernohan index (the ratio of the area of the lumen to the entire area of the vessel) was (22.90±0.18) %. The height of the endotheliocytes of these vessels reached (6.12±0.06) μm, the diameter of their nuclei – (3.12±0.03) μm. The nuclear-cytoplasmic ratio in the studied cells was equal to (0.260±0.003).
 The thickness of the media of small caliber arteries on day 7 of the experiment increased from (6.38±0.06) μm to (6.60±0.06) μm, i.e. by 3.4 % (р<0.05), and adventitia thickness – by 24.7 % (р<0.001). Thus, the Wogenvoort index increased from (436.20±4.20) % to (513.90±4.50) %, i.e. by 77.7 %. It is also worth noting that the given morphometric indicators were statistically significantly (p<0.001) different from each other.
 On day 14 of the experiment, the relative volume of damaged endothelial cells was (4.20±0.03) %. This morphometric parameter exceeded the similar control by 1.8 times (p<0.001). The morphometric parameters of the small-caliber arteries of the soft tissues of the anterior abdominal wall of male Vietnamese pigs on day 21 of the experiment did not differ significantly from similar quantitative morphological indicators of the control observations.
 Conclusions. On day 14 of the experiment, the structural rearrangement of small-caliber arteries did not significantly differ from similar vessels of control observations. The relative volume of damaged endothelial cells was equal to (4.20±0.03) %. The morphometric parameters of the small-caliber arteries of the soft tissues of the anterior abdominal wall of male Vietnamese pigs on day 21 of the experiment did not differ significantly from similar quantitative morphological indicators of the control observations.
 On day 21 of the experiment, the nuclear-cytoplasmic ratio in the studied muscle structures was equal to (0.055±0.002), and the given morphometric parameter did not differ from the similar control, which indicated the preservation and stability of cellular structural homeostasis.

Neuroprotective Activity of 5-ethoxy-2-ethylthiobenzimidazole (Etomerzol) and 2-ethylthiobenzimidazole (Bemitil) in a Model of Middle Cerebral Artery Occlusion

Introduction. Cerebrovascular diseases (CVD) are one of the most pressing medical and social problems due to the high rate of mortality and disability. Stroke is the leading cause of CVD. About 15 million strokes are registered annually in the world according to the World Federation of Neurological Societies. It should be noted that CVD of ischemic origin has a tendency to rejuvenation and growth. Traditionally, in clinical practice, antihypoxic, antioxidant agents, as well as drugs with neuroprotective and neurorehabilitation effects are used to treat CVD. In connection with the increase in the incidence of CVD, there is an urgent need to search for promising neuroprotectors.Aim. To study the neuroprotective activity of 5-ethoxy-2-ethylthiobenzimidazole (etomerzol) and 2-ethylthiobenzimidazole (bemityl) in a model of middle cerebral artery occlusion.Materials and methods. Occlusion of the middle cerebral artery (ОMCA) was used as a model. The object of the study was inbred male rats of the Dark Agouti line, randomized into five groups: a group of intact animals and four groups with OSMA: a control group, a group with the introduction of 5-ethoxy-2-ethylthiobenzimidazole (etomerzole, 25 mg/kg), a group with the introduction 2-ethylthiobenzimidazole (bemityl, 25 mg/kg) and a group with the reference drug dimethyl fumarate (DMF, 100 mg/kg). On the 1st, 3rd, and 7th days after the operation, the "Limb Stimulation" (SC) test was performed. On the 7th day, the tests "Open field" (OP) and "Elevated plus maze" (EPM) were performed. Euthanasia was performed on the 7th day in an induction chamber (Bioscape GmbH, CO2 box for euthanasia, Germany).Results and discussion. The study showed a pronounced pharmacological effect of etomerzole in a model of acute ischemic stroke. The introduction of bemitil, etomerzol and DMF significantly reduced the neurological deficit 24 hours after OSMA and improved the psycho-functional state on the 7th day. Thus, in the etomerzol group on the first day after surgery, the neurological deficit was reduced by 1.9 times (p < 0.05), and by 3 and 2.0 times (p < 0.05) compared with the control group. While bemityl and DMF reduced the index by 1.3 (p < 0.05) and 1.4 times (p < 0.05) on the 3rd day and by 1.5 times (p < 0.05) on the 7th day. In the OP test in the etomerzol group, an increase in HDA by 2.7 times (p < 0.05) was observed compared with the control. The rate of peering into minks in the etomersol and bemitil groups was higher than in the control group by 2.6 (p < 0.05) and 3.4 times (p < 0.05), respectively. Search and research activity was increased in the same groups by 2.0 (p < 0.05) and 2.2 times (p < 0.05) compared to control animals. In the PCL test, the etomerzol and DMF groups showed an increase in the number of uprights in the sleeves compared to the control group by 2.7 (p < 0.05) and 3.8 times (p < 0.05), respectively. In the etomerzol and bemityl groups, there was an increase in the number of overhangs by 3.4 (p < 0.05) and 6.2 times (p < 0.05).Conclusion. The data obtained during the study of the activity of benzimidazoles after ischemic stroke indicate a significant increase in the motor activity of animals in the OP tests, which may indicate a psychostimulating and potential nootropic effect of etomerzole and bemitil, and an increase in the number of racks and hangings in the PCL test can be regarded as manifestation of anxiolytic activity. In our study, the severity of the pharmacological effects of DMF was inferior to benzimidazoles. However, a significant decrease in neurological deficit in the SC test and an increase in the number of uprights in the sleeves in the PCL may indicate the presence of anxiolytic activity in the drug. The results obtained underline the importance of conducting additional studies to evaluate the effectiveness of DMF in OSMA.

