Animal model construction method for simulating crowd flood trapped fatigue and application

Abstract

The invention discloses an animal model construction method for simulating crowd flood trapped fatigue and application, and belongs to the technical field of fatigue animal model establishment. The whole environment (temperature, humidity and illumination intensity) is controlled through the full-automatic artificial climate box so as to simulate the high-temperature and high-humidity environment, food shortage in flood disasters is simulated in combination with a calorie intake mode, and therefore a novel animal fatigue model consistent with flood trapped fatigue induction factors is established. The modeling method is simple and effective, the environment of victims in flood disasters is simulated, a closer animal model is provided for research on reasons, mechanisms and progress of fatigue caused by flood trapping, a certain basis is provided for subsequent development of food functional ingredients for relieving fatigue under the condition of flood trapping, and the modeling method is suitable for use and popularization.

Description

Technical Field

[0001] The invention relates to a method for constructing an animal model simulating fatigue of people trapped by floods and waterlogging and application thereof, belonging to the technical field of establishing fatigue animal models. Background Art

[0002] There are many definitions of fatigue, such as "a protective physiological phenomenon in which the body avoids damage to tissues caused by excessive exercise or overwork", "a temporary decrease in exercise capacity or body function caused by exercise", etc. The most unified definition is the one proposed at the Fifth International Biochemical Exercise Conference in 1982: "Fatigue is the inability of the body's physiological process to continue its function at a certain level or to maintain a predetermined exercise intensity". And the current mechanism of fatigue is believed to be that fatigue is caused by one or more factors such as depletion of energy storage substances, oxidative stress in the body, and accumulation of metabolic products, which lead to decreased exercise capacity and reduced desire for exploration. The World Health Organization has listed fatigue as a major factor endangering human life and health.

[0003] In recent years, natural disasters have occurred frequently due to climate change. As people explore natural disasters in depth, it is found that people will experience fatigue during floods, which not only reduces the survival probability of victims but also increases the difficulty of subsequent rescue. In order to develop anti-fatigue functional ingredients that can be suitable for people affected by disasters, the efficacy verification experiment of animal models must be carried out first. At present, a variety of animal models simulating fatigue have been established, mainly small animals (mice and rats) as model objects. Macroscopically, they can be divided into three categories: exercise fatigue model, sleep deprivation fatigue model and restraint fatigue model. Exercise fatigue models can be divided into: rat exhaustion swimming model, treadmill fatigue model, sleep deprivation model is mostly through the water platform method and sleep deprivation instrument method, and the most commonly used method of restraint fatigue model is the long-term restraint method of restraint tube. However, the above traditional models are all animal models of single inducement, and for the actual flood-trapped people, the fatigue inducements that affect their occurrence are multiple and superimposed, and the existing animal fatigue models are not suitable, which seriously affects the development of anti-fatigue functional ingredients suitable for people affected by disasters.

[0004] In order to better develop anti-fatigue functional ingredients suitable for disaster-stricken people and understand the mechanism of fatigue among people trapped by floods, it is necessary to establish an animal model that is consistent with the causes of fatigue caused by floods in reality, thereby providing a solid foundation for subsequent research and development work. Summary of the invention

[0005] The purpose of the present invention is to provide a method for establishing an animal model of fatigue caused by simulating a trapped environment caused by floods. The trapped environment of mice is simulated by restraining cages, and the mice are exposed to a high temperature and high humidity environment based on the trapped environment in combination with a fully automatic artificial climate chamber. The calorie intake restriction is then combined to simulate the food shortage scenario in the flood-stricken area, thereby establishing an animal model of fatigue caused by simulating a trapped environment caused by floods, which can be used for the needs of related fields.

[0006] The purpose described in the present invention is achieved through the following technical solutions:

[0007] A method for constructing an animal model simulating fatigue caused by a flood-trapped environment, wherein the animals are first raised in a normal environment, their average daily calorie intake is calculated, and then they are transferred to a restraint cage adjusted according to the body size of mice, and the restrained mice are transferred to a high temperature and high humidity environment for modeling for 3 to 7 days. Every day, 30 to 60% of the mass percentage of the normal mouse food intake is fed according to the weight of the mice.

