Device for conducting experimental studies of interval normobaric hypoxic training on small laboratory animals and method for conducting interval hypoxic training on small laboratory animals

Abstract

FIELD: medical equipment.SUBSTANCE: group of inventions relates to medical equipment, namely, to a device for conducting experimental studies of interval normobaric hypoxic training on small laboratory animals and a method for conducting interval hypoxic training on them. The device contains a transparent chamber divided by a removable lattice partition into individual compartments for small laboratory animals, a removable lid that can be used to close the transparent chamber, a computer with control software, and a compressed nitrogen cylinder equipped with a reducer. The removable cover contains a pressure regulator with mechanical pressure adjustment and a nitrogen gas inlet valve, valves and pipes with fans for purging the transparent chamber with atmospheric air, an electronic control unit, a digital oxygen level sensor in the transparent chamber, and a fan that can mix the gas mixture in the transparent chamber. A pressure-regulating gear and a nitrogen gas inlet valve are connected by a supply hose to a compressed nitrogen cylinder, and a computer is connected by cables to a digital oxygen level sensor in a transparent chamber and an electronic control unit. In the method, animals are placed in a transparent chamber, and the values of the exposure interval with a reduced oxygen concentration level, the exposure interval with a normal oxygen concentration level, the period and duration of purging during exposure with a normal oxygen concentration level, the number of exposure cycles with a reduced and normal oxygen concentration level, and the levels of normal and reduced oxygen concentration are set. Next, experimental studies of interval normobaric hypoxic training on animals are conducted with control of the operation of fans and valves for introducing nitrogen gas and atmospheric air. Then, the animals are removed from the transparent chamber with removal of the removable lattice partition and the removable lid. Next, the transparent chamber, the removable lattice partition, and the removable lid are cleaned and disinfected, followed by drying and assembly.EFFECT: technical result is to provide the possibility, convenience, and safety of conducting experimental studies of interval normobaric hypoxic training on animals.2 cl, 3 dwg.

Description

[0001] The invention relates to a group of inventions in the field of experimental physiology and medicine, in particular to a device for simulating certain environmental conditions in an experiment, namely to an automated installation for conducting experimental studies on small laboratory rodents (mice, rats) of periodic hypoxic training by simulating short-term states of normobaric hypoxia alternating with periods of normoxia and the method of its operation.

[0002] Hypoxic training can be used for therapeutic, health-improving, and preventative purposes, as activators of immunity and adaptive potential, and can also be used to increase strength and endurance in healthy individuals and athletes. However, expanding the scope of application, namely, selecting optimal regimens and studying the mechanisms involved, is impossible without experimental studies on laboratory animals.

[0003] Known hypoxia simulation devices for hypoxic training of athletes, the prevention of certain human diseases, or experimental purposes, positioned on the medical hypoxicator market as of the filing date, typically utilize the principle of membrane gas separation to create hypoxic mixtures of a specific composition. Chambers, tents, face masks, and portable artificial respiration apparatus are used to deliver these mixtures to the patient [1-4]. The main drawback limiting their experimental use, in addition to their high cost, is that these devices are exclusively designed for human use.

[0004] As of the filing date, nitrogen gas is used in experimental animal setups to reduce the oxygen content in hypoxic mixtures. These setups are typically flow-through chambers, which results in high gas consumption. Devices suitable for certain hypoxic training options for small laboratory animals are known. To create a hypoxic mixture in such devices, either an air compressor and gas mixing chamber [5-6] must be installed outside the main chamber of the hypoxicator, in addition to a nitrogen cylinder, or an oxygen cylinder [7] must also be installed in addition to the nitrogen cylinder, significantly increasing the complexity, size, and cost of the design.The main disadvantage of these devices is the lack of automation; the required oxygen level in the chamber is maintained by purging it with an appropriate gas mixture at a rate determined by the readings of the rotameters on each line and manually adjusted by taps and valves for precise gas supply, which significantly complicates the study of interval hypoxic training modes with short (5 min) and multiple (3-12 episodes) periods of hypoxia.

[0005] Close to the declared technical solution in terms of purpose are: the commercially available device “Hypoxia Isolator ProOx-100” manufactured by “Shanghai TOW Intelligent Technology” [8] and a commercial hypoxic chamber, previously offered by the now defunct group of companies “Vivarium” [9], which are intended for keeping animals in conditions with reduced or increased oxygen content, but have an additional option for the automated creation of interval hypoxia, and allow for periodic hypoxic training in some modes.