Physicochemical characterization and biological activity of polysaccharides from the seeds of the turnip brassica rapa

Analysis of the monosaccharide composition showed that the composition of the neutral polysaccharide BSP-1-1 is represented by monosaccharides in the following composition: ribose – 5.05%, arabinose – 56.38%, mannose – 5.87%, glucose – 8.63% and galactose – 24.05%. The composition of the anionic polysaccharide BSP-2-1 is represented by monosaccharides: ribose – 6.35%, arabinose – 60.15%, mannose – 7.19%, glucose – 4.12% and galactose – 22.16%. It was determined that the isolated polysaccharides consist mainly of arabinose (BSP-1-1 – 56.3%, BSP-2-1 – 60%) and galactose (BSP-1-1 – 24%, BSP-2-1 – 22%). Based on the data obtained, it can be assumed that the studied polysaccharides from the seeds of the turnip Brassica rapa belong to the type of arabinogalactans. According to the results of studying the properties of acute toxicity of the BSP polysaccharide from the seeds of the turnip Brassica rapa, it was found that this substance belongs to class V compounds - practically non-toxic substances. With intragastric administration of inulin at doses of 25, 50, and 75 mg/kg and BSP polysaccharide from the seeds of the turnip Brassica rapa at doses of 10, 20 and 30 mg/kg 14 days after the induction of diabetes mellitus, the indicator was close to that of the intact group of animals (intact - 3.07 ± 0.25, inulin 2.99 - 3.14 ± 0.23, and BSP polysaccharide from the seeds of the turnip Brassica rapa 2.53-3.14 ± 0.20), while the indicator of the control group was - 8.40 ± 0.35. This study will provide an opportunity to plan and conduct studies to study the action of substances in the metabolism of glucose and lipids, which examines the enzymes that regulate the lipid activity of the liver, as well as the morphology of the liver and adipose tissue.

Long-term effects of cytostatic agents on germ cells of testicular tissue (experimental study)

