CN111011310A

CN111011310A - Construction method of hypoxia quick habituation animal model

Info

Publication number: CN111011310A
Application number: CN201911377882.2A
Authority: CN
Inventors: 王小波; 余志斌; 赵汝舟; 程九华; 蒋帅
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2020-04-17

Abstract

The invention discloses a method for constructing a rapid hypoxia habituated animal model, which is simple in modeling method, only needs to be fasted in advance to adapt to rats, and then carries out habituation at an air pressure of 4500m in a low-pressure cabin, and has the advantages of low cost, short period, high modeling rate which can reach 100% after 34h, good repeatability and stability, and can gradually transit to human body experiments.

Description

Construction method of hypoxia quick habituation animal model

Technical Field

The invention belongs to the field of biology, and particularly relates to a construction method of a hypoxia quick habituation animal model.

Background

The plateau area of China is vast and occupies about 1/4 of the land area of China, wherein the average altitude of the Qinghai-Tibet plateau is more than 4000m, and the altitude of Himalayan mountains forming the middle seal boundary is more than 7000 m. It is known that for every 5km increase in altitude, the partial pressure of oxygen decreases as the atmospheric pressure decreases to about 1/2; at an altitude of 7500m, the atmospheric pressure is reduced from 760mmHg to 287mmHg, the oxygen partial pressure is reduced from 150mmHg to about 50mmHg, the requirement of the organism is difficult to meet, acute high altitude hypoxia is caused, for mammals without hypoxia habituation, the mammal can stay for only a few minutes at the altitude, and the prolonged stay time can cause the loss of consciousness until death. In these areas, emergency situations may occur, and people living in low altitude areas for a long time may need to quickly enter the plateau area to perform tasks, and the emergency entry of people into the plateau area is likely to cause altitude reaction of people who enter the plateau initially due to the influence of low air pressure and hypoxia, and even threatens life safety. At present, the research aiming at preventing altitude stress is more and more, and the main prevention measures comprise the steps, oral medicines and the like. The current accepted conventional high-altitude clothes scheme is step-up, which is time-consuming and labor-consuming although the effect is obvious and is difficult to deal with sudden situations. In addition, a great deal of research suggests that the rhodiola rosea has a certain prevention effect by oral administration in advance, but the effectiveness is unstable, and the individual difference of curative effect is large. Therefore, how to realize fast habituation of hypoxia is still a hot problem to be solved urgently at present. It was found that SD rats have a high similarity in their signal pathways to low-pressure hypoxic stimuli and to the human body. Therefore, the SD rat model is used for researching the fast hypoxia habituation, and effective experimental basis can be provided for the fast hypoxia habituation of a human body. Establishing an effective hypoxia fast habituation SD rat model has important significance for the research in the field.

The current study uses various hypoxia regimens in SD rats, but most of them achieve the desired height by a step-up. For example, Ahmad Y et al found that SD rats all died due to acute hypoxia in an environment of 7600m, while rats were habituated at 4500m for 10h, then left for 1h under normoxic conditions, then immediately placed at 7600m for 24h before all survived. In addition, there are models of hypoxia at atmospheric pressure, circulatory hypoxia, blood hypoxia, tissue hypoxia, and the like. These models suggest that SD rats have the ability to rapidly habituate hypoxia, however, we found some problems in repeated experiments: the animal model of AhmadY et al, the effect was not stable, and after SD rats were treated with these treatments (the experimental conditions were strictly performed according to the reported parameters of AhmadY et al), 100% survival could not be achieved in 7600m environment.

Disclosure of Invention

The invention aims to overcome the defects, provides a construction method of a rapid hypoxia habituation animal model, solves the problems of instability, poor repeatability and the like in the conventional model, and provides experimental data and good reference for the next step of exploring a rapid hypoxia habituation scheme.

