RU2796316C1 - Method of simulation of physical load for evaluation of workability of laboratory rats with dysbiotic intestinal disorders - Google Patents

Abstract

FIELD: experimental medicine.

SUBSTANCE: invention can be used to study changes in exercise tolerance of the organism of small laboratory rodents and to test methods for its correction by factors of various nature under conditions of dysbiotic intestinal disorders. The method includes oral administration to rats of Gentamicin antimicrobial drug at a dose of 30 mg once a day for 5 days, starting from the 1st day of the experiment. Also, from the 1st day of the experiment, the animals are subjected to forced swimming with a load attached to the tail and constituting 7% of the animal's body weight, by four forced swimming with an interval of 1 time in 5 days. An installation is used, which is a pool of opaque material, 98 cm high and 50 cm in diameter, the height of the water column in the pool is 60 cm; the temperature is maintained at 29–30°C. The animals are subjected to swimming until they are completely exhausted, the criterion of which is the unsuccessful attempts of the animal to rise to the surface of the water for more than 10 s and/or sink to the bottom of the pool.

EFFECT: invention provides for the development of a method that allows modeling physical activity under conditions of dysbiotic disorders in laboratory rats with an assessment of changes in physical performance.

1 cl, 2 tbl

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

SUBSTANCE: invention relates to experimental medicine and can be used to study changes in exercise tolerance of the organism of small laboratory rodents and to test methods for its correction by factors of various nature under conditions of dysbiotic intestinal disorders.

BACKGROUND OF THE INVENTION

Elucidation of the patterns of the body's response to physical activity, the study of their consequences is one of the urgent problems of modern biology and medicine. Known experimental models of post-traumatic stress disorders in laboratory animals using intense physical activity.

It is known to provide physical activity by means of forced swimming of the animal in a container of water; in which the load is carried out in two stages: at the first stage, experimental desynchronosis is induced by keeping rats for 12 days around the clock under artificial light or in complete darkness, after which the animals are subjected to forced swimming under natural light in a rectangular glass aquarium with dimensions of 100 cm × 100 cm × 20 cm, divided into compartments connected at the bottom, and equipped with two valves - one at the top and bottom of one of the side walls, with a load attached to the tail, the weight of which is 10% of the animal's body weight, at a water temperature of 26-28 °С for 5 days until complete fatigue, the criterion of which is three unsuccessful attempts to ascend to the surface or the refusal of such attempts to sink to the bottom. (Description of the invention to the patent of the Russian Federation No.

In this technical solution, the task is to simulate physical overwork in rats under conditions of desynchronosis, during the formation of which the animals reach a state of fitness and resistance to stress. The resulting model adequately reflects the problems of sports physiology, the creation of forced physical activity is the mode of exposure 1 time in 24 hours, which corresponds more to sports medicine; i.e., it limits the range of use of this model in other areas of research, namely the use of physical activity in intestinal dysbacteriosis to study the problems of sports physiology.

The effect of the antibacterial drug gentamicin on the intestinal microflora is known, and the determination of the number of viable microorganisms of the intestinal microflora at the beginning and end of the experiment, and comparing their numerical values, while creating a selective pressure of the antibacterial drug gentamicin when administered orally to laboratory animals 1 time per day for 5 days in doses exceeding its daily therapeutic dose when administered parenterally by 4.8 times in mice and 4 times in guinea pigs. (Description of the invention to the patent of the Russian Federation No. 2477894, IPC G09B 23/28, published on 03/20/2013).

The difference lies in the fact that the determination of viable intestinal microorganisms is carried out only at the beginning and at the end of the experiment without assessing the intensity of the natural restoration of microflora after a long period of time after the administration of the last dose of the antimicrobial drug. Modeling of physical overwork in animals under conditions of desynchronosis is not used for comparative analysis.

The proposed technical solution makes it possible to create such an experimental model that allows studying the effect of a violation of the normal intestinal microflora on physical performance and finding ways and means to prevent and correct a decrease in physical performance under the influence of factors leading to a violation of the intestinal microflora.

DISCLOSURE OF THE INVENTION

The objective of the invention is to develop a method that allows simulating physical activity under conditions of dysbiotic disorders in laboratory rats with an assessment of changes in physical performance.