Currently, cytostatic drugs are widely used not only in cancer treatment, but also in the treatment of autoimmune infammatory diseases. A favorable prognosis of the disease, ability to reproduce, young age and the absence of children serve as an incentive to decide on the need for childbearing. There is concern, that the mutagenic effects of chemotherapy in germ cells, the ability to induce epigenetic changes in them, may have phenotypic manifestations in offspring. Conception in the early stages after treatment (impact on mature and differentiating germ cells) has been proven to increase the risk of defective offspring. Data on the health of the offspring of patients conceived in the long term after treatment (impact on stem spermatogenic cells) are contradictory. The aim of the study was to assess long-term toxic effects of cytostatic drugs in the male rat offspring copulated in terms corresponding to the effect on stem spermatogonial cells (SSCs). Material and Methods. The experiments were carried out on autobred male Wistar rats (n=140), aged 2.5 months, 70 of which made up the group of intact animals. The effect of cytostatic drugs (etoposide, irinotecan, cisplatin, carboplatin, methotrexate, farmorubicin, and paclitaxel) injected 3 and 6 months before mating was assessed on the offspring of intact female and male rats. Results. The male rat offspring treated with cytostatic drugs was found to be viable. Gross external developmental anomalies were detected in 2 cases. In several offspring, a slowdown in physical development, decrease in the rate of formation of sensory-motor refexes and learning ability were observed. The most toxic drugs were etoposide and paclitaxel. Conclusion. The offspring of rats treated with cytostatic drugs in terms corresponding to the effect on the SSCs is at risk. The degree of severity of long-term effects varies signifcantly and depends on the type of the drugs used. A decrease in the ability to learn is the most frequently detected abnormalities in offspring. Judging by the timing of conception after cytostatic exposure, a signifcant increase in the period of time after the administration of the drug before mating is not always justifed.

Safety Study of an Original Mesenchymal Stromal Cell Secretome-Based Medicinal Product for Spermatogenesis Restoration

SCIENTIFIC RELEVANCE. Currently, there are no effective and safe medicinal products for idiopathic male infertility. Previous studies in two animal models of infertility (short-term cryptorchidism in rats and doxorubicin-induced testicular injury in mice) have shown the effectiveness of an originator medicinal product based on the mesenchymal stromal cell (MSC) secretome.AIM. The aim of the study was to evaluate the toxicity profile of the MSC secretome-based medicinal product in rats after local intratesticular or intramuscular administration.MATERIALS AND METHODS. The MSC secretome is a combination of factors secreted by MSCs in low-glucose Dulbecco’s modified Eagle’s medium (DMEM-LG) for MSC conditioning. In the single-dose toxicity study, the MSC secretome-based medicinal product was injected under the testicular tunica albuginea of male Wistar rats (15 per group) at doses of 15 and 25 relative units (RU) per animal, which are 1.5 and 2.5 times higher than the therapeutic dose (10 RU). In the repeat-dose toxicity study, male Wistar rats (10 per group) received intramuscular thigh injections of the medicinal product on days 1, 6, and 12 at doses of 15 and 25 RU per animal. The local tolerance study involved histopathological examination of the testes and thighs at the injection site. All studies included control groups of intact animals and animals similarly injected with blank DMEM-LG. The early follow-up period was 14 days, and the late follow-up period was 42 days.RESULTS. The rats showed no changes in the general condition after single and repeated doses of the MSC secretome-based medicinal product. Single subtunical doses induced moderate irritation; its signs included pathological changes in individual seminiferous tubules: epithelial atrophy (70% of the animals on day 14; 55% at late follow-up) and sperm stasis (70% of the animals). Similar changes were observed in the blank DMEM-LG group (up to 80% of the animals). There were no pathological changes in the tissues after repeated injections. A transient increase in alkaline phosphatase activity was detected in animals after their third intramuscular injection at a dose of 25 RU; the other biochemical parameters were normal in all study groups.CONCLUSIONS. The MSC secretome-based medicinal product has a favourable safety profile following both intratesticular and intramuscular administration, as it does not cause any permanent changes in the studied organs and tissues.

Effects of leonardite and lignite on blood parameters in rats exposed to lead acetate