[0008] The present invention uses restraint cages to place mice in an environment with limited living space, and combines a fully automatic artificial climate chamber to provide mice with a high temperature and high humidity environment with a circadian rhythm, so that the mice can be exposed to conditions more similar to the flood-trapped environment. In addition, the calorie intake of mice is restricted to simulate the food shortage phenomenon in the disaster area, thereby constructing an animal fatigue model caused by the flood-trapped environment.

[0009] In one embodiment, the caloric intake is measured as caloric intake per gram of body weight.

[0010] In one embodiment, the animal restraint cage is preferably one that does not affect the free water and food intake of mice.

[0011] In one embodiment, the space of the restraint cage is a space in which the animal is placed in a volume of 5-7 cubic centimeters per gram of body weight.

[0012] In one embodiment, the model animal is a mouse or a rat.

[0013] In one embodiment, the mouse is a male ICR mouse.

[0014] In one embodiment, the selected model animal is 8-12 weeks old.

[0015] In one embodiment, after the adaptive feeding is completed and before the formal modeling begins, all animals need to be raised under the same conditions, and the daily calorie intake per gram of the animals is counted.

[0016] In one embodiment, the circadian rhythm in the fully automatic artificial climate box should be consistent with that of the blank group, generally with a 12 / 12h day and night alternation.

[0017] In one embodiment, the modeling temperature in the fully automatic artificial climate box needs to be switched according to the circadian rhythm, with high temperature during the day and low temperature or normal temperature at night.

[0018] In one embodiment, the daytime temperature is controlled at 33-36°C and the nighttime temperature is controlled at 20-24°C.

[0019] In one embodiment, the humidity is controlled to be 90-100%.

[0020] In one embodiment, since the mice in the model group are in a high temperature and high humidity environment, the mouse feed and drinking water need to be replaced every day.

[0021] In one embodiment, after the model is established, the core temperature of the animals needs to be monitored every 1-2 days to prevent the mice from experiencing severe heat exhaustion.

[0022] The flood-trapped fatigue animal model obtained by the method of the present invention is characterized by decreased weight, decreased body fat rate, accumulation of metabolites, and reduced spontaneous movement behavior. The inducing stimulus factors and clinical pathological behavioral manifestations of the model are closer to the actual flood-trapped reaction.

[0023] The present invention also provides application of the method in testing and / or screening functional products for individuals trapped by floods.

[0024] Beneficial Effects

[0025] The present invention constructs an animal fatigue model for flood-trapped environments, which is more in line with the fatigue characteristics of people under flood-trapped conditions than traditional fatigue construction methods such as exhausting exercise and sleep deprivation.

[0026] The model mice constructed by the present invention showed fatigue symptoms such as shortened spontaneous movement distance, decreased movement speed, and reduced desire for independent exploration; they also showed decreased body fat content, increased free water content, and decreased content of energy reserve substances such as muscle glycogen and liver glycogen, which are consistent with the characteristics of people trapped by floods.

[0027] The animal model constructed by the present invention can provide a targeted evaluation method for the screening and development of functional ingredients of anti-fatigue emergency foods, fill the gap in the current evaluation method of functional ingredients of anti-fatigue emergency foods, and serve as the basis for the industrialization of functional ingredients of anti-fatigue emergency foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Figure 3 shows the weight changes (a), body temperature detection (b), body imaging before and after modeling (c), and body composition images (df) of mice in the blank group and mice trapped in the flood modeling.

[0029] Figure 2The spontaneous movement trajectory (a), moving distance (b), movement speed (c), center area trajectory proportion (d), area change times (e), slow movement time proportion (f) and movement stop time proportion (g) of the blank group mice and the flood-trapped model mice.

[0030] Figure 3 Blood lactate (a), blood ammonia (b), blood urea nitrogen (c), muscle glycogen (d), and liver glycogen (e) contents of mice in the blank group and mice trapped by floods. DETAILED DESCRIPTION

[0031] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or components having the same or similar functions.

