5-aminolevulinic acid-induced accumulation of protoporphyrin IX in rat brain tissue

The aim of the study was to compare the level of accumulation of protoporphyrin IX (ППIX) in the brain of rats in normal conditions and in experimental C6 glioma. Materials and methods. In an experiment on 15 rats, one group of animals (n=5) was intracranially implanted with rat glioma of the C6 line. 14 days after tumor implantation, the animals were injected into the lateral vein of the tail with a photosensitizer — a preparation of 5-aminolevulinic acid (5-ALA) Alasens at a dose of 100 mg / kg. Another group consisted of 5 intact rats, which were also injected with Alasens. The rats were euthanized 4–5 hours after the injection of the photosensitizer, and fluorescent metabolic navigation was performed with illumination of the brain with light with wavelengths of 417 and 435 nm. For objectification, fluorescence biospectroscopy was performed. Similar manipulations were performed with animals of another group (n=5) — intact rats that did not receive Alasens. Results. In contrast to humans, in rats, the 5-ALA metabolite — PPIX accumulates in healthy brain tissue, while the fluorescence intensity does not differ from that visualized in the tumor area. It was also noted that the light of the blue spectrum promotes weak fluorescence of the white matter of the rat brain in the absence of exogenous 5-ALA, which can potentially be explained by the activation of endogenous PPIX or other fluorophores. Conclusion. After the administration of Alasens (5-ALA preparation), the accumulation of PPIX by the rat brain tissue occurs not only by malignant cells, but also by normal brain tissue without signs of malignancy or other pathological changes. A more thorough study of this phenomenon is required, since significant differences in the metabolism of 5-ALA in humans and laboratory animals will call into question the correctness of translation of experimental results into clinical practice.

INFLUENCE OF HEAVY METALS ON ANTIOXIDANT SYSTEM AND BIOCHEMICAL INDEXES IN RATS

The study was undertaken to examine the effect of heavy metals on antioxidant system and biochemical indexes in the organism of rats. The influence of heavy metals on indexes The influence of heavy metals on the indicators of the antioxidant system (activity of the antioxidant system enzymes – glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase) and the processes of lipid peroxidation (content of hydroperoxides and products of thiobarbituric acid) was determined. It is established, that the antioxidant system functions more intensively in blood and liver of rats under the action of heavy metals. The study of enzyme activity showed the activation of the latter under conditions of heavy metal intoxication in 1.5–2.0 times (depending on heavy metal) compared with the control. We found that blood levels of total and direct bilirubin, creatinine, and urea increased in intoxicated rats from all experimental groups compared with intact animals. However, a decrease in the content of albumin, total protein, cholesterol, and triglycerides was also found in all experimental groups, in comparison with intact rats. Under the action of heavy metals, the activity of total α-amylase, lactate dehydrogenase, and glucose concentration increases in blood of rats. According to the results of studies in intoxicated animals compared with the intact group, there was a change in the cation-anion pool, in particular, a tendency to decrease the content of sodium and inorganic phosphorus and increase chlorides, magnesium, calcium, and potassium.

STRESS-PROTECTIVE EFFECT OF SUCCINATE-CONTAINING DRUG ON THE LEUKOCYTE COMPOSITION OF RAT BLOOD UNDER THE INFLUENCE OF HYPOXIA