In order to achieve the above object, the present invention comprises the steps of:

randomly selecting a plurality of rats, fasting for 24 hours in advance, and only providing drinking water to adapt the rats;

step two, placing the rat in a constant-temperature variable-pressure cabin, closing a cabin door, simulating the air pressure height rising to the altitude of 4500m at the speed of 10m/s, and only providing food and drinking water for the rat so as to enable the rat to learn;

step three, slowly reducing the altitude to the current altitude at the speed of 20m/s, and opening the cabin door for placing so that the rat can learn to wear;

step four, closing the cabin door, increasing the air pressure to the altitude of 7600m at the speed of 10m/s, providing food and water for the rat in the cabin, and observing the anoxia endurance of the rat;

and step five, slowly reducing the altitude to the current altitude at the speed of 20m/s, and opening the cabin door to complete the construction of the animal model.

In the first step, the rat is a healthy SPF male SD rat, and the weight of the rat is 200-250 g.

In the first step, the rat is adapted to an environment temperature of 22 +/-5 ℃ and a humidity of 50 +/-5%.

In the second step, after the cabin door is closed, the constant temperature in the cabin is kept at 22 +/-5 ℃, the humidity is kept at 50 +/-5%, and the airflow speed is 4L/min.

In the second step, the atmospheric pressure at an altitude of 4500m is maintained for 10 h.

In the third step, the cabin door is opened and placed for 1 h.

In the fourth step, after the cabin door is closed, the constant temperature in the cabin is kept at 22 +/-5 ℃, the humidity is kept at 50 +/-5%, and the airflow speed is 4L/min.

In step four, the barometric pressure altitude of 7600m above sea level is maintained for 24 h.

Compared with the prior art, the modeling method of the hypoxia fast habituated animal model constructed by the invention is simple, rats are adapted only by pre-fasting, then the hypoxia habituated animal model is subjected to air pressure habituation of 4500m in a low-pressure cabin, the cost is low, the period is short, the model can be modeled within 34h at the fastest speed, the modeling rate is high and can reach 100%, and the model has good repeatability and stability and can be gradually transited to human body experiments.

Drawings

FIG. 1 is a statistical chart comparing the present invention with the prior art.

Detailed Description

The invention will be further explained with reference to the drawings.

The invention comprises the following steps:

randomly selecting a plurality of healthy SPF male SD rats with the weight of 200-250 g, fasting in advance, providing drinking water, feeding the SD rats in an IVC cage with the ambient temperature of 22 +/-5 ℃ and the humidity of 50 +/-5%, adapting the rats, and maintaining for 24 hours;

step two, placing the rat in a constant-temperature pressure-changing cabin, closing a cabin door, simulating the air pressure rising to the altitude of 4500m at the speed of 10m/s, keeping the constant temperature in the cabin at 22 +/-5 ℃, the humidity at 50 +/-5% and the airflow speed at 4L/min, only providing drinking water for the rat, and keeping for 10 hours to enable the rat to get habit;

step three, slowly reducing the altitude to the current altitude at the speed of 20m/s, opening the cabin door and placing for 1h to enable the rat to learn;

step four, closing the cabin door, increasing the air pressure to 7600m above sea level at the speed of 10m/s, keeping the constant temperature in the cabin at 22 +/-5 ℃, the humidity at 50 +/-5% and the airflow rate at 4L/min, providing food and water for the rat in the cabin, maintaining 24h, observing the anoxia endurance of the rat, and determining the habituation effect;

1. Selecting healthy SPF male Sprague-Dawley (SD) rats with similar age (8 weeks of age) and weight of 200-250 g. SD rats were housed in IVC cages at 22 + -5 deg.C and 50 + -5% humidity. Maintaining a 12 hour day-night cycle. Each cage was housed with 3 rats, food and water.

2. After 3 days of feeding adaptation, the group was randomly divided into a normal Control group (CON), a Fasting group (FA), a hypoxia habituation group (HA), and a Fasting complex hypoxia habituation group (F & H).

3. The fasted group was fasted beforehand and provided with water only for 24 h.

4. The hypoxia habituation group places rats in a low-pressure oxygen chamber of a small animal with a customized design, closes the chamber door, simulates the rat to rise to the altitude of 4500m at the speed of 10m/s, keeps constant temperature and humidity in the chamber at 22 +/-5 ℃ and 50 +/-5%, and keeps 4L/min of airflow, and only provides drinking water for the rats in the chamber for 10 hours. After the preset habit taking time is finished, the air inlet valve is adjusted, the altitude is slowly reduced to the normal altitude at the speed of 20m/s, the cabin door is opened, and the clothes are placed for 1 hour at room temperature and normal pressure (the pressure of the Western-An is about 730 mmHg).