[0006] The applicant's prototype is the "ProOx-100 Hypoxia Isolator," which is designed to simulate hypoxic / hyperoxic conditions. This device requires cylinders of compressed nitrogen and oxygen. It consists of an animal chamber and a separate control unit, including a built-in automatic gas dosing system with associated software and a 7-inch touchscreen, an oxygen concentration detector and controller with temperature compensation, and detectors and controllers for temperature, humidity, pressure, and carbon dioxide concentration.

[0007] The disadvantages of the known technical solution are:

[0008] - the need for an oxygen cylinder in addition to the nitrogen cylinder for operation, which, among other things, increases the danger for the experimenter and tightens the requirements for the premises and the qualifications of the personnel;

[0009] - the time required to reduce oxygen levels from 21% to 9% in such chambers is more than two minutes, and to increase the level from 9% to 21% requires at least 4.5 minutes. This significantly distorts such common hypoxic interval training regimens as three- or five-time cycles of 5 minutes of hypoxia (9-16% O2) / 15 minutes of reoxygenation at normoxia (21% O2);

[0010] - excessive technical complexity (and cost) for interval hypoxic training of devices containing, in addition to an oxygen sensor, sensors and regulators of temperature, humidity, carbon dioxide concentration and a system for its removal, relief valves and pressure regulators, etc.;

[0011] - inability to train animals in home cages.

[0012] The technical problem that is solved by the stated technical solution is the long duration of the reduction and normalization of oxygen content to a given level, which distorts training modes, especially the most effective short and intense ones, as well as the increased danger when using compressed oxygen.

[0013] The technical objective of the claimed technical solution is to develop an efficient, reliable, convenient, safe to operate and inexpensive automated installation for conducting experimental studies of periodic normobaric hypoxic training by modeling short-term states of normobaric hypoxia alternating with periods of normoxia on laboratory animals, and a method for its use.

[0014] The stated technical problem is solved by using a device, which is an automatic system controlled by a computer via a control unit and valve system. It simulates normobaric hypoxia in a semi-hermetic chamber by displacing atmospheric oxygen with nitrogen supplied to the chamber. Normoxia in the chamber is maintained by purging with outside air. The device consists of a small transparent chamber (which can be replaced with a standard home cage) divided into individual compartments by a removable lattice partition. A tightly fitting lid houses a pressure regulator and nitrogen inlet valve, valves and pipes with fans for purging the chamber with outside air, a digital oxygen level sensor, a control electronic unit, and a fan for mixing the gas mixture in the chamber.The device is connected by cables to a computer with specially developed software and a supply hose with a cylinder of compressed nitrogen equipped with a reducer.

[0015] The technical result consists of enabling the convenient, safe, and feasible conduct of experimental studies of interval normobaric hypoxic training in animals. At the same time, purging with atmospheric air allows for the rapid normalization of oxygen levels in the semi-hermetic chamber, eliminating the need for compressed oxygen cylinders, additional valves, and sensors. The chamber's small size and internal fan accelerate the achievement of the desired hypoxia level. This, combined with automation, allows for precise adherence to even brief and intense hypoxic training regimens, increasing the repeatability of research results and their translational potential.

[0016] A distinctive feature of this setup is the ability to fine-tune and control the creation of hypoxic episodes alternating with normoxia within the chamber, taking into account their short duration. The proposed design enables the modeling of various regimens of periodic hypoxic training on small laboratory rodents due to the ability to controllably vary the oxygen concentration in the air environment inside the chamber with animals within the range of 1-21% (optimally 9-21%) with an accuracy of 0.1%, the duration of hypoxic (optimally 5 min) and normoxic (optimally 15 min) episodes within the range of 0-99 min with an accuracy of 1 s, and the number of hypoxia / reoxygenation cycles from 1 to 99 (optimally 3-10 per day).

[0017] Fig. 1 shows the general view of the developed device.

[0018] The claimed device consists of the following structural elements:

[0019] 1 - Animal camera;

[0020] 2 - removable lattice partition;

[0021] 3 - top removable cover;

[0022] 4 - reducer with mechanical pressure regulation and nitrogen gas inlet valve;

[0023] 5 - valves and pipes with multi-directional fans;

[0024] 6 - electronic control unit;

[0025] 7 - digital oxygen level sensor in the chamber;

[0026] 8 - fan;

[0027] 9 - cable from O2 level sensor;

[0028] 10 - cable from the control unit;

[0029] 11 - computer;

[0030] 12 - supply hose;

[0031] 13 - compressed nitrogen cylinder;

[0032] 14 - gearbox.

