Experience of Antiseptic Application for Processing of Rat Lung Biological Matrices.

To select the optimum method for disinfecting scaffolds before recellularization, the effects of octenisept and chlorhexidine at different concentrations on lung biological matrices before and after decellularization were studied by using morphological methods (studies of biomechanical strength of extracellular matrix fibers) and by analyzing chemiluminescence in rats. Chlorhexidine diluted 1 : 10 had the least damage on the matrix properties and to the greatest extent contributed to disinfection of scaffolds for their further storage and experimental studies.

Estimation of Decellularization and Recellularization Quality of Tissue-Engineered Constructions by the Chemiluminescence Method.

It was found that the chemiluminescence intensity in native and recellularized tissues of rat muscular organs as well as in their decellularized scaffolds can serve as an express criterion that, along with ultrastructural analysis, makes it possible to perform quantitative assessment of the viability of cellular structures in biological samples of the diaphragm.

Mathematical Modeling of Open States in DNA Molecule Depending on the Deuterium Concentration in the Surrounding Liquid Media at Different Values of Hydrogen Bond Disruption Energy.

The effect of the deuterium concentration in the surrounding liquid medium on the appearance of open states between the nitrogen bases of a double-stranded DNA molecule was investigated. It is shown that the nature of this effect depends on the magnitude of the energy of the hydrogen bond break.

Changes in the functional activity of mitochondria isolated from the liver of rat that passed the preadaptation to ultra-low deuterium concentration.

The incubation in deuterium-depleted medium of mitochondria isolated from the liver of rats that consumed drinking diet with depleted deuterium (46 ppm) revealed a higher (by 35%) generation of hydrogen peroxide in comparison with the mitochondria (isolated from the liver of rats that consumed drinking diet with 152 ppm deuterium) incubated in medium that contained 152 ppm deuterium. Succinate addition to the reaction system led to an increase in the production of hydrogen peroxide in isolated mitochondria by 44-81%, whereas the difference in the generation of H2O2 between the organelles incubated in mediums 46 and 152 ppm was reduced by 14%. The revealed change in the functional activity of mitochondria suggests the ability of the organism to adapt to the deuterium-depleted drinking diet, which is probably due to the formation of the D/H isotope transmembrane gradient.

Dynamics of the Pro-Oxidant/Antioxidant System Parameters in Wound Discharge and Plasma in Experimental Purulent Wound during Its Technological Liquid Phase Treatment.

The dynamics of the pro-oxidant/antioxidant system of rabbits was studied at the local (in exudate) and systemic (blood) levels during therapy of purulent wounds in liquid medium with the use of a programmed device and the efficiency of the proposed technological method was compared with that of classical methods for the treatment of these wounds. More rapid recovery of the indicators of the pro-oxidant/antioxidant system to physiological values in the postoperative period was observed after treatment by the proposed method (7 days vs. 10 days after standard treatment), these results attest to strengthening of the adaptation potential in laboratory animals. The intensity of free radical oxidation in the exudate decreased by 26.6% in animals treated by the technological method. These data indicated acceleration of the regenerative processes at the local level.

[Experimental development and rationale for a renal decellularization protocol with subsequent comprehensive assessment of the biological scaffold].

Chronic renal failure (CRF) is one of the most challenging problems of contemporary medicine. Patients with chronic renal failure usually need renal replacement therapy as either hemodialysis, peritoneal dialysis or a kidney transplant. The latter is the most promising option for end-stage kidney disease. However, the shortage of donor organs, the complexity of their delivery, the difficulty in finding an immunologically compatible donor and the need for lifelong immunosuppression triggered advances in modern tissue engineering. In this field, the primary priority is focused on developing bioengineered scaffolds with subsequent recellularization with autologous cells. Using such constructs would allow for solving both ethical and immunological problems of transplantation. The aim of this pilot study was to develop a new method of renal decellularization using small laboratory animals. MATERIALS AND METHODS: The study investigated the morphological structure of the obtained decellularized matrix and quantitatively tested DNA residues in the resulting scaffold. We proposed a new biophysical method for assessing the matrix quality using the EPR spectroscopy and conducted experiments on the matrix recellularization with mesenchymal multipotent stem cells to estimate cytotoxicity, cell viability and metabolic activity. RESULTS: The obtained decellularized renal matrix retained the native tissue architecture after a complete removal of the cell material, had no cytotoxic properties and supported cell adhesion and proliferation. CONCLUSION: All the above suggests that the proposed decellularization protocol is a promising method to produce tissue-engineered kidney constructs with possible clinical application in the foreseeable future.