[0032] The following examples are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention. On the contrary, the purpose of providing these examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. And unless otherwise specified, all experimental animals, consumables, and reagents can be obtained through general commercial channels.

[0033] Example 1 Construction of model animals

[0034] The steps include:

[0035] S1. Experimental animals: This example uses 8-week-old ICR mice weighing 35-40g. Before the modeling experiment, the mice were placed in an SPF animal room for adaptive feeding for one week. The feeding conditions for the adaptive feeding for one week were a temperature of 22°C, a humidity of 55%, and a light exposure time of 12h. The mice were given SPF feed and bedding, and sterilized drinking water and SPF bedding were replaced every day.

[0036] S2. Blank control group: After the adaptive feeding, the mice in the blank group were kept at a temperature of 22°C, a humidity of 55%, and a light exposure of 12h. The mice were given SPF-grade feed and bedding, and sterilized drinking water and SPF-grade bedding were replaced every day.

[0037] S3. Model establishment: In this example, mice were placed in a restraint (6 cm*6 cm) environment with 40% of normal diet for 3-5 days, and were alternated between day and night every 12 hours. The temperature and humidity conditions during the day were set to T=34(±2)°C, RH=95(±5)%, and the temperature and humidity conditions at night were set to T=22(±2)°C, RH=95(±5)%. Mice were given SPF-grade feed (40% of normal caloric intake) and bedding, and sterilized drinking water and SPF-grade bedding were replaced every day.

[0038] The spatial restraint of mice can be adjusted according to the weight of the mice so that each gram of body weight occupies 5-7 cubic centimeters of space.

[0039] Example 2 Characterization of the state of model animals

[0040] 1. Physiological characteristics detection

[0041] Weight loss and body fat content reduction are physiological characteristics of people trapped by floods. Based on this, the weight and body composition monitoring of the model mice constructed by the present invention includes the following steps:

[0042] S1. Body weight monitoring: Specific implementation method Referring to Example 1, the body weight of mice was monitored every day after the animal adaptation period.

[0043] S2. Body composition monitoring: In this embodiment, a special cage for body composition measurement of mice made of polytetrafluoroethylene (inner diameter: 2.3 cm, outer diameter: 3.0 cm) is used to fix the moving mice and ensure that the mice can breathe freely during the test. The mouse cage is placed in the center of the radio frequency coil of the nuclear magnetic resonance imaging analyzer, and the CPMG sequence is used at 32°C to measure the transverse relaxation information of the mice. Test parameters: P1 (!s) = 15, P2 (!s) = 29, SW (kHz) = 200, TW (ms) = 3000, NECH = 5000, NS = 8, DL1 (ms) = 0.100.

[0044] The statistical results showed that the body weight of the animals in Example 1 decreased by 19.25% ( Figure 1 a) There was no significant difference in body temperature ( Figure 1 b). Compared with the blank group, the body composition of the model group ( Figure 1 df), the muscle content had no significant change, the fat content decreased (P<0.001), and the free water content increased (P<0.0001).

[0045] 2. Behavioral testing of model animals

[0046] When the human body is in a state of fatigue, there will be a decrease in exercise ability and exploration behavior, including the following steps:

[0047] S1. Open field experiment: Specific implementation method: Refer to Example 1. After the modeling is completed, the model animals and the control animals are placed in a 50*50*50 cm acrylic box, and the animal behavior monitoring system is used to record the animal's movement trajectory, movement speed, number of grid crossings, fast / slow movement time and other data within 3 minutes.

[0048] Before the experiment officially begins, the animals need to be transferred to the experimental room to adapt for 1-2 hours. When the above behavioral experiments are carried out, it is best for the experimenters and animals to be in two different rooms to reduce human interference. After each animal monitoring is completed, the above acrylic box is thoroughly cleaned with 75% ethanol to prevent odor and other factors from interfering with subsequent experiments.