[0033] Fig. 2 shows a photograph illustrating a variant of the design and application of the developed device for performing periodic hypoxic training in laboratory rats.

[0034] Fig. 3 shows an example of the user interface of the device control software.

[0035] The animal chamber (1) (Fig. 1) is made of materials that do not absorb odor and are resistant to urine and alcohol, with the minimum possible internal volume to quickly create hypoxia, but sufficient for comfortable placement of animals in the chamber, while the upper edge coincides with standard home cages for the possibility of replacement. In the embodiment of the chamber for 6-12 rats, the overall dimensions are recommended to be within the range of no less than 450 × 200 × 150 mm and no more than 600 × 300 × 300 mm. The chamber can be conveniently divided into individual compartments by a removable lattice partition (2), which does not interfere with air mixing in the chamber and is made of material resistant to rodent teeth and urine, which ensures the same conditions for each animal and facilitates observation. The chamber or home cage is closed from above with a tightly fitting removable lid (3), providing conditional tightness. In the embodiment shown in Fig. 2, the cover is provided with a sealing ring (in Fig.2 not indicated) and is made heavy enough so that the animals inside the chamber cannot move it, without additional locking elements, which allows for the release of excess pressure, and is also completely removable, which provides easy access to the interior of the chamber for changing animals and cleaning the chamber. On the cover of the device there is a reducer with mechanical pressure regulation and a valve for inlet of gaseous nitrogen (4), which replaces oxygen in the gas mixture; valves and pipes (5) with multi-directional fans for through-purging of the chamber with atmospheric air, which allow for rapid creation of normoxia due to the release of the hypoxic gas mixture from the chamber and its replacement with air from the environment; an electronic control unit (6) and a digital sensor of the oxygen level in the chamber (7), which allow for monitoring and automatic regulation of the O2 level; a fan (8) for mixing the gas mixture in the chamber is mounted under the cover.The device is connected via a cable (9) from the O2 level sensor and a cable (10) from the control unit to a computer (11) running specially developed software. A supply hose (12) connects it to a compressed nitrogen cylinder (13) equipped with a reducer (14). The pipe for purging the chamber with atmospheric air (5) can be connected to the ventilation system via a corrugated air exhaust pipe. All sensors, pipes, and other system components are mounted on the removable upper lid (3) of the chamber (1), allowing the use of home cages as a hypoxic training chamber. All sensors and pipes leading into the chamber, as well as the internal fan, are covered with grates to prevent animals from damaging them or getting into the channels.

[0036] Figure 3 shows a version of the control program user interface. The "O2 Level" window displays current readings from the oxygen concentration sensor in the chamber; the "Current Status" window displays information about the action currently being performed by the device (nitrogen supply, air purging, exposure to low or normal O2 concentration, waiting for the start or end of operation); and the "Time to completion" window displays the number of minutes remaining in the cycle phase. The "Low O2 Exposure Interval," "Normal O2 Exposure Interval," and "Number of Cycles" input fields allow the user to specify hypoxia / reoxygenation time intervals and the number of their repetitions with precision down to the second.In the "Low O2 Level" field, the experimenter enters the desired oxygen concentration in the hypoxic mixture. When the "Start" button is pressed, the hardware and software system supplies nitrogen to the animal chamber and operates the internal fan until the O2 sensor reaches this level. It then automatically closes the nitrogen inlet valve and begins counting the duration of the hypoxic episode. After the specified hypoxic time has elapsed, the control program opens the external valves and activates the inlet and outlet fans to purge the chamber with atmospheric air until the oxygen concentration specified in the "Normal O2 Level" field is reached. Changing this value allows the device to be used at ambient air oxygen concentrations other than 21%.During normoxia, when the O2 level in the chamber drops by 1% below the set value, automatic purges are initiated. The frequency and duration of additional purges during reoxygenation can also be pre-set in the corresponding fields. Thus, after the set oxygen level is reached, the unit begins counting down the time. After the set time has elapsed, the chamber automatically moves to the next interval, the number and duration of which are determined by the researcher. Figure 3 also shows that manual control of all valves and fans is also available.

[0037] The claimed method of interval hypoxic training of small laboratory animals is implemented by performing the following sequence of actions:

[0038] 1. Laboratory animals, such as rats, are placed in the chamber.

[0039] 2. Using the user interface of the software product, set the values ​​of the low O2 holding interval, the normal O2 holding interval, the additional purge period during holding with normal O2, the number of cycles, the low O2 level, the normal O2 level, and the additional purge duration during holding with normal O2 by pressing the "Start" button.