The study of the dynamics of white blood cells in male rats under the conditions of normoxia and in the simulation of hypoxia before and after the use of the succinate-containing drug Cytoflavin ®was carried out. The animals were divided into three groups: two control groups of animals: group 1 – intact rats kept in standard vivarium conditions, and group 2, which were subjected to hourly hypoxic exposure, and group 3 - experimental animals subjected to daily training in a hypoxicator (O2-14%) and received the drug immediately before training. The multidirectional shifts characterizing the morphofunctional state of the white blood of rats under experimental conditions were determined. In group 2 animals, compared to group 3, there was a decrease in the number of white blood cells due to an increase in the pool of granulocyte cells, and an unchanged level of agranlocytes with a tendency to reduce the number of lymphocytes. In-group 2, compared with group 3, the change in the number of white blood cells and their subpopulation composition significantly differed from the control group 1, intact rats, and there was a decrease in the number of phagocytic mononuclears and an increase in granulocyte cells. The differences in the cellular composition of blood in group 2 animals from the data of group 3 are considered as the effect of the succinate-containing drug Cytoflavin ® on the preparation of the body for the implementation of the protective function under the influence of extreme factors and the stress of the body's resistance to load, since the nature of the redistribution of the leukocyte composition of blood in group 2 has a significant similarity in indicators with animals in group 3. The practical significance of the research is related to the search for new biologically active substances that optimally affect the ability of the animal body to adapt to conditions of low oxygen content. New data on the mechanism of redistribution of animal blood, which is realized under the action of the succinate-containing drug Cytoflavin ® in conditions of oxygen deficiency, can be used to study the regularities of the manifestation of an urgent adaptive effect.

EFFECT OF IMUNOPHAN ON MORPHOMETRIC PARAMETERS AND HISTOLOGICAL STRUCTURE OF PEYER'S PATCHES IN SMALL INTESTINE OF RATS UNDER IMMUNOSUPPRESSION INDUCED BY CYCLOPHOSPHAN

The aim of the study is to analyze morphometric parameters and histological structure of Peyer's patches in the small intestine of rats after imunofan administration under immunosuppression caused by cyclophosphan. Materials and Methods. The study was carried out on 36 white male rats (210–250 gramms). The animals were divided into two groups. Rats of Group 1 were injected with cyclophosphamide on the 1st day of the experiment (200 mg/kg). On the 2nd, 4th, 6th, 8th and 10th days they received imunofan (0.7 μg/kg). Group 2 consisted of intact rats. The animals were withdrawn from the experiment on the 7th, 30th, and 90th days after imunofan administration. The authors measured the length and width of Peyer's patches, and the distance from the ileocecal angle to the first patch. We also examined the height and width of lymph nodes and internodal zones on histological sections. Results. On the 7th and 30th days of our study the length and width of Peyer's patches increased by 18.71 %/16.94 % and 3.56 %/24.22 % respectively in mature animals, compared with the intact rats. At the same time, the distance from the ileocecal angle to the 1st Peyer’s patch decreased by 6.15 % and 19.80 %. The micrography showed that the height and width of the lymph nodes of Peyer's patches in experimental animals increased by 29.79 %/12.73 % (Day 7) and 10.49 %/12.46 % (Day 30) in comparison with the intact rats. The linear sizes of internodal zones increased by 39.35 %/10.33 % (Day 7) and 4.77 %/4.25 % (Day 30). By the 90th day, the macroscopic parameters of Peyer's patches, the height and width of their lymph nodules and internodal zones approxomated those of intact animals. Conclusion. When using imunofan under experimental immunosuppression, macro- and micromorphometric parameters of Peyer's patches of the small intestine of mature rats gradually approximated those in intact rats, which indicated the leveling of cyclophosphan immunosuppressive effect. Key words: rats, small intestine, Peyer's patches, imunofan, cyclophosphan. Цель исследования – изучение морфометрических параметров и гистологического строения пейеровых бляшек тонкой кишки крыс после введения имунофана на фоне иммуносупрессии, вызванной циклофосфаном. Материалы и методы. Исследование проводилось на 36 белых крысах-самцах массой 210–250 г. Животные были разделены на две группы. В I группе крысам в 1-е сут эксперимента вводился циклофосфан в дозе 200 мг/кг, на 2, 4, 6, 8, 10-е сут – имунофан в дозе 0,7 мкг/кг массы тела; II группу составили интактные крысы. Животные выводились из эксперимента на 7, 30, 90-е сут после завершения введения имунофана. Измерялись длина и ширина пейеровых бляшек, а также расстояние от илеоцекального угла до первой из них, а на гистологических срезах – высота и ширина их лимфатических узелков и межузелковых зон. Результаты. У половозрелых животных на 7-е и 30-е сут наблюдения длина и ширина пейеровых бляшек увеличивалась на 18,71 и 16,94 % и 3,56 и 24,22 % соответственно по сравнению с данными интактных крыс, а расстояние от илеоцекального угла до первой из них уменьшалось на 6,15 и 19,80 %. При микроскопическом исследовании высота и ширина лимфатических узелков пейеровых бляшек были больше по сравнению с данными интактных крыс на 29,79 и 12,73 % (7-е сут), 10,49 и 12,46 % (30-е сут), а линейные размеры межузелковых зон – на 39,35 и 10,33 % (7-е сут), 4,77 и 4,25 % (30-е сут). К 90-м сут макроскопические параметры пейеровых бляшек, а также высота и ширина их лимфатических узелков и межузелковых зон приближались к данным интактных животных. Выводы. При использовании имунофана на фоне экспериментальной иммуносупрессии значения исследуемых макро- и микроморфометрических параметров пейеровых бляшек тонкой кишки половозрелых крыс в ходе эксперимента постепенно приближались к аналогичным показателям интактных крыс, что свидетельствует о нивелировании иммуносупрессивного эффекта циклофосфана. Ключевые слова: крысы, тонкая кишка, пейеровы бляшки, имунофан, циклофосфан.