The study aimed to research in vivo changes in the hematological and biochemical parameters of laboratory rats' blood when they were continuously exposed to a moderate dose of lead acetate while receiving humic feed additives made of leonardite and lignite. Two-month-old white rats were used to assess the detoxifying properties of the humic compounds leonardite and lignite. The analog pair approach was used to create four groups of eight animals from 32 male laboratory rats for the study. Rats received a pre-meal injection of lead acetate at a dose of 7 mg/100 g of animal weight (1/110 LD50) using a veterinary feeding needle. By creating solutions from lignite and leonardite at a dosage of 18 and 25 mg/kg depending on the active ingredient, humic feed additives were supplied to animals. It has been demonstrated that lignite- and leonardite-based feed additives may affect the morphological parameters of lead acetate-treated rats' blood, including hemoglobin, hematocrit, erythrocyte, and platelet count. These parameters were very near to the levels of the intact rats, which suggests that these humic feed additives may have an anti-anemic impact. The effects of feed additives from leonardite and lignite on laboratory rats in groups that were also exposed to a toxic agent for all 21 days of the experiment led to the normalization of markers of the state of protein metabolism in the group of intact animals (serum protein, albumins, urea, creatinine), particularly activity of the enzyme’s aspartate aminotransferase and alanine aminotransferase. As one of the most crucial indicators of lipid metabolism together with cholesterol, the concentration of triglycerides was slightly lower in the humic substances treated groups than in the intact animals. It was discovered that using a feed supplement made from leonardite in a dosage of 18 mg/kg, which contained more fulvic acids than lignite, led to more favorable blood test results in the research group.

Experimental study of the effectiveness of ademethionine and inosine glycyl-cysteinyl-glutamate disodium in the treatment of dichloroethane-induced toxic hepatitis

Introduction. Currently, in the search for drugs for the toxic liver damage treatment, considerable attention is paid to glutathione preparations and its metabolic precursors. There are separate reports in the literature on the hepatoprotective and antifibrotic effects of glutathione disulfide in viral liver lesions, which gives grounds for their additional study when exposed to hepatotropic substances.
 Material and methods. Performing a comparative assessment of the hepatoprotective effect of inosine glycyl-cysteinyl-glutamate disodium and ademetionine on the model of dichloroethane-induced toxic hepatitis in rats was carried out with the formation of the following experimental groups of animals: no treatment; with the introduction of physiological saline; with the introduction of inosine glycyl-cysteinyl-glutamate disodium; with the introduction of ademetionine, as well as a group of intact animals. The study drugs were administered intraperitoneally for 10 days. In the course of the study, biochemical indicators of cytolytic and cholestatic syndromes of liver damage were determined, the activity of its detoxification system and plastic function were assessed.
 Results. In groups that did not receive treatment, but received physiological saline, the lethality of experimental animals by the 20th day of observation was 40%, in the group treated with ademetionine – 
 10%. In the group treated with inosine glycyl-cysteinyl-glutamate disodium, no death of animals was observed. The use of inosine glycyl-cysteinyl-glutamate disodium led to a more significant decrease in the indicators of cytolytic and cholestatic syndromes, as well as normalization of the functional activity of the liver (restoration of the content of glutathione in the liver tissue) and its plastic function (restoration 
 of the level of total blood serum protein and glycogen content in the liver parenchyma) to a greater extent than with ademetionine.
 limitation. The experimental study was carried out on outbred male rats weighing 180–220 g, kept under standard vivarium conditions in one cage, no more than 6 individuals, divided into groups by randomization with the exclusion of weakened and sick animals from the study.
 Conclusion. The study provided an experimental justification for further study of the hepatoprotective effect of inosine glycylcysteinylglutamate disodium in toxic liver damage.

Application of the Se NPs-Chitosan molecular complex for the correction of selenium deficiency in rats model