[0049] The statistical results showed that compared with the control group, the spontaneous movement distance of the mice in Example 1 was shortened by 60.88% ( Figure 2 b, P<0.001), movement speed decreased by 23.51% ( Figure 2 c, P<0.001), the proportion of slow exercise time increased by 102.80% ( Figure 2 f, P < 0.01) showed that the animals' motor ability decreased, and the number of area changes during the test period decreased by 42.71% ( Figure 2 e, P<0.0001), the central trajectory ratio was shortened by 3.57% ( Figure 2 d, P<0.001), and the exercise cessation time was prolonged by 252.61% ( Figure 2 g, P<0.001) indicates that the animals' desire for exploratory activities is reduced.

[0050] 3. Monitoring of biochemical indicators of model animals

[0051] The increase in the content of metabolites such as urea nitrogen and blood ammonia in the body and the decrease in the content of energy storage substances such as muscle glycogen and liver glycogen can lead to fatigue in the body, including the following steps:

[0052] S1. Biochemical detection: Specific implementation method: Refer to Example 1. After the model animal is anesthetized intraperitoneally with isoflurane, the blood of the model animal is collected by removing the eyeball. After standing at room temperature for 60 minutes, it is centrifuged at a centrifugal force of 3500g for 10 minutes, and the upper serum is carefully aspirated into a sterile EP tube to obtain the model animal serum sample, which is stored in a -80°C refrigerator.

[0053] The levels of urea nitrogen, blood ammonia, and lactic acid in serum were tested, and the levels of muscle glycogen and liver glycogen were tested using tissue supernatant.

[0054] The statistical results showed that compared with the control group, the serum urea nitrogen ( Figure 3 c, P<0.05), blood ammonia levels increased by 83.79% and 51.83% ( Figure 3 b, P<0.05), lactic acid content decreased by 23.04% ( Figure 3 c, P<0.0001). Liver ( Figure 3 d, P<0.01) and muscle ( Figure 3 e, P<0.0001) decreased by 56.58% and 14.41%, respectively.

[0055] The above results indicate that the method for rapidly and specifically constructing a fatigue mouse model provided by the embodiments of the present invention can rapidly obtain an ideal flood-trapped fatigue mouse model.

[0056] Comparative Example 1:

[0057] Specific implementation method is as in Example 1, except that the temperature of the artificial climate box is adjusted to 36 (± 2) ° C during the day, the other conditions are the same as the blank control group, and the test is carried out according to the method of Example 2, and the results are as follows:

[0058] The mortality rate of mice in Comparative Example 1 exceeded 50% 24 hours after the start of modeling. To meet the ethical requirements of animal experiments, the remaining animals in the group were euthanized to end the experiment.

[0059] Comparative Example 2:

[0060] The specific implementation method is as in Example 1, except that the animals are subject to the same intake restriction as in Example 1 every day, and the other conditions are the same as the blank control group, and the test is carried out with reference to Example 2, and the results are as follows:

[0061] Compared with the blank control group, the body weight of mice in comparison example 2 decreased by 15.52%, the body temperature did not change significantly, the muscle content increased, the fat content decreased, and the free water content did not change significantly (P>0.05).

[0062] There were no significant changes in moving distance, average speed, center area trajectory, number of area changes, slow movement time, and stop movement time (P>0.05).

[0063] The muscle and liver glycogen contents decreased by 8.53% and 39.04% respectively, the blood ammonia content increased by 16.60%, and the serum lactic acid and serum urea nitrogen contents had no significant changes (P>0.05).

[0064] Comparative Example 3:

[0065] Specific implementation method is as in Example 1, except that the animals are raised in the same high temperature and high humidity environment as in Example 1, and the other conditions are the same as the blank control group, and the test is carried out with reference to Example 2, and the results are as follows:

[0066] Compared with the blank control group, the weight of mice in comparison example 2 decreased by 7.70%, the body temperature did not change significantly, the muscle content decreased, the fat content decreased, and the free water content increased.