EXPRESS: Excitotoxic lesions of the perirhinal cortex leave intact rats’ gustatory sensory preconditioning

We report findings from two sensory preconditioning experiments in which rats consumed two flavoured solutions, each with two gustatory components (AX and BY), composed of sweet, bitter, salt and acid elements. After this pre-exposure, rats were conditioned to X by pairing with lithium chloride. Standard sensory preconditioning was observed: Consumption of flavour A was less than that of B. We found that sensory preconditioning was maintained when X was added to A and B. Both experiments included one group of rats with lesions of the perirhinal cortex, which did not influence sensory preconditioning. We discuss our findings in the light of other sensory preconditioning procedures that do involve the perirhinal cortex and conclude that differences in experimental variables invoke different mechanisms of sensory preconditioning, which vary in their requirement of the perirhinal cortex.

Age-related features of developing insulin resistance and adipocyte sensitivity to insulin in rats with induced metabolic syndrome

Old age and insulin resistance accompanying metabolic syndrome are significant factors in the development of cardiovascular pathology.Aim. The aim of the study was to identify the age-related characteristics of developing insulin resistance on the body level and insulin resistance in rat adipocytes in animals with induced metabolic syndrome.Material and Methods. The study was carried out on male Wistar rats, which were divided into the following groups: group 1 (n = 14) comprised intact rats aged 150 days at the end of study; group 2 (n = 14) comprised rats aged 150 days at the end of 90-day period on diet with high contents of carbohydrates and fats (HCHFD); group 3 (n = 14) comprised intact rats aged 540 days at the end of the study; group 4 (n = 14) comprised rats aged 540 days after the end of 90-day HCHFD. Diet composition was as follows: 16% proteins, 21% fats, 46% carbohydrates including 17% fructose, 0.125% cholesterol, and replacement of drinking water with a 20%-fructose solution. At the end of the diet, body and organ weights were measured, and contents of glucose, insulin, triglycerides, serum leptin, and liver triglycerides were assessed. Epididymal adipose tissue adipocytes were isolated enzymatically. The content of reactive oxygen species (ROS) was investigated using 2,3-dihydrodichlorofluorescein diacetate. Increased ROS production and lipolysis inhibition in response to insulin were observed.Results. The signs of metabolic syndrome were observed in both age groups of HCHFD animals and included visceral obesity, hyperglycemia, and insulin resistance measured by homeostatic model assessment of insulin resistance (HOMA-IR). However, the obesity indicators were more pronounced in the group of young rats, whereas the signs of insulin resistance prevailed in older rats. Leptinemia directly correlated with HOMA-IR (rSp = 0.485, p = 0.03). Insulin resistance of adipocytes was observed in rats older than 540 days after the HCHFD.Conclusion. The obtained results suggested that age was a risk factor for the development of insulin resistance on the body level. Aging resulted in a decrease in the adipocyte sensitivity to insulin in metabolic syndrome. An increase in leptin may be a possible mechanism for worsening of insulin resistance with age.