[0040] 3. In automatic mode, the software controls the operation of fans and valves for inlet of nitrogen and atmospheric air.

[0041] 4. In the manual mode of the device, fans and valves are controlled by selecting the corresponding interface button.

[0042] 5. After the experiment, the animals are removed from the chamber, which is disassembled by removing the removable lattice partition and the removable top cover.

[0043] 6. The chamber, removable grille, and top removable cover are cleaned of animal feces and urine, and all components are washed and disinfected. After drying, the device is reassembled.

[0044] This invention enables the safe and convenient automated simulation of hypoxic normobaric conditions and the implementation of intermittent hypoxic training in various modes. The efficiency, reliability, and convenience of the developed automated setup reduce labor costs during experimental studies of intermittent hypoxia in small laboratory rodents, which, for example, has made a significant contribution to solving such a pressing physiological problem as the development of non-drug methods for correcting stress-induced disorders and improving adaptation to stress. The invention is also successfully used to study the physiological mechanisms underlying the effects of intermittent hypoxic training, a pressing task due to the high therapeutic potential of this method.

[0045] For example, the developed automatic setup was used to simultaneously create a 5-minute normobaric hypoxia followed by 15-minute reoxygenation for an entire group of rats, three or more episodes of hypoxia / reoxygenation (5 / 15 min) per day in a row every day for 3 days. The effects of five interval training modes were experimentally studied: three five-minute hypoxia sessions at 16% O2 per day, three sessions at 12% O2, seven sessions at 12% O2, 15 sessions at 12% O2, and three sessions at 9% O2. The effects of these test hypoxic effects were studied in experimental models of psychoemotional stress and post-stress anxiety-depressive states in rats. As a result of the conducted work, it was found that periodic hypoxia significantly increases stress resistance in models of anxiety-depressive pathologies. The most noticeable influence on the severity of the effect is exerted by the intensity of hypoxic exposure.The 3×9% O2 regimen proved to be the most effective of all those tested. When the hypoxia level was reduced to 12% O2, the effectiveness of the antidepressant or anxiolytic effect was partially or completely preserved by regimens with an increasing number of sessions (3, 7, and 15). However, an increase in the number of sessions from 3 to 7 (in the 3×12% O2 and 7×12% O2 regimens) did not lead to an increase in the anxiolytic effect, so such an increase is inappropriate. A decrease in the intensity to 16% O2 leads to a significant decrease in the main stress-protective properties of hypoxia in these pathology models

[10] .

[0046] After testing several hypoxic training regimens with different durations, frequency, and intensity for their adaptive effectiveness, we selected three regimens, categorized as the most, medium, and least effective. The selected regimens were identical in duration—3 days—and frequency—3 hypoxic episodes of 5 minutes per day. They differed in intensity—the oxygen concentration in the hypoxic mixture was 9%, 12%, or 16%. In animals from the group with the highest intensity, the oxygen content in the hypoxic gas mixture was 9%—3×9% O2; in the medium-effective group, the O2 content during training was 12%—3×12% O2; and in the group with the least effective IHT regimen, it was 3×16% O2.The most intense and effective regimen, 3x9% O2, was shown to have the greatest impact on clinical blood test parameters in rats, in terms of duration and amplitude. It triggered an increase in red blood cell counts, reduced variability in their volumes, and shifted the balance of lymphokine and monokine effects toward a calm activation response. During the first 24 hours after training, total serum antioxidant capacity significantly decreased at 9 and 12% oxygen, followed by a rapid normalization. This is consistent with the dynamic response of pro- and antioxidant systems to non-damaging hypoxia. A stimulating effect characteristic of conditioning was observed for all studied interval training regimens on the basal and stress-related activity of the hypothalamic-pituitary-adrenocortical axis. All observed post-training changes can be attributed to basic adaptive mechanisms that contribute to increased resilience to adverse factors.Based on a comparative analysis of protocols with different exposure intensities, a regime was identified that caused the greatest pro-adaptive shifts in the studied blood parameters

[11] .

[0047] The claimed device was developed within the framework of the Federal project “Biomedical and cognitive technologies of the future” of the National project “New technologies for saving health” (No. FMMU-2025-0003).

[0048] An additional advantage of the proposed invention over hypoxicators is that it is a device specially adapted for periodic normobaric hypoxic training of small laboratory animals, and is therefore significantly cheaper and easier to manufacture and operate.