[0067] There were no significant changes in moving distance, average speed, center area trajectory, slow movement time, and stop movement time (P>0.05), and the number of area changes decreased by 21.29%.

[0068] Serum lactic acid and liver glycogen levels decreased by 23.71% and 20.73% respectively, blood ammonia level increased by 16.60%, and muscle glycogen and serum urea nitrogen levels showed no significant changes (P>0.05).

[0069] Comparative Example 4:

[0070] Specific implementation method is as in Example 1, except that the animals are raised in the same space restraint environment as in Example 1, and other conditions are the same as the blank control group, and the test is carried out with reference to Example 2, and the results are as follows:

[0071] Compared with the blank control group, the body weight of mice in comparison example 2 decreased by 5.21%, the body temperature did not change significantly, the muscle content decreased, and the fat and free water content did not change significantly (P>0.05).

[0072] There were no significant changes in the moving distance, average speed, number of area changes, slow movement time, and stop movement time (P>0.05), and the trajectory of the central area decreased by 2.08%.

[0073] Serum lactic acid and muscle glycogen levels decreased by 28.41% and 6.62%, respectively, while liver glycogen, blood ammonia and serum urea nitrogen levels increased by 80.61%, 21.71% and 34.94%, respectively.

[0074] Comparative Example 5:

[0075] The specific implementation method is the same as Example 1, except that taurine is administered to the animals by gavage at a fixed time every day, and the other items are the same as Example 1, and the test is carried out with reference to Example 2, and the results are as follows:

[0076] Compared with the blank control group, the body weight of mice in comparison example 2 decreased by 19.61%, the body temperature did not change significantly, the fat content decreased, and the muscle content and free water content did not change significantly (P>0.05).

[0077] The moving distance to the center, the area trajectory, and the number of area changes decreased by 28.36%, 3.51%, and 33.86%, respectively. The slow movement time was prolonged by 115.62%, and there was no significant change in the stop movement time and the average speed (P>0.05).

[0078] Serum lactic acid and muscle and liver glycogen levels decreased by 16.02%, 8.53%, and 39.04%, respectively; blood ammonia level increased by 16.60%; serum urea nitrogen level showed no significant change (P>0.05).

[0079] The above results show that the modeling intensity of Example 1 is too high, which may cause the death of animals. Examples 2-4 show fatigue characteristics, but they are not enough to cover the fatigue characteristics of people trapped by floods. Example 1 successfully constructs an animal fatigue model based on a flood-trapped scenario. Through Example 5, it is found that taurine, a traditional anti-fatigue substance, can alleviate the fatigue symptoms caused by the present invention. The present invention proves the effectiveness of the model from three perspectives: appearance, prediction and structure.

[0080] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for constructing an animal model simulating fatigue caused by a flood-trapped environment, characterized in that: Animal models of fatigue are obtained through dietary restriction, temperature regulation, humidity regulation, and space restraint.

2. The method according to claim 1, characterized in that: The model construction time is 3 to 7 days.

3. The method according to claim 2, characterized in that: The restricted diet refers to a feeding amount that is 30-60% of the mass percentage of the normal diet of the animal.

4. The method according to claim 3, characterized in that: The temperature adjustment refers to controlling the daytime temperature to 33-36°C and the nighttime temperature to 20-24°C.

5. The method according to claim 4, characterized in that: The humidity adjustment refers to controlling the humidity to be 90-100%.

6. The method according to claim 5, characterized in that The spatial restraint is performed without affecting the normal drinking and eating of the animals.

7. The method according to claim 6, characterized in that The space restraint refers to placing the animal in a space with a volume of 5-7 cubic centimeters per gram of body weight.

8. The method according to claim 7, characterized in that: The animals include, but are not limited to, mice or rats.

9. The method according to claim 8, characterized in that The fatigue animal model has the following characteristics: (1) Weight loss; (2) Decreased body fat percentage; (3) accumulation of metabolites; (4) Decreased spontaneous motor behavior.

10. Use of the method according to any one of claims 1 to 9 in testing and / or screening functional products for individuals trapped by floods.

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