THE DYNAMICS OF LUNG HISTOPATHOLOGY IN ACUTE BACLOFEN POISONING

Baclofen (sold under the name Baclosan©, Lioresal©) is a muscle relaxant. This drug is chemically different from other muscle relaxants. Due to its pronounced psychotropic effect the drug is often a subject of abuse especially among young people. The article deals with the dynamics of lung histopathology in acute baclofen poisoning. Experimental studies were performed on 15 Wistar rats. The animals were divided into 3 groups (the controls and two experimental groups). The controls included 5 intact rats. Each experimental group included 5 animals. Both groups were treated with baclofen at a dosage of 85 mg/kg. The duration of the experiment was 3 and 24 hours, respectively. We revealed a complex of pathological changes in the lungs of the rats. There were circulatory disorders in all the elements of the microcirculatory bed, areas of emphysema, atelectasis and dystelectasis, WBC infiltration of intralveolar septa and their thickening due to edema. The changes were even more severe 24 hours after the drug administration. These results along with the results of chemical analysis will be useful in establishing the fact of baclofen intoxication and the exact moment of the intoxication.

Xenogeneic lymphocytic RNA stimulates skeletal muscle regeneration

Objective: to find evidence of the existence of distant lymphocytic RNA control of physiological myogenesis as a way to control the muscle tissue regeneration process.Materials and methods. The study was conducted on male Wistar rats, n=33. In the first part of the experiment, 12 rats were subjected to regular 40-day physical activity (swimming), half of them were intraperitoneally injected 4 times with total RNA isolated from pig spleen lymphocytes at 30 days of age; 6 rats made up the intact control group. In histological preparations of different skeletal muscle groups, the width and cross-sectional area of muscle fibers, the area of nuclei, and the number of myocytes and myosatellite cells were evaluated. In the second part of the experiment, 15 intact rats were injected with the studied xenogeneic RNA and the amounts of ribonucleic acids in peripheral blood lymphocytes, spleen lymphocytes, and skeletal muscles were determined 2 hours and 24 hours after injection.Results. After the 40- day physical activity, the width of the fibers and the area of myocyte nuclei in the skeletal muscles increased; the absolute number of myosatellite cells and the area of their nuclei did not change. After administration of xenogenic RNA in the trained rats, in addition to an increase in the thickness and cross-sectional area of muscle fibers, the absolute number of myosatellite cells in m. biceps femoris, in m. triceps brachii, and in m. pectoralis major increased 1.4-fold, 1.3-fold, and 1.4-fold, respectively; the area of myosatellite nuclei increased on average by 7%. In intact rats, two hours after xenogeneic RNA injection, the amount of RNA in skeletal muscles remained unchanged, it increased by 19% in spleen lymphocytes, and by 16% in peripheral blood lymphocytes. At 24 hours, the RNA amount in the lymphocytes remained significantly higher than the control values, while in the muscle tissue, it didn’t differ from the control.Conclusion. Xenogeneic lymphocytic RNA stimulates physiological myogenesis by activating myosatellite cell proliferation.

Molecular Pathomechanisms of Impaired Flow-Induced Constriction of Cerebral Arteries Following Traumatic Brain Injury: A Potential Impact on Cerebral Autoregulation