5. The fasting compound hypoxia habitual group is treated according to the fasting group and then the hypoxia habitual group.

6. Four groups of rats are distributed into four chambers according to a random control principle, are marked and distinguished by basic fuchsin, simultaneously close the chambers, simulate to rise to an altitude of 7600m at a speed of 10m/s, keep constant temperature and humidity in the chambers at 22 +/-5 ℃ and 50 +/-5 percent respectively, and provide 4L/min of airflow, and provide food and water for the rats in the chambers for 24 hours. And finally, adjusting an air inlet valve, slowly reducing the air inlet valve to the normal altitude at the speed of 20m/s, opening a cabin door, counting the number of the surviving rats, and calculating the survival rate.

TABLE 17600 m survival rate of rats after 24h of hypoxic treatment

Grouping	Survival	Death was caused by death	Total of	Survival rate
					Control group (CON)	16	22	38	42.1％
Fasting group (FA)	34	9	43	79.1％
					Low oxygen garment (HA)	19	13	32	59.4％
Fasting complex hypoxia habituation group (F)&H)	31	0	31	100.0％

The above experimental data were analyzed using grapgppad Prism 8.0 software. Comparing the groups by adopting R multiplied by C table chi-square test analysis, and further comparing every two groups by adopting a Tukey method if the difference between the groups has statistical significance.

Referring to fig. 1, compared to CON, the survival rates of rats in FA, HA and F & H groups were statistically significantly or very significantly different (P <0.05 or P < 0.01); FA was not statistically different (P >0.05) compared to HA group; f & H had a very significant statistical difference compared to the FA group (P < 0.01); f & H had a very significant statistical difference compared to the HA group (P < 0.01).

The above results show that: the survival rate of the fasted compound hypoxia habituated group rats under the severe hypoxia condition reaches 100 percent, and the fasted compound hypoxia habituated group rats can be repeated and are rat models capable of fast hypoxia habituation.

Experimental research shows that SD rats subjected to fasting composite hypoxia habituation for a certain time can completely survive in 7600m of extreme hypoxia environment, and fast hypoxia habituation and 100% survival are realized. The rat model of fast hypoxia habituation is not reported in the literature so far.

Claims

1. A construction method of a hypoxia quick habituation animal model is characterized by comprising the following steps:

randomly selecting a plurality of rats, fasting in advance, and only providing drinking water to adapt the rats;

step two, placing the rat in a constant-temperature low-pressure cabin, closing a cabin door, simulating the air pressure height rising to the altitude of 4500m at the speed of 10m/s, and providing food and water for the rat to habitually take the rat;

2. The method for constructing a hypoxia quick habituation animal model according to claim 1, wherein in the first step, the rat is a healthy SPF male SD rat, and the weight of the rat is 200-250 g.

3. The method for constructing a hypoxia animal model, according to claim 1, wherein in the first step, the rat is acclimatized to an ambient temperature of 22 ± 5 ℃ and a humidity of 50 ± 5%.

4. The method for constructing a hypoxia animal model, according to claim 1, wherein in the second step, after the door is closed, the constant temperature in the chamber is kept at 22 ± 5 ℃, the humidity is kept at 50 ± 5%, and the air flow rate is 4L/min.

5. The method for constructing a hypoxia animal model, according to claim 1, wherein in the second step, the air pressure at an altitude of 4500m is maintained for 10 hours.

6. The method for constructing a hypoxia animal model, according to claim 1, wherein in the third step, the cage door is opened and the animal model is left for 1 hour.

7. The method for constructing a rapid hypoxia animal model according to claim 1, wherein in the fourth step, after the door is closed, the constant temperature in the chamber is kept at 22 ± 5 ℃, the humidity is kept at 50 ± 5%, and the airflow rate is 4L/min.

8. The method for constructing a hypoxia animal model for rapid habituation, according to claim 1, wherein in the fourth step, the air pressure at an altitude of 7600m is maintained for 24 hours.