This task is achieved by combining the introduction of gentamicin with physical swimming exercise, which leads to modification of the intestinal microflora of animals (rats). The method for modeling physical activity in rats with dysbiotic disorders of the intestine is carried out by oral administration of the antimicrobial drug gentamicin at a dose of 30 mg 1 time per day for 5 days, starting from the 1st day of the experiment; animals are also subjected to forced swimming with a load attached to the tail and constituting 7% of the animal's body weight, by four times forced swimming with an interval of 1 time in 5 days; use the installation, which is a pool of opaque material, 98 cm high and 50 cm in diameter, the height of the water column in the pool is 60 cm; the temperature is maintained at 29-30°C; the animals are subjected to swimming until they are completely exhausted, the criterion of which is the unsuccessful attempts of the animal to rise to the surface of the water for more than 10 seconds and / or sink to the bottom of the pool.

IMPLEMENTATION OF THE INVENTION

The method is carried out as follows.

In experimental animals, the state of intestinal dysbacteriosis was modeled by oral administration of the antimicrobial drug gentamicin at a dose of 30 mg once a day for 5 days, starting from the 1st day of the experiment. From the 1st day of the experiment, the animals also began to be subjected to forced physical activity with monitoring of changes in working capacity, for which the test "Forced swimming with a load" was used. For the test, an installation was used, which is a pool of opaque material, 98 cm high and 50 cm in diameter. The height of the water column in the pool was 60 cm, the temperature was maintained at 29-30°C. Lead weights were used as weights. The weighting agent was attached to the base of the animal's tail by means of a rubber ligature and fishing line. The length of the fishing line from the point of attachment to the weighting agent approximately corresponded to the length of the animal's tail, at which the “weight” did not slip off the tail during the test, and the rats did not lower the weighting agent to the bottom of the pool to relieve themselves of the load. The used weight of the weight, which was 7% of the individual body weight of the animal. The animals are subjected to swimming until they are completely exhausted, the criterion of which is the unsuccessful attempts of the animal to rise to the surface of the water for more than 10 seconds and/or sink to the bottom of the pool. Animals were subjected to forced swimming with weighting four times with an interval of 1 time in 5 days. The total swimming time of rats was used as a parameter for assessing physical endurance.

The experimental study was carried out on 16 sexually mature non-linear male rats weighing 438[423; 496] g, kept under standard conditions of a conventional vivarium at a temperature of 23 ± 2 ° C and a humidity of 65% with natural lighting on a complete commercial granulated feed for keeping (Laboratorkorm LLC, Moscow) with free access to drinking tap water and food. All manipulations were carried out in accordance with Directive 2010/63/EU of the European Parliament and of the Council of the European Union of September 22, 2010 on the protection of animals used for scientific purposes.

All animals were divided into 3 groups of 5-6 individuals:

1) rats that were not exposed to the action of an antimicrobial drug and exercise; intact group;

2) rats that were subjected to physical activity to overwork; group #1;

3) rats in which dysbiotic disturbances in the intestine were modeled and which were subjected to physical activity to overwork; group number 2.

Physical activity in animals was realized by 4-fold forced swimming with an interval of 5 days in a round opaque pool with a load of 7% of body weight at a water temperature of 29-30°C until complete fatigue. Induction of experimental intestinal dysbiosis in animals of group No. 2 was carried out by oral administration of a commercial 4% solution of gentamicin sulfate using an insulin syringe without a needle at a dose of 30 mg per day for 5 days, starting from the first day of the experiment. To monitor changes in the intestinal microflora under the influence of physical activity and an antimicrobial drug on days 1, 6 and 16 of the experiment, i.e. before administration, after the completion of the course administration and 10 days after the completion of the administration of gentamicin, respectively, feces were individually selected from each animal, weighed, and then suspended in 9.0 ml of isotonic 0.9% sodium chloride solution and sown in different dilutions on selective nutrient media. After incubation, the number of grown colonies was counted. Taking into account the mass of feces from each animal and the number of grown colonies, the total number of microorganisms (TMC), bifidobacteria, lactobacilli and Escherichia per 1 g of feces (CFU·g ^-1 ) was recalculated. The collection of biomaterial was carried out in accordance with MU 4.2.2039-05. The identification of colonic microorganisms was carried out in accordance with the Order of the Ministry of Health of the USSR dated April 22, 1985 No. 535 "On the unification of microbiological (bacteriological) research methods used in clinical diagnostic laboratories of medical institutions" and MU "Bacteriological diagnosis of intestinal dysbacteriosis", according to the method developed by R.V. Epstein-Litvak and F.L. Vilshanskaya (1977). The final result of the quantitative content of bacteria per gram of faeces was expressed as lgCOE/g.