[The metabolic correction of the experimental alloxan diabetes by means of the antioxidant remedies].

The research concerning the effect of the remedies with the different antioxidant mechanisms on the indices of carbohydrate and lipid metabolism as well as the thiol link of the antioxidant system and the functional detoxication system in modeling conditions of the decompensated alloxan diabetes mellitus have been presented. The research was performed on 105 white nonlinear male-rats with initial body weight (b.w.) 200-230 g, divided into seven groups. Group 1 has consisted of the intact animals. Group 2 (the comparison group) has consisted of the animals suffering from the severe alloxan diabetes mellitus (intraperitoneal administration of alloxan monohydrate at 10 mg/100 g b.w. three times, with an interval of one day, against fasting) without correction. In groups 3-7 diabetes mellitus has also been modeled. The animals of group 3 were fed by the diet with the supplement containing the antioxidant complex with high content of carotenoid substances (1 mg lutein per day, orally for a month before simulations of diabetes mellitus and for a month after). The animals of group 4 have been given the sodium dichloroacetate (15 mg/100g bw) with drinking water for the month before and a month after the modeling of diabetes. The animals of group 5 have been injected with the reduced glutathione (15 mg/100 g of b.w.) daily one week prior to administration of alloxan and until the end of the experiment, the animals of group 6 have been injected with the quercithin (10 mg/100 g of b.w.) and the rats of group 7 have been given the water with the reduced content of deuterium (91 mg/l) instead of the usual drinking water. The development of experimental alloxan diabetes mellitus in rats has been accompanied by the increase in blood plasma of glucose concentration by 6.9-fold, cholesterol and lipoproteins of low density by 66 and 100% respectively. The compensatory activity increase of the antiradical protective enzymes (glutathione peroxidase and glutathione reductase) as well as the concentration decrease of reduced glutathione in erythrocytes by 11% have been revealed. According to the data concerning the level of the molecules with medium and low mass in blood plasma as well as in erythrocytes, the evident endogenous intoxication has taken place. The performed researches have proved the presence of the significant hypoglycemic effect of the usage of sodium dichloroacetate, of the water with the reduced content of deuterium and of quercithin - the glucose concentration in these groups was lower than in the comparison group by 36, 33 and 47% respectively. In the same groups as well as in the group of rats injected with glutathione the hypolipidemic effect of various manifestation degree has been detected, the most evident in animals from group 7 in which all the indices except triglycerides haven't differed from indices of the control group. The usage of various corrective methods has also assisted the adequate functioning of the antioxidant system that has been proved by the normalization of glutathione content except of groups 3 and 6 in which its concentration was lower than the control indices by 25 and 12% respectively. Endogenous intoxication has been significantly lower in groups 4, 5 and 7 in which the level of molecular of medium and low mass in blood plasma was normal and only in erythrocyte mass was increased by 49-74% than in the control group. Thus, the data obtained prove the usage availability of the antioxidant remedies in the complex therapy in patients suffering from the diabetes mellitus. The most interesting is the usage of antioxidants with the indirect mechanism of action, i.e. the water with the reduced content of deuterium and sodium dichloroacetate. Quercithin and the reduced glutathione deserve attention as well, and considering the hypoglycemic effect of the first one and the metabolic effects of glutathione aimed at the functioning maintenance of the non-specific organism resistance, the possibility of the combined usage of both antioxidants should be taken into account.

EPR spectroscopy solutions for assessment of decellularization of intrathoracic organs and tissues.

Using EPR spectroscopy it was established that the determination of the concentration of paramagnetic centers in lyophilized tissues allows indirect evaluation of the quality of decellularization of intrathoracic organs (diaphragm, heart, and lungs), since the content of paramagnetic particles in them can serve as a criterion of cell viability and points to the necessity to repeat decellularization. Experiments in rats showed that the EPR spectra of the native thoracic organs contained paramagnetic centers with g-factor values ranging from 2.007 to 2.011 at a concentration of 10(-8) to 6.62 × 10(-7) mol/g of lyophilized tissue, whereas in all decellularized tissues of the same organs paramagnetic particles were not detected.

[Influence of linseed oil and deiterium depleted water on isotopic D/H composition and functional antioxidant defense of the hepatobiliary system in rabbits with carbon tetrachloride intoxication].

The article presents results of studying the influence of linseed oil and drinking diet with a modified isotopic composition with low deuterium content on indicators of prooxidant-antioxidant system during modeling of liver toxicity. The research was performed on 36 rabbits (weighing 3.1-3.5 kg) which were divided into 4 groups. Group 1 consisted of control animals; in group 2, 3 and 4 in rabbits the liver toxicity was modeled by administration of CCl4 (intraperitoneally, in the form of a 50% oil solution, 1 ml per kg bw, 2 times a week for 30 days); nutritional correction using flaxseed oil (0.1 ml per 100 g bw) and drinking diet with deuterium depleted water (50 ppm) was carried out in animals from groups 3 and 4 respectively, for 30 days prior to simulation of toxic hepatitis and more throughout the experiment. Using the method of nuclear magnetic resonance and mass spectrometry the influence of deuterium depleted water on D/H composition of the blood plasma, bile and liver tissues was determined: the deuterium concentration in these biological materials significant decreased. The most significant decrease in the deuterium content (30.2% compared with the control group) was found in bloodplasma in animals from group 4. The study of the state of prooxidant-antioxidant balance of the liver and bile showed oxidative stress at the local level, with the toxic effects of carbon tetrachloride. This was followed by EPR spectroscopy data pronounced increase of the number of paramagnetic centers in the hepatocytes by 5.4, 1.9 and 2.8 fold in animals of 2, 3 and 4 groups, respectively (compared to the indicators of the first group). There was also increase in the intensity of free radical oxidation processes in the bile with a simultaneous reduction of its antioxidant activity, which was significantly less distinct (on average 51.18-59.8%, p<0.05) in animals treated with nutritional correction, indicating that higher functional activity of protective systems involved in recycling prooxidant factors using dietary lipophilic antioxidants and water with low deuterium content. Overall, the results of the present study indicate that existing in the liver and bile autonomous mechanisms of regulation of the state of prooxidant-antioxidant systems are quite sensitive to the effects of antioxidant factors of lipophilic nature and shifts of isotopic D/H gradient, and suggest usefulness of the products that can affect these indicators to increase adaptive capabilities of the organism during intoxication.

[Correction of Metabolic Processes in Rats during Chronic Endotoxicosis using Isotope (D/H) Exchange Reactions].

The effect of isotope exchange reactions (deuterium/protium, D/H) on morphofunctional indices and the state of the antioxidant blood system in rats was studied under physiological conditions and during experimental chronic endotoxicosis of hepatorenal genesis. It was demonstrated that introduction of water with a decreased content of deuterium in the food rations of rats results in a decrease in its concentration in the blood plasma by 32-36% (to 98-106 ppm) and in lyophilized liver, kidney, and heart tissues by 13-17% (to 123-128 ppm). It was noted that it is accompanied by correction of metabolic processes, an increase in the functional activity of nonspecific protection system, and an increase in the body weight growth by the 42nd day in the group of animals that passed (for 14 days) the stage of preliminary adaptation with a change in the D/H ratio in the organism.