Selenium is an integral component of vital biologically active compounds of the human body. Currently, the population of many countries is characterized by selenium deficiency. In this regard, many preparations of inorganic and organic forms of selenium have been developed. Nevertheless, it is evident that the most effective solution to the problem is to enrich the diet with bioavailable forms of selenium. Thus, this work aimed to synthesize and study the antioxidant and immunomodulatory effects of the molecular complex of selenium nanoparticles (Se NPs) and chitosan in laboratory rats with induced hyposelenosis. During the experiment with animals, we found that as a result of 70-day consumption of food with a low selenium content, rats develop an alimentary selenium deficiency state, as evidenced by a significant decrease in the content of this trace element in control group rats to 48.2 ±6.71 µg/kg versus 149.3 ±21.63 µg/kg in intact animals. Course, administration of the molecular complex Se NPs- Chitosan to rats of the experimental group, contributed to the replenishment of selenium deficiency: its concentration in the blood of animals was 96.6 ±3.57 µg/kg. Thus, in animals of the control group, there was a decrease in the total number of lymphocytes by 2.7 times, T-lymphocytes – by 1.8 times, and B-lymphocytes – by 2.3 times compared with similar data in intact animals. In the context of hyposelenosis, it is worth mentioning that there was a slight increase in the content of T-helper cells and cytotoxic T-lymphocytes. The synthesized Se NPs – Chitosan complex administration during hyposelenosis demonstrated a notable immunomodulatory effect by restoring the body's immune response indicators. Thus, the total number of lymphocytes increased by 3 times, T-lymphocytes – by 1.9 times, and B-lymphocytes – by 2 times. The number of T-helper cells and cytotoxic T-lymphocytes increased by 1.9 times compared to the group of intact animals and 1.6 times compared to selenium-deficient rats. Thus, the course introduction of the molecular complex Se NPs – Chitosan against the background of selenium deficiency was accompanied by inhibition of free radical oxidation processes, activation of the antioxidant system and restoration of the immune status of the organism of laboratory animals.

MICROSCOPIC CHANGES IN RAT’S GINGIVA UNDER CONDITIONS OF EXPERIMENTAL PERIODONTITIS AND THE USE OF MESENCHYMAL STEM CELLS

MICROSCOPIC CHANGES IN RAT’S GINGIVA UNDER CONDITIONS OF EXPERIMENTAL PERIODONTITIS AND THE USE OF MESENCHYMAL STEM CELLS

MORPHOLOGICAL CHANGES OF THE RABBIT CORNEA IN THE LATE TERMS AFTER THE SIMULATION OF A MECHANICAL NON-PENETRATING CORNEA INJURY AND ITS CORRECTION USING DECELLULARIZED XENOSTROMA

Abstract. The global incidence of vision loss is estimated at 36 million per year, and one of the fi ve leading causes is injury or disease aff ecting the cornea. In some cases, damage to the cornea requires surgical intervention using xenografts.This study aims to investigate the peculiarities of the morphological changes of the rabbit cornea in the dynamics of experimental mechanical non-penetrating corneal injury and its correction with decellularized xenocorneal stroma.Material and methods. Experiments were performed on Chinchilla rabbits in compliance with the rules of bioethics. The animals were divided into three groups: I – animals without simulated pathology, II – animals with simulated mechanical nonenetrating corneal trauma, III – animals with corneal trauma that underwent surgical correction using pig corneal stroma obtained by the decellularization method. Animals were removed from the experiment 14 and 28 days after the beginning of pathology simulation. Rabbits of the second and third groups were subjected to a concentric epithelial incision in the upper half of the cornea of both eyes under local anaesthesia, within which the anterior epithelium was removed together with the upper layer of the corneal stroma with a disposable ophthalmic scalpel. The rabbits of the third group underwent corneal injury correction by closing the defect with decellularized pig xenocorneal stroma. For histological examination, pieces of the cornea were processed according to generally accepted methods. Para ffi n sections of the cornea were stained with hematoxylin and eosin. The thickness of the anterior corneal epithelium in the central and peripheral areas of the injury, the stroma’s thickness, and the cornea’s general thickness were determined morphometrically. The normal distribution determined the reliability of the diff erence between the data according to the student’s t-test. Diff erences were considered signifi cant at p<0,05.Results. Morphometrically established, the dynamic increase in the thickness of the anterior epithelium in the marginal zones up to 103.49±5.17 μm on the 14th day of the experiment, which is 1.44 times greater (p˂0,001) compared to the norm, and the thickness of the cornea, with the maximum value in the same period, to 759.42±27.12 μm, which exceeded the intact indicator by 2.39 times (p˂0.001). On the 28th day of the experiment, under the conditions of simulation of mechanical injury at the microscopic level, the closure of the defect with an epithelium of average thickness 60.27±2.01 μm with an uneven relief of the basal surface and areas of detachment from the substantia propria was revealed. In the upper layers of the stroma, signs of maturation of young connective tissue with the presence of fi broblastic and leukocyte cells, capillaries and disordered orientation of collagen fi bers are microscopically revealed. Morphometrically, on the 28th day, the substantia propria thickness of the second group animals is signifi cantly (p˂0.001) bigger than the norm but 1.56 times less compared to the 14th day of the experiment. Microscopically and morphometrically, it was established that under the condition of using a decellularized pig cornea for the correction of mechanical corneal trauma, hyperplasia of the epithelium of the marginal areas was most pronounced on the 14th day 126.37±5.06 μm, which resulted in the closure of the defect with a well-structured epithelial regenerate 35.20±1.07 μm without areas of exfoliation. These morphometric parameters signifi cantly (p˂0.001) exceeded those of similar groups of animals in the cornea without correction. In the same term, the dominance of cells of the fi broblastic series and the absence of morphological manifestations of infl ammation were recorded in the granulation tissue. It was histologically investigated that using a decellularized pig cornea 28 days after correction ensures the restoration of the typical structure of the cornea with the approximation of morphometric indicators to the group of intact animals. In contrast to the second experimental group, where no correction was performed, the renewed anterior epithelium of the central corneal zone of the animals of the third group had a clear diff erentiation into layers, an even relief of the basal surface and a contoured Bowman’s membrane. Its thickness 54.19±2.09 μm was unreliably diff erent from the norm. During this period of the experiment, when correction was applied, mature connective tissue and single capillaries in a state of reduction were histologically detected in the stroma, while the phenomena of maturation of connective tissue components were often observed in the cornea of the animals of the second experimental group. Microscopically, in the substantia propria, bundles of collagen fi brils were oriented parallel, which is one of the factors that ensure the cornea’s transparency.Conclusions. Microscopically and morphometrically, it was established that the use of decellularized pig cornea for the correction of mechanical corneal trauma improves the dynamics of regeneration, accelerates the maturation of granulation tissue, the healing of the defect, contributes to the structuring and restoration of the corneal morphology as close to normal as possible.