The swimming of the animals was carried out in a special installation, which is a pool of opaque material, 98 cm high and 50 cm in diameter. The height of the water column in the pool was 60 cm, the temperature was maintained at 29-30°C. Lead weights were used as weights. The weighting agent was attached to the base of the animal's tail by means of a rubber ligature and fishing line. The length of the fishing line from the point of attachment to the weighting agent approximately corresponded to the length of the animal's tail, at which the “weight” did not slip off the tail during the test, and the rats did not lower the weighting agent to the bottom of the pool to relieve themselves of the load. The used weight of the weight, which was 7% of the individual body weight of the animal. Animals are subjected to swimming until complete fatigue, the criterion of which was unsuccessful attempts to float to the surface of the water for more than 10 seconds and/or sink to the bottom of the pool, after which the rat was removed from the pool.

Two days after the last day of physical activity, rats were taken out of the experiment by simultaneous decapitation and blood was collected to assess changes in energy metabolism in terms of plasma lactate levels using the Lactic Acid-Olvex reagent kit (OlvexDiagnosticum LLC, St. Petersburg, Russia.) according to instructions.

Statistical data processing was carried out using STATISTICA 8.0 software. To identify statistical differences between two independent groups, the Mann–Whitney test (p≤0.05) was used. As a preliminary statistical method for multiple comparison of dependent groups, the Friedman test was used, followed by pairwise comparison using the Wilcoxon test, when comparing independent groups, the Kruskal–Wallis H-test, followed by pairwise comparison using the Mann–Whitney test. Differences were considered statistically significant at p≤0.017 (after recalculation by the number of comparisons). The results were presented as Me [ICR] (where Me is the median and IQI is the interquartile range).

Table 1

Dynamics of changes in the physical performance of laboratory animals in research groups

Group name Swimming on the 1st day of the experiment, with Swimming on the 6th day of the experiment, with Swimming on the 11th day of the experiment, with Swimming on the 16th day of the experiment, with Group #1 109.0[89.6; 109.9] 98.0 [83.0; 105.0] 133.0 [115.0; 154.0] 157.0 [140.0; 167.0] Group #2 87.0 [79.0; 95.0] 93.9 [83.0; 120.0] 88.0 [79.0; 98.0] • 105.0 [89.0; 133.0]

•p=0.019 according to the Mann-Whitney U-test when compared with group No. 1 on the 11th day of the experiment

table 2

Dynamics of changes in the microbiota of the large intestine of rats during modeling of physical activity and dysbacteriosis

MO groups,
lg cfu/g Int. group 1 day 6 days 16 day #1 #2 #1 #2 #1 #2 Bifidobacterium 3[3; 5] 3[3;4] 4[3;6] 3[3;3] 3[3;4] 3[3;3] 3[3;5] Lactobacillus 9[9; 9] 9[9;9] 9[7;9] 9[7;9] 9[7;9] 6[3;9] 7[4;9] E. coli 4.9[0;5.5] 4.6 [2.3;5.0] 5.3 [5.1;5.5] 4.2 [2.0;4.6] 0 [0;0]x 2.2[0.0; 4.5] 0 [0;0]x OMC 8.4[8.0;8.6] 8.5 [8.3;8.6] 8.6 [8.5;8.6] 7.7 [7.5;7.7]^,* 0 [0;0]^,x 8.2 [8.0;8.4]*, & 8.2 [7.9; 8,2]х,#

Note: MO - Microorganisms, TMC - Total microbial count, No. 1 - group No. 1, No. 2 - Group No. 2;

^ p≤0.013 by Mann-Whitney test when compared with intact rats;

* p<0.012 according to the Wilcoxon test when compared with group No. 1 on day 1;

^x p<0.012 according to the Wilcoxon test when compared with group No. 2 on day 1;

^& p<0.012 according to the Wilcoxon test when compared with group No. 1 on day 6;

^# p<0.012 according to the Wilcoxon test when compared with group No. 2 on the 6th day.

As experiments have shown, with physical swimming load until complete fatigue in the mode of 1 time in 5 days without modeling dysbiotic disorders in group No. 1, by the 16th day of the experiment, there is a tendency to increase the time of active swimming (Table 1). The obtained results indicate that the selected regimen of exposure does not contribute to the rapid development of the state of fitness and resistance to exercise in animals, which are necessary conditions for assessing the role of the microbiotic state of the intestine in adapting to increasing physical exertion.