[Effect of water isotopic composition on Rhodococcus erythropolis biomass production].

The effect of water isotopic composition on concentration of oil-oxidizing actinobacteria Rhodococcus erythropolis VKM Ac-2017D cellular biomass is observed during cultivation in liquid nutrient media. The value for the effect was determined by experimental conditions, including consecutive use of hydrophilic and hydrophobic nutritious substrates--saccharose and hexadecane. It is shown, that when Rhodococcus erythropolis VKM Ac-2017D cells, cultivated in media with sucrose and 71 and 98 ppm deuterium content in water were inoculated into the similar in mineral and isotopic composition media with hexadecane, a considerable increase in cellular biomass production is observed if compared to a control sample in which water with 150 ppm deuterium concentration was used.

Content of deuterium in biological fluids and organs: Influence of deuterium depleted water on D/H gradient and the process of adaptation.

It is found that consumption of deuterium depleted water reduces not only the content of deuterium in biological fluids but also more than 2 times reduces the D/H gradient value along the line: mixed saliva > blood plasma. The experimental data showed that a physiological solution prepared on deuterium depleted water during induced apoptosis activates the DNA repair system, significantly reducing the number of single-stranded DNA breaks, which, in general, indicates an increase in the efficiency of defensive systems of the cell.

[Comparative characteristics of the isotopic D/H composition and antioxidant activity of freshly squeezed juices from fruits and vegetables grown in different geographical regions].

Data presented in this paper reflect changes in antioxidant activity, the content of prooxidant factors and deuterium concentration in freshly squeezed juices from fruits and vegetables grown in different climatic regions (10 samples of juices from wholesale and retail trade network of 8 kinds of vegetables and fruits, 28 manufacturers from 14 countries). Determination of the concentration of deuterium was performed using a nuclear magnetic resonance spectrometer. Total antioxidant activity of fresh juices was determined amperometrically after dilution in 2.2 mM H3PO4 in a ratio of 1:100. Prooxidant performance was evaluated by a maximum and area of flash of chemiluminescence induced by the introduction of 0.3% hydrogen peroxide. It was found that the antioxidant activity of fresh juice from fruits and vegetables grown within the same climatic region can differ by several times. In this case, most of the fruits and vegetables of russian producers were not inferior, than antioxidant activity of the fresh juices from the same plant products grown abroad. It should be noted that the indicators of the antioxidant activity of fresh juice from Russian pears exceeded this indicator of all fresh juices from pears, imported from Argentina, South Africa and the United States of America by 21.1, 30.4 and 32.7%, respectively. In assessing the prooxidant properties of fresh juices should be noted the almost complete absence of factors with prooxidant nature only in 36% of the studied fresh juices, whose maximum performance and area of flash of chemiluminescence were less than 0.1%, including a pear and apple juices from the russian production. It should be noted that the area of chemiluminescence of the juice from potatoes, grown in Russia, was at 103.1 and 115.2% lower than in juice obtained respectively from potatoes produced in Israel and Egypt (p<0.05), indicating a higher safety of consumption of potatoes produced in Russia. When studying--the isotopic D/H composition of fresh juices it was found that the highest deuterium content was in the juice from the pears, imported from Argentina (deltaD = -72% per hundred), while the lowest concentration of deuterium was observed in the juice from the Egyptian potatoes (delta = -358% per hundred). In general, significantly lower deuterium content was determined in fresh juices made from potatoes and cabbage grown in different countries, in comparison with other fresh juices from fruits and vegetables. The smallest range of differences in the isotopic D/H was composed in freshjuices from tomato, pomegranate and oranges of Turkish manufacturers (deuterium concentration ranged in them from -221 to -214% per hundred), that can be used to confirm the geographical origin of fruits and vegetables grown in Turkey. The data reflecting the antioxidant activity, the content of prooxidant factors and deuterium concentration in the juices, allow us to recommend the latter as additional criteria when assessing the quality of food products.

[Influence of deuterium depleted water on freeze-dried tissue isotopic composition and morphofunctional body performance in rats of different generations].

The influence of deuterium depleted water on the body of different rats generations was investigated in physiological conditions. As a result of this study it was established that the most significant and rapid reduction in D/H equilibrium was observed in plasma (by 36.2%), and lyophilized kidney tissues (by 15.8%). Less pronounced deuterium decrease was characteristic of liver tissue (9.3%) and heart (8.5%). Stabilization of the isotopic exchange reaction rate was fixed in the blood and tissues of rats, starting from the second generation. At the same time when deuterium depleted water (40 ppm) was used in dietary intake, the change in morphological and functional parameters in laboratory animals associated with the processes of adaptation to the effects of substress isotopic D/H gradient was also noted. The study shows that modification of:only drinking water intake regime can't significantly change the deuterium content in tissues of metabolically active organs, because of the concurrent deuterium receipt in food substances of plant and animal origin.

[Influence of deuterium depleted water on the organism of laboratory animals in various functional conditions of nonspecific protective systems].

The influence of deuterium depleted water with the modified isotope composition on an organism of animals is studied in physiological conditions and development of chronic endogenous intoxication of hepatorenal genesis. The influence of this water on isotope composition of plasma and tissues (the liver and kidneys) in laboratory animals is shown. The impact of this water on biochemical indicators (AST, ALT, alkaline phosphatase, creatinine, bilirubin) and dynamics in body weight of laboratory animals within 42 days is established. As a result of studies the possibility of the preventive use of deuterium depleted water for correction of metabolic processes is shown in various conditions of the functional system of the body's detoxification.

[Determination of deuterium concentration in foods and influence of water with modified isotopic composition on oxidation parameters and heavy hydrogen isotopes content in experimental animals].

The article presents the results of the study of the deuterium (D) content in food products as well as the influence of deuterium depleted water (DDW) on the concentration of heavy hydrogen isotopes in the blood and lyophilized tissues of rats. The most significant difference in the content of D was found between potato and pork fat, which indexes the standard delta notation (δ) D in promille, related to the international standard SMOW (Standard Mean Ocean of Water) amounted to -83,2 per thousand and -250,7 per thousand, respectively (p<0,05). Among the investigated samples of water deuterium concentration ranged from -75,5 per thousand (Narzan) to +72,1 per thousand (Kubai), that indicates the ability of some food products to increase the concentration of heavy hydrogen atoms in the body. The data obtained in the experimental modeling of the diet of male Wistar rats in the age of 5-6 mo (weight 235 ± 16 g) using DDW (δD = -743,2 per thousand) instead of drinking water (δD = -37,0 per thousand) with identical mineral composition showed that after 2 weeks significant (p <0,05) formation of isotopic (deuterium-protium, D/H) gradient in the body is possible. Changing the direction of isotopic D/H gradient in laboratory animals in comparison with its physiological indicators (72-127 per thousand, "plasma>>tissue") is due to different rates ofisotopic exchange reactions in plasma and tissues (liver, kidney, heart), which can be explained by entering into the composition of a modified diet of organic substrates with more than DDW concentration D, which are involved in the construction of cellular structures and eventually lead to a redistribution of D and change direction of D/H gradient "plasma<

[The effect of water with modified isotope content on the parameters of free radical oxidation in vivo].

With the use of nuclear magnetic resonance we investigated the effect of consumption of water with the modified isotope content on the composition of deuterium in the blood, assessed the state of pro-oxidant-antioxidant system in the blood and lyophilized tissues (liver, kidneys) under prolonged oxidative stress. Possible mechanisms of direct and indirect antioxidant effects of the water with modified isotope content and the perspectives of its use for nutritional correction of abnormalities of oxidative metabolism during special physiological conditions and in clinical practice are discussed.