ULTRASTRUCTURAL ORGANIZATION OF THE RETINA LAYERS OF THE RAT IN NORM

The use of experimental animals with the aim of studying the dynamics of morphological changes requires deep knowledge of the initial indicators of the structure of the studied object in norm. In scientifi c medical literature, great importance belonges to the study of the retina in norm and the processes of changes in its structure under the infl uence of various nosologies. Considering the complexity of the retina structure and its role in the receiving information from the surrounding environment, it is important to understand the peculiarities of its layered organization in order to create a morphological basis for further comparing the experimental material and comparing it with the initial indicators of the norm with the results obtained at diff erent periods of the experimentally reproduced pathology. The aim of the study was to fi nd out the peculiarities of the layered structure of the cellular elements of the retina on the ultrastructural level in norm. Material and methods. The research material was 10 sexually mature, purebred male rats, weighing 160 g, aged 4.5 months. Before taking the material of the experimental area, the animal was removed from the experiment with the help of diethyl ether. Rat eyeballs were used as material for ultrastructural research. Ultrastructural samples were prepared according to the generally accepted method. As a result of our study of the retina of an intact group of animals, its layers preserved a clear stratifi cation architecture. The layer of the pigment epithelium was localized on Bruch’s membrane, choroidal vessels contained single erythrocytes and blood plasma. The nuclei of the pigment epithelium were preserved, the basal folds of the plasmolemma were clearly contoured. Small phagosomes were located in the cytoplasm of the pigment epithelium in one or two rows. The apical microvilli of the pigment epithelium were preserved, the interphotoreceptor space was hermetic. The membrane discs of the outer segments of the rods were clearly contoured. The cytoplasmic isthmus between the outer and inner segments of photoreceptors was intact. The outer boundary membrane was intensively osmophilic, preserved. Photoreceptor nuclei in the outer nuclear layer, clearly contoured. The synaptic zone of the outer reticular layer contained synaptic membranes and synaptic vesicles. The nuclei of horizontal and bipolar cells, the nuclei of Mьller cells and the nuclei of amacrine cells were located in the inner nuclear layer. The lumens of the vessels of the inner reticular layer were not dilated, they contained single erythrocytes. Nuclei and perikaryons of ganglion cells were arranged in one or two rows. Cytoplasmic structures of large gangloinary cells were preserved.