In group No. 2, the dynamics of physical performance indicators on the 11th day of the experiment was disturbed. A statistically significant decrease in the swimming time of the animals was observed (Table 1). This is due to antibiotic-associated intestinal dysbacteriosis in rats, the development of which was confirmed by bacteriological analysis (Table 2).

Bacterial analysis showed that physical swimming loads led to the modulation of the intestinal microflora: on the 6th day there was a statistically significant decrease in the TMC index by 0.7-0.8 orders of magnitude compared with intact rats and relative to their initial values before swimming, and on the 16th day of the experiment there was a statistically significant recovery of the TMC index relative to the level on day 6, but a tendency to a decrease in the content of E. coli. Such changes are probably part of the body's natural adaptive reactions during the stage of physiological stress (Kartysheva, S. I. Physiology of physical exercises: educational manual for students studying in the specialty 050720 "Physical culture" and the direction "Pedagogical education", profile "Physical culture" / S. I. Kartysheva. - Voronezh: Voronezh State Pedagogical University, 2012. - 176 p.; Gubert C., Kong G., Renoir T., Hannan A. J. Exercise, dietandstressasmodulatorsofgutmicrobiota: Implicationsforneurodegenerativediseases // NeurobiologyofDisease. 2020 Vol. 134. No. 104621. DOI: 10.1016/j.nbd.2019.104621). 12 hours after the completion of the course administration of gentamicin, the TMC indicator statistically significantly decreased to an undetectable level both relative to intact animals and its initial values. At the same time, the content of E. coli in the large intestine decreased statistically significantly compared to the values before the administration of the drug, and there was also a tendency for a decrease in the content of bifidobacteria (Table 2). It should be noted that 10 days after the completion of the administration of the antimicrobial drug, a spontaneous quantitative restoration of the microflora of the large intestine of rats was observed, however, with a mixture of the proportion of its individual representatives: E. coli was not recorded, but a tendency towards a decrease in lactobacilli was characteristic.

Thus, a dysbiotic disorder, characterized by a general decrease in the number of representatives of the intestinal microflora and a pronounced imbalance in the number of Escherichia, caused a violation of adaptation to physical activity, expressed in a decrease in the swimming time of rats on the 11th day of the experiment. The data obtained indicate that dysbacteriosis accelerated the development of physical fatigue in rats in the swimming test.

The participation of microflora in maintaining the physical performance of animals was also confirmed by the parallel recovery of these two indicators by the 16th day of the experiment (Tables 1 and 2).

The study of metabolic changes in the recovery period after 4 sessions of physical activity showed a tendency to reduce the concentration of lactate in the blood plasma of animals of group No. 1 (2.1 [1.6; 2.9] mmol/l) compared with intact (4.4 [ 3.4; 5.4] mmol/l; p = 0.050, Mann-Whitney U-test). At the same time, changes in energy metabolism on the background of physical activity in violation of the intestinal microbiome were more pronounced: the concentration of lactic acid in the blood plasma by 18- On the first day of the experiment, it was statistically significantly lower (1.3 [1.2; 1.7] mmol/L) by 3 times compared with intact animals (p = 0.014, Mann-Whitney U-test). These data indicate that with intestinal dysbiosis, the rate of recovery of energy metabolism after physical exertion decreases.

Thus, the proposed method allows you to create a model, during the formation of which in animals there is a violation of the normal adaptive shift in the body when exposed to physical activity. The resulting model adequately reflects the problems of changes in the physiology of labor associated with a violation of the adaptive processes of the body due to dysbiotic disorders of the intestine, and to a greater extent can serve to find ways and means of preventing and correcting the decline in physical performance under the influence of factors, including harmful factors of the labor process. leading to disruption of the intestinal microflora.

Claims (2)

A method for modeling physical activity in rats with dysbiotic disorders of the intestine is carried out by oral administration of the antimicrobial drug gentamicin at a dose of 30 mg 1 time per day for 5 days, starting from the 1st day of the experiment; animals are also subjected to forced swimming with a load attached to the tail and constituting 7% of the animal's body weight, by four times forced swimming with an interval of 1 time in 5 days; use the installation, which is a pool of opaque material, 98 cm high and 50 cm in diameter, the height of the water column in the pool is 60 cm; the temperature is maintained at 29-30°C; the animals are subjected to swimming until they are completely exhausted, the criterion of which is the unsuccessful attempts of the animal to rise to the surface of the water for more than 10 s and / or sink to the bottom of the pool.