[0049] List of used literature

[0050] 1. Serebrovskaya, T. V., & Xi, L. (2016). Intermittent hypoxia training as non-pharmacologic therapy for cardiovascular diseases: Practical analysis on methods and equipment. Experimental biology and medicine (Maywood, NJ), 241(15), 1708-1723. https: / / doi.org / 10.1177 / 1535370216657614.

[0051] 2. US Patent US 5799652 (A) "Hypoxic room, as well as equipment for hypoxic training and therapy at normal atmospheric pressure" (A61G10 / 00, A61G10 / 04, A61M16 / 10, B01D53 / 02, B01D53 / 047, published 01.09.1998).

[0052] 3. US Patent US 5964222 (A) "Hypoxic Tent" (A61G10 / 04, B01D53 / 02, B01D53 / 047, published 12.10.1999).

[0053] 4. Russian Federation Patent RU 2301686 C1 “Device for hypo-, hyperoxytherapy” (A61M 16 / 00 (2006.01), published 06 / 27 / 2007).

[0054] 5. Russian Federation Patent RU 178743 U1 “Device for modeling hypoxia in small laboratory animals” (G09B 23 / 28 (2006.01), published 04 / 18 / 2018).

[0055] 6. Russian Federation Patent RU 201120 U1 “Setup for conducting experimental studies on animals under hypoxic conditions at low temperatures” (G09B 23 / 28 (2006.01), A61M 16 / 00 (2006.01), published 11 / 27 / 2020).

[0056] 7. Labyntseva O.M. Combined effect of normobaric hypoxia and pulsed magnetic field on nonspecific resistance and tolerance of the rat organism to acute hypoxic hypoxia: Abstract of a PhD thesis. Cand. Biol. Sci. - Nizhny Novgorod: 2008. - 24 p.

[0057] 8. https: / / www.medicalexpo.ru / prod / shanghai-tow-intelligent-technology / product-298330-1051137.html (accessed - 01.2024).

[0058] 9. https: / / www.vivariy.com / products / gipoksicheskie-kamery-dlya-zhivotnyh (accessed - 12.2022).

[0059] 10. Zenko M.Yu., Baranova K.A., Kukina M.V., Rybnikova E.A. Effects of different modes of interval hypoxic training in experimental models of anxiety-depressive states in rodents / / Pavlov Journal of Higher Nervous Activity. 2023. Vol. 73. No. 6. P. 845-856.

[0060] 11. Baranova K.A., Zenko M.Yu., Rybnikova E.A. The effect of interval hypoxic training in different modes on blood parameters of rats / / I.M. Sechenov Russian Physiological Journal. 2024. Vol. 110. No. 1. P. 122-135. https: / / doi.org / 10.31857 / S0869813924010087.

Claims

1. A device for conducting experimental studies of interval normobaric hypoxic training on small laboratory animals, comprising a transparent chamber divided by a removable lattice partition into individual compartments for small laboratory animals, a removable lid configured to close the transparent chamber, a computer with control software, a cylinder with compressed nitrogen equipped with a reducer; wherein on the removable lid there is a reducer with mechanical pressure regulation and a valve for inlet of gaseous nitrogen, valves and pipes with fans for purging the transparent chamber with atmospheric air, an electronic control unit, a digital oxygen level sensor in the transparent chamber and a fan configured to mix the gas mixture in the transparent chamber;wherein the reducer with mechanical pressure regulation and the nitrogen gas inlet valve are connected by a supply hose to a cylinder with compressed nitrogen, and the computer is connected by cables to a digital oxygen level sensor in a transparent chamber and an electronic control unit.

2. A method for conducting interval hypoxic training of small laboratory animals using a device for conducting experimental studies of interval normobaric hypoxic training on small laboratory animals according to paragraph 1, characterized in that small laboratory animals are placed in a transparent chamber; the values ​​of the exposure interval with a reduced oxygen concentration level, the exposure interval with a normal oxygen concentration level, the period and duration of purging during exposure with a normal oxygen concentration level, the number of exposure cycles with reduced and normal oxygen concentration levels, and the levels of normal and reduced oxygen concentration are established using a computer with control software; experimental studies of interval normobaric hypoxic training on small laboratory animals are conducted using the device according to paragraph1 with control of the operation of fans and valves for the introduction of gaseous nitrogen and atmospheric air; small laboratory animals are removed from the transparent chamber by removing the removable lattice partition and removable lid; the transparent chamber, removable lattice partition and removable lid are cleaned and disinfected, followed by drying and assembly.

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