(1) Background: Traumatic brain injury (TBI) frequently occurs worldwide, resulting in high morbidity and mortality. Here, we hypothesized that TBI impairs an autoregulatory mechanism, namely the flow-induced constriction of isolated rat middle cerebral arteries (MCAs). (2) Methods: TBI was induced in anaesthetized rats by weight drop model, and then MCAs were isolated and transferred into a pressure-flow chamber. The internal diameter was measured by a video-microscopy. (3) Results: In MCAs from intact rats, increases in flow and pressure + flow elicited constrictions (−26 ± 1.9 µm and −52 ± 2.8 µm, p < 0.05), which were significantly reduced after TBI or in the presence of thromboxane-prostanoid (TP receptor) antagonist SQ 29,548. Flow-induced constrictions were significantly reduced by HET0016, inhibitor of cytochrome P450 4A (CYP450 4A). Arachidonic acid, (AA, 10−7 M), and CYP-450 4A metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) elicited constrictions of intact MCA (−26 ± 2.3% and −31 ± 3.6%), which were significantly reduced after TBI (to 11 ± 1.3% and −16 ±2.5%). The TP receptor agonist U46619 (10−7 M) elicited substantial constrictions of MCA from intact rats (−21 ± 3.3%), which were also significantly reduced, after TBI (to −16 ± 2.4%). (4) Conclusions: Flow-induced constrictor response of MCA is impaired by traumatic brain injury, likely due to the reduced ability of cytochrome P450 4A to convert arachidonic acid to constrictor prostaglandins and the mitigated sensitivity of thromboxane-prostanoid receptors.

Levels of Hsp60 in periodontal tissue of rats: influence of injections of hyaluronic acid

Heat shock protein 60 (Hsp60) is considered as one of the possible autoantigens involved in the pathogenesis of a number of chronic diseases including periodontal diseases. The application of hyaluronic acid or hyaluronan (HA) in the treatment of periodontitis has been evaluated in several clinical trials, however, the effect of hyaluronic acid on heat shock protein 60 level in periodontal soft tissues has not been studied. The aim of this work was to evaluate the effect of HA injections on levels of Hsp60 in periodontal tissue of the rats. Samples of periodontal tissue of mandibular incisors of adult male Wistar rats at 10-12 months of age were investigated. Rats were distributed into the control group and the periodontitis group. Visual manifestations of hyperemia of the gums around the incisors were the criterion for selecting animals into the periodontitis group. There were two subgroups in the control group: intact rats (I); intact rats after HA “hyaDENT BG” 1.0 MDa (BioScience GmbH, Germany) treatment (I+“G-1.0”). There were four subgroups in the periodontitis group: rats with periodontitis (P); rats with periodontitis after HA “hyaDENT BG” 1.0 MDa (BioScience GmbH, Germany) treatment (P+“G-1.0”); rats with periodontitis after HA “SERTOBEC” 2.4 MDa (S.C. Rompharm Company S.R.L., Romania) treatment (P+“S-2.4”); rats with periodontitis after HA “SERTOBEC Tendon” 2.4 МDа (S.C. Rompharm Company S.R.L., Romania) treatment (P+“ST-2.4”). There were three animals in each subgroup. Rats were injected 0.05 ml HA in the area of alveolar processus of central incisors once a week, three times. Levels of Hsp60 in total lysates of periodontal tissue were tested by Western blotting method before and after the treatment with HA (one month after the last injection). There was no significant difference between levels of Hsp60 in total lysates of periodontal tissue of intact rats and rats with periodontitis before treatment (p>0.05). Rats with periodontitis showed decreased inflammation in the periodontal tissue after treatment with HA with different molecular weight. Intact rats and rats with periodontitis which were treated with HA “hyaDENT BG” 1.0 MDa showed reduced levels of Hsp60 in total lysates of periodontal tissue comparatively with levels of Hsp60 before treatment (by 15.4 and 10.7 times respectively, p<0.001). Rats with periodontitis which were treated with HA “SERTOBEC” 2.4 MDa or HA “SERTOBEC Tendon” 2.4 MDa also showed reduced levels of Hsp60 in total lysates of periodontal tissue com­paratively with levels of Hsp60 before treatment (by 21.3 and 16.4 times respectively, p<0.001). Rats with pe­riodontitis showed the decrease in inflammation in periodontal tissue after treatment with HA with different molecular weight. Injections of HA has contributed to reduce levels of Hsp60 in periodontal tissue of intact rats and rats with